Cardiovascular Dysfunction and Altered Lysosomal Signaling in a Murine Model of Acid Sphingomyelinase Deficiency

Cardiovascular Dysfunction and Altered Lysosomal Signaling in a Murine Model of Acid Sphingomyelinase Deficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cardiovascular Dysfunction and Altered Lysosomal Signaling in a Murine Model of Acid Sphingomyelinase Deficiency Yun-Ting Wang, Alexandra K Moura, Rui Zuo, Kiana Roudbari, Jenny Z Hu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5154105/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Journal of Molecular Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Niemann-Pick Disease (NPD) is a rare autosomal recessive lysosomal storage disorder (LSD) caused by the deficiency of acid sphingomyelinase (ASMD), which is encoded by the Smpd1 gene. ASMD impacts multiple organ systems in the body, including the cardiovascular system. This study is the first to characterize cardiac pathological changes in ASMD mice under baseline conditions, offering novel insights into the cardiac implications of NPD. Using histological analysis, biochemical assays, and echocardiography, we assessed cardiac pathological changes and function in Smpd1 −/− mice compared to Smpd1 +/+ littermate controls. Immunofluorescence and biochemical assays demonstrated that ASMD induced lysosomal dysfunction, as evidenced by the accumulation of lysosomal-associated membrane proteins, lysosomal protease, and autophagosomes in pericytes and cardiomyocytes. This lysosomal dysfunction was accompanied by pericytes and cardiomyocytes inflammation, characterized by increased expression of caspase1 and inflammatory cytokines, and infiltration of inflammatory cells in the cardiac tissues of Smpd1 −/− mice. In addition, histological analysis revealed increased lipid deposition and cardiac steatosis, along with pericyte-to-myofibroblast transition (PMT) and interstitial fibrosis in Smpd1 −/− mice. Moreover, echocardiography further demonstrated that Smpd1 −/− mice developed coronary microvascular dysfunction (CMD), as evidenced by decreased coronary blood flow velocity and increased coronary arteriolar wall thickness. Additionally, these mice exhibited significant impairments in systolic and diastolic cardiac function, as shown by a reduced ejection fraction and prolonged left ventricular relaxation time constant (Tau value). These findings suggest that ASMD induces profound pathological changes and vascular dysfunction in the myocardium, potentially driven by mechanisms involving lysosomal dysfunction as well as both pericytes and cardiac inflammation. Niemann-Pick Disease acid sphingomyelinase deficiency lysosome storage disorder coronary microvascular dysfunction cardiac pathology pericyte-to-myofibroblast transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Niemann-Pick disease (NPD) is a rare autosomal recessive disorder caused by acid sphingomyelinase (ASM) activity deficiency (ASMD) 1 . ASM, encoded by the sphingomyelin phosphodiesterase 1 (Smpd1) gene, is a lysosomal hydrolase that breaks down sphingomyelin into phosphorylcholine and ceramide 2 . Mutations in Smpd1 caused ASMD leads to the accumulation of sphingomyelin in lysosomes, resulting in lysosomal storage disorder (LSD) 3 . The initial accumulation of lysosomal sphingomyelin in ASMD triggers the subsequent buildup of other lipids within lysosomes, with cholesterol being the most prominent 4 – 6 . Furthermore, lipids derived from sphingomyelin, including ceramide and its downstream metabolites (e.g., sphingosine), lysosphingomyelin (sphingosylphosphorylcholine), glycosphingolipids, and bis(monoacylglycero)phosphate, also accumulate 6 . Over time, these accumulated lipids are recycled to the plasma membrane and redistributed to other cellular compartments, disrupting cellular homeostasis and causing secondary abnormalities across multiple organelles 6 , 7 . There are two subtypes of NPD caused by ASMD, NPD type A (NPD A) and NPD type B (NPD B). NPD A patients have less than 1% of normal enzyme activity, leading to the early onset of rapid systemic and neurodegenerative symptoms, typically reducing their lifespan to around 3 years 8 . In contrast, NPD B patients have 5–10% of normal enzyme activity, do not exhibit neurodegenerative symptoms, and often survive into adulthood 9 . However, these patients present with a highly variable clinical phenotype and disease-related morbidities affecting multiple organ systems, including respiratory and cardiovascular systems 10 . Cardiac disease accounts for 7.2% of deaths among NPD B patients, making it the fifth leading cause of mortality, following respiratory disease (27.7%), liver disease (27.7%), and bleeding (9.6%) 10 . While NPD B patients have numerous cardiac conditions, valvular heart disease and coronary artery disease are the most common 11 . Previous studies have shown that ASM activation and ceramide accumulation contribute to the development of cardiomyopathy and heart failure in conditions such as high-fat diet (HFD), myocardial infarction (MI), and sepsis 12 – 16 . However, no comprehensive study has been conducted on the nature and extent of cardiac dysfunction and related cardiovascular events resulting from ASMD. Proper sphingolipid homeostasis is essential for maintaining health, and disruption of this finely regulated balance can result in various diseases. This phenomenon is evident in several LSDs, such as Gaucher and Farber diseases, where inherited lysosomal defects lead to sphingolipid accumulation and subsequent pathogenesis 17 . In this study, we use the Smpd1 knockout mouse model of ASMD to identify cardiac pathologies and dysfunction under baseline conditions. Our findings indicate that ASMD leads to perturbed cardiac function. ASMD-induced cardiac pathological changes encompass lysosomal dysfunction, cardiac inflammation, steatosis, fibrosis, coronary microvascular dysfunction (CMD), and overall cardiac dysfunction. These findings provide novel insights into the cardiac complications associated with ASMD. Materials and Methods Mice All experimental protocols received approval from the University of Houston Institutional Animal Care and Use Committee. We used female wild-type ( Smpd1 +/+ ) and acid sphingomyelinase deficient ( Smpd1 −/− ) mice, aged 5–6 months and of the C57BL/6J strain, for all experiments. The mice were kept in a temperature-controlled room with a 12-hour light-dark cycle and had free access to standard rodent chow and water. Following euthanasia, heart samples were collected and stored at -80°C for subsequent analysis. Echocardiography Transthoracic echocardiography was conducted using a Vevo 3100 micro-ultrasound imaging system with an MX550D-0073 probe (VisualSonics Inc., Canada). Mice were anesthetized with 1.5–2% isoflurane (ISO) in 100% oxygen and maintained at 1–1.5% isoflurane throughout the procedure. M-mode images were acquired from the parasternal short-axis or apical four-chamber views and analyzed using Vevo LAB 2.1.0. The flow velocity of the left anterior descending coronary artery was measured in pulsed-wave Doppler mode under baseline conditions and after induction of hyperemia with 1.0% and 2.5% isoflurane, respectively. Left ventricular relaxation time constant (Tau value) = (IVRT + IVCT)/MV ET. Antibodies and reagents Primary antibodies: NG2 (Abcam ab275024, Miltenyi Biotec 130-123-964), Troponin I (Abcam AB56357-1001), LAMP1 (BD 553792), LAMP2A (abcam ab18528), Cathepsin B (Abcam ab58802), LC3 (CST 12741S), p62 (Abcam ab109012), HMGB1 (Abcam ab79823), RAGE (Abcam ab3611), CD45 (Abcam ab25386), F4/80 (BD 565411), PLIN2 (Proteintech 15294-1-AP), TGFβ (CST 3711S), PDGFRα (CST 3174T, R&D AF1062-SP), FSP1 (CST 13018S), Pan-cadherin (Abcam ab51034), Vimentin (Abcam ab92547), Collagen 1 (Abcam ab21286), FITC-αSMA (Sigma F3777), Ceramide (Enzo, ALX-804-196-T050), β-actin (CST 3700S), GADPH (CST 2118S). Secondary antibody for immunofluorescence: anti-Mouse IgG, Alexa Fluor 488 conjugate (Thermo Fisher A-21202), Alexa Fluor 488 conjugate anti-Rabbit IgG (Thermo Fisher A21206), Alexa Fluor 555 conjugate anti-Mouse IgG (Thermo Fisher A-31570), Alexa Fluor 555 conjugate anti-Rabbit IgG (Thermo Fisher A-31572). Secondary antibody for western blot: IRDye® 800CW anti-Mouse IgG (LICOR 926-32212), IRDye® 800CW anti-Rabbit IgG (LICOR 926-32213), anti-Mouse IgG, HRP (Thermo Fisher A16011), stabilized peroxidase conjugated anti-rabbit (Invitrogen 32460), anti-rat IgG-HRP (Fisher 629520). Reagents: Oil red O (VWR BT135140-100G), Sirius red (Sigma 365548-5g), Isolectin GS-IB4 (Fisher I21411), WGA-FITC (Sigma L4895), Aurum Total RNA Mini Kits (Bio-Rad, 732–6820), iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 1708841), iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121). Immunofluorescence staining Frozen heart section slides were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After washing with PBS, the tissues were blocked and permeabilized using 5% BSA and 0.3% Triton X-100 in PBS for 1 hour at room temperature, then incubated with primary antibodies overnight at 4°C. Following washing with PBST (PBS with 0.05% Tween-20), the slides were incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 555 for 1 hour at room temperature. After another wash with PBST, the slides were mounted with DAPI mounting solution and analyzed using an Olympus IX73 or Leica STED 8 imaging system. Pearson correlation coefficient (PCC) of fluorescence image was quantified using Image-Pro Plus 6.0 software, as previously described 18 . Frozen heart sections were stained with WGA-FITC (5 µg/ml in PBS) for 30 minutes at room temperature, followed by staining with Troponin I using the immunofluorescence staining protocol described above. The cardiomyocyte outlines were identified through Troponin I-positive staining and WGA-FITC labeling of cell membranes. Cardiomyocytes with centrally localized nuclei should be used for analysis to ensure consistent orthogonal cross-sectional measurements, minimizing the influence of obliquely oriented cardiomyocytes. Cardiomyocyte size was then measured using Image-Pro Plus 6.0 software. Hematoxylin and eosin (H&E) staining H&E staining was carried out using a hematoxylin and eosin staining kit (Teomics HAE-1). Frozen heart sections were fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After washing with distilled water, the tissues were stained with Mayer’s hematoxylin solution for 5 minutes, followed by two washes with distilled water to remove excess stain. A bluing reagent was then applied for 10–15 seconds, followed by two additional washes with distilled water. The slides were then dipped in absolute alcohol to remove excess moisture and stained with Eosin Y solution for 2–3 minutes. After rinsing with absolute alcohol, the slides were cleared and mounted in synthetic resin, followed by prompt imaging with the Olympus IX73 system. Oil Red O staining Frozen heart sections were prepared at a thickness of 8 µm. The sections were air-dried on slides, fixed in 4% paraformaldehyde (PFA) for 15 minutes, and rinsed immediately in three changes of distilled water before being soaked for 20 minutes. To prevent water carryover, the sections were then washed with 60% isopropanol for 1 minute. Following staining with Oil Red O working solution for 30 minutes, the sections were rinsed with 60% isopropanol for 2 minutes and then rinsed twice with distilled water. Finally, the sections were mounted in mounting media, and images were captured promptly using the Olympus IX73 imaging system. Sirius red and fast green staining Frozen heart section slides were fixed with Kahle fixative solution for 15 minutes at room temperature. After washing with PBS, the slides were stained with 0.1% Sirius Red and Fast Green in a saturated aqueous solution of picric acid at room temperature for 60 minutes. The slides were then washed in two changes of acidified water (0.1N HCl in ddH2O) for 1 minute each. After a final wash with ddH2O, the sections were mounted in mounting media, and images were captured promptly using the Olympus IX73 imaging system. Quantitative Real-time PCR Total RNA was isolated from heart tissue using Aurum Total RNA Mini Kits (Bio-Rad, 732–6820). cDNA was synthesized from the isolated RNA using iScript Reverse Transcription Supermix (Bio-Rad, 1708841). Real-time PCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121) on the Bio-Rad CFX Connect Real-Time System. Primers for Nlrp3, Asc, Caspase-1, IL1β, IL18, IL6, IL8, TNF, Icam1, and Vcam1 were obtained from Bio-Rad. Additional primers used in this study are listed in the Supplementary Material Table 1. Cycle threshold (Ct) values were converted to relative gene expression levels using the 2-ΔΔCt method, and the data were normalized to the internal controls β-actin or GAPDH. Western blotting Heart tissue homogenates were lysed in Laemmli sample buffer (Bio-Rad, 161–0737), boiled at 95°C for 10 minutes, and then placed in an ice-cold ultrasonic bath for 5 minutes. The prepared samples were separated by 10–15% sodium dodecyl sulfate-polyacrylamide gels. Proteins were then transferred electrophoretically onto PVDF membranes at 35 V at 4°C overnight. The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween 20. After washing, the membranes were incubated with the appropriate primary antibodies, following the manufacturer's instructions. Following incubation, the membranes were washed with PBST and subsequently incubated with corresponding secondary antibodies for 1 hour at room temperature. Finally, the bands were washed with PBST, visualized, and analyzed using the LI-COR® Odyssey Fc System. Statistics analysis Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons between two groups were made using either a Student's t-test or the Mann-Whitney test. All analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). A p-value of less than 0.05 was considered statistically significant. Results Lysosomal and autophagosome accumulation in cardiac pericyte and myocardium Lysosomal storage disorder (LSD) is a hallmark and major contributor to lysosomal dysfunction in NPD 19 . To investigate the presence of lysosomal dysfunction, we first performed immunofluorescence analyses of lysosome or autophagy-related proteins on cardiac sections focusing on cardiac pericyte and cardiomyocytes of Smpd1 −/− mice in comparison to Smpd1 +/+ mice. The results showed lysosomal membrane protein LAMP1 (Fig. 1 A) and LAMP2A (Fig. 1 B), lysosomal protease cathepsin B (Fig. 1 C), and the autophagosome membrane protein LC3 (Fig. 1 D) were upregulated in cardiac pericyte of Smpd1 −/− mice. In addition to cardiac pericytes, these proteins were also elevated in the cardiomyocytes of Smpd1-/- mice ( Supplementary Fig. 1A-D ). These findings were further validated by immunoblot analyses (Fig. 1 E, 1 F). p62/Sqstm1, a macroautophagy receptor, is typically degraded during autophagy maturation, where autophagosomes fuse with lysosomes, leading to the degradation of their contents, including LC3 and p62/Sqstm1. Lysosomal dysfunction is often associated with impaired autophagic flux, resulting in the accumulation of LC3 and p62/Sqstm1. Consistent with the increased LC3 protein expression, the p62/Sqstm1 protein was also elevated in ASMD (Fig. 1 E, 1 F). However, the mRNA quantification of cardiac tissues showed that ASMD did not affect the gene expression of Lamp1 , Lc3 , and p62 , but did lead to increased Lamp2a levels (Fig. 1 G). The mRNA quantification confirmed the absence of the Smpd1 gene, but not the Smpd2 gene, (encoding neutral sphingomyelinase/NSM) in the cardiac tissues of Smpd1 −/− mice ( Supplementary Fig. 2A ). Serine palmitoyltransferase (SPT), a multi-subunit enzyme, catalyzes the first and rate-limiting step in de novo ceramide synthesis 20 . SPT activity is negatively regulated by ORMDL1-3, which are activated in response to increased ceramide levels 21 . ASMD had no effect on gene expression of SPT subunit Sptlc1 or Ormdl1-3 in cardiac tissues but led to upregulation of Sptlc2 ( Supplementary Fig. 2A ). Surprisingly, no significant difference in ceramide levels was observed between Smpd1 +/+ and Smpd1 −/− mice in either cardiac pericytes ( Supplementary Fig. 2B ) or cardiomyocytes ( Supplementary Fig. 2C ). These results suggest that while ASMD significantly reduces Smpd1 gene in cardiac tissues, it had compensatory upregulation of Sptlc2 , but minimal impact on the NSM-dependent ceramide or other de novo ceramide synthesis pathways. Cardiac inflammation and cellular infiltration Cardiac inflammation can lead to severe health conditions, including coronary artery disease, cardiac fibrosis, and heart failure 22 , 23 . Immunofluorescence studies were first conducted on cardiac sections of Smpd1 −/− and Smpd1 +/+ mice. The results revealed significantly increased infiltration of CD45-positive leukocytes (Fig. 2 A) and CD206-positive macrophage (Fig. 2 B) in the cardiac tissues in Smpd1 −/− mice compared to Smpd1 +/+ mice. However, no increase in F4/80-positive macrophage infiltration was observed in Smpd1 −/− mice (Fig. 2 C). To further characterize the inflammation in specific cell types, we analyzed the expression of HMGB1 (high mobility group box 1), a DAMP (damage-associated molecular pattern) linked to sterile inflammation, and its receptor RAGE (receptor for advanced glycation end-products) 24 . We observed that ASMD-induced HMGB1 upregulation was primarily localized in non-cardiac cells, such as pericytes (Fig. 2 D), while RAGE upregulation was predominantly observed in cardiomyocytes (Fig. 2 E). In addition, mRNA quantification (Fig. 2 F) from cardiac tissues of Smpd1 −/− mice showed increased expression of inflammation-related genes, including inflammatory Caspase-1 , Interleukin 18 (IL18) , Vcam1 (vascular cell adhesion molecule 1), and IL6 . However, no change was detected for other genes, including inflammasome component Asc (PYD and CARD domain-containing), Nlrp3 (NLR family pyrin domain-containing 3), IL1β , Icam1 (intercellular adhesion molecule 1), IL8 , and Tnfα . Cardiac steatosis and lipid accumulation To investigate whether ASMD-induced lysosomal dysfunction is associated with altered lipid storage in the cardiac tissues, we performed Oil Red O staining and immunostained for the lipid droplet-associated protein perilipin 2 (PLIN2) in cardiac tissue sections from Smpd1 −/− and Smpd1 +/+ mice. As shown in Fig. 3 A, a significant increase in Oil Red O staining was observed in the cardiac tissues of Smpd1 −/− mice, whereas no positive staining was detected in Smpd1 +/+ mice. Similarly, PLIN2 expression was significantly upregulated in Smpd1 −/− mice (Fig. 3 B-D). We further confirmed that ASMD-induced PLIN2 accumulation was localized in cardiac pericytes (Fig. 3 B), cardiac fibroblasts (Fig. 3 C), and cardiomyocytes (Fig. 3 D). We also examined the expression of genes involved in lipid droplet biogenesis, including Gpat4 (glycerol-3-phosphate acyltransferase 4), Agpat2 (1-acylglycerol-3-phosphate O-acyltransferase 2), Dgat1/2 (diacylglycerol O-acyltransferase 1 and 2), and Plin2/3 . As shown in Fig. 3 E, the mRNA levels of most of these genes were comparable between Smpd1 −/− and Smpd1 +/+ mice, except for a decrease in Dgat2 expression. These findings suggest that ASMD leads to lipid deposition in the myocardium, resulting in cardiac steatosis, which is not associated with increased lipid droplet biogenesis. Cardiac fibrosis and pericyte-to-myofibroblast transition While inflammation and steatosis were the most prominent cardiac changes observed in tissue samples from Smpd1 −/− mice, fibrosis was also evident. This was assessed using immunostaining for cardiac pericyte, fibroblast and myofibroblast markers, as well as Sirius red staining to visualize total collagen in cardiac tissue samples from Smpd1 −/− and Smpd1 +/+ mice (Fig. 4 A-F). Immunostaining demonstrated significant increases in the expression of proteins associated with pericyte-to-myofibroblast transition (PMT), a key process in cardiac fibrosis development 25 , 26 . We demonstrated that ASMD increased transforming growth factor beta (TGF-β) (Fig. 4 A), platelet-derived growth factor receptor alpha (PDGFRα) (Fig. 4 B), fibroblast-specific protein 1 (FSP1) (Fig. 4 C), vimentin (Fig. 4 D) and collagen 1 (Fig. 4 E) in cardiac pericytes. Sirius red staining confirmed an increase in collagen in the cardiac interstitial areas (Fig. 4 F). Coronary microvascular dysfunction and arteriolar remodeling Echocardiography was conducted to assess coronary microvascular function by measuring the coronary blood flow velocity of Smpd1 −/− and Smpd1 +/+ mice at baseline conditions (1.0% isoflurane) as well as under hyperemic conditions (2.5% isoflurane). Representative images and summarized data (Fig. 5 A and 5 B) demonstrated a significant decrease in coronary blood flow velocity at baseline in Smpd1 −/− mice compared to Smpd1 +/+ mice. However, no differences were observed under hyperemic conditions. Immunohistological analysis revealed increased coronary arteriolar wall thickness in Smpd1 −/− mice, which was attributed to increased media thickness, without evidence of neointima formation (Fig. 5 C). Further immunofluorescence analysis was conducted to evaluate the density of coronary arterioles (Fig. 5 D, α-SMA + arterioles) and myocardial endothelial cells to pericytes coverage in capillaries (Fig. 5 E, isolectin-IB4-labeled endothelial cells, NG2-labeled pericytes) in cardiac sections. These results showed no significant differences in the number of coronary arterioles and endothelial cells to pericytes coverage in capillaries between Smpd1 −/− and Smpd1 +/+ mice. These findings suggest that ASMD leads to coronary arteriolar remodeling and impaired coronary microvascular function. Cardiac systolic and diastolic dysfunction Finally, echocardiography was performed to investigate whether the cardiac pathologies and coronary microvascular dysfunction (CMD) observed in Smpd1 −/− mice are associated with changes in cardiac function and remodeling. Representative images and summarized data (Fig. 6 A, 6 B) revealed that Smpd1 −/− mice developed cardiac systolic dysfunction compared to Smpd1 +/+ mice. Specifically, Smpd1 −/− mice exhibited reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), along with increased systolic left ventricular internal diameter (LVID; s) and systolic left ventricle volume (LV Vol; s). However, there were no significant differences in diastolic left ventricular internal diameter (LVID; d) or diastolic left ventricle volume (LV Vol; d). As summarized in Fig. 6 C, no significant cardiac remodeling was observed in Smpd1 −/− mice, as evidenced by the similar calculated left ventricular mass, diastolic left ventricular anterior wall thickness (LVAW; d), systolic left ventricular anterior wall thickness (LVAW; s), diastolic left ventricular posterior wall thickness (LVPW; d), and systolic left ventricular posterior wall thickness (LVPW; s) between the groups. In addition, there was no significant difference in cardiomyocytes size between Smpd1 +/+ and Smpd1 −/− mice (Fig. 6 D). Cardiac diastolic function of Smpd1 −/− and Smpd1 +/+ mice was also assessed by measuring the left ventricular relaxation time constant (Tau value), isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), mitral valve ejection time (MV ET), and mitral valve E/A (MV E/A) peak (Fig. 6 E, 6 F). The results indicated the development of cardiac diastolic dysfunction in Smpd1 −/− mice, as shown by increased IVRT, IVCT, and MV ET, alongside a reduction in MV E peak, with no change in MV A peak. Together, these results suggest that ASMD leads to heart failure characterized by both cardiac systolic and diastolic dysfunction. Discussion The clinical manifestations of ASMD in patients with NPD are diverse and wide-ranging. In NPD A, the most frequent symptoms are central nervous system degeneration and respiratory failure. NPD B presents a broader range of clinical manifestations, with the most severely affected organs being the respiratory system, liver, and tissues associated with an atherogenic lipid profile 9 . In addition, an increasing number of studies have reported cardiovascular diseases associated with NPD B 3 , 9 , 10 . The Smpd1 knockout mouse serve as a well-established murine model of NPD. In this study, we identified significant cardiac pathology in this model, showing that ASMD results in lysosomal dysfunction, cardiac inflammation, steatosis, fibrosis, CMD, and overall cardiac dysfunction. LSDs encompass a group of diseases caused by deficiencies in lysosomal enzymes, membrane transporters, or other proteins critical for lysosomal function. These disorders are categorized based on the type of accumulated material, including lipids, mucopolysaccharidoses, and glycoproteinoses 17 , 27 . ASM plays a key role in hydrolyzing sphingomyelin to ceramide within lysosomes, and in ASMD, this process is disrupted, leading to alterations in the ‘sphingolipid rheostat’ 28 . This disruption results in imbalanced levels of sphingolipid species, such as sphingomyelin, ceramide, sphingosine, and their phosphorylated metabolites. These imbalances reorganize lysosomal membrane composition by forming sphingolipid-enriched microdomains 29 , which in turn redistribute membrane-associated ion channels or proteins, influencing either their stability or function. Sphingolipids like sphingomyelin and sphingosine may also directly interact with lysosomal membrane ion channels, modulating their activities and contributing to lysosomal dysfunction. For example, the lysosomal membrane cation channel TRPML1 is primarily responsible for mediating lysosomal Ca 2+ release. Previous studies have shown that TRPML1-mediated lysosomal Ca 2+ release is markedly reduced in cells from NPD patients 30 , likely due to the inhibitory effects of sphingomyelin accumulation within lysosomal membranes in ASMD. Our previous studies also confirmed that ASMD inhibits TRPML1-mediated lysosomal Ca 2+ release, which impairs dynein-dependent autophagosome trafficking, resulting in defective lysosome-autophagosome fusion and diminished autophagic flux in vascular smooth muscle cells from Smpd1 −/− mice 31 – 33 . These findings suggest that sphingolipids serve as major regulators for Ca 2+ -dependent lysosomal trafficking function and that ASMD disrupts this process, reducing the turnover of autophagosomes and lysosomes along with their contents. In the present study, we observed that ASMD increased the expression of lysosomal proteins LAMP1/2, cathepsin B, and autophagic proteins LC3 in cardiac pericytes and cardiomyocytes of Smpd1 −/− mice. Interestingly, ASMD had minimal effects on their mRNA levels, indicating that lysosome or autophagosome biogenesis is not substantially altered. Together, our findings reinforce the view that ASMD induces lysosomal dysfunction, leading to impaired autophagic flux in cardiac cells in a mouse model of NPD. Notably, there was no significant decrease in ceramide levels in ASMD mice in either cardiac pericytes or cardiomyocytes. Previous studies have reported higher ceramide levels in ASMD type B organoids compared to controls 34 . This elevation in ceramide levels may result from the activity of other sphingomyelinases located in non-lysosomal compartments, as described in ASM knockout mice by other researchers 35 . Furthermore, our data demonstrated an upregulation of the Sptlc2 gene in ASMD mice (Supplementary Fig. 2A), which could enhance the de novo synthesis of ceramide, providing a compensatory mechanism. Sphingosine, a vital bioactive sphingolipid, is generated from ceramide through the enzymatic action of ceramidases and is subsequently phosphorylated by sphingosine kinases to produce sphingosine-1-phosphate (S1P), a molecule that promotes cell survival 36 . The dynamic balance among sphingomyelin, ceramide, sphingosine, and S1P is crucial in modulating the effects of ASMD and determining the fate of the cell 37 , 38 . Future research will aim to quantify sphingomyelin, ceramide, sphingosine, and S1P levels using lipidomics to clarify their roles in ASMD and their contributions to cellular dysfunction, including inflammation, lipid accumulation, and PMT. This strategy will address current limitations and provide critical insights into the sphingolipid-driven mechanisms underlying ASMD. Accumulating evidence indicates that lysosomal dysfunction is intimately linked to inflammation through several mechanisms. Impaired autophagic flux caused by lysosomal dysfunction can lead to the accumulation of damaged organelles (e.g. damaged mitochondria), protein aggregates, and other undegraded cellular materials. In LSDs, severe dysfunction may cause lysosomal membrane destabilization or rupture which releases lysosomal enzymes and harmful substances, such as cathepsins, into the cytosol. These accumulated substances or lysosomal contents can be recognized by the immune system as DAMPs, triggering inflammatory responses through activation of pattern recognition receptors (PRRs). One of the best studied connections between the lysosomal-autophagy pathways and inflammation is the NLRP3 inflammasome pathway, which plays a key role in the progression of cardiovascular diseases 22 , 39 . NLRP3 inflammasome activation leads to increased inflammatory caspase-1 activity and production of pro-inflammatory cytokines, such as IL-1β and IL-18. It can also activate inflammatory caspase-11 and induce GSDMD-dependent pore formation in the plasma membrane, causing pyroptotic cell death. Additionally, HMGB1 is increasingly recognized as a key DAMP molecule actively released by immune cells or passively released from dying cells during infection or sterile inflammation 40 . HMGB1 is a multifunctional protein that exerts proinflammatory activity primarily by binding to RAGE 24 . The HMGB1/RAGE axis is critical in the pathogenesis of inflammatory cardiomyopathy 24 , 41 . Early inflammatory responses include the upregulation of adhesion molecules, such as VCAM-1 and ICAM-1, which facilitate the adhesion of CD45-positive leukocytes (e.g. lymphocytes, monocytes, eosinophils, and basophils) and CD206-positive macrophages to the myocardium 42 – 44 . In this study, cardiac tissue from Smpd1 −/− mice showed a significant increase in the number of CD45-positive leukocytes, CD206-positive macrophages and protein expression of HMGB1 and RAGE, along with elevated expression of inflammation-related genes, including Caspase1 , IL18 , Vcam1 , and IL6 . These findings align with previous research showing that vascular smooth muscle cells from Smpd1 −/− mice exhibit higher gene expression of IL18 and IL6 , as well as increased monocyte adhesion at basal levels or after PDGF stimulation 31 . Similarly, Elyse et al. reported that ASM knockdown in bronchial epithelial cells increased inflammation and neutrophil recruitment under both basal and infected conditions 45 . In contrast, ASM was shown to mediate pro-atherogenic stimulus-induced inflammation in macrophages and endothelial cells 46 , 47 . Furthermore, ASM activation can lead to excess ceramides, driving inflammation in various diseases, including cystic fibrosis (CF) 48 , 49 . Therefore, both the loss of ASM activity leading to lysosomal dysfunction and the activation of ASM resulting in excess ceramide production can trigger inflammatory responses depending on the stimulus or disease context. Nonetheless, our findings provide evidence that lysosomal dysfunction is linked to cardiac inflammation in ASMD. Cardiac steatosis refers to the abnormal accumulation of lipid droplets (mainly neutral triglycerides) within the myocardium. It has increasingly been recognized as a causative factor for cardiomyopathy, particularly in conditions such as severe aortic stenosis 50 and dilated cardiomyopathy 51 . Selective overexpression of DGAT1 (a key gene in lipogenesis) in cardiomyocytes induces cardiac steatosis and fibrosis 52 , exacerbating angiotensin II-induced heart failure in mice 53 . Although cardiac steatosis is often associated with metabolic conditions such as obesity, diabetes, and fatty liver disease, it can also occur in other pathological conditions, such as LSDs 3 . In some LSDs, such as Gaucher disease and Farber disease, abnormal lipid metabolism results in lipid storage within cells, such as hepatocytes 54 . Though not directly reported as a common symptom in LSDs, lipid droplet accumulation may occur in cardiomyocytes or other cell types in the myocardium and subsequently induce cardiac steatosis. Additionally, impaired autophagy caused by lysosomal dysfunction can exacerbate lipid accumulation by hindering the normal breakdown of lipid droplets. Indeed, in ASMD, cholesterol-enriched lipid droplets accumulate in human or murine macrophages, contributing to foam cell formation, a key mechanism in atherogenesis in metabolic disorders 55 . However, the link between ASMD and cardiac steatosis has not previously been reported in murine models. In this study, we show for the first time that Smpd1 −/− mice exhibit a significant increase in cardiac steatosis without elevated expression of lipogenesis-related genes. This suggests that cardiac steatosis in ASMD is primarily due to impaired lipid droplet degradation rather than increased lipid droplet biogenesis. Additionally, we observed the accumulation of the lipid droplet-associated protein PLIN2 not only in cardiomyocytes but also in cardiac pericytes and fibroblasts. The role of lipid droplets in the function of cardiac pericytes and fibroblasts remains largely unexplored and warrants further investigation. Our findings suggest that lipid droplets may play a pivotal role in the PMT process, which should be further studied in future research using Smpd1 pericyte- or fibroblast-specific knockout mice. Cardiac fibrosis, a complex pathophysiological process marked by excessive extracellular matrix accumulation, plays a key role in cardiac dysfunction and heart failure 25 . A key cellular mechanism driving fibrosis is the pericyte-to-myofibroblast transition (PMT) 25 , 26 . Fibrosis in various organs, such as the lung, liver, and endocardium, has been observed in patients with LSDs, including Farber disease and NPD B 3 , 9 . In this study, Smpd1 −/− mice exhibited elevated expression of TGF-β in pericyte, increased PMT, and fibrotic myofibroblast markers such as FSP1, vimentin, and collagen 1 in pericyte, and increased interstitial collagen deposition in cardiac tissue. These findings suggest that ASMD may promote cardiac fibrosis through the activation of PMT. Recent studies have identified pericytes as a significant source of myofibroblast via PMT 26 , 56 . Interestingly, our recent findings showed that vascular smooth muscle cells from Smpd1 −/− mice underwent a myofibrogenic transition when stimulated by PDGF, a process driven by lysosomal dysfunction, impaired autophagic flux, and the activation of p62/Nrf2 signaling axis 31 . Since pericytes and vascular smooth muscle cells originate from the same perivascular progenitors, it is reasonable that ASMD in pericytes promotes PMT, contributing to cardiac fibrosis. M2 macrophages derived from monocytes are known to play a critical role in cardiac fibrosis, primarily by promoting the activation of myofibroblasts through the secretion of profibrotic factors such as TGF-β1 and IL-1β 57 , 58 . Therefore, ASMD-induced cardiac fibrosis may result from the infiltration of CD206-positive M2 macrophages. Further studies are required to elucidate the mechanisms by which ASMD drives PMT, using pericyte-specific Smpd1 knockout mouse models and cultured pericytes. In addition, previous studies have implicated ASM activation in fibrosis, suggesting that targeting ASM activity could be a therapeutic approach. For instance, imipramine or cardiomyocyte-specific Smpd1 knockout has been shown to attenuate HFD-induced cardiac fibrosis via downregulating NADPH oxidase 4 (NOX4) 12 . Similarly, astaxanthin has been found to reduce myocardial infarction-induced cardiac fibrosis by inhibiting ASM activity 13 . Thus, like its role in cardiac inflammation, both ASMD and ASM overactivation can lead to fibrotic activation, depending on the specific stimulus or disease context. The findings from this study suggest that lysosomal dysfunction is closely associated with the development of cardiac fibrosis in ASMD. Coronary microvascular dysfunction (CMD) refers to functional or structural abnormalities in the coronary microcirculation that lead to impaired myocardial perfusion 59 , 60 . CMD can occur as a clinical condition independent of heart failure, and it may result in myocardial ischemia 61 , 62 . This ischemia may manifest as symptoms such as chest pain (angina) and can contribute to the progression of heart failure over time. Previous studies have identified CMD in LSDs including Fabry disease 63 – 66 and Danon disease 67 . In this study, non-invasive echocardiographic analysis of Smpd1 −/− mice revealed a significant reduction in coronary blood perfusion at baseline (1.0% isoflurane); however, no difference was observed under hyperemic conditions (2.5% isoflurane) (Fig. 5 ). Interestingly, this reduction at baseline was associated with structural remodeling of coronary arterioles, characterized by thickened arteriolar walls, while the number of mature coronary arterioles and endothelial cells to pericytes coverage in capillary remained unchanged (Fig. 5 ). Our findings of thickened coronary arteriolar walls align with previous studies showing coronary artery disease in NPD B 11 , as well as increased inflammation and dedifferentiation of vascular smooth muscle cell from Smpd1 −/− mice 31 , 33 . These findings support the view that ASMD promotes coronary arteriolar wall remodeling, resulting in CMD and impaired myocardial perfusion. Additionally, PMT and collagen 1 expression were increased in pericytes of interstitial capillary from Smpd1 −/− mice (Fig. 4 ), suggesting that extracellular matrix remodeling may increase capillary stiffness and exacerbate vasomotor dysfunction in thickened coronary arterioles. The precise mechanisms by which ASMD induces structural and functional changes in coronary microcirculation deserve further investigation. Cardiac diseases, including hypertrophic and dilated cardiomyopathy, and valvular diseases, are commonly found in LSDs 3 . Although cardiac disease accounts for 7.2% of deaths in NPD B patients, with a few reported cases of heart failure 11 , 68 , 69 , cardiac dysfunction has not been extensively investigated in murine models of NPD or ASMD. In this study, non-invasive echocardiographic analysis of Smpd1 −/− mice showed both systolic and diastolic cardiac dysfunction under baseline conditions. Myocardial relaxation is one of the earliest manifestations of mechanical dysfunction of the human LV. The time constant tau (Ƭ) is higher in the elderly and patients with hypertrophic cardiomyopathy (HCM), coronary artery disease (CAD), and cardiomyopathies 70 . Studies have shown that the lifespan of ASMD mice is typically less than 1 year, whereas wild-type mice generally live for over 2 years 71 , 72 . Therefore, it is reasonable to consider the approximately 6-month-old ASMD mice in our study as aged mice, particularly since they exhibit upregulation of lysosomal accumulation, inflammation, and fibrosis—hallmarks commonly associated with aging 73 – 75 . This ASMD-induced cardiac dysfunction was not associated with significant cardiac remodeling. Although ASMD induces significant cardiac fibrosis, it does not lead to notable cardiomyocyte hypertrophy. It would be valuable to investigate whether ASMD exacerbates cardiac remodeling in classical cardiac fibrosis models, such as isoproterenol-induced myocardial fibrosis. Our findings provide the first evidence linking ASMD to cardiac dysfunction in a murine model of NPD. While this study did not aim to further investigate the underlying mechanisms of ASMD and lysosomal dysfunction driving cardiac dysfunction, a lysosome-centered mechanistic model is proposed based on the observed cardiac pathologies (Fig. 7 ). In ASMD or NPD, the abnormal accumulation of lysosomal sphingomyelin leads to LSDs and lysosomal dysfunction in cardiac pericyte or cardiomyocyte. These lysosomal abnormalities may contribute to cardiac pericyte or cardiomyocyte inflammation/inflammasome activation, steatosis, PMT/fibrosis, and CMD. These pathological changes, either alone or in combination, ultimately lead to cardiac dysfunction. In future studies, we will implement targeted therapeutic strategies to address specific pathological changes in ASMD mice, aiming to identify the key contributors to ASMD-induced cardiac dysfunction. Although our study provides significant evidence that ASMD induces a wide range of cardiac pathological changes, there are several limitations. First, the specific cell types involved in these pathological changes remain unclear and may vary depending on the context of the observed pathology. Future studies should explore the role of ASMD by employing tissue-specific Smpd1 knock-out murine models in various cardiovascular cells, such as pericytes, cardiomyocytes, or fibroblasts, both under basal conditions and in disease models. This approach would provide a better understanding of how specific cell types contribute to the progression of ASMD-induced cardiac pathology. Second, although we propose a general mechanism by which ASMD-induced LSD leads to cardiac pathology, the lack of detailed mechanistic studies in the specific cell types limits the depth of our conclusions. Future in vivo or in vitro studies would provide definitive mechanistic insights into understanding the precise cellular and molecular pathways underlying ASMD-induced cardiac dysfunction. In conclusion, the present study provides novel insights into the cardiac pathology in mice with ASMD. The most notable pathological changes reveal a complex interplay between lysosomal dysfunction, inflammation/inflammasome activation, lipid accumulation, cardiac fibrosis, CMD, and overall cardiac dysfunction. The profound ASMD-induced CMD and cardiac dysfunction observed in murine models suggest that similar mechanisms may contribute to cardiovascular complications in patients with NPD B or ASMD, highlighting the need for early diagnosis and intervention to improve patient outcomes. These findings lay the foundation for future research aimed at developing therapeutic targets for cardiovascular complications in patients with NPD B or ASMD, while also calling for further investigation into the complex factors contributing to ASMD-induced cardiovascular dysfunction. Abbreviations NPD Niemann-Pick disease NPD A Niemann-Pick disease type A NPD B Niemann-Pick disease types B LSD Lysosome storage disorder ASMase Acid sphingomyelinase ASMD Acid sphingomyelinase deficiency Smpd1 Sphingomyelin phosphodiesterase 1 Tau Left ventricular relaxation time constant ISO Isoflurane CMD Coronary microvascular dysfunction H&E staining Hematoxylin and eosin staining PFA Paraformaldehyde LVAW Left ventricular anterior wall LVID Left ventricular internal end LVPW Left ventricular posterior wall LVEF Left ventricular ejection fraction LVFS Left ventricular shortening fraction LV Vol Left ventricle volume IVRT Isovolumic relaxation time IVCT Isovolumic contraction time MV ET Mitral valve ejection time α-SMA Alpha-smooth muscle actin LD Lipid droplet PLIN2 Perilipin 2 GPAT4 Glycerol-3-phosphate acyltransferase 4 AGPAT2 1-acylglycerol-3-phosphate O-acyltransferase 2 DGAT1 Diacylglycerol O-acyltransferase 1 DGAT2 Diacylglycerol O-acyltransferase 2 PMT Pericyte-to-myofibroblast transition TGF-β Transforming growth factor beta FSP1 Fibroblast-specific protein 1 HMGB1 High mobility group box 1 RAGE Receptor for advanced glycation end-products ASC PYD and CARD domain containing NLRP3 NLR family pyrin domain containing 3 IL1β Interleukin 1 beta IL18 Interleukin 18 VCAM1 Vascular cell adhesion molecule 1 ICAM1 Intercellular adhesion molecule 1 IL6 Interleukin 6 IL8 Interleukin 8 TNF-α Tumor necrosis factor alpha LAMP1 Lysosomal-associated membrane protein 1 LAMP2A Lysosome-associated membrane protein type 2A LC3A/B Microtubule-associated proteins 1A/1B light chain 3A/B p62 p62/SQSTM1 SPT Serine palmitoyltransferase SPTLC1 Serine palmitoyltransferase long chain base subunit 1 SPTLC2 Serine palmitoyltransferase long chain base subunit 2 ORMDL1 ORMDL Sphingolipid Biosynthesis Regulator 1 ORMDL2 ORMDL Sphingolipid Biosynthesis Regulator 2 ORMDL3 ORMDL Sphingolipid Biosynthesis Regulator 3 HFD High fat diet DAMP Damage-associated molecular pattern MI Myocardial infarction NG2 Nerve/glial antigen 2 Declarations Ethical approval and consent to participate Not applicable. Availability of data and material The authors declare that the data supporting the findings of this study are available within the paper, and its supplementary information files. Competing interests The authors declare no competing or conflicting interests. Funding This work was supported by the grants from the National Institutes of Health (R01HL150007, R01HL122937). Authors' contributions X.L. and Y.Z. designed the research and wrote the manuscript; Y-T.W. performed the experiments and contributed to writing the manuscript; Y-T.W., A.M., and R.Z. analyzed the data; K.R., J.H., S.K., Z.C.W., Y.P.S.T., M.W., P-L.L., and J.K.H. reviewed and edited the manuscript; and all authors have read and approved the final manuscript. References Schuchman EH. The pathogenesis and treatment of acid sphingomyelinase-deficient Niemann-Pick disease. J Inherit Metab Dis . 2007;30:654-663. doi: 10.1007/s10545-007-0632-9 Gorelik A, Illes K, Heinz LX, Superti-Furga G, Nagar B. 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Rev Med Interne . 2017;38:291-299. doi: 10.1016/j.revmed.2016.10.387 Fotoulaki M, Schuchman EH, Simonaro CM, Augoustides-Savvopoulou P, Michelakakis H, Panagopoulou P, Varlamis G, Nousia-Arvanitakis S. Acid sphingomyelinase-deficient Niemann-Pick disease: novel findings in a Greek child. J Inherit Metab Dis . 2007;30:986. doi: 10.1007/s10545-007-0557-3 Hirota Y. A clinical study of left ventricular relaxation. Circulation . 1980;62:756-763. doi: 10.1161/01.cir.62.4.756 Horinouchi K, Erlich S, Perl DP, Ferlinz K, Bisgaier CL, Sandhoff K, Desnick RJ, Stewart CL, Schuchman EH. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease. Nat Genet . 1995;10:288-293. doi: 10.1038/ng0795-288 Hagemann N, Mohamud Yusuf A, Martiny C, Zhang X, Kleinschnitz C, Gunzer M, Kolesnick R, Gulbins E, Hermann DM. Homozygous Smpd1 deficiency aggravates brain ischemia/ reperfusion injury by mechanisms involving polymorphonuclear neutrophils, whereas heterozygous Smpd1 deficiency protects against mild focal cerebral ischemia. Basic Res Cardiol . 2020;115:64. doi: 10.1007/s00395-020-00823-x Baechle JJ, Chen N, Makhijani P, Winer S, Furman D, Winer DA. Chronic inflammation and the hallmarks of aging. Mol Metab . 2023;74:101755. doi: 10.1016/j.molmet.2023.101755 Tan JX, Finkel T. Lysosomes in senescence and aging. EMBO Rep . 2023;24:e57265. doi: 10.15252/embr.202357265 Selman M, Pardo A. Fibroageing: An ageing pathological feature driven by dysregulated extracellular matrix-cell mechanobiology. Ageing Res Rev . 2021;70:101393. doi: 10.1016/j.arr.2021.101393 Supplementary Files 01KeymessagesJMMER2.docx SFigure1.jpg Supplementary material figure 1. Lysosome components and autophagosome accumulation in cardiomyocytes of ASMD mice. Representative immunofluorescence staining images for LAMP1/ Troponin I and summary of their PCC (A), LAMP2A/Troponin I and summary of their PCC (B), lysosomal protease cathepsin B/ Troponin I and summary of their PCC (C), and autophagosome marker LC3/ Troponin I and summary of their PCC (D). NG2 is a marker for cardiomyocytes. DAPI stains nuclei. PCC, Pearson correlation coefficient; Scale bar=10 µm, *P< 0.05, NS. No Significance, (n=6). SFigure2.jpg Supplementary material figure 2. Transcriptional changes in ceramide synthesis pathways. A, mRNA levels of Smpd1, Smpd2, Sptlc1, Sptlc2, Ormdl1, Ormdl2, and Ormdl3 . Representative immunofluorescence staining images Ceramide/NG2 and summary of their PCC (B), and Ceramide/Troponin I and summary of their PCC (C). NG2 is a marker for pericytes, Troponin I is a marker for cardiomyocytes. DAPI stains nuclei. PCC, Pearson correlation coefficient; Scale bar=10 µm, * P < 0.05, NS. No Significance, (n=4-6). 01Table1Primerlist.docx Supplementary material table 1. Primer list. SuncroppedWB.jpg Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2025 Read the published version in Journal of Molecular Medicine → Version 1 posted Editorial decision: Accept as is 02 Apr, 2025 Reviewers agreed at journal 19 Mar, 2025 Reviewers invited by journal 19 Mar, 2025 Editor assigned by journal 19 Mar, 2025 First submitted to journal 18 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5154105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431206823,"identity":"bd86206b-ad68-45b0-8173-1a2d33bf1b79","order_by":0,"name":"Yun-Ting Wang","email":"","orcid":"","institution":"University of Houston","correspondingAuthor":false,"prefix":"","firstName":"Yun-Ting","middleName":"","lastName":"Wang","suffix":""},{"id":431206824,"identity":"706bfa88-638f-4684-8e89-b3353ea0ec55","order_by":1,"name":"Alexandra K Moura","email":"","orcid":"","institution":"University of 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1","display":"","copyAsset":false,"role":"figure","size":25044242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLysosome components and autophagosome accumulation in pericyte and myocardium of ASMD mice. \u003c/strong\u003eRepresentative immunofluorescence staining images for lysosomal-associated membrane protein LAMP1/NG2and summary of their PCC (\u003cstrong\u003eA\u003c/strong\u003e), LAMP2A/NG2 and summary of their PCC (\u003cstrong\u003eB\u003c/strong\u003e), lysosomal protease cathepsin B/NG2 and summary of their PCC(\u003cstrong\u003eC\u003c/strong\u003e), and autophagosome marker LC3/NG2 and summary of their PCC (\u003cstrong\u003eD\u003c/strong\u003e). NG2 is a marker for pericytes. DAPI stains nuclei. \u003cstrong\u003eE and F\u003c/strong\u003e, Representative immunoblots and summarized data for LAMP1, LAMP2A, Cathepsin B, LC3, and p62. \u003cstrong\u003eG,\u003c/strong\u003e mRNA levels of \u003cem\u003eLamp1, Lamp2a, Lc3, and p62\u003c/em\u003e. PCC, Pearson correlation coefficient; Scale bar=10 µm, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=4-6).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/b2b98bed8415cd11e2d1c201.jpg"},{"id":78867877,"identity":"0e8a2e16-3041-4315-b65b-e8b30d618362","added_by":"auto","created_at":"2025-03-20 04:52:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19483941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac inflammation and cellular infiltration in myocardium of ASMD mice. \u003c/strong\u003eCardiac inflammatory cells infiltration was indicated by CD45-positive leukocyte and summary of positive cell numbers per view \u003cstrong\u003e(A)\u003c/strong\u003e, CD206-positive macrophage and summary of positive cell numbers per view \u003cstrong\u003e(B), \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eF4/80-positive macrophage and summary of positive cell numbers per view\u003cstrong\u003e(C)\u003c/strong\u003e. Representative immunofluorescence staining images for inflammation markers: HMGB1/NG2 and summary of their PCC (\u003cstrong\u003eD\u003c/strong\u003e), and RAGE/Troponin Iand summary of their PCC (\u003cstrong\u003eE\u003c/strong\u003e). NG2 is a marker for pericytes, Troponin I is a marker for cardiomyocytes. DAPI stains nuclei. \u003cstrong\u003eF, \u003c/strong\u003emRNA levels of inflammasome and inflammation related genes: \u003cem\u003eAsc, Nlrp3, Caspase-1, IL1β, IL18, IL6, IL8, Vcam1, Icam1, and Tnfα\u003c/em\u003e. PCC, Pearson correlation coefficient; Scale bar=10 µm, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=4-6).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/be49a9967287b765931f6fdc.jpg"},{"id":78868263,"identity":"0adbee49-d1f7-4c4e-a879-60abe34e3afc","added_by":"auto","created_at":"2025-03-20 05:00:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22983160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac steatosis and lipid accumulation in myocardium of ASMD mice. A, \u003c/strong\u003eRepresentative Oil Red O staining for lipid deposition in cardiac tissues and summary ratio of Oil Red O positive areas over total view area under 40x lens.\u003cstrong\u003e \u003c/strong\u003eRepresentative immunofluorescence staining images for lipid droplets associated protein PLIN2/NG2 and summary of their PCC (\u003cstrong\u003eB\u003c/strong\u003e), PLIN2/PDGFRα and summary of their PCC (\u003cstrong\u003eC\u003c/strong\u003e), and PLIN2/Troponin I and summary of their PCC (\u003cstrong\u003eD\u003c/strong\u003e). NG2 is a marker for pericytes, PDGFRα is a marker for fibroblasts, Troponin I is a marker for cardiomyocytes. DAPI stains nuclei. \u003cstrong\u003eE, \u003c/strong\u003emRNA levels of lipogenesis related genes: \u003cem\u003eGpat4, Agpat2, Dgat1, Dgat2, Plin2, and Plin3\u003c/em\u003e. PCC, Pearson correlation coefficient; Scale bar:20 µm for \u003cstrong\u003eA\u003c/strong\u003e, 10 µm for \u003cstrong\u003eB-D\u003c/strong\u003e, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=4-6).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/1fc873f9b5a8d741c2898905.jpg"},{"id":78867888,"identity":"90c79ab5-e6f4-4a7b-ace7-0894643de588","added_by":"auto","created_at":"2025-03-20 04:52:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26319830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac fibrosis and pericyte-to-myofibroblast transition in myocardium of ASMD mice. A-D, \u003c/strong\u003eRepresentative immunofluorescence staining images for pericyteto myofibroblast transition markers: TGF-β/NG2 and summary of their PCC (\u003cstrong\u003eA\u003c/strong\u003e), PDGFRα/NG2 and summary of their PCC (B), FSP1/NG2 and summary of their PCC (\u003cstrong\u003eC\u003c/strong\u003e), vimentin/NG2 and summary of their PCC (\u003cstrong\u003eD\u003c/strong\u003e), and collagen 1/NG2 and summary of their PCC (\u003cstrong\u003eE\u003c/strong\u003e). NG2 is a marker for pericytes. DAPI stains nuclei. Representative Sirius red and fast green staining images in cardiac interstitial, and their summary ratio of Sirius red staining positive areas over total view area under 40x lens (\u003cstrong\u003eF\u003c/strong\u003e). PCC, Pearson correlation coefficient; Scale bar:10 for \u003cstrong\u003eA-E\u003c/strong\u003e, 20 µm for \u003cstrong\u003eF\u003c/strong\u003e, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=6).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/cca1405c335e36a194f0f728.jpg"},{"id":78868265,"identity":"bf8c0455-718f-48c2-889d-b7a517408985","added_by":"auto","created_at":"2025-03-20 05:00:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9398653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoronary microvascular dysfunction and arteriolar remodeling in ASMD mice. A, \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eultrasound images of isoflurane-induced vasodilation of the left anterior descending coronary artery at basal (1.0% ISO) and hyperemia (2.5% ISO) levels. \u003cstrong\u003eB, \u003c/strong\u003eSummarized data of diastolic blood flow velocity peeks at 1.0 % ISO and 2.5% ISO. \u003cstrong\u003eC,\u003c/strong\u003e Representative H\u0026amp;E staining image and summary of coronary artery media thickness (black arrow). \u003cstrong\u003eD,\u003c/strong\u003e α-SMA-labeled mature coronary artery and number summary per view under 4x lens. \u003cstrong\u003eE, \u003c/strong\u003eRepresentative Isolectin-IB-4 labeled endothelial cells and NG2 labeled pericytes in capillary and summary in IB4/NG2 intensity ratio. DAPI stains nuclei. Scale bar:20 µm for \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003e, 200 µm for \u003cstrong\u003eD\u003c/strong\u003e, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=6).\u003c/p\u003e","description":"","filename":"Figure51.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/5af2ab01911e13c9895e441e.jpg"},{"id":78868269,"identity":"01cedc21-32a4-4494-8676-939115a7a028","added_by":"auto","created_at":"2025-03-20 05:00:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18754252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac systolic and diastolic dysfunction in ASMD mice.\u003c/strong\u003e \u003cstrong\u003eA, \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eM-mode\u003cstrong\u003e \u003c/strong\u003eimages of left ventricular in parasternal short-axis view. \u003cstrong\u003eB, \u003c/strong\u003eSummarized data of echocardiographic parameters in cardiac systolic function: left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), diastolic left ventricular internal end (LVID; d), systolic left ventricular internal end (LVID; s), diastolic left ventricle volume (LV Vol; d), and systolic left ventricle volume (LV Vol; s). \u003cstrong\u003eC, \u003c/strong\u003eSummarized data of echocardiographic parameters in cardiac remodeling: left ventricle mass (LV Mass), diastolic left ventricular anterior wall (LVAW; d), systolic left ventricular anterior wall (LVAW; s), diastolic left ventricular posterior wall (LVPW; d), and systolic left ventricular posterior wall (LVPW; s). \u003cstrong\u003eD, \u003c/strong\u003eRepresentative immunofluorescence staining images for Troponin I labeled cardiomyocytes and WGA-FITC labeled cell membrane, and summary in cardiomyocyte size. DAPI stains nuclei. \u003cstrong\u003eE, \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003ePW doppler mode images in apical four chamber view. \u003cstrong\u003eF, \u003c/strong\u003eSummarized data of echocardiographic parameters in cardiac diastolic function: Left ventricular relaxation time constant Tau value, isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), mitral valve ejection time (MV ET), MV E peak and MV A peak. Scale bar=10 µm, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=6).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/dd487c52c2a870f196e8fc05.jpg"},{"id":78867881,"identity":"6ad88ac0-709a-471e-a5b3-3de377e7fd07","added_by":"auto","created_at":"2025-03-20 04:52:34","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10703297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanisms by which ASM deficiency induces cardiovascular dysfunction in ASMD mice\u003c/strong\u003e \u003cstrong\u003e(see discussion for detail description).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/700f19c68dc3f71261fbd01b.jpg"},{"id":81051336,"identity":"78ce3b01-05a6-457b-9bb7-e2891ef271a7","added_by":"auto","created_at":"2025-04-21 16:10:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":134056557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/fc764c2b-a4cb-4fa3-9a03-1e9e7459cd0d.pdf"},{"id":78867875,"identity":"27a6ac87-fedf-4d72-aca7-bf2da45c3400","added_by":"auto","created_at":"2025-03-20 04:52:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13546,"visible":true,"origin":"","legend":"","description":"","filename":"01KeymessagesJMMER2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/679eb7d45755903666fa555d.docx"},{"id":78868267,"identity":"98d95ede-2677-42e6-b4e3-268a2e482146","added_by":"auto","created_at":"2025-03-20 05:00:35","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31576241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material figure 1. Lysosome components and autophagosome accumulation in cardiomyocytes of ASMD mice. \u003c/strong\u003eRepresentative immunofluorescence staining images for LAMP1/ Troponin I and summary of their PCC (A), LAMP2A/Troponin I and summary of their PCC (B), lysosomal protease cathepsin B/ Troponin I and summary of their PCC (C), and autophagosome marker LC3/ Troponin I and summary of their PCC (D). NG2 is a marker for cardiomyocytes. DAPI stains nuclei. \u0026nbsp;PCC, Pearson correlation coefficient; Scale bar=10 µm, *P\u0026lt; 0.05, NS. No Significance, (n=6).\u003c/p\u003e","description":"","filename":"SFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/3abee55f524f46ee5af14625.jpg"},{"id":78867886,"identity":"13971e70-c25c-4f68-8f68-33b53265efdf","added_by":"auto","created_at":"2025-03-20 04:52:34","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13704878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material figure 2. Transcriptional changes in ceramide synthesis pathways. A\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003emRNA levels of \u003cem\u003eSmpd1, Smpd2, Sptlc1, Sptlc2, Ormdl1, Ormdl2, and Ormdl3\u003c/em\u003e. Representative immunofluorescence staining images Ceramide/NG2 and summary of their PCC (\u003cstrong\u003eB\u003c/strong\u003e), and Ceramide/Troponin I and summary of their PCC (\u003cstrong\u003eC\u003c/strong\u003e). NG2 is a marker for pericytes, Troponin I is a marker for cardiomyocytes. DAPI stains nuclei. PCC, Pearson correlation coefficient; Scale bar=10 µm, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, NS. No Significance, (n=4-6).\u003c/p\u003e","description":"","filename":"SFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/eb6bb4fa6222739250b2f2d0.jpg"},{"id":78867876,"identity":"f28094d0-c428-44e5-b4d5-8359f7b4cda0","added_by":"auto","created_at":"2025-03-20 04:52:34","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material table 1. Primer list.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"01Table1Primerlist.docx","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/b8e4897930f41d847915ee03.docx"},{"id":78868262,"identity":"84d77481-51d8-4f44-a16a-d35e9241d497","added_by":"auto","created_at":"2025-03-20 05:00:34","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2231593,"visible":true,"origin":"","legend":"","description":"","filename":"SuncroppedWB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5154105/v1/c759f9ee1fff16669dd65e7e.jpg"}],"financialInterests":"","formattedTitle":"Cardiovascular Dysfunction and Altered Lysosomal Signaling in a Murine Model of Acid Sphingomyelinase Deficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNiemann-Pick disease (NPD) is a rare autosomal recessive disorder caused by acid sphingomyelinase (ASM) activity deficiency (ASMD) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. ASM, encoded by the sphingomyelin phosphodiesterase 1 (Smpd1) gene, is a lysosomal hydrolase that breaks down sphingomyelin into phosphorylcholine and ceramide \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Mutations in Smpd1 caused ASMD leads to the accumulation of sphingomyelin in lysosomes, resulting in lysosomal storage disorder (LSD) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The initial accumulation of lysosomal sphingomyelin in ASMD triggers the subsequent buildup of other lipids within lysosomes, with cholesterol being the most prominent \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Furthermore, lipids derived from sphingomyelin, including ceramide and its downstream metabolites (e.g., sphingosine), lysosphingomyelin (sphingosylphosphorylcholine), glycosphingolipids, and bis(monoacylglycero)phosphate, also accumulate \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Over time, these accumulated lipids are recycled to the plasma membrane and redistributed to other cellular compartments, disrupting cellular homeostasis and causing secondary abnormalities across multiple organelles \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. There are two subtypes of NPD caused by ASMD, NPD type A (NPD A) and NPD type B (NPD B). NPD A patients have less than 1% of normal enzyme activity, leading to the early onset of rapid systemic and neurodegenerative symptoms, typically reducing their lifespan to around 3 years \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In contrast, NPD B patients have 5\u0026ndash;10% of normal enzyme activity, do not exhibit neurodegenerative symptoms, and often survive into adulthood \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, these patients present with a highly variable clinical phenotype and disease-related morbidities affecting multiple organ systems, including respiratory and cardiovascular systems \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCardiac disease accounts for 7.2% of deaths among NPD B patients, making it the fifth leading cause of mortality, following respiratory disease (27.7%), liver disease (27.7%), and bleeding (9.6%) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While NPD B patients have numerous cardiac conditions, valvular heart disease and coronary artery disease are the most common \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that ASM activation and ceramide accumulation contribute to the development of cardiomyopathy and heart failure in conditions such as high-fat diet (HFD), myocardial infarction (MI), and sepsis \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, no comprehensive study has been conducted on the nature and extent of cardiac dysfunction and related cardiovascular events resulting from ASMD.\u003c/p\u003e \u003cp\u003eProper sphingolipid homeostasis is essential for maintaining health, and disruption of this finely regulated balance can result in various diseases. This phenomenon is evident in several LSDs, such as Gaucher and Farber diseases, where inherited lysosomal defects lead to sphingolipid accumulation and subsequent pathogenesis \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this study, we use the Smpd1 knockout mouse model of ASMD to identify cardiac pathologies and dysfunction under baseline conditions. Our findings indicate that ASMD leads to perturbed cardiac function. ASMD-induced cardiac pathological changes encompass lysosomal dysfunction, cardiac inflammation, steatosis, fibrosis, coronary microvascular dysfunction (CMD), and overall cardiac dysfunction. These findings provide novel insights into the cardiac complications associated with ASMD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003e All experimental protocols received approval from the University of Houston Institutional Animal Care and Use Committee. We used female wild-type (\u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e) and acid sphingomyelinase deficient (\u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mice, aged 5\u0026ndash;6 months and of the C57BL/6J strain, for all experiments. The mice were kept in a temperature-controlled room with a 12-hour light-dark cycle and had free access to standard rodent chow and water. Following euthanasia, heart samples were collected and stored at -80\u0026deg;C for subsequent analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEchocardiography\u003c/h3\u003e\n\u003cp\u003eTransthoracic echocardiography was conducted using a Vevo 3100 micro-ultrasound imaging system with an MX550D-0073 probe (VisualSonics Inc., Canada). Mice were anesthetized with 1.5\u0026ndash;2% isoflurane (ISO) in 100% oxygen and maintained at 1\u0026ndash;1.5% isoflurane throughout the procedure. M-mode images were acquired from the parasternal short-axis or apical four-chamber views and analyzed using Vevo LAB 2.1.0. The flow velocity of the left anterior descending coronary artery was measured in pulsed-wave Doppler mode under baseline conditions and after induction of hyperemia with 1.0% and 2.5% isoflurane, respectively. Left ventricular relaxation time constant (Tau value) = (IVRT\u0026thinsp;+\u0026thinsp;IVCT)/MV ET.\u003c/p\u003e\n\u003ch3\u003eAntibodies and reagents\u003c/h3\u003e\n\u003cp\u003ePrimary antibodies: NG2 (Abcam ab275024, Miltenyi Biotec 130-123-964), Troponin I (Abcam AB56357-1001), LAMP1 (BD 553792), LAMP2A (abcam ab18528), Cathepsin B (Abcam ab58802), LC3 (CST 12741S), p62 (Abcam ab109012), HMGB1 (Abcam ab79823), RAGE (Abcam ab3611), CD45 (Abcam ab25386), F4/80 (BD 565411), PLIN2 (Proteintech 15294-1-AP), TGFβ (CST 3711S), PDGFRα (CST 3174T, R\u0026amp;D AF1062-SP), FSP1 (CST 13018S), Pan-cadherin (Abcam ab51034), Vimentin (Abcam ab92547), Collagen 1 (Abcam ab21286), FITC-αSMA (Sigma F3777), Ceramide (Enzo, ALX-804-196-T050), β-actin (CST 3700S), GADPH (CST 2118S).\u003c/p\u003e \u003cp\u003eSecondary antibody for immunofluorescence: anti-Mouse IgG, Alexa Fluor 488 conjugate (Thermo Fisher A-21202), Alexa Fluor 488 conjugate anti-Rabbit IgG (Thermo Fisher A21206), Alexa Fluor 555 conjugate anti-Mouse IgG (Thermo Fisher A-31570), Alexa Fluor 555 conjugate anti-Rabbit IgG (Thermo Fisher A-31572). Secondary antibody for western blot: IRDye\u0026reg; 800CW anti-Mouse IgG (LICOR 926-32212), IRDye\u0026reg; 800CW anti-Rabbit IgG (LICOR 926-32213), anti-Mouse IgG, HRP (Thermo Fisher A16011), stabilized peroxidase conjugated anti-rabbit (Invitrogen 32460), anti-rat IgG-HRP (Fisher 629520).\u003c/p\u003e \u003cp\u003eReagents: Oil red O (VWR BT135140-100G), Sirius red (Sigma 365548-5g), Isolectin GS-IB4 (Fisher I21411), WGA-FITC (Sigma L4895), Aurum Total RNA Mini Kits (Bio-Rad, 732\u0026ndash;6820), iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 1708841), iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eFrozen heart section slides were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After washing with PBS, the tissues were blocked and permeabilized using 5% BSA and 0.3% Triton X-100 in PBS for 1 hour at room temperature, then incubated with primary antibodies overnight at 4\u0026deg;C. Following washing with PBST (PBS with 0.05% Tween-20), the slides were incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 555 for 1 hour at room temperature. After another wash with PBST, the slides were mounted with DAPI mounting solution and analyzed using an Olympus IX73 or Leica STED 8 imaging system. Pearson correlation coefficient (PCC) of fluorescence image was quantified using Image-Pro Plus 6.0 software, as previously described \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Frozen heart sections were stained with WGA-FITC (5 \u0026micro;g/ml in PBS) for 30 minutes at room temperature, followed by staining with Troponin I using the immunofluorescence staining protocol described above. The cardiomyocyte outlines were identified through Troponin I-positive staining and WGA-FITC labeling of cell membranes. Cardiomyocytes with centrally localized nuclei should be used for analysis to ensure consistent orthogonal cross-sectional measurements, minimizing the influence of obliquely oriented cardiomyocytes. Cardiomyocyte size was then measured using Image-Pro Plus 6.0 software.\u003c/p\u003e\n\u003ch3\u003eHematoxylin and eosin (H\u0026E) staining\u003c/h3\u003e\n\u003cp\u003eH\u0026amp;E staining was carried out using a hematoxylin and eosin staining kit (Teomics HAE-1). Frozen heart sections were fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After washing with distilled water, the tissues were stained with Mayer\u0026rsquo;s hematoxylin solution for 5 minutes, followed by two washes with distilled water to remove excess stain. A bluing reagent was then applied for 10\u0026ndash;15 seconds, followed by two additional washes with distilled water. The slides were then dipped in absolute alcohol to remove excess moisture and stained with Eosin Y solution for 2\u0026ndash;3 minutes. After rinsing with absolute alcohol, the slides were cleared and mounted in synthetic resin, followed by prompt imaging with the Olympus IX73 system.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOil Red O staining\u003c/h2\u003e \u003cp\u003eFrozen heart sections were prepared at a thickness of 8 \u0026micro;m. The sections were air-dried on slides, fixed in 4% paraformaldehyde (PFA) for 15 minutes, and rinsed immediately in three changes of distilled water before being soaked for 20 minutes. To prevent water carryover, the sections were then washed with 60% isopropanol for 1 minute. Following staining with Oil Red O working solution for 30 minutes, the sections were rinsed with 60% isopropanol for 2 minutes and then rinsed twice with distilled water. Finally, the sections were mounted in mounting media, and images were captured promptly using the Olympus IX73 imaging system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSirius red and fast green staining\u003c/h3\u003e\n\u003cp\u003eFrozen heart section slides were fixed with Kahle fixative solution for 15 minutes at room temperature. After washing with PBS, the slides were stained with 0.1% Sirius Red and Fast Green in a saturated aqueous solution of picric acid at room temperature for 60 minutes. The slides were then washed in two changes of acidified water (0.1N HCl in ddH2O) for 1 minute each. After a final wash with ddH2O, the sections were mounted in mounting media, and images were captured promptly using the Olympus IX73 imaging system.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from heart tissue using Aurum Total RNA Mini Kits (Bio-Rad, 732\u0026ndash;6820). cDNA was synthesized from the isolated RNA using iScript Reverse Transcription Supermix (Bio-Rad, 1708841). Real-time PCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121) on the Bio-Rad CFX Connect Real-Time System. Primers for \u003cem\u003eNlrp3, Asc, Caspase-1, IL1β, IL18, IL6, IL8, TNF, Icam1, and Vcam1\u003c/em\u003e were obtained from Bio-Rad. Additional primers used in this study are listed in the Supplementary Material Table\u0026nbsp;1. Cycle threshold (Ct) values were converted to relative gene expression levels using the 2-ΔΔCt method, and the data were normalized to the internal controls β-actin or GAPDH.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eHeart tissue homogenates were lysed in Laemmli sample buffer (Bio-Rad, 161\u0026ndash;0737), boiled at 95\u0026deg;C for 10 minutes, and then placed in an ice-cold ultrasonic bath for 5 minutes. The prepared samples were separated by 10\u0026ndash;15% sodium dodecyl sulfate-polyacrylamide gels. Proteins were then transferred electrophoretically onto PVDF membranes at 35 V at 4\u0026deg;C overnight. The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween 20. After washing, the membranes were incubated with the appropriate primary antibodies, following the manufacturer's instructions. Following incubation, the membranes were washed with PBST and subsequently incubated with corresponding secondary antibodies for 1 hour at room temperature. Finally, the bands were washed with PBST, visualized, and analyzed using the LI-COR\u0026reg; Odyssey Fc System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistics analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical comparisons between two groups were made using either a Student's t-test or the Mann-Whitney test. All analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLysosomal and autophagosome accumulation in cardiac pericyte and myocardium\u003c/h2\u003e \u003cp\u003eLysosomal storage disorder (LSD) is a hallmark and major contributor to lysosomal dysfunction in NPD \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To investigate the presence of lysosomal dysfunction, we first performed immunofluorescence analyses of lysosome or autophagy-related proteins on cardiac sections focusing on cardiac pericyte and cardiomyocytes of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice in comparison to \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. The results showed lysosomal membrane protein LAMP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and LAMP2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), lysosomal protease cathepsin B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and the autophagosome membrane protein LC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) were upregulated in cardiac pericyte of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. In addition to cardiac pericytes, these proteins were also elevated in the cardiomyocytes of Smpd1-/- mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A-D\u003c/b\u003e). These findings were further validated by immunoblot analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). p62/Sqstm1, a macroautophagy receptor, is typically degraded during autophagy maturation, where autophagosomes fuse with lysosomes, leading to the degradation of their contents, including LC3 and p62/Sqstm1. Lysosomal dysfunction is often associated with impaired autophagic flux, resulting in the accumulation of LC3 and p62/Sqstm1. Consistent with the increased LC3 protein expression, the p62/Sqstm1 protein was also elevated in ASMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). However, the mRNA quantification of cardiac tissues showed that ASMD did not affect the gene expression of \u003cem\u003eLamp1\u003c/em\u003e, \u003cem\u003eLc3\u003c/em\u003e, and \u003cem\u003ep62\u003c/em\u003e, but did lead to increased \u003cem\u003eLamp2a\u003c/em\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mRNA quantification confirmed the absence of the \u003cem\u003eSmpd1\u003c/em\u003e gene, but not the \u003cem\u003eSmpd2\u003c/em\u003e gene, (encoding neutral sphingomyelinase/NSM) in the cardiac tissues of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). Serine palmitoyltransferase (SPT), a multi-subunit enzyme, catalyzes the first and rate-limiting step in \u003cem\u003ede novo\u003c/em\u003e ceramide synthesis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. SPT activity is negatively regulated by ORMDL1-3, which are activated in response to increased ceramide levels \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. ASMD had no effect on gene expression of SPT subunit \u003cem\u003eSptlc1\u003c/em\u003e or \u003cem\u003eOrmdl1-3\u003c/em\u003e in cardiac tissues but led to upregulation of \u003cem\u003eSptlc2\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). Surprisingly, no significant difference in ceramide levels was observed between \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice in either cardiac pericytes (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e) or cardiomyocytes (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C\u003c/b\u003e). These results suggest that while ASMD significantly reduces \u003cem\u003eSmpd1\u003c/em\u003e gene in cardiac tissues, it had compensatory upregulation of \u003cem\u003eSptlc2\u003c/em\u003e, but minimal impact on the NSM-dependent ceramide or other \u003cem\u003ede novo\u003c/em\u003e ceramide synthesis pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCardiac inflammation and cellular infiltration\u003c/h2\u003e \u003cp\u003eCardiac inflammation can lead to severe health conditions, including coronary artery disease, cardiac fibrosis, and heart failure \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Immunofluorescence studies were first conducted on cardiac sections of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. The results revealed significantly increased infiltration of CD45-positive leukocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and CD206-positive macrophage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) in the cardiac tissues in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. However, no increase in F4/80-positive macrophage infiltration was observed in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To further characterize the inflammation in specific cell types, we analyzed the expression of HMGB1 (high mobility group box 1), a DAMP (damage-associated molecular pattern) linked to sterile inflammation, and its receptor RAGE (receptor for advanced glycation end-products) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We observed that ASMD-induced HMGB1 upregulation was primarily localized in non-cardiac cells, such as pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), while RAGE upregulation was predominantly observed in cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In addition, mRNA quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) from cardiac tissues of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed increased expression of inflammation-related genes, including inflammatory \u003cem\u003eCaspase-1\u003c/em\u003e, \u003cem\u003eInterleukin 18 (IL18)\u003c/em\u003e, \u003cem\u003eVcam1\u003c/em\u003e (vascular cell adhesion molecule 1), and \u003cem\u003eIL6\u003c/em\u003e. However, no change was detected for other genes, including inflammasome component \u003cem\u003eAsc\u003c/em\u003e (PYD and CARD domain-containing), \u003cem\u003eNlrp3\u003c/em\u003e (NLR family pyrin domain-containing 3), \u003cem\u003eIL1β\u003c/em\u003e, \u003cem\u003eIcam1\u003c/em\u003e (intercellular adhesion molecule 1), \u003cem\u003eIL8\u003c/em\u003e, and \u003cem\u003eTnfα\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCardiac steatosis and lipid accumulation\u003c/h2\u003e \u003cp\u003eTo investigate whether ASMD-induced lysosomal dysfunction is associated with altered lipid storage in the cardiac tissues, we performed Oil Red O staining and immunostained for the lipid droplet-associated protein perilipin 2 (PLIN2) in cardiac tissue sections from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, a significant increase in Oil Red O staining was observed in the cardiac tissues of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, whereas no positive staining was detected in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. Similarly, PLIN2 expression was significantly upregulated in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). We further confirmed that ASMD-induced PLIN2 accumulation was localized in cardiac pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), cardiac fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We also examined the expression of genes involved in lipid droplet biogenesis, including \u003cem\u003eGpat4\u003c/em\u003e (glycerol-3-phosphate acyltransferase 4), \u003cem\u003eAgpat2\u003c/em\u003e (1-acylglycerol-3-phosphate O-acyltransferase 2), \u003cem\u003eDgat1/2\u003c/em\u003e (diacylglycerol O-acyltransferase 1 and 2), and \u003cem\u003ePlin2/3\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, the mRNA levels of most of these genes were comparable between \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice, except for a decrease in \u003cem\u003eDgat2\u003c/em\u003e expression. These findings suggest that ASMD leads to lipid deposition in the myocardium, resulting in cardiac steatosis, which is not associated with increased lipid droplet biogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCardiac fibrosis and pericyte-to-myofibroblast transition\u003c/h2\u003e \u003cp\u003eWhile inflammation and steatosis were the most prominent cardiac changes observed in tissue samples from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, fibrosis was also evident. This was assessed using immunostaining for cardiac pericyte, fibroblast and myofibroblast markers, as well as Sirius red staining to visualize total collagen in cardiac tissue samples from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F). Immunostaining demonstrated significant increases in the expression of proteins associated with pericyte-to-myofibroblast transition (PMT), a key process in cardiac fibrosis development \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We demonstrated that ASMD increased transforming growth factor beta (TGF-β) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), platelet-derived growth factor receptor alpha (PDGFRα) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), fibroblast-specific protein 1 (FSP1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and collagen 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) in cardiac pericytes. Sirius red staining confirmed an increase in collagen in the cardiac interstitial areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCoronary microvascular dysfunction and arteriolar remodeling\u003c/h2\u003e \u003cp\u003eEchocardiography was conducted to assess coronary microvascular function by measuring the coronary blood flow velocity of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice at baseline conditions (1.0% isoflurane) as well as under hyperemic conditions (2.5% isoflurane). Representative images and summarized data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) demonstrated a significant decrease in coronary blood flow velocity at baseline in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. However, no differences were observed under hyperemic conditions. Immunohistological analysis revealed increased coronary arteriolar wall thickness in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, which was attributed to increased media thickness, without evidence of neointima formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Further immunofluorescence analysis was conducted to evaluate the density of coronary arterioles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, α-SMA\u003csup\u003e+\u003c/sup\u003e arterioles) and myocardial endothelial cells to pericytes coverage in capillaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, isolectin-IB4-labeled endothelial cells, NG2-labeled pericytes) in cardiac sections. These results showed no significant differences in the number of coronary arterioles and endothelial cells to pericytes coverage in capillaries between \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. These findings suggest that ASMD leads to coronary arteriolar remodeling and impaired coronary microvascular function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCardiac systolic and diastolic dysfunction\u003c/h2\u003e \u003cp\u003eFinally, echocardiography was performed to investigate whether the cardiac pathologies and coronary microvascular dysfunction (CMD) observed in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice are associated with changes in cardiac function and remodeling. Representative images and summarized data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) revealed that \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice developed cardiac systolic dysfunction compared to \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice. Specifically, \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), along with increased systolic left ventricular internal diameter (LVID; s) and systolic left ventricle volume (LV Vol; s). However, there were no significant differences in diastolic left ventricular internal diameter (LVID; d) or diastolic left ventricle volume (LV Vol; d). As summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, no significant cardiac remodeling was observed in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, as evidenced by the similar calculated left ventricular mass, diastolic left ventricular anterior wall thickness (LVAW; d), systolic left ventricular anterior wall thickness (LVAW; s), diastolic left ventricular posterior wall thickness (LVPW; d), and systolic left ventricular posterior wall thickness (LVPW; s) between the groups. In addition, there was no significant difference in cardiomyocytes size between \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Cardiac diastolic function of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice was also assessed by measuring the left ventricular relaxation time constant (Tau value), isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), mitral valve ejection time (MV ET), and mitral valve E/A (MV E/A) peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). The results indicated the development of cardiac diastolic dysfunction in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, as shown by increased IVRT, IVCT, and MV ET, alongside a reduction in MV E peak, with no change in MV A peak. Together, these results suggest that ASMD leads to heart failure characterized by both cardiac systolic and diastolic dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe clinical manifestations of ASMD in patients with NPD are diverse and wide-ranging. In NPD A, the most frequent symptoms are central nervous system degeneration and respiratory failure. NPD B presents a broader range of clinical manifestations, with the most severely affected organs being the respiratory system, liver, and tissues associated with an atherogenic lipid profile \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In addition, an increasing number of studies have reported cardiovascular diseases associated with NPD B \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eSmpd1\u003c/em\u003e knockout mouse serve as a well-established murine model of NPD. In this study, we identified significant cardiac pathology in this model, showing that ASMD results in lysosomal dysfunction, cardiac inflammation, steatosis, fibrosis, CMD, and overall cardiac dysfunction.\u003c/p\u003e \u003cp\u003eLSDs encompass a group of diseases caused by deficiencies in lysosomal enzymes, membrane transporters, or other proteins critical for lysosomal function. These disorders are categorized based on the type of accumulated material, including lipids, mucopolysaccharidoses, and glycoproteinoses \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. ASM plays a key role in hydrolyzing sphingomyelin to ceramide within lysosomes, and in ASMD, this process is disrupted, leading to alterations in the \u0026lsquo;sphingolipid rheostat\u0026rsquo; \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This disruption results in imbalanced levels of sphingolipid species, such as sphingomyelin, ceramide, sphingosine, and their phosphorylated metabolites. These imbalances reorganize lysosomal membrane composition by forming sphingolipid-enriched microdomains \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, which in turn redistribute membrane-associated ion channels or proteins, influencing either their stability or function. Sphingolipids like sphingomyelin and sphingosine may also directly interact with lysosomal membrane ion channels, modulating their activities and contributing to lysosomal dysfunction. For example, the lysosomal membrane cation channel TRPML1 is primarily responsible for mediating lysosomal Ca \u003csup\u003e2+\u003c/sup\u003e release. Previous studies have shown that TRPML1-mediated lysosomal Ca \u003csup\u003e2+\u003c/sup\u003e release is markedly reduced in cells from NPD patients \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, likely due to the inhibitory effects of sphingomyelin accumulation within lysosomal membranes in ASMD. Our previous studies also confirmed that ASMD inhibits TRPML1-mediated lysosomal Ca\u003csup\u003e2+\u003c/sup\u003e release, which impairs dynein-dependent autophagosome trafficking, resulting in defective lysosome-autophagosome fusion and diminished autophagic flux in vascular smooth muscle cells from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice \u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These findings suggest that sphingolipids serve as major regulators for Ca\u003csup\u003e2+\u003c/sup\u003e-dependent lysosomal trafficking function and that ASMD disrupts this process, reducing the turnover of autophagosomes and lysosomes along with their contents. In the present study, we observed that ASMD increased the expression of lysosomal proteins LAMP1/2, cathepsin B, and autophagic proteins LC3 in cardiac pericytes and cardiomyocytes of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Interestingly, ASMD had minimal effects on their mRNA levels, indicating that lysosome or autophagosome biogenesis is not substantially altered. Together, our findings reinforce the view that ASMD induces lysosomal dysfunction, leading to impaired autophagic flux in cardiac cells in a mouse model of NPD. Notably, there was no significant decrease in ceramide levels in ASMD mice in either cardiac pericytes or cardiomyocytes. Previous studies have reported higher ceramide levels in ASMD type B organoids compared to controls \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This elevation in ceramide levels may result from the activity of other sphingomyelinases located in non-lysosomal compartments, as described in ASM knockout mice by other researchers \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Furthermore, our data demonstrated an upregulation of the \u003cem\u003eSptlc2\u003c/em\u003e gene in ASMD mice (Supplementary Fig.\u0026nbsp;2A), which could enhance the de novo synthesis of ceramide, providing a compensatory mechanism. Sphingosine, a vital bioactive sphingolipid, is generated from ceramide through the enzymatic action of ceramidases and is subsequently phosphorylated by sphingosine kinases to produce sphingosine-1-phosphate (S1P), a molecule that promotes cell survival \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The dynamic balance among sphingomyelin, ceramide, sphingosine, and S1P is crucial in modulating the effects of ASMD and determining the fate of the cell \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Future research will aim to quantify sphingomyelin, ceramide, sphingosine, and S1P levels using lipidomics to clarify their roles in ASMD and their contributions to cellular dysfunction, including inflammation, lipid accumulation, and PMT. This strategy will address current limitations and provide critical insights into the sphingolipid-driven mechanisms underlying ASMD.\u003c/p\u003e \u003cp\u003eAccumulating evidence indicates that lysosomal dysfunction is intimately linked to inflammation through several mechanisms. Impaired autophagic flux caused by lysosomal dysfunction can lead to the accumulation of damaged organelles (e.g. damaged mitochondria), protein aggregates, and other undegraded cellular materials. In LSDs, severe dysfunction may cause lysosomal membrane destabilization or rupture which releases lysosomal enzymes and harmful substances, such as cathepsins, into the cytosol. These accumulated substances or lysosomal contents can be recognized by the immune system as DAMPs, triggering inflammatory responses through activation of pattern recognition receptors (PRRs). One of the best studied connections between the lysosomal-autophagy pathways and inflammation is the NLRP3 inflammasome pathway, which plays a key role in the progression of cardiovascular diseases \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. NLRP3 inflammasome activation leads to increased inflammatory caspase-1 activity and production of pro-inflammatory cytokines, such as IL-1β and IL-18. It can also activate inflammatory caspase-11 and induce GSDMD-dependent pore formation in the plasma membrane, causing pyroptotic cell death. Additionally, HMGB1 is increasingly recognized as a key DAMP molecule actively released by immune cells or passively released from dying cells during infection or sterile inflammation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. HMGB1 is a multifunctional protein that exerts proinflammatory activity primarily by binding to RAGE \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The HMGB1/RAGE axis is critical in the pathogenesis of inflammatory cardiomyopathy \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Early inflammatory responses include the upregulation of adhesion molecules, such as VCAM-1 and ICAM-1, which facilitate the adhesion of CD45-positive leukocytes (e.g. lymphocytes, monocytes, eosinophils, and basophils) and CD206-positive macrophages to the myocardium \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In this study, cardiac tissue from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed a significant increase in the number of CD45-positive leukocytes, CD206-positive macrophages and protein expression of HMGB1 and RAGE, along with elevated expression of inflammation-related genes, including \u003cem\u003eCaspase1\u003c/em\u003e, \u003cem\u003eIL18\u003c/em\u003e, \u003cem\u003eVcam1\u003c/em\u003e, and \u003cem\u003eIL6\u003c/em\u003e. These findings align with previous research showing that vascular smooth muscle cells from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibit higher gene expression of \u003cem\u003eIL18\u003c/em\u003e and \u003cem\u003eIL6\u003c/em\u003e, as well as increased monocyte adhesion at basal levels or after PDGF stimulation \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Similarly, Elyse et al. reported that ASM knockdown in bronchial epithelial cells increased inflammation and neutrophil recruitment under both basal and infected conditions \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In contrast, ASM was shown to mediate pro-atherogenic stimulus-induced inflammation in macrophages and endothelial cells \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, ASM activation can lead to excess ceramides, driving inflammation in various diseases, including cystic fibrosis (CF) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Therefore, both the loss of ASM activity leading to lysosomal dysfunction and the activation of ASM resulting in excess ceramide production can trigger inflammatory responses depending on the stimulus or disease context. Nonetheless, our findings provide evidence that lysosomal dysfunction is linked to cardiac inflammation in ASMD.\u003c/p\u003e \u003cp\u003eCardiac steatosis refers to the abnormal accumulation of lipid droplets (mainly neutral triglycerides) within the myocardium. It has increasingly been recognized as a causative factor for cardiomyopathy, particularly in conditions such as severe aortic stenosis \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and dilated cardiomyopathy\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Selective overexpression of \u003cem\u003eDGAT1\u003c/em\u003e (a key gene in lipogenesis) in cardiomyocytes induces cardiac steatosis and fibrosis \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, exacerbating angiotensin II-induced heart failure in mice \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Although cardiac steatosis is often associated with metabolic conditions such as obesity, diabetes, and fatty liver disease, it can also occur in other pathological conditions, such as LSDs \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In some LSDs, such as Gaucher disease and Farber disease, abnormal lipid metabolism results in lipid storage within cells, such as hepatocytes \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Though not directly reported as a common symptom in LSDs, lipid droplet accumulation may occur in cardiomyocytes or other cell types in the myocardium and subsequently induce cardiac steatosis. Additionally, impaired autophagy caused by lysosomal dysfunction can exacerbate lipid accumulation by hindering the normal breakdown of lipid droplets. Indeed, in ASMD, cholesterol-enriched lipid droplets accumulate in human or murine macrophages, contributing to foam cell formation, a key mechanism in atherogenesis in metabolic disorders \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. However, the link between ASMD and cardiac steatosis has not previously been reported in murine models. In this study, we show for the first time that \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibit a significant increase in cardiac steatosis without elevated expression of lipogenesis-related genes. This suggests that cardiac steatosis in ASMD is primarily due to impaired lipid droplet degradation rather than increased lipid droplet biogenesis.\u003c/p\u003e \u003cp\u003eAdditionally, we observed the accumulation of the lipid droplet-associated protein PLIN2 not only in cardiomyocytes but also in cardiac pericytes and fibroblasts. The role of lipid droplets in the function of cardiac pericytes and fibroblasts remains largely unexplored and warrants further investigation. Our findings suggest that lipid droplets may play a pivotal role in the PMT process, which should be further studied in future research using \u003cem\u003eSmpd1\u003c/em\u003e pericyte- or fibroblast-specific knockout mice.\u003c/p\u003e \u003cp\u003eCardiac fibrosis, a complex pathophysiological process marked by excessive extracellular matrix accumulation, plays a key role in cardiac dysfunction and heart failure \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. A key cellular mechanism driving fibrosis is the pericyte-to-myofibroblast transition (PMT) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Fibrosis in various organs, such as the lung, liver, and endocardium, has been observed in patients with LSDs, including Farber disease and NPD B \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In this study, \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited elevated expression of TGF-β in pericyte, increased PMT, and fibrotic myofibroblast markers such as FSP1, vimentin, and collagen 1 in pericyte, and increased interstitial collagen deposition in cardiac tissue. These findings suggest that ASMD may promote cardiac fibrosis through the activation of PMT. Recent studies have identified pericytes as a significant source of myofibroblast via PMT \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Interestingly, our recent findings showed that vascular smooth muscle cells from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice underwent a myofibrogenic transition when stimulated by PDGF, a process driven by lysosomal dysfunction, impaired autophagic flux, and the activation of p62/Nrf2 signaling axis \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Since pericytes and vascular smooth muscle cells originate from the same perivascular progenitors, it is reasonable that ASMD in pericytes promotes PMT, contributing to cardiac fibrosis. M2 macrophages derived from monocytes are known to play a critical role in cardiac fibrosis, primarily by promoting the activation of myofibroblasts through the secretion of profibrotic factors such as TGF-β1 and IL-1β \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Therefore, ASMD-induced cardiac fibrosis may result from the infiltration of CD206-positive M2 macrophages. Further studies are required to elucidate the mechanisms by which ASMD drives PMT, using pericyte-specific \u003cem\u003eSmpd1\u003c/em\u003e knockout mouse models and cultured pericytes. In addition, previous studies have implicated ASM activation in fibrosis, suggesting that targeting ASM activity could be a therapeutic approach. For instance, imipramine or cardiomyocyte-specific \u003cem\u003eSmpd1\u003c/em\u003e knockout has been shown to attenuate HFD-induced cardiac fibrosis via downregulating NADPH oxidase 4 (NOX4) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Similarly, astaxanthin has been found to reduce myocardial infarction-induced cardiac fibrosis by inhibiting ASM activity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Thus, like its role in cardiac inflammation, both ASMD and ASM overactivation can lead to fibrotic activation, depending on the specific stimulus or disease context. The findings from this study suggest that lysosomal dysfunction is closely associated with the development of cardiac fibrosis in ASMD.\u003c/p\u003e \u003cp\u003eCoronary microvascular dysfunction (CMD) refers to functional or structural abnormalities in the coronary microcirculation that lead to impaired myocardial perfusion \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. CMD can occur as a clinical condition independent of heart failure, and it may result in myocardial ischemia \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This ischemia may manifest as symptoms such as chest pain (angina) and can contribute to the progression of heart failure over time. Previous studies have identified CMD in LSDs including Fabry disease \u003csup\u003e\u003cspan additionalcitationids=\"CR64 CR65\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and Danon disease \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. In this study, non-invasive echocardiographic analysis of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice revealed a significant reduction in coronary blood perfusion at baseline (1.0% isoflurane); however, no difference was observed under hyperemic conditions (2.5% isoflurane) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, this reduction at baseline was associated with structural remodeling of coronary arterioles, characterized by thickened arteriolar walls, while the number of mature coronary arterioles and endothelial cells to pericytes coverage in capillary remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our findings of thickened coronary arteriolar walls align with previous studies showing coronary artery disease in NPD B \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, as well as increased inflammation and dedifferentiation of vascular smooth muscle cell from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These findings support the view that ASMD promotes coronary arteriolar wall remodeling, resulting in CMD and impaired myocardial perfusion. Additionally, PMT and collagen 1 expression were increased in pericytes of interstitial capillary from \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that extracellular matrix remodeling may increase capillary stiffness and exacerbate vasomotor dysfunction in thickened coronary arterioles. The precise mechanisms by which ASMD induces structural and functional changes in coronary microcirculation deserve further investigation.\u003c/p\u003e \u003cp\u003eCardiac diseases, including hypertrophic and dilated cardiomyopathy, and valvular diseases, are commonly found in LSDs \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although cardiac disease accounts for 7.2% of deaths in NPD B patients, with a few reported cases of heart failure \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, cardiac dysfunction has not been extensively investigated in murine models of NPD or ASMD. In this study, non-invasive echocardiographic analysis of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed both systolic and diastolic cardiac dysfunction under baseline conditions. Myocardial relaxation is one of the earliest manifestations of mechanical dysfunction of the human LV. The time constant tau (Ƭ) is higher in the elderly and patients with hypertrophic cardiomyopathy (HCM), coronary artery disease (CAD), and cardiomyopathies \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the lifespan of ASMD mice is typically less than 1 year, whereas wild-type mice generally live for over 2 years \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Therefore, it is reasonable to consider the approximately 6-month-old ASMD mice in our study as aged mice, particularly since they exhibit upregulation of lysosomal accumulation, inflammation, and fibrosis\u0026mdash;hallmarks commonly associated with aging \u003csup\u003e\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. This ASMD-induced cardiac dysfunction was not associated with significant cardiac remodeling. Although ASMD induces significant cardiac fibrosis, it does not lead to notable cardiomyocyte hypertrophy. It would be valuable to investigate whether ASMD exacerbates cardiac remodeling in classical cardiac fibrosis models, such as isoproterenol-induced myocardial fibrosis. Our findings provide the first evidence linking ASMD to cardiac dysfunction in a murine model of NPD. While this study did not aim to further investigate the underlying mechanisms of ASMD and lysosomal dysfunction driving cardiac dysfunction, a lysosome-centered mechanistic model is proposed based on the observed cardiac pathologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In ASMD or NPD, the abnormal accumulation of lysosomal sphingomyelin leads to LSDs and lysosomal dysfunction in cardiac pericyte or cardiomyocyte. These lysosomal abnormalities may contribute to cardiac pericyte or cardiomyocyte inflammation/inflammasome activation, steatosis, PMT/fibrosis, and CMD. These pathological changes, either alone or in combination, ultimately lead to cardiac dysfunction. In future studies, we will implement targeted therapeutic strategies to address specific pathological changes in ASMD mice, aiming to identify the key contributors to ASMD-induced cardiac dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough our study provides significant evidence that ASMD induces a wide range of cardiac pathological changes, there are several limitations. First, the specific cell types involved in these pathological changes remain unclear and may vary depending on the context of the observed pathology. Future studies should explore the role of ASMD by employing tissue-specific \u003cem\u003eSmpd1\u003c/em\u003e knock-out murine models in various cardiovascular cells, such as pericytes, cardiomyocytes, or fibroblasts, both under basal conditions and in disease models. This approach would provide a better understanding of how specific cell types contribute to the progression of ASMD-induced cardiac pathology. Second, although we propose a general mechanism by which ASMD-induced LSD leads to cardiac pathology, the lack of detailed mechanistic studies in the specific cell types limits the depth of our conclusions. Future \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e studies would provide definitive mechanistic insights into understanding the precise cellular and molecular pathways underlying ASMD-induced cardiac dysfunction.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study provides novel insights into the cardiac pathology in mice with ASMD. The most notable pathological changes reveal a complex interplay between lysosomal dysfunction, inflammation/inflammasome activation, lipid accumulation, cardiac fibrosis, CMD, and overall cardiac dysfunction. The profound ASMD-induced CMD and cardiac dysfunction observed in murine models suggest that similar mechanisms may contribute to cardiovascular complications in patients with NPD B or ASMD, highlighting the need for early diagnosis and intervention to improve patient outcomes. These findings lay the foundation for future research aimed at developing therapeutic targets for cardiovascular complications in patients with NPD B or ASMD, while also calling for further investigation into the complex factors contributing to ASMD-induced cardiovascular dysfunction.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"639\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eNPD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eNiemann-Pick\u0026nbsp;disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eNPD A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eNiemann-Pick\u0026nbsp;disease\u0026nbsp;type A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eNPD B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eNiemann-Pick\u0026nbsp;disease\u0026nbsp;types\u0026nbsp;B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLysosome storage disorder\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eASMase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eAcid sphingomyelinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eASMD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eAcid sphingomyelinase deficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eSmpd1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eSphingomyelin phosphodiesterase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eTau\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricular relaxation time constant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eISO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eIsoflurane\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eCMD \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eCoronary microvascular dysfunction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eH\u0026amp;E\u0026nbsp;staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eHematoxylin and eosin staining\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003ePFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eParaformaldehyde\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLVAW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricular anterior wall\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLVID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricular internal end \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLVPW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricular posterior wall \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLVEF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricular ejection fraction\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLVFS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricular shortening fraction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLV Vol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLeft ventricle volume\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eIVRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eIsovolumic relaxation time\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eIVCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eIsovolumic contraction time\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eMV ET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eMitral valve ejection time\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003e\u0026alpha;-SMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eAlpha-smooth muscle actin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLipid droplet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003ePLIN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003ePerilipin 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eGPAT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eGlycerol-3-phosphate acyltransferase 4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eAGPAT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003e1-acylglycerol-3-phosphate O-acyltransferase 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eDGAT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eDiacylglycerol O-acyltransferase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eDGAT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eDiacylglycerol O-acyltransferase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003ePMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003ePericyte-to-myofibroblast transition\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eTGF-\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eTransforming growth factor beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eFSP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eFibroblast-specific protein 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eHMGB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eHigh mobility group box 1 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eRAGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eReceptor\u0026nbsp;for\u0026nbsp;advanced\u0026nbsp;glycation\u0026nbsp;end-products\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eASC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003ePYD and CARD domain containing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eNLR family pyrin domain containing 3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eIL1\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eInterleukin 1 beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eIL18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eInterleukin 18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eVCAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eVascular cell adhesion molecule 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eICAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eIntercellular adhesion molecule 1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eIL6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eInterleukin 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eIL8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eInterleukin 8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eTumor necrosis factor alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLAMP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLysosomal-associated membrane protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLAMP2A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eLysosome-associated membrane protein type 2A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eLC3A/B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eMicrotubule-associated proteins 1A/1B light chain 3A/B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003ep62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003ep62/SQSTM1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eSPT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eSerine palmitoyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eSPTLC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eSerine palmitoyltransferase long chain base subunit 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eSPTLC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eSerine palmitoyltransferase long chain base subunit 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eORMDL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eORMDL Sphingolipid Biosynthesis Regulator 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eORMDL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eORMDL Sphingolipid Biosynthesis Regulator\u0026nbsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eORMDL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eORMDL Sphingolipid Biosynthesis Regulator\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eHFD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eHigh fat diet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eDAMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eDamage-associated molecular pattern\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eMyocardial infarction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eNG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 453px;\"\u003e\n \u003cp\u003eNerve/glial antigen 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper, and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing or conflicting interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the grants from the National Institutes of Health (R01HL150007, R01HL122937).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.L. and Y.Z. designed the research and wrote the manuscript; Y-T.W. performed the experiments and contributed to writing the manuscript; Y-T.W., A.M., and R.Z. analyzed the data; K.R., J.H., S.K., Z.C.W., Y.P.S.T., M.W., P-L.L., and J.K.H. reviewed and edited the manuscript; and all authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchuchman EH. 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Homozygous Smpd1 deficiency aggravates brain ischemia/ reperfusion injury by mechanisms involving polymorphonuclear neutrophils, whereas heterozygous Smpd1 deficiency protects against mild focal cerebral ischemia. \u003cem\u003eBasic Res Cardiol\u003c/em\u003e. 2020;115:64. doi: 10.1007/s00395-020-00823-x\u003c/li\u003e\n\u003cli\u003eBaechle JJ, Chen N, Makhijani P, Winer S, Furman D, Winer DA. Chronic inflammation and the hallmarks of aging. \u003cem\u003eMol Metab\u003c/em\u003e. 2023;74:101755. doi: 10.1016/j.molmet.2023.101755\u003c/li\u003e\n\u003cli\u003eTan JX, Finkel T. Lysosomes in senescence and aging. \u003cem\u003eEMBO Rep\u003c/em\u003e. 2023;24:e57265. doi: 10.15252/embr.202357265\u003c/li\u003e\n\u003cli\u003eSelman M, Pardo A. Fibroageing: An ageing pathological feature driven by dysregulated extracellular matrix-cell mechanobiology. \u003cem\u003eAgeing Res Rev\u003c/em\u003e. 2021;70:101393. doi: 10.1016/j.arr.2021.101393\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Niemann-Pick Disease, acid sphingomyelinase deficiency, lysosome storage disorder, coronary microvascular dysfunction, cardiac pathology, pericyte-to-myofibroblast transition","lastPublishedDoi":"10.21203/rs.3.rs-5154105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5154105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNiemann-Pick Disease (NPD) is a rare autosomal recessive lysosomal storage disorder (LSD) caused by the deficiency of acid sphingomyelinase (ASMD), which is encoded by the \u003cem\u003eSmpd1\u003c/em\u003e gene. ASMD impacts multiple organ systems in the body, including the cardiovascular system. This study is the first to characterize cardiac pathological changes in ASMD mice under baseline conditions, offering novel insights into the cardiac implications of NPD. Using histological analysis, biochemical assays, and echocardiography, we assessed cardiac pathological changes and function in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e littermate controls. Immunofluorescence and biochemical assays demonstrated that ASMD induced lysosomal dysfunction, as evidenced by the accumulation of lysosomal-associated membrane proteins, lysosomal protease, and autophagosomes in pericytes and cardiomyocytes. This lysosomal dysfunction was accompanied by pericytes and cardiomyocytes inflammation, characterized by increased expression of caspase1 and inflammatory cytokines, and infiltration of inflammatory cells in the cardiac tissues of \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. In addition, histological analysis revealed increased lipid deposition and cardiac steatosis, along with pericyte-to-myofibroblast transition (PMT) and interstitial fibrosis in \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Moreover, echocardiography further demonstrated that \u003cem\u003eSmpd1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice developed coronary microvascular dysfunction (CMD), as evidenced by decreased coronary blood flow velocity and increased coronary arteriolar wall thickness. Additionally, these mice exhibited significant impairments in systolic and diastolic cardiac function, as shown by a reduced ejection fraction and prolonged left ventricular relaxation time constant (Tau value). These findings suggest that ASMD induces profound pathological changes and vascular dysfunction in the myocardium, potentially driven by mechanisms involving lysosomal dysfunction as well as both pericytes and cardiac inflammation.\u003c/p\u003e","manuscriptTitle":"Cardiovascular Dysfunction and Altered Lysosomal Signaling in a Murine Model of Acid Sphingomyelinase Deficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-20 04:52:29","doi":"10.21203/rs.3.rs-5154105/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2025-04-02T05:08:07+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-19T22:20:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-19T18:29:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-19T12:44:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2025-03-18T12:30:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5b46f717-dd95-492f-b235-25460f1f38e3","owner":[],"postedDate":"March 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:04:45+00:00","versionOfRecord":{"articleIdentity":"rs-5154105","link":"https://doi.org/10.1007/s00109-025-02542-z","journal":{"identity":"journal-of-molecular-medicine","isVorOnly":false,"title":"Journal of Molecular Medicine"},"publishedOn":"2025-04-15 15:56:56","publishedOnDateReadable":"April 15th, 2025"},"versionCreatedAt":"2025-03-20 04:52:29","video":"","vorDoi":"10.1007/s00109-025-02542-z","vorDoiUrl":"https://doi.org/10.1007/s00109-025-02542-z","workflowStages":[]},"version":"v1","identity":"rs-5154105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5154105","identity":"rs-5154105","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}