Promotion of Pathological Cardiac Remodeling by Excessive Mitochondrial Fission in Sedentary Lifestyle-Associated Myocardial Infarction

Promotion of Pathological Cardiac Remodeling by Excessive Mitochondrial Fission in Sedentary Lifestyle-Associated Myocardial Infarction | 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 Article Promotion of Pathological Cardiac Remodeling by Excessive Mitochondrial Fission in Sedentary Lifestyle-Associated Myocardial Infarction Masashi Miyao, Hikaru Oshima, Chihiro Kawai, Shota Furukawa, Hirokazu Kotani, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6508918/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract A sedentary lifestyle such as prolonged periods of sitting is one of the most important modifiable risk factors for morbidity and mortality in myocardial infarction (MI). Recently, mitochondrial dynamics (fusion and fission) have gained considerable attention as imbalanced dynamics may play central roles in various organ injuries, including MI. This study aimed to elucidate whether imbalanced mitochondrial dynamics of cardiomyocytes are involved in sedentary lifestyle-associated MI using mouse models. An MI model created by permanent coronary artery ligation showed a decreased survival rate, and the hearts of mice developed cardiac necrosis in the apex, cardiomyocyte hypertrophy, reduced ejection fraction, inflammation, and fibrosis. Ultrastructural analysis revealed increased mitochondrial fission, abnormal cardiac remodeling such as sarcomere disruption, and increased mRNA expression of cardiac injury and mitochondrial fission markers. Compared to the simple MI model, the combined sedentary lifestyle model created by narrow cage breeding and the MI model showed a further decrease in survival rate, cardiac hypertrophy, increased mitochondrial fission in cardiomyocytes, and myofibroblast activation with cardiac fibrosis. These findings suggest that mitochondrial fission could be involved in sedentary lifestyle-associated MI via abnormal crosstalk between cardiomyocytes and fibroblasts. Our study highlights the importance of developing novel mitochondrial-dynamics-rebalancing treatments in patients with MI. Health sciences/Cardiology Health sciences/Pathogenesis Coronary artery disease metabolic syndrome mitochondrial dynamics mitochondrial dysfunction physical inactivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Myocardial infarction (MI) is recognized as the most common cause of death globally. 1 Despite extensive efforts to develop novel treatments for MI to improve patient outcomes, the mortality rate has not changed in the last decade. 2 Recently, systemic therapies (pharmacological treatment and lifestyle interventions), rather than heart-targeted therapies, have been emphasized to be important for improving prognosis. 3 The efficacy of heart-targeted therapies, such as percutaneous coronary intervention, in increasing life expectancy is limited to only 12 h after the onset of ST-segment elevation MI, 4 and most patients with MI have other co-morbidities or systemic diseases (e.g., dyslipidemia, hypertension). Over the past several decades, statins and blood pressure-lowering medications have been widely used for the primary and secondary prevention of cardiovascular disease (CVD) owing to their high effectiveness and safety. 5 Nevertheless, there is an urgent need to develop novel systemic therapies to prevent further CVD events. From the viewpoint of sudden cardiac death (SCD) cases, most events occur in the general population (composed of “healthy population”) rather than patients with coronary artery disease (the epidemiological paradox in SCD). 6 Moreover, it is thought that a majority of individuals with SCD are not treated with medications in time owing to the high prevalence and “low risk” estimations of atherosclerotic CVD events before SCD. Additionally, in statin therapy, continued administration for at least 2.5 years is needed to prevent future CVD events. 7 Therefore, lifestyle interventions (e.g., healthy diet, exercise, and smoking cessation) are extremely important for reducing future SCD events. Sedentary behavior, defined as walking behavior with low energy expenditure (≤ 1.5 metabolic equivalents) while in a sitting or reclining posture, is the leading modifiable risk factor for CVD and all-cause mortality. 8 A sedentary lifestyle with prolonged sedentary behavior is observed in 27.5% of the global population, with high-income western countries showing a higher prevalence at 42.3%. 9 However, the strength and specificity of the recommendations to reduce sedentary behavior are limited, probably because of the difficulties in experimental modeling and the scarcity of evidence on sedentary lifestyle-associated MI. To date, numerous types of animal models of sedentary lifestyle (e.g., hindlimb unloading models) have been reported. 10 Recently, Kim et al. demonstrated a new mouse model using a narrow cage that recapitulated the natural human sedentary lifestyle without severe mental stress. 11 Nonetheless, the association between the sedentary lifestyle and MI remains largely unknown. In the present study, we sought to clarify the mechanisms of sedentary lifestyle-associated MI by investigating the influence of sedentary behavior on MI development, particularly on mitochondrial dynamics, using a combined sedentary lifestyle and MI mouse model. 12 Mitochondria generate the majority of energy required to maintain cellular function and are consequently referred to as the powerhouse of the cells. 13 Therefore, they play a central role in maintaining optimal organ performance throughout the body. 14 , 15 Because the heart is the core organ with the highest energy demand, mitochondrial dysfunction of the cardiomyocytes, including an imbalance in the dynamics of fusion and fission, has been reported to be causatively involved in CVD. 16 Furthermore, numerous studies using animal and in vitro models have suggested that dysregulated mitochondrial dynamics of cells in the whole body, especially increased fission and decreased fusion, could be associated with sedentary behavior. 17 Conversely, exercise and decreased sedentary behavior may have beneficial roles in rebalancing the mitochondrial dynamics of cardiomyocytes. 18 Accordingly, the question arises as to whether dysregulated mitochondrial dynamics in the heart could contribute to the pathologies of both sedentary behavior and MI. The present study aimed to clarify whether increased mitochondrial fission occur in cardiomyocytes during MI pathogenesis and whether a sedentary lifestyle could worsen MI pathologies through further aggravation of excessive mitochondrial fission in cardiomyocytes. This study focused on sedentary lifestyle-associated MI, given that a better understanding of sedentary behaviors in MI could lead to substantial improvements in MI-related morbidity and mortality by enhancing motivation to decrease the sedentary behaviors in the healthy populations and the developing novel systemic treatments for patients with MI. Results Coronary Ligation Leads to Myocardial Ischemia and Compensatory Cardiac Hypertrophy To evaluate chronological changes in the acute myocardial infarction (AMI) model, 12 we first performed macroscopic and survival analyses of mice in which the left anterior coronary artery was ligated after 1 and 4 weeks (Fig. 1 A, B). At 1 and 4 weeks after AMI (hereafter called “AMI 1w”and “AMI 4w”, respectively), brownish discoloration of the apex and a pale yellowish change in the left ventricular wall were observed, indicating necrosis and incomplete ischemia (penumbra), respectively. Moreover, the hearts of the AMI model were enlarged compared to those of the controls, suggesting compensatory cardiac hypertrophy. The survival rate of mice in the AMI 4w model was significantly reduced compared with that of control mice. Generally, AMI model mice died even several days after post-MI due to sudden cardiac arrest or cardiac rupture, 12 whereas no mouse died after perisurgical period in the study, likely due to high mortality in the current study (about 50% in the study vs 70% in the previous study). As most autopsied mice showed intrathoracic hemorrhage, the common cause of death could be hemorrhagic shock. All mice in the mock operation group survived, and no pathological changes were observed in the mock group by macroscopic or histological analysis (Supplementary Fig. 3). We next performed serum biochemical analysis in each mouse to assess lipid and glucose metabolism, but no significant changes were found in the AMI model compared to the controls, except for a slight increase in the total cholesterol level in the AMI 4w model (Fig. 1 C). The volumes of food and water intake were similar among the control groups, mock operation group, and AMI 1 w model (Supplementary Fig. 2). To evaluate the functional changes in the heart in AMI pathology, we performed echocardiography in mice (Fig. 1 D, E). The ejection fraction and fraction shortening parameters, which are markers of cardiac function, were significantly lower in the AMI model than in the controls. Although left ventricular end diastolic dimension of the ischemic heart usually shows an increase by compensatory cardiac dilatation, those of the current study showed a decrease, likely due to worsened shape of left ventricular cavity by increased necrotic area and the consequent exacerbated cardiac hypertrophy. These findings suggest that the AMI model accurately reflects human MI development without the severe effects of dyslipidemia, insulin resistance, and hyperphagia. Coronary Ligation Leads to Fibrotic Changes and Cardiomyocyte Enlargement To further evaluate the chronological changes in the AMI model, we performed histological analyses in mice. Hematoxylin and eosin staining of heart sections in the AMI model revealed discolored, thinner, and fibrotic changes in the apex in a time dependent manner (Fig. 2 A, Supplementary Fig. 4A–C). Conversely, the cardiac wall in the interventricular septum and basal area enlarged in a time dependent manner, indicating compensatory cardiac hypertrophy. Reticulin staining of heart sections from the AMI model showed that gray-stained areas (immature type III collagen) peaked at 1 week and that, thick black areas (mature type I collagen) worsened over time (Fig. 2 B). The AMI models at both 1 and 4 weeks after ligation exhibited slight lymphocyte infiltration; however, no neutrophils were found in the interstitial regions of cardiac muscles. We confirmed substantial increases in neutrophils and lymphocytes around necrotic cardiomyocytes and occluded coronary arteries during the super acute phase (at 1 day after MI) (Supplementary Fig. 4A–C). Immunohistochemical staining of heart sections in the AMI model for α-SMA, a marker of collagen-secreting myofibroblasts, showed that positive-stained cells were increased around occluded coronary arteries, especially in the penumbra lesions, peaked at 1 week after coronary ligation, and slightly decreased at 4 weeks after MI. Immunofluorescence staining of heart sections in the AMI model for wheat germ agglutinin revealed that the sizes of cardiomyocytes in the penumbra areas were considerably increased at 1 and 4 weeks after MI, confirming cardiomyocyte hypertrophy. These findings suggest that inflammatory cell infiltration first occurs after an ischemic event, followed by active fibrotic changes by myofibroblast proliferation. Ultimately, mature fibrosis and consequent cardiac hypertrophy further worsen over time in MI pathology. Physical Inactivity Aggravates Coronary Artery Ligation-Induced Myocardial Necrosis and Hypertrophy To clarify the association between sedentary lifestyle and MI development, we conducted macroscopic and survival analyses in the combined sedentary lifestyle and AMI model (Fig. 3 A-D). To imitate the natural sedentary lifestyle in humans and avoid unnecessary mental stress, we created narrow cages using acrylic plates, and mice were bred in the cages for up to 2 weeks.1 To clarify the contribution of a sedentary lifestyle in the treatable phase of MI development (before scar fibrosis maturation), we chose the AMI 1w model, because this phase showed the most drastic changes in immature fibrosis (before scar maturation). 19 To evaluate the physical activity levels of the sedentary model, we performed a behavioral analysis of the control and sedentary model mice before and after MI. The results showed that the moving distance in the sedentary model was significantly reduced by 15-fold compared to controls, regardless of the coronary ligation procedure. Although the moving distances in both groups showed reduced tendencies after MI, these changes were negligible. Most importantly, the survival rate of the sedentary lifestyle model at 1 week after MI showed a decreasing tendency compared to the simple AMI model. Moreover, the hearts of the sedentary lifestyle model mice after MI showed expanded areas of necrotic and penumbral lesions along with significant cardiac hypertrophic changes compared to those of the simple AMI group (Fig. 3 A, C, and Supplementary Fig. 4). However, no pathological changes were found in the sedentary lifestyle model mice after MI based on food and water intake, and serum biochemical analyses (Fig. 3 D and Supplementary Fig. 2). Macroscopic and histological analyses confirmed that the sedentary lifestyle model mice without coronary ligation exhibited no significant pathological changes (Supplementary Fig. 3). These findings suggest that although a sedentary lifestyle itself does not induce severe cardiac damage, it does contribute to the further deterioration of MI pathologies, such as the expansion of necrotic areas and cardiac hypertrophic remodeling. Physical Inactivity Aggravates Coronary Artery Ligation-Induced Fibrotic Changes and Cardiomyocyte Enlargement To further evaluate the association between a sedentary lifestyle and MI development, we conducted a histological analysis using a combined sedentary lifestyle and AMI model. Hematoxylin and eosin staining of heart sections from the sedentary lifestyle model after MI showed significant aggravation of cardiac necrotic and fibrotic areas compared to the simple AMI model (Fig. 4 A, B). Azan-, reticulin-, and α-SMA-immunohistochemical-staining heart sections of sedentary lifestyle model after MI showed that substantial proliferations of myofibroblasts with mature collagen depositions compared to simple AMI model. Moreover, immunofluorescent staining for wheat germ agglutinin showed significant aggravation of cardiomyocyte enlargement in the sedentary lifestyle model after MI compared with simple MI. In contrast, sedentary lifestyle model without coronary ligation showed neither excessive collagen deposition, enlarged cardiomyocytes, nor active proliferation of spindle-shaped cells suggestive of myofibroblasts in the interstitial regions of the hearts (Supplementary Fig. 3). These findings suggest that a sedentary lifestyle induces excessive proliferation of myofibroblasts and subsequent cardiac fibrosis as well as abnormal cardiomyocyte hypertrophy. Physical Inactivity Aggravates Coronary Artery Ligation-Induced Aberrant Gene Expression Changes in Immune Cells, Cardiomyocytes, and Mitochondrial Dynamics Markers To understand the cellular and molecular mechanisms underlying sedentary lifestyle-associated MI aggravation, we conducted qRT-PCR analysis using a combined sedentary lifestyle and AMI model. Generally, changes in mRNA expression precede morphological changes in cells, especially during inflammatory changes. 20 Therefore, we conducted qRT-PCR analysis at 1 day after MI in the models (Fig. 5 A). Morphologically, no significant change was observed in mice at 1 day after MI in the combined sedentary lifestyle and AMI model compared with the simple AMI model (Supplementary Fig. 4A–C). As expected, mRNA expression changes including markers of cardiomyocyte injury, activated myofibroblasts, inflammation, mitochondrial fusion, and endoplasmic reticulum stress (ER stress), already emerged at 1 day after MI. Notably, the combined sedentary lifestyle and AMI model showed significant changes in the markers of inflammation, endothelial injury, mitochondrial fission/fusion, and ER stress compared to the simple AMI model. Interestingly, most mRNA markers did not show significant changes between the controls and AMI 4w model, except for markers of cardiomyocyte injury and endothelial injury, suggesting active compensatory reactions or chronic “burnout” changes (e.g., reduced numbers and functions of TNF-α-expressing cardiomyocytes due to replacement by collagen fibers, extracellular matrix, and myofibroblasts) after 4 weeks of MI. The gaps in most mRNA markers between the control group and AMI 1w model were smaller than those between the control group and AMI 1d model. Contrary to our expectations, the combined sedentary lifestyle and AMI 1w model showed improvements in the markers of cardiomyocyte injury, fibrosis, inflammation, macrophage activation, mitochondrial biogenesis, mitochondrial fusion, and ER stress compared to the simple AMI model (Fig. 5 B). Given that most mRNA expression levels of markers in the combined sedentary lifestyle and AMI 1w model were between those of simple AMI 1w and 4w models, these seemingly contradictory improved changes in mRNA expressions could be explained by active compensatory reactions or chronic “burnout” changes. These results suggest that a sedentary lifestyle could accelerate pathological cardiac remodeling in MI by heightening the levels and shortening the duration of dysregulated gene expression changes in inflammation, mitochondrial dynamics, and ER stress. Physical Inactivity Aggravates Coronary Artery Ligation-Induced Excessive Mitochondrial Fissions in Cardiomyocytes Sedentary behavior can induce an imbalance in mitochondrial dynamics in cardiomyocytes; in contrast, exercise can delay or ameliorate the pathogenesis of CVD. 8 , 18 However, the role of mitochondrial dynamics in sedentary lifestyle-associated MI remains unclear. To elucidate the mitochondrial ultrastructural changes in the pathologies, transmission electron microscopic analysis was conducted on the combined sedentary lifestyle and AMI model (Fig. 6 A, B). To exclude bias due to positional heterogeneity in the morphology and function of mitochondria and sarcomeres, we analyzed the penumbra lesions of the left ventricular walls in the MI model and the corresponding areas in controls. As expected, time-dependent aggravation of decreased individual mitochondrial sizes and mitochondrial areas, as well as increased numbers of mitochondria and fractions of malformed mitochondria were found in cardiomyocytes from simple AMI 1w- and 4w-models, indicating excessive mitochondrial fission. Ultrastructural changes in the sarcomere of cardiomyocytes also showed time dependent aggravations in sarcomere length and dispersion degree, indicating sarcomere disruption and abnormal sarcomere orientation (polarity disturbance), respectively. Consistent with the increased mRNA expression of Drp1, the primary regulator of mitochondrial fission, and those of cardiomyocyte injury markers during the super-acute phase (at 1 day after MI) in the combined sedentary lifestyle and AMI model (Fig. 6 A, B), the combined model showed further aggravation of excessive mitochondrial fission compared to the simple AMI model. Although the mean widths of sarcomeres in all groups were similar, the variabilities in the widths of the three AMI groups were larger than those of the control group, suggesting that MI could induce not only detrimental sarcomere disruption and pathological dedifferentiation of cardiomyocytes but also beneficial re-differentiation of surviving or transiently hibernated cardiomyocytes to functional and ischemia-tolerant cardiomyocytes. 21 Taken together, our findings suggest that a sedentary lifestyle could lead to aggravation of excessive mitochondrial fission and abnormal sarcomere remodeling in ischemic cardiomyocytes and subsequent activation of collagen-producing myofibroblasts after MI, after entering a vicious cycle, eventually leading to heart failure and SCD (Fig. 6 C and Supplementary Fig. 5). Discussion To the best of our knowledge, the current investigation is the first study to demonstrate that a sedentary lifestyle could lead to a further decrease in survival rate after MI and further deterioration of cardiac pathologies, including cardiac hypertrophy, inflammation, fibroblast activation, fibrosis, and excessive mitochondrial fission in cardiomyocytes. Taken together, our findings suggest that sedentary lifestyle contributes to the aggravation of MI pathologies and SCD through a vicious cycle of excessive mitochondrial fission in ischemic cardiomyocytes, myofibroblast activation, and fibrosis (Fig. 6 C, Supplementary Fig. 5). Furthermore, our study underscores the potential therapeutic implications of pharmaceutical therapies focusing on mitochondrial dynamics and lifestyle modifications, such as decreased duration of sedentary behaviors in healthy individuals and patients with coronary artery disease even after MI events. Whether sedentary behavior could have greater roles in causing MI onset rather than in aggravating MI development or vice versa has long been in question because data on the contribution of sedentary behavior to MI have been scarce. 22 The current study using the combined sedentary lifestyle and AMI model clearly supports the latter hypothesis because 2-week physical inactivity in mice did not cause severe cardiac damage but significantly worsened MI development. If this hypothesis is true, shortening the duration of sedentary behavior may be more effective post-MI than pre-MI. This theory further highlights the clinical importance of earlier and progressive non-pharmacotherapies, such as decreased sedentary behavior and increased levels and durations of physical activity, even after the MI event. 23 Indeed, a recent meta-analysis of eight clinical trials that studied people with coronary heart disease at long-term follow-up (over 3 years) showed that exercise-based cardiac rehabilitation may result in a large reduction in cardiovascular mortality (RR 0.58, 95% CI 0.43 to 0.78). 24 A combination of exercise and decreased sedentary behavior could plausibly further improve patient’ outcomes. Therefore, to maximize the beneficial effects of controlling physical activity in patients, both exercise and decreased sedentary behavior are needed. The data from the current study suggest that sedentary behavior could first promote mitochondrial fission in cardiomyocytes immediately after MI and then lead to compensatory host cellular reactions, such as enhanced mitochondrial fusion and biogenesis, to prevent the deterioration of cardiac injuries. If sedentary behavioral stress persists in patients, it could further result in mitochondrial fission, a decreased mitochondrial fraction, and ER stress in cardiomyocytes. Thereafter, unfavorable organellar injuries in cardiomyocytes caused by prolonged sedentary behavior during MI can lead to permanent cardiac hypertrophy, inflammation with pathological macrophage polarization, myofibroblast activation, and cardiac fibrosis. Considering that damaged cardiomyocytes and excessive myofibroblast activation could mutually influence pathological responses and lead to cardiomyocyte apoptosis and necrosis, 25 we propose that a sedentary lifestyle could induce a vicious cycle of cardiomyocyte injury and fibrosis, eventually causing heart failure, arrhythmogenesis, and SCD. 26 The findings of this study have some clinical implications. First, they suggest that decreased sedentary behavior and increased physical activity could help improve complications associated with MI by inhibiting the vicious cycle and prevent SCD even after MI events. Second, medications that inhibit fission and/or promote fusion of mitochondria in cardiomyocytes could ameliorate MI development, especially in the acute phase. Nevertheless, this study has some limitations. First, since no mouse model can completely replicate human disease, the findings cannot be completely generalized to humans. To resolve this issue, we used sedentary lifestyle and AMI model that closely reflect human disease pathologies. However, we did not investigate whether the proposed mechanism could be valid in different models. Therefore, future studies with different situations (duration, time point, and degree of physical activity and ischemic cardiac injury) should be conducted to clarify the precise roles of sedentary behavior in MI pathologies. Second, whether the inhibition of fission or the promotion of fusion in cardiomyocytes could ameliorate disease progression could not be clarified. Therefore, whether excessive mitochondrial fission in cardiomyocytes is the cause or a secondary phenomenon in the pathogenesis remains unknown. Hence, future studies using pharmacological treatments that directly influence mitochondrial dynamics are warranted. In conclusion, our study provides novel insights into sedentary lifestyle-associated MI. Our findings suggest that prolonged periods of sedentary lifestyle and excessive mitochondrial fission in ischemic cardiomyocytes are associated with aggravated MI. Therefore, combined lifestyle and pharmacological interventions, such as reducing sedentary behavior and mitochondria-targeted medications, could be promising novel strategies for the prevention of MI and MI-related deaths. Methods Animals Male 8–10 weeks old C57BL/6 mice (SHIMIZU Laboratory Supplies Co., Ltd., Kyoto, Japan) were used in this study. The experimental animals were euthanized by cervical dislocation under anesthesia with an intraperitoneal injections of a combination anesthetics (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol) at the indicated time points. Blood was collected by cardiac puncture after a 2-h fast. Tissues were harvested for further analyses. For electron microscopy analysis, the hearts were fixed with 2% glutaraldehyde and 4% paraformaldehyde. At least three mice were treated and analyzed for each time period and treatment. All mice were housed in cages under specific pathogen-free conditions with food and water ad libitum. All protocols were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (Med Kyo 24043) and were performed according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. All the work involving the animals were performed in the Kyoto University Graduate School of Medicine. This study is reported in accordance with the ARRIVE guidelines 2.0. Acute myocardial infarction model AMI model (1 to 4 weeks) was made by permanent ligation of left anterior descending artery with minor modifications (Supplementary Fig. 1). 12 Briefly, mice were firstly anesthetized with an intraperitoneal injections of a combination anesthetics (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol). The fur was shaved and skin cut was made over the left chest. A small hole was made at the fourth intercostal space, and the heart was exposed through the hole. The left anterior descending artery was located and ligated at a site 2 mm from its origin. After ligation, the heart was immediately placed back with an evacuation of air. Thoracic cavity was closed and the skin was sutured. The mice were continuously monitored at least 30 min after the operation. Mock operation mice underwent the same surgical procedure except that the coronary artery was not occluded. No mice died in the mock operation model. Sedentary lifestyle model Sedentary lifestyle model (2 weeks) was made by narrow cages using acrylic plates with minor modifications. 11 Briefly, sedentary lifestyle model mice were housed in the narrow cages, residual space of a mouse was 10 cm in length, 6 cm in width, and 12 cm in height. To avoid unnecessary isolation stress, the mice could visually see the other mice in the next cages though transparent acrylic plate, and could touch each other’s noses through a 1 cm hole in the wall. For the combination of AMI- and sedentary lifestyle models, mice were firstly bred in the narrow cages for 1 week, then the left coronary artery of the mice was ligated, the mice were bred in the narrow cages for additionally 1 week. Control mice were housed in the standard cages, residual space of a mouse was 25 cm in length, 18 cm in width, and 12 cm in height. No mice died both in the control and the sedentary lifestyle model without coronary ligation. Quantitative reverse transcription polymerase chain reaction Total RNA was extracted from heart tissues using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total RNA using SuperScript III reverse transcriptase (Invitrogen, Waltham, USA). Real-time PCR was performed using FastStart SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland) and a Rotor-Gene Q (Qiagen, Venlo, Netherlands) instrument. The primer pairs are shown in Supplementary Table 1. Relative target gene expression was normalized to Gapdh expression as the mRNA expressions of Gapdh in different experimental conditions were stable than those of β-actin by the preliminary study. Serum biochemical analyses Serum samples were stored at − 80°C until analyses were performed. Total cholesterol (T-chol), triglyceride (TG), free fatty acid (FFA), and glucose levels were analyzed using a Hitachi 7180 analyzer (Hitachi, Tokyo, Japan). Levels of insulin and TNF-α were analyzed with Enzyme-linked immuno-sorbent assay kits (Morinaga Institute of Biological Science, Yokohama, Japan M1104 for insulin; R&D, Asaka, Japan, DTA00D for TNF-α). Homeostasis model assessment of insulin resistance, the index of insulin resistance, was calculated by fasting plasma glucose (mmol/L) x fasting insulin (mIU/L)/22.5. Histopathology Formaldehyde-fixed paraffin-embedded tissue sections of 4-µm thickness were cut and stained with hematoxylin and eosin (H&E), Azan, and reticulin. The histological features of the specimens were independently assessed by experienced pathologists (MM and CK) in a blinded fashion, and a consensus diagnosis was obtained for each sample. For quantitative assessment, the staining of each specimen was captured in 15 randomly selected fields and quantified using the ImageJ software (NIH, MD, USA). Immunohistochemistry and immunofluorescent analyses Immunohistochemical and immunofluorescent staining of the specimens was performed as previously described 20 . Briefly, antigen retrieval was performed in a pressure cooker by boiling in 10 mM citrate buffer (pH 6.0), followed by washing with phosphate-buffered saline. For immunohistochemical staining, endogenous peroxidase was quenched with 3% H2O2 for 10 min. After rinsing, the slides were incubated overnight at 4°C with a negative control reagent or the following optimally diluted primary antibody: α-smooth muscle actin (α-SMA; rabbit; 1:400; Abcam, ab5694). Next, the slides were incubated at 25°C with a secondary antibody (MBL, Nagoya, Japan) for 1 h. Permanent Red was used as a chromogen, followed by counterstaining with hematoxylin. For analysis of cardiomyocyte sizes in each mouse, heart tissues were frozen in optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan, 45833) and stored at -80°C before being sectioned at a thickness of 10 µm on a microtome. Sections were pre-warmed at room temperature and then fixed with pre-chilled 4% formaldehyde for 10 min, and stained with FITC conjugated-wheat germ agglutinin (Invitrogen, MA, USA, W11261) for 30 min before nuclear counterstaining with DAPI (Vector Laboratories, CA, USA, H-1200). Cardiomyocytes fluoresced in green. The immunofluorescent images were captured using a fluorescence microscope (Keyence, Osaka, Japan, BZ-X810). For quantitative assessment, the staining of each specimen was captured in 15 randomly selected fields and quantified using the ImageJ software. Moving trajectories and moving distance measurements To evaluate physical activity levels in each mouse, moving trajectories were recorded and the moving distance were calculated in the controls and sedentary lifestyle model mice. To avoid as much as confounding effects by acute psychological stress, anesthesia, and the coronary ligation procedures, the mice were analyzed at 1 day before coronary artery ligation, and at 6 days after the ligation. All behavioral tests were performed in the light phase, between 10:00am and 01:00pm (light time: 09:00am − 09:00pm). Quantitative reverse transcription polymerase chain reaction Total RNA was extracted from heart tissues using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total RNA using SuperScript III reverse transcriptase (Invitrogen). Real-time PCR was performed using FastStart SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland) and a Rotor-Gene Q (Qiagen, Venlo, Netherlands) instrument. The primer pairs are shown in Supplementary Table 1. Relative target gene expression was normalized to Gapdh expression as the mRNA expressions of Gapdh in different experimental conditions were stable than those of β-actin by the preliminary study. Transmission electron microscopy analysis Fixed hearts were cut into 1-mm sections. These sections were subsequently immersed in 1% glutaraldehyde at 4°C overnight. The specimens were extensively washed with phosphate buffered saline, postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Epon. Ultra-thin sections (80 nm) were cut on an Ultra microtome EM UC6 (Leica, Vienna, Austria), stained with 1% uranyl acetate, counterstained using the Reynolds method, and examined using an H-7650 electron microscope (Hitachi, Tokyo, Japan). For the assessments of the mitochondrial content and mitochondrial area in each cell, areas of lipid particles and nucleus were excluded to avoid the confounding effects. Deformed mitochondria were defined by at least one positive sign of the following features; abnormally enlarged mitochondria or mega-mitochondria (a minor radius > 1000 nm), loss of cristae, autophagosomes or autolysosome with the destructed mitochondria, and irregular shaped mitochondria (e.g. abnormally blanching, donut-shaped). Individual sarcomere lengths were measured as the distance between adjacent Z-bands. For quantitative assessment, 15 randomly selected fields were captured for each group (n = 3), and quantification was performed using the ImageJ software. Statistical analysis Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test or analysis of variance with Tukey’s post-hoc test for multiple comparisons. For all analyses, statistical significance was set at P < .05. Declarations Acknowledgments We express our gratitude to Kanako Maruo, Kumiko Kokuryo, Yuji Hamaguchi, Haruyasu Kohda, Keiko Furuta, and Tatsuya Katsuno for providing technical assistance, as well as Editage for carefully reviewing the manuscript for English editing. We also acknowledge the technical assistance and histopathological analyses performed by the members of the Center for Anatomical, Pathological, and Forensic Research at the Graduate School of Medicine, Kyoto University. Author Contributions M.M. and H.O. contributed to the study concept and design, collected the data, performed statistical analysis, and wrote the paper. C.K., S.F., H.K., and Y.N. conducted the data acquisition, analysis, and interpretation and reviewed and revised the paper. H.M., H.A., H.N., H.Y., K.O., and K.T. provided technical and material support, developed the methodology and writing, and reviewed and revised the paper. All authors have read and approved the final manuscript. Data availability The datasets and/or analyses of the current study are available from the corresponding author upon reasonable request. Additional information Detailed methodology is included in the Supplementary information. Funding This work was supported by the Japan Society for the Promotion of Science, KAKENHI under grant numbers 23K09763 (MM) and 24K13544 (CK). Declaration of Competing Interests The authors report no conflicts of interest. References Global burden of 288 causes of death and life expectancy decomposition. in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet 403 (10440), 2100–2132. https://doi.org/10.1016/s0140-6736(24)00367-2 (2024). Woodruff, R. C. et al. Trends in Cardiovascular Disease Mortality Rates and Excess Deaths, 2010–2022. Am. J. Prev. Med. 66 (4), 582–589. https://doi.org/10.1016/j.amepre.2023.11.009 (2024). Beatty, A. L. et al. A New Era in Cardiac Rehabilitation Delivery: Research Gaps, Questions, Strategies, and Priorities. Circulation 147 (3), 254–266. https://doi.org/10.1161/circulationaha.122.061046 (2023). Lawton, J. S. et al. 2021 ACC/AHA/SCAI Guideline for Coronary Artery Revascularization: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 145 (3), e18–e114. https://doi.org/10.1161/cir.0000000000001038 (2022). Arnett, D. K. & ACC/AHA Guideline on the Primary Prevention of Cardiovascular Disease. : Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation. 140(11), e563-e595. (2019). https://doi.org/10.1161/cir.0000000000000677 (2019). Marijon, E. et al. The Lancet Commission to reduce the global burden of sudden cardiac death: a call for multidisciplinary action. Lancet 402 (10405), 883–936. https://doi.org/10.1016/s0140-6736(23)00875-9 (2023). Yourman, L. C. et al. Evaluation of Time to Benefit of Statins for the Primary Prevention of Cardiovascular Events in Adults Aged 50 to 75 Years: A Meta-analysis. JAMA Intern. Med. 181 (2), 179–185. https://doi.org/10.1001/jamainternmed.2020.6084 (2021). Lavie, C. J., Ozemek, C., Carbone, S., Katzmarzyk, P. T. & Blair, S. N. Sedentary Behavior, Exercise, and Cardiovascular Health. Circ. Res. 124 (5), 799–815. https://doi.org/10.1161/circresaha.118.312669 (2019). Guthold, R., Stevens, G. A., Riley, L. M. & Bull, F. C. Worldwide trends in insufficient physical activity from 2001 to 2016: a pooled analysis of 358 population-based surveys with 1·9 million participants. Lancet Glob Health . 6 (10), e1077–e1086. https://doi.org/10.1016/s2214-109x(18)30357-7 (2018). Reidy, P. T., Monnig, J. M., Pickering, C. E., Funai, K. & Drummond, M. J. Preclinical rodent models of physical inactivity-induced muscle insulin resistance: challenges and solutions. J. Appl. Physiol. (1985) . 130 (3), 537–544. https://doi.org/10.1152/japplphysiol.00954.2020 (2021). Kim, J., Park, J. & Mikami, T. R. Low-Intensity Exercise Prevents Cognitive Decline and a Depressive-Like State Induced by Physical Inactivity in Mice: A New Physical Inactivity Experiment Model. Front. Behav. Neurosci. 16 , 866405. https://doi.org/10.3389/fnbeh.2022.866405 (2021). Gao, E. et al. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circ. Res. 107 (12), 1445–1453. https://doi.org/10.1161/circresaha.110.223925 (2010). Tian, R. et al. Unlocking the Secrets of Mitochondria in the Cardiovascular System: Path to a Cure in Heart Failure—A Report from the 2018 National Heart, Lung, and Blood Institute Workshop. Circulation 140 (14), 1205–1216. https://doi.org/10.1161/circulationaha.119.040551 (2019). Miyao, M. et al. Mitochondrial fission in hepatocytes as a potential therapeutic target for nonalcoholic steatohepatitis. Hepatol. Res. 52 (12), 1020–1033. https://doi.org/10.1111/hepr.13832 (2022). Forrester, S. J. et al. Mitochondrial Fission Mediates Endothelial Inflammation. Hypertension 76 (1), 267–276. https://doi.org/10.1161/hypertensionaha.120.14686 (2020). Vásquez-Trincado, C. et al. Mitochondrial dynamics, mitophagy and cardiovascular disease. J. Physiol. 594 (3), 509–525. https://doi.org/10.1113/jp271301 (2016). Hernandez-Resendiz, S. et al. Targeting mitochondrial shape: at the heart of cardioprotection. Basic. Res. Cardiol. 118 (1), 49. https://doi.org/10.1007/s00395-023-01019-9 (2023). Zhang, H., Zhang, Y., Zhang, J. & Jia, D. Exercise Alleviates Cardiovascular Diseases by Improving Mitochondrial Homeostasis. J. Am. Heart Assoc. 13 (19), e036555. https://doi.org/10.1161/jaha.124.036555 (2024). Prabhu, S. D. & Frangogiannis, N. G. The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis. Circ. Res. 119 (1), 91–112. https://doi.org/10.1161/circresaha.116.303577 (2016). Miyao, M. et al. Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression. Lab. Invest. 95 (10), 1130–1144. https://doi.org/10.1038/labinvest.2015.95 (2015). Zhu, Y., Do, V. D., Richards, A. M. & Foo, R. What we know about cardiomyocyte dedifferentiation. J. Mol. Cell. Cardiol. 152 , 80–91. https://doi.org/10.1016/j.yjmcc.2020.11.016 (2021). Duran, A. T., Garber, C. E., Cornelius, T., Schwartz, J. E. & Diaz, K. M. Patterns of Sedentary Behavior in the First Month After Acute Coronary Syndrome. J. Am. Heart Assoc. 8 (15), e011585. https://doi.org/10.1161/jaha.118.011585 (2019). Dalal, H. M., Doherty, P. & Taylor, R. S. Cardiac rehabilitation. BMJ 351 , h5000. https://doi.org/10.1136/bmj.h5000 (2015). Dibben, G. et al. Exercise-based cardiac rehabilitation for coronary heart disease. Cochrane Database Syst. Rev. 11 (11). https://doi.org/10.1002/14651858.CD001800.pub4 (2021). Cd001800. Nicin, L., Wagner, J. U. G., Luxán, G. & Dimmeler, S. Fibroblast-mediated intercellular crosstalk in the healthy and diseased heart. FEBS Lett. 596 (5), 638–654. https://doi.org/10.1002/1873-3468.14234 (2022). Hall, C., Gehmlich, K., Denning, C. & Pavlovic, D. Complex Relationship Between Cardiac Fibroblasts and Cardiomyocytes in Health and Disease. J. Am. Heart Assoc. 10 (5), e019338. https://doi.org/10.1161/jaha.120.019338 (2021). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 May, 2025 Reviews received at journal 25 May, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 11 May, 2025 Reviewers agreed at journal 09 May, 2025 Reviewers invited by journal 09 May, 2025 Editor assigned by journal 09 May, 2025 Editor invited by journal 06 May, 2025 Submission checks completed at journal 05 May, 2025 First submitted to journal 23 Apr, 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6508918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":455115468,"identity":"d566d4f6-d343-4443-8e67-652700c0fca0","order_by":0,"name":"Masashi Miyao","email":"data:image/png;base64,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","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Miyao","suffix":""},{"id":455115469,"identity":"f698c613-34f6-4902-9f39-f3d958b6e0cd","order_by":1,"name":"Hikaru Oshima","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hikaru","middleName":"","lastName":"Oshima","suffix":""},{"id":455115470,"identity":"412b7a58-69ec-449c-bf9c-59fbe623fcb5","order_by":2,"name":"Chihiro Kawai","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chihiro","middleName":"","lastName":"Kawai","suffix":""},{"id":455115471,"identity":"3cb53534-23cb-41bb-82b8-0b69b33d9bb3","order_by":3,"name":"Shota Furukawa","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shota","middleName":"","lastName":"Furukawa","suffix":""},{"id":455115472,"identity":"4e1812f7-7524-49f5-910e-7bf609d9e143","order_by":4,"name":"Hirokazu Kotani","email":"","orcid":"","institution":"Mie University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hirokazu","middleName":"","lastName":"Kotani","suffix":""},{"id":455115473,"identity":"bb121951-cd96-478b-bc44-7ef3c5ce589f","order_by":5,"name":"Hirozo Minami","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hirozo","middleName":"","lastName":"Minami","suffix":""},{"id":455115474,"identity":"3ae98767-b60c-4bab-80b1-57785b5dea5f","order_by":6,"name":"Hitoshi Abiru","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hitoshi","middleName":"","lastName":"Abiru","suffix":""},{"id":455115475,"identity":"e1e114a2-350d-402a-abb5-2251b4988416","order_by":7,"name":"Hideki Nagai","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Nagai","suffix":""},{"id":455115476,"identity":"0742102f-e50d-4b29-9b55-7d42aed15b59","order_by":8,"name":"Hiromu Yanagisawa","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiromu","middleName":"","lastName":"Yanagisawa","suffix":""},{"id":455115477,"identity":"b7b70d03-7c6b-4e94-b4c4-062a1e71202f","order_by":9,"name":"Koh Ono","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Koh","middleName":"","lastName":"Ono","suffix":""},{"id":455115478,"identity":"e0d3c4d2-0976-443c-ad39-4f8f21951d8e","order_by":10,"name":"Keiji Tamaki","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Keiji","middleName":"","lastName":"Tamaki","suffix":""},{"id":455115480,"identity":"75eb154a-1101-455c-bb06-a17d8704967d","order_by":11,"name":"Yoko Nishitani","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoko","middleName":"","lastName":"Nishitani","suffix":""}],"badges":[],"createdAt":"2025-04-23 05:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6508918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6508918/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82850184,"identity":"37cd103b-6ea3-4062-86f5-0ef5dd74a627","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":978497,"visible":true,"origin":"","legend":"\u003cp\u003eCoronary ligation leads to myocardial ischemia and compensatory cardiac hypertrophy. (A) Study protocol, macroscopic appearance, and Kaplan–Meier analysis in each mouse. Scale bar = 5 mm. (B) Bodyweight, heart/body weight, liver/body weight, spleen/body weight, and fat/body weight-ratios in each mouse. (C) Serum biochemical analyses in each mouse. (D) Echocardiographic images and analyses in each mouse. Data are presented as mean±SEM. Statistical analyses were performed by unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA with Tukey’s post-hoc test, where appropriate. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;.001. \u003cem\u003en\u003c/em\u003e≥4 for all groups.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/72761518dff12df6015e5ead.jpeg"},{"id":82850185,"identity":"a0afc8a9-87ab-4834-b0a7-fb8a30a8efee","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":689635,"visible":true,"origin":"","legend":"\u003cp\u003eCoronary ligation leads to fibrotic changes and cardiomyocyte enlargement. (A) Histological images and (B) quantitative analyses of each mouse. Scale bars = 1 mm in H\u0026amp;E-stained sections and 50 μm in Azan-, reticulin-, α-SMA-, and wheat germ agglutinin-stained sections. Data are presented as mean±SEM. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;.001. \u003cem\u003en\u003c/em\u003e=3 for all groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/4df958830a26f54cb1d9f8a2.jpeg"},{"id":82850188,"identity":"cd476e5a-4b32-4f77-b7ef-b42f61f82eeb","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":525701,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical inactivity aggravates coronary artery ligation-induced myocardial necrosis and hypertrophy. (A) Study protocol, representative images of standard and narrow cages, macroscopic images of hearts, and Kaplan–Meier analysis in each mouse. (B) Moving activity trajectories and moving distance in pre- and post-coronary artery ligation in each mouse. (C) Body weight, heart/body weight, liver/body weight, spleen/body weight, and fat/body weight ratios in each mouse. (D) Serum biochemical analyses in each mouse. Data are presented as mean±SEM. Statistical analyses were performed by unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA with Tukey’s post-hoc test, where appropriate. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;.001, #\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;.001. \u003cem\u003en\u003c/em\u003e≥4 for all groups.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/9a002bc2afea274442f6f867.jpeg"},{"id":82850193,"identity":"2182ba26-b9d7-4598-8102-ad866a580787","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":672259,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical inactivity aggravates coronary artery ligation-induced fibrotic changes and cardiomyocyte enlargement. (A) Histological images and (B) quantitative analyses of each mouse. Scale bars = 1 mm in H\u0026amp;E-stained sections and 50 μm in Azan-, reticulin-, α-SMA-, and wheat germ agglutinin-stained sections. Data are presented as mean±SEM. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;.001, #\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;.001. \u003cem\u003en\u003c/em\u003e=3 for all groups.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/cb7a2c9c1c6e64bf534beaf6.jpeg"},{"id":82850192,"identity":"ef3a1153-701e-4f95-9deb-c0f98b350a7e","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":501433,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical inactivity aggravates coronary ligation-induced aberrant gene expression changes in immune cells and cardiomyocytes as well as mitochondrial dynamics markers. (A, B) Quantitative reverse transcription-polymerase chain reaction analysis of hearts in each mouse. Data are presented as mean±SEM. Statistical analyses were performed by unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA with Tukey’s post-hoc test, where appropriate. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;.001, #\u003cem\u003eP\u003c/em\u003e\u0026lt;.05. ##\u003cem\u003eP\u003c/em\u003e\u0026lt;.01. \u003cem\u003en\u003c/em\u003e≥3 for all groups.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/1e70185112b625cbfaa7c35c.jpeg"},{"id":82850196,"identity":"f2b6c90f-3f8d-48e5-9ad7-69b4472239d6","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":670435,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical inactivity aggravates coronary ligation-induced excessive mitochondrial fissions in cardiomyocytes. (A) Transmission electron microscopic images and (B) quantitative analyses of cardiomyocytes in each mouse. Scale bars = 500 nm in low-magnification photographs and 200 nm in high-magnification photographs. Data are presented as mean±SEM. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;.001, #\u003cem\u003eP\u003c/em\u003e\u0026lt;.05. \u003cem\u003en\u003c/em\u003e=3 for all groups.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/22c9f7ca989b61d96936ffd7.jpeg"},{"id":82851187,"identity":"c4981198-2f49-4bbf-88d5-3d29f7485c0d","added_by":"auto","created_at":"2025-05-16 03:22:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5012706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/790bcaf2-805c-4371-aedc-55e24f3270e9.pdf"},{"id":82850183,"identity":"785a6f33-470f-4996-8636-6ff733462f79","added_by":"auto","created_at":"2025-05-16 03:06:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1536398,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6508918/v1/772ca8647b634a858ebf9fb4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Promotion of Pathological Cardiac Remodeling by Excessive Mitochondrial Fission in Sedentary Lifestyle-Associated Myocardial Infarction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyocardial infarction (MI) is recognized as the most common cause of death globally.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Despite extensive efforts to develop novel treatments for MI to improve patient outcomes, the mortality rate has not changed in the last decade.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Recently, systemic therapies (pharmacological treatment and lifestyle interventions), rather than heart-targeted therapies, have been emphasized to be important for improving prognosis.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The efficacy of heart-targeted therapies, such as percutaneous coronary intervention, in increasing life expectancy is limited to only 12 h after the onset of ST-segment elevation MI,\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and most patients with MI have other co-morbidities or systemic diseases (e.g., dyslipidemia, hypertension). Over the past several decades, statins and blood pressure-lowering medications have been widely used for the primary and secondary prevention of cardiovascular disease (CVD) owing to their high effectiveness and safety.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Nevertheless, there is an urgent need to develop novel systemic therapies to prevent further CVD events.\u003c/p\u003e \u003cp\u003eFrom the viewpoint of sudden cardiac death (SCD) cases, most events occur in the general population (composed of \u0026ldquo;healthy population\u0026rdquo;) rather than patients with coronary artery disease (the epidemiological paradox in SCD).\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Moreover, it is thought that a majority of individuals with SCD are not treated with medications in time owing to the high prevalence and \u0026ldquo;low risk\u0026rdquo; estimations of atherosclerotic CVD events before SCD. Additionally, in statin therapy, continued administration for at least 2.5 years is needed to prevent future CVD events.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Therefore, lifestyle interventions (e.g., healthy diet, exercise, and smoking cessation) are extremely important for reducing future SCD events.\u003c/p\u003e \u003cp\u003eSedentary behavior, defined as walking behavior with low energy expenditure (\u0026le;\u0026thinsp;1.5 metabolic equivalents) while in a sitting or reclining posture, is the leading modifiable risk factor for CVD and all-cause mortality.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e A sedentary lifestyle with prolonged sedentary behavior is observed in 27.5% of the global population, with high-income western countries showing a higher prevalence at 42.3%.\u003csup\u003e9\u003c/sup\u003e However, the strength and specificity of the recommendations to reduce sedentary behavior are limited, probably because of the difficulties in experimental modeling and the scarcity of evidence on sedentary lifestyle-associated MI.\u003c/p\u003e \u003cp\u003eTo date, numerous types of animal models of sedentary lifestyle (e.g., hindlimb unloading models) have been reported.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Recently, Kim et al. demonstrated a new mouse model using a narrow cage that recapitulated the natural human sedentary lifestyle without severe mental stress.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Nonetheless, the association between the sedentary lifestyle and MI remains largely unknown. In the present study, we sought to clarify the mechanisms of sedentary lifestyle-associated MI by investigating the influence of sedentary behavior on MI development, particularly on mitochondrial dynamics, using a combined sedentary lifestyle and MI mouse model.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMitochondria generate the majority of energy required to maintain cellular function and are consequently referred to as the powerhouse of the cells.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Therefore, they play a central role in maintaining optimal organ performance throughout the body.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Because the heart is the core organ with the highest energy demand, mitochondrial dysfunction of the cardiomyocytes, including an imbalance in the dynamics of fusion and fission, has been reported to be causatively involved in CVD.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Furthermore, numerous studies using animal and in vitro models have suggested that dysregulated mitochondrial dynamics of cells in the whole body, especially increased fission and decreased fusion, could be associated with sedentary behavior.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Conversely, exercise and decreased sedentary behavior may have beneficial roles in rebalancing the mitochondrial dynamics of cardiomyocytes.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Accordingly, the question arises as to whether dysregulated mitochondrial dynamics in the heart could contribute to the pathologies of both sedentary behavior and MI.\u003c/p\u003e \u003cp\u003eThe present study aimed to clarify whether increased mitochondrial fission occur in cardiomyocytes during MI pathogenesis and whether a sedentary lifestyle could worsen MI pathologies through further aggravation of excessive mitochondrial fission in cardiomyocytes. This study focused on sedentary lifestyle-associated MI, given that a better understanding of sedentary behaviors in MI could lead to substantial improvements in MI-related morbidity and mortality by enhancing motivation to decrease the sedentary behaviors in the healthy populations and the developing novel systemic treatments for patients with MI.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCoronary Ligation Leads to Myocardial Ischemia and Compensatory Cardiac Hypertrophy\u003c/h2\u003e \u003cp\u003eTo evaluate chronological changes in the acute myocardial infarction (AMI) model,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e we first performed macroscopic and survival analyses of mice in which the left anterior coronary artery was ligated after 1 and 4 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). At 1 and 4 weeks after AMI (hereafter called \u0026ldquo;AMI 1w\u0026rdquo;and \u0026ldquo;AMI 4w\u0026rdquo;, respectively), brownish discoloration of the apex and a pale yellowish change in the left ventricular wall were observed, indicating necrosis and incomplete ischemia (penumbra), respectively. Moreover, the hearts of the AMI model were enlarged compared to those of the controls, suggesting compensatory cardiac hypertrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe survival rate of mice in the AMI 4w model was significantly reduced compared with that of control mice. Generally, AMI model mice died even several days after post-MI due to sudden cardiac arrest or cardiac rupture,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e whereas no mouse died after perisurgical period in the study, likely due to high mortality in the current study (about 50% in the study vs 70% in the previous study). As most autopsied mice showed intrathoracic hemorrhage, the common cause of death could be hemorrhagic shock. All mice in the mock operation group survived, and no pathological changes were observed in the mock group by macroscopic or histological analysis (Supplementary Fig.\u0026nbsp;3). We next performed serum biochemical analysis in each mouse to assess lipid and glucose metabolism, but no significant changes were found in the AMI model compared to the controls, except for a slight increase in the total cholesterol level in the AMI 4w model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The volumes of food and water intake were similar among the control groups, mock operation group, and AMI 1 w model (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eTo evaluate the functional changes in the heart in AMI pathology, we performed echocardiography in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). The ejection fraction and fraction shortening parameters, which are markers of cardiac function, were significantly lower in the AMI model than in the controls. Although left ventricular end diastolic dimension of the ischemic heart usually shows an increase by compensatory cardiac dilatation, those of the current study showed a decrease, likely due to worsened shape of left ventricular cavity by increased necrotic area and the consequent exacerbated cardiac hypertrophy.\u003c/p\u003e \u003cp\u003eThese findings suggest that the AMI model accurately reflects human MI development without the severe effects of dyslipidemia, insulin resistance, and hyperphagia.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCoronary Ligation Leads to Fibrotic Changes and Cardiomyocyte Enlargement\u003c/h3\u003e\n\u003cp\u003eTo further evaluate the chronological changes in the AMI model, we performed histological analyses in mice. Hematoxylin and eosin staining of heart sections in the AMI model revealed discolored, thinner, and fibrotic changes in the apex in a time dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;4A\u0026ndash;C). Conversely, the cardiac wall in the interventricular septum and basal area enlarged in a time dependent manner, indicating compensatory cardiac hypertrophy. Reticulin staining of heart sections from the AMI model showed that gray-stained areas (immature type III collagen) peaked at 1 week and that, thick black areas (mature type I collagen) worsened over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The AMI models at both 1 and 4 weeks after ligation exhibited slight lymphocyte infiltration; however, no neutrophils were found in the interstitial regions of cardiac muscles. We confirmed substantial increases in neutrophils and lymphocytes around necrotic cardiomyocytes and occluded coronary arteries during the super acute phase (at 1 day after MI) (Supplementary Fig.\u0026nbsp;4A\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemical staining of heart sections in the AMI model for α-SMA, a marker of collagen-secreting myofibroblasts, showed that positive-stained cells were increased around occluded coronary arteries, especially in the penumbra lesions, peaked at 1 week after coronary ligation, and slightly decreased at 4 weeks after MI. Immunofluorescence staining of heart sections in the AMI model for wheat germ agglutinin revealed that the sizes of cardiomyocytes in the penumbra areas were considerably increased at 1 and 4 weeks after MI, confirming cardiomyocyte hypertrophy.\u003c/p\u003e \u003cp\u003eThese findings suggest that inflammatory cell infiltration first occurs after an ischemic event, followed by active fibrotic changes by myofibroblast proliferation. Ultimately, mature fibrosis and consequent cardiac hypertrophy further worsen over time in MI pathology.\u003c/p\u003e\n\u003ch3\u003ePhysical Inactivity Aggravates Coronary Artery Ligation-Induced Myocardial Necrosis and Hypertrophy\u003c/h3\u003e\n\u003cp\u003eTo clarify the association between sedentary lifestyle and MI development, we conducted macroscopic and survival analyses in the combined sedentary lifestyle and AMI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). To imitate the natural sedentary lifestyle in humans and avoid unnecessary mental stress, we created narrow cages using acrylic plates, and mice were bred in the cages for up to 2 weeks.1 To clarify the contribution of a sedentary lifestyle in the treatable phase of MI development (before scar fibrosis maturation), we chose the AMI 1w model, because this phase showed the most drastic changes in immature fibrosis (before scar maturation).\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the physical activity levels of the sedentary model, we performed a behavioral analysis of the control and sedentary model mice before and after MI. The results showed that the moving distance in the sedentary model was significantly reduced by 15-fold compared to controls, regardless of the coronary ligation procedure. Although the moving distances in both groups showed reduced tendencies after MI, these changes were negligible. Most importantly, the survival rate of the sedentary lifestyle model at 1 week after MI showed a decreasing tendency compared to the simple AMI model. Moreover, the hearts of the sedentary lifestyle model mice after MI showed expanded areas of necrotic and penumbral lesions along with significant cardiac hypertrophic changes compared to those of the simple AMI group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C, and Supplementary Fig.\u0026nbsp;4). However, no pathological changes were found in the sedentary lifestyle model mice after MI based on food and water intake, and serum biochemical analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;2). Macroscopic and histological analyses confirmed that the sedentary lifestyle model mice without coronary ligation exhibited no significant pathological changes (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eThese findings suggest that although a sedentary lifestyle itself does not induce severe cardiac damage, it does contribute to the further deterioration of MI pathologies, such as the expansion of necrotic areas and cardiac hypertrophic remodeling.\u003c/p\u003e\n\u003ch3\u003ePhysical Inactivity Aggravates Coronary Artery Ligation-Induced Fibrotic Changes and Cardiomyocyte Enlargement\u003c/h3\u003e\n\u003cp\u003eTo further evaluate the association between a sedentary lifestyle and MI development, we conducted a histological analysis using a combined sedentary lifestyle and AMI model. Hematoxylin and eosin staining of heart sections from the sedentary lifestyle model after MI showed significant aggravation of cardiac necrotic and fibrotic areas compared to the simple AMI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Azan-, reticulin-, and α-SMA-immunohistochemical-staining heart sections of sedentary lifestyle model after MI showed that substantial proliferations of myofibroblasts with mature collagen depositions compared to simple AMI model. Moreover, immunofluorescent staining for wheat germ agglutinin showed significant aggravation of cardiomyocyte enlargement in the sedentary lifestyle model after MI compared with simple MI. In contrast, sedentary lifestyle model without coronary ligation showed neither excessive collagen deposition, enlarged cardiomyocytes, nor active proliferation of spindle-shaped cells suggestive of myofibroblasts in the interstitial regions of the hearts (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings suggest that a sedentary lifestyle induces excessive proliferation of myofibroblasts and subsequent cardiac fibrosis as well as abnormal cardiomyocyte hypertrophy.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhysical Inactivity Aggravates Coronary Artery Ligation-Induced Aberrant Gene Expression Changes in Immune Cells, Cardiomyocytes, and Mitochondrial Dynamics Markers\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo understand the cellular and molecular mechanisms underlying sedentary lifestyle-associated MI aggravation, we conducted qRT-PCR analysis using a combined sedentary lifestyle and AMI model. Generally, changes in mRNA expression precede morphological changes in cells, especially during inflammatory changes.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Therefore, we conducted qRT-PCR analysis at 1 day after MI in the models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Morphologically, no significant change was observed in mice at 1 day after MI in the combined sedentary lifestyle and AMI model compared with the simple AMI model (Supplementary Fig.\u0026nbsp;4A\u0026ndash;C). As expected, mRNA expression changes including markers of cardiomyocyte injury, activated myofibroblasts, inflammation, mitochondrial fusion, and endoplasmic reticulum stress (ER stress), already emerged at 1 day after MI. Notably, the combined sedentary lifestyle and AMI model showed significant changes in the markers of inflammation, endothelial injury, mitochondrial fission/fusion, and ER stress compared to the simple AMI model. Interestingly, most mRNA markers did not show significant changes between the controls and AMI 4w model, except for markers of cardiomyocyte injury and endothelial injury, suggesting active compensatory reactions or chronic \u0026ldquo;burnout\u0026rdquo; changes (e.g., reduced numbers and functions of TNF-α-expressing cardiomyocytes due to replacement by collagen fibers, extracellular matrix, and myofibroblasts) after 4 weeks of MI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe gaps in most mRNA markers between the control group and AMI 1w model were smaller than those between the control group and AMI 1d model. Contrary to our expectations, the combined sedentary lifestyle and AMI 1w model showed improvements in the markers of cardiomyocyte injury, fibrosis, inflammation, macrophage activation, mitochondrial biogenesis, mitochondrial fusion, and ER stress compared to the simple AMI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Given that most mRNA expression levels of markers in the combined sedentary lifestyle and AMI 1w model were between those of simple AMI 1w and 4w models, these seemingly contradictory improved changes in mRNA expressions could be explained by active compensatory reactions or chronic \u0026ldquo;burnout\u0026rdquo; changes.\u003c/p\u003e \u003cp\u003eThese results suggest that a sedentary lifestyle could accelerate pathological cardiac remodeling in MI by heightening the levels and shortening the duration of dysregulated gene expression changes in inflammation, mitochondrial dynamics, and ER stress.\u003c/p\u003e\n\u003ch3\u003ePhysical Inactivity Aggravates Coronary Artery Ligation-Induced Excessive Mitochondrial Fissions in Cardiomyocytes\u003c/h3\u003e\n\u003cp\u003eSedentary behavior can induce an imbalance in mitochondrial dynamics in cardiomyocytes; in contrast, exercise can delay or ameliorate the pathogenesis of CVD.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, the role of mitochondrial dynamics in sedentary lifestyle-associated MI remains unclear. To elucidate the mitochondrial ultrastructural changes in the pathologies, transmission electron microscopic analysis was conducted on the combined sedentary lifestyle and AMI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). To exclude bias due to positional heterogeneity in the morphology and function of mitochondria and sarcomeres, we analyzed the penumbra lesions of the left ventricular walls in the MI model and the corresponding areas in controls. As expected, time-dependent aggravation of decreased individual mitochondrial sizes and mitochondrial areas, as well as increased numbers of mitochondria and fractions of malformed mitochondria were found in cardiomyocytes from simple AMI 1w- and 4w-models, indicating excessive mitochondrial fission.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUltrastructural changes in the sarcomere of cardiomyocytes also showed time dependent aggravations in sarcomere length and dispersion degree, indicating sarcomere disruption and abnormal sarcomere orientation (polarity disturbance), respectively. Consistent with the increased mRNA expression of Drp1, the primary regulator of mitochondrial fission, and those of cardiomyocyte injury markers during the super-acute phase (at 1 day after MI) in the combined sedentary lifestyle and AMI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), the combined model showed further aggravation of excessive mitochondrial fission compared to the simple AMI model. Although the mean widths of sarcomeres in all groups were similar, the variabilities in the widths of the three AMI groups were larger than those of the control group, suggesting that MI could induce not only detrimental sarcomere disruption and pathological dedifferentiation of cardiomyocytes but also beneficial re-differentiation of surviving or transiently hibernated cardiomyocytes to functional and ischemia-tolerant cardiomyocytes.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTaken together, our findings suggest that a sedentary lifestyle could lead to aggravation of excessive mitochondrial fission and abnormal sarcomere remodeling in ischemic cardiomyocytes and subsequent activation of collagen-producing myofibroblasts after MI, after entering a vicious cycle, eventually leading to heart failure and SCD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, the current investigation is the first study to demonstrate that a sedentary lifestyle could lead to a further decrease in survival rate after MI and further deterioration of cardiac pathologies, including cardiac hypertrophy, inflammation, fibroblast activation, fibrosis, and excessive mitochondrial fission in cardiomyocytes. Taken together, our findings suggest that sedentary lifestyle contributes to the aggravation of MI pathologies and SCD through a vicious cycle of excessive mitochondrial fission in ischemic cardiomyocytes, myofibroblast activation, and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;5). Furthermore, our study underscores the potential therapeutic implications of pharmaceutical therapies focusing on mitochondrial dynamics and lifestyle modifications, such as decreased duration of sedentary behaviors in healthy individuals and patients with coronary artery disease even after MI events.\u003c/p\u003e \u003cp\u003eWhether sedentary behavior could have greater roles in causing MI onset rather than in aggravating MI development or vice versa has long been in question because data on the contribution of sedentary behavior to MI have been scarce.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The current study using the combined sedentary lifestyle and AMI model clearly supports the latter hypothesis because 2-week physical inactivity in mice did not cause severe cardiac damage but significantly worsened MI development. If this hypothesis is true, shortening the duration of sedentary behavior may be more effective post-MI than pre-MI. This theory further highlights the clinical importance of earlier and progressive non-pharmacotherapies, such as decreased sedentary behavior and increased levels and durations of physical activity, even after the MI event.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Indeed, a recent meta-analysis of eight clinical trials that studied people with coronary heart disease at long-term follow-up (over 3 years) showed that exercise-based cardiac rehabilitation may result in a large reduction in cardiovascular mortality (RR 0.58, 95% CI 0.43 to 0.78).\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e A combination of exercise and decreased sedentary behavior could plausibly further improve patient\u0026rsquo; outcomes. Therefore, to maximize the beneficial effects of controlling physical activity in patients, both exercise and decreased sedentary behavior are needed.\u003c/p\u003e \u003cp\u003eThe data from the current study suggest that sedentary behavior could first promote mitochondrial fission in cardiomyocytes immediately after MI and then lead to compensatory host cellular reactions, such as enhanced mitochondrial fusion and biogenesis, to prevent the deterioration of cardiac injuries. If sedentary behavioral stress persists in patients, it could further result in mitochondrial fission, a decreased mitochondrial fraction, and ER stress in cardiomyocytes. Thereafter, unfavorable organellar injuries in cardiomyocytes caused by prolonged sedentary behavior during MI can lead to permanent cardiac hypertrophy, inflammation with pathological macrophage polarization, myofibroblast activation, and cardiac fibrosis. Considering that damaged cardiomyocytes and excessive myofibroblast activation could mutually influence pathological responses and lead to cardiomyocyte apoptosis and necrosis,\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e we propose that a sedentary lifestyle could induce a vicious cycle of cardiomyocyte injury and fibrosis, eventually causing heart failure, arrhythmogenesis, and SCD.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe findings of this study have some clinical implications. First, they suggest that decreased sedentary behavior and increased physical activity could help improve complications associated with MI by inhibiting the vicious cycle and prevent SCD even after MI events. Second, medications that inhibit fission and/or promote fusion of mitochondria in cardiomyocytes could ameliorate MI development, especially in the acute phase.\u003c/p\u003e \u003cp\u003eNevertheless, this study has some limitations. First, since no mouse model can completely replicate human disease, the findings cannot be completely generalized to humans. To resolve this issue, we used sedentary lifestyle and AMI model that closely reflect human disease pathologies. However, we did not investigate whether the proposed mechanism could be valid in different models. Therefore, future studies with different situations (duration, time point, and degree of physical activity and ischemic cardiac injury) should be conducted to clarify the precise roles of sedentary behavior in MI pathologies. Second, whether the inhibition of fission or the promotion of fusion in cardiomyocytes could ameliorate disease progression could not be clarified. Therefore, whether excessive mitochondrial fission in cardiomyocytes is the cause or a secondary phenomenon in the pathogenesis remains unknown. Hence, future studies using pharmacological treatments that directly influence mitochondrial dynamics are warranted.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provides novel insights into sedentary lifestyle-associated MI. Our findings suggest that prolonged periods of sedentary lifestyle and excessive mitochondrial fission in ischemic cardiomyocytes are associated with aggravated MI. Therefore, combined lifestyle and pharmacological interventions, such as reducing sedentary behavior and mitochondria-targeted medications, could be promising novel strategies for the prevention of MI and MI-related deaths.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale 8\u0026ndash;10 weeks old C57BL/6 mice (SHIMIZU Laboratory Supplies Co., Ltd., Kyoto, Japan) were used in this study. The experimental animals were euthanized by cervical dislocation under anesthesia with an intraperitoneal injections of a combination anesthetics (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol) at the indicated time points. Blood was collected by cardiac puncture after a 2-h fast. Tissues were harvested for further analyses. For electron microscopy analysis, the hearts were fixed with 2% glutaraldehyde and 4% paraformaldehyde. At least three mice were treated and analyzed for each time period and treatment. All mice were housed in cages under specific pathogen-free conditions with food and water ad libitum. All protocols were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (Med Kyo 24043) and were performed according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. All the work involving the animals were performed in the Kyoto University Graduate School of Medicine. This study is reported in accordance with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAcute myocardial infarction model\u003c/h2\u003e \u003cp\u003eAMI model (1 to 4 weeks) was made by permanent ligation of left anterior descending artery with minor modifications (Supplementary Fig.\u0026nbsp;1).\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Briefly, mice were firstly anesthetized with an intraperitoneal injections of a combination anesthetics (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol). The fur was shaved and skin cut was made over the left chest. A small hole was made at the fourth intercostal space, and the heart was exposed through the hole. The left anterior descending artery was located and ligated at a site 2 mm from its origin. After ligation, the heart was immediately placed back with an evacuation of air. Thoracic cavity was closed and the skin was sutured. The mice were continuously monitored at least 30 min after the operation. Mock operation mice underwent the same surgical procedure except that the coronary artery was not occluded. No mice died in the mock operation model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSedentary lifestyle model\u003c/h2\u003e \u003cp\u003eSedentary lifestyle model (2 weeks) was made by narrow cages using acrylic plates with minor modifications.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Briefly, sedentary lifestyle model mice were housed in the narrow cages, residual space of a mouse was 10 cm in length, 6 cm in width, and 12 cm in height. To avoid unnecessary isolation stress, the mice could visually see the other mice in the next cages though transparent acrylic plate, and could touch each other\u0026rsquo;s noses through a 1 cm hole in the wall. For the combination of AMI- and sedentary lifestyle models, mice were firstly bred in the narrow cages for 1 week, then the left coronary artery of the mice was ligated, the mice were bred in the narrow cages for additionally 1 week. Control mice were housed in the standard cages, residual space of a mouse was 25 cm in length, 18 cm in width, and 12 cm in height. No mice died both in the control and the sedentary lifestyle model without coronary ligation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from heart tissues using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total RNA using SuperScript III reverse transcriptase (Invitrogen, Waltham, USA). Real-time PCR was performed using FastStart SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland) and a Rotor-Gene Q (Qiagen, Venlo, Netherlands) instrument. The primer pairs are shown in Supplementary Table\u0026nbsp;1. Relative target gene expression was normalized to Gapdh expression as the mRNA expressions of Gapdh in different experimental conditions were stable than those of β-actin by the preliminary study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum biochemical analyses\u003c/h2\u003e \u003cp\u003eSerum samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analyses were performed. Total cholesterol (T-chol), triglyceride (TG), free fatty acid (FFA), and glucose levels were analyzed using a Hitachi 7180 analyzer (Hitachi, Tokyo, Japan). Levels of insulin and TNF-α were analyzed with Enzyme-linked immuno-sorbent assay kits (Morinaga Institute of Biological Science, Yokohama, Japan M1104 for insulin; R\u0026amp;D, Asaka, Japan, DTA00D for TNF-α). Homeostasis model assessment of insulin resistance, the index of insulin resistance, was calculated by fasting plasma glucose (mmol/L) x fasting insulin (mIU/L)/22.5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eFormaldehyde-fixed paraffin-embedded tissue sections of 4-\u0026micro;m thickness were cut and stained with hematoxylin and eosin (H\u0026amp;E), Azan, and reticulin. The histological features of the specimens were independently assessed by experienced pathologists (MM and CK) in a blinded fashion, and a consensus diagnosis was obtained for each sample. For quantitative assessment, the staining of each specimen was captured in 15 randomly selected fields and quantified using the ImageJ software (NIH, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and immunofluorescent analyses\u003c/h2\u003e \u003cp\u003eImmunohistochemical and immunofluorescent staining of the specimens was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, antigen retrieval was performed in a pressure cooker by boiling in 10 mM citrate buffer (pH 6.0), followed by washing with phosphate-buffered saline. For immunohistochemical staining, endogenous peroxidase was quenched with 3% H2O2 for 10 min. After rinsing, the slides were incubated overnight at 4\u0026deg;C with a negative control reagent or the following optimally diluted primary antibody: α-smooth muscle actin (α-SMA; rabbit; 1:400; Abcam, ab5694). Next, the slides were incubated at 25\u0026deg;C with a secondary antibody (MBL, Nagoya, Japan) for 1 h. Permanent Red was used as a chromogen, followed by counterstaining with hematoxylin. For analysis of cardiomyocyte sizes in each mouse, heart tissues were frozen in optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan, 45833) and stored at -80\u0026deg;C before being sectioned at a thickness of 10 \u0026micro;m on a microtome. Sections were pre-warmed at room temperature and then fixed with pre-chilled 4% formaldehyde for 10 min, and stained with FITC conjugated-wheat germ agglutinin (Invitrogen, MA, USA, W11261) for 30 min before nuclear counterstaining with DAPI (Vector Laboratories, CA, USA, H-1200). Cardiomyocytes fluoresced in green. The immunofluorescent images were captured using a fluorescence microscope (Keyence, Osaka, Japan, BZ-X810). For quantitative assessment, the staining of each specimen was captured in 15 randomly selected fields and quantified using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMoving trajectories and moving distance measurements\u003c/h2\u003e \u003cp\u003eTo evaluate physical activity levels in each mouse, moving trajectories were recorded and the moving distance were calculated in the controls and sedentary lifestyle model mice. To avoid as much as confounding effects by acute psychological stress, anesthesia, and the coronary ligation procedures, the mice were analyzed at 1 day before coronary artery ligation, and at 6 days after the ligation. All behavioral tests were performed in the light phase, between 10:00am and 01:00pm (light time: 09:00am \u0026minus;\u0026thinsp;09:00pm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from heart tissues using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total RNA using SuperScript III reverse transcriptase (Invitrogen). Real-time PCR was performed using FastStart SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland) and a Rotor-Gene Q (Qiagen, Venlo, Netherlands) instrument. The primer pairs are shown in Supplementary Table\u0026nbsp;1. Relative target gene expression was normalized to Gapdh expression as the mRNA expressions of Gapdh in different experimental conditions were stable than those of β-actin by the preliminary study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy analysis\u003c/h2\u003e \u003cp\u003eFixed hearts were cut into 1-mm sections. These sections were subsequently immersed in 1% glutaraldehyde at 4\u0026deg;C overnight. The specimens were extensively washed with phosphate buffered saline, postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Epon. Ultra-thin sections (80 nm) were cut on an Ultra microtome EM UC6 (Leica, Vienna, Austria), stained with 1% uranyl acetate, counterstained using the Reynolds method, and examined using an H-7650 electron microscope (Hitachi, Tokyo, Japan). For the assessments of the mitochondrial content and mitochondrial area in each cell, areas of lipid particles and nucleus were excluded to avoid the confounding effects. Deformed mitochondria were defined by at least one positive sign of the following features; abnormally enlarged mitochondria or mega-mitochondria (a minor radius\u0026thinsp;\u0026gt;\u0026thinsp;1000 nm), loss of cristae, autophagosomes or autolysosome with the destructed mitochondria, and irregular shaped mitochondria (e.g. abnormally blanching, donut-shaped). Individual sarcomere lengths were measured as the distance between adjacent Z-bands. For quantitative assessment, 15 randomly selected fields were captured for each group (n\u0026thinsp;=\u0026thinsp;3), and quantification was performed using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was determined using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or analysis of variance with Tukey\u0026rsquo;s post-hoc test for multiple comparisons. For all analyses, statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our gratitude to Kanako Maruo, Kumiko Kokuryo, Yuji Hamaguchi, Haruyasu Kohda, Keiko Furuta, and Tatsuya Katsuno for providing technical assistance, as well as Editage for carefully reviewing the manuscript for English editing. We also acknowledge the technical assistance and histopathological analyses performed by the members of the Center for Anatomical, Pathological, and Forensic Research at the Graduate School of Medicine, Kyoto University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.M. and H.O. contributed to the study concept and design, collected the data, performed statistical analysis, and wrote the paper. C.K., S.F., H.K., and Y.N. conducted the data acquisition, analysis, and interpretation and reviewed and revised the paper. H.M., H.A., H.N., H.Y., K.O., and K.T. provided technical and material support, developed the methodology and writing, and reviewed and revised the paper. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets and/or analyses of the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed methodology is included in the Supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science, KAKENHI under grant numbers 23K09763 (MM) and 24K13544 (CK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlobal burden of 288 causes of death and life expectancy decomposition. in 204 countries and territories and 811 subnational locations, 1990\u0026ndash;2021: a systematic analysis for the Global Burden of Disease Study 2021. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e403\u003c/b\u003e (10440), 2100\u0026ndash;2132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0140-6736(24)00367-2\u003c/span\u003e\u003cspan address=\"10.1016/s0140-6736(24)00367-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodruff, R. C. et al. Trends in Cardiovascular Disease Mortality Rates and Excess Deaths, 2010\u0026ndash;2022. \u003cem\u003eAm. J. Prev. Med.\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e (4), 582\u0026ndash;589. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.amepre.2023.11.009\u003c/span\u003e\u003cspan address=\"10.1016/j.amepre.2023.11.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeatty, A. L. et al. A New Era in Cardiac Rehabilitation Delivery: Research Gaps, Questions, Strategies, and Priorities. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e147\u003c/b\u003e (3), 254\u0026ndash;266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/circulationaha.122.061046\u003c/span\u003e\u003cspan address=\"10.1161/circulationaha.122.061046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawton, J. S. et al. 2021 ACC/AHA/SCAI Guideline for Coronary Artery Revascularization: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e145\u003c/b\u003e (3), e18\u0026ndash;e114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/cir.0000000000001038\u003c/span\u003e\u003cspan address=\"10.1161/cir.0000000000001038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnett, D. K. \u0026amp; ACC/AHA Guideline on the Primary Prevention of Cardiovascular Disease. : Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. \u003cem\u003eCirculation.\u003c/em\u003e 140(11), e563-e595. (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/cir.0000000000000677\u003c/span\u003e\u003cspan address=\"10.1161/cir.0000000000000677\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarijon, E. et al. The Lancet Commission to reduce the global burden of sudden cardiac death: a call for multidisciplinary action. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e402\u003c/b\u003e (10405), 883\u0026ndash;936. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0140-6736(23)00875-9\u003c/span\u003e\u003cspan address=\"10.1016/s0140-6736(23)00875-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYourman, L. C. et al. Evaluation of Time to Benefit of Statins for the Primary Prevention of Cardiovascular Events in Adults Aged 50 to 75 Years: A Meta-analysis. \u003cem\u003eJAMA Intern. Med.\u003c/em\u003e \u003cb\u003e181\u003c/b\u003e (2), 179\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/jamainternmed.2020.6084\u003c/span\u003e\u003cspan address=\"10.1001/jamainternmed.2020.6084\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLavie, C. J., Ozemek, C., Carbone, S., Katzmarzyk, P. T. \u0026amp; Blair, S. N. Sedentary Behavior, Exercise, and Cardiovascular Health. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cb\u003e124\u003c/b\u003e (5), 799\u0026ndash;815. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/circresaha.118.312669\u003c/span\u003e\u003cspan address=\"10.1161/circresaha.118.312669\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuthold, R., Stevens, G. A., Riley, L. M. \u0026amp; Bull, F. C. Worldwide trends in insufficient physical activity from 2001 to 2016: a pooled analysis of 358 population-based surveys with 1\u0026middot;9 million participants. \u003cem\u003eLancet Glob Health\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e (10), e1077\u0026ndash;e1086. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s2214-109x(18)30357-7\u003c/span\u003e\u003cspan address=\"10.1016/s2214-109x(18)30357-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReidy, P. T., Monnig, J. M., Pickering, C. E., Funai, K. \u0026amp; Drummond, M. J. Preclinical rodent models of physical inactivity-induced muscle insulin resistance: challenges and solutions. \u003cem\u003eJ. Appl. Physiol. (1985)\u003c/em\u003e. \u003cb\u003e130\u003c/b\u003e (3), 537\u0026ndash;544. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/japplphysiol.00954.2020\u003c/span\u003e\u003cspan address=\"10.1152/japplphysiol.00954.2020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, J., Park, J. \u0026amp; Mikami, T. R. Low-Intensity Exercise Prevents Cognitive Decline and a Depressive-Like State Induced by Physical Inactivity in Mice: A New Physical Inactivity Experiment Model. \u003cem\u003eFront. Behav. Neurosci.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 866405. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnbeh.2022.866405\u003c/span\u003e\u003cspan address=\"10.3389/fnbeh.2022.866405\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, E. et al. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e (12), 1445\u0026ndash;1453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/circresaha.110.223925\u003c/span\u003e\u003cspan address=\"10.1161/circresaha.110.223925\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, R. et al. Unlocking the Secrets of Mitochondria in the Cardiovascular System: Path to a Cure in Heart Failure\u0026mdash;A Report from the 2018 National Heart, Lung, and Blood Institute Workshop. \u003cem\u003eCirculation\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e (14), 1205\u0026ndash;1216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/circulationaha.119.040551\u003c/span\u003e\u003cspan address=\"10.1161/circulationaha.119.040551\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyao, M. et al. Mitochondrial fission in hepatocytes as a potential therapeutic target for nonalcoholic steatohepatitis. \u003cem\u003eHepatol. Res.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e (12), 1020\u0026ndash;1033. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/hepr.13832\u003c/span\u003e\u003cspan address=\"10.1111/hepr.13832\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForrester, S. J. et al. Mitochondrial Fission Mediates Endothelial Inflammation. \u003cem\u003eHypertension\u003c/em\u003e \u003cb\u003e76\u003c/b\u003e (1), 267\u0026ndash;276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/hypertensionaha.120.14686\u003c/span\u003e\u003cspan address=\"10.1161/hypertensionaha.120.14686\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026aacute;squez-Trincado, C. et al. Mitochondrial dynamics, mitophagy and cardiovascular disease. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cb\u003e594\u003c/b\u003e (3), 509\u0026ndash;525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1113/jp271301\u003c/span\u003e\u003cspan address=\"10.1113/jp271301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHernandez-Resendiz, S. et al. Targeting mitochondrial shape: at the heart of cardioprotection. \u003cem\u003eBasic. Res. Cardiol.\u003c/em\u003e \u003cb\u003e118\u003c/b\u003e (1), 49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00395-023-01019-9\u003c/span\u003e\u003cspan address=\"10.1007/s00395-023-01019-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H., Zhang, Y., Zhang, J. \u0026amp; Jia, D. Exercise Alleviates Cardiovascular Diseases by Improving Mitochondrial Homeostasis. \u003cem\u003eJ. Am. Heart Assoc.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (19), e036555. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/jaha.124.036555\u003c/span\u003e\u003cspan address=\"10.1161/jaha.124.036555\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrabhu, S. D. \u0026amp; Frangogiannis, N. G. The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cb\u003e119\u003c/b\u003e (1), 91\u0026ndash;112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/circresaha.116.303577\u003c/span\u003e\u003cspan address=\"10.1161/circresaha.116.303577\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyao, M. et al. Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression. \u003cem\u003eLab. Invest.\u003c/em\u003e \u003cb\u003e95\u003c/b\u003e (10), 1130\u0026ndash;1144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/labinvest.2015.95\u003c/span\u003e\u003cspan address=\"10.1038/labinvest.2015.95\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, Y., Do, V. D., Richards, A. M. \u0026amp; Foo, R. What we know about cardiomyocyte dedifferentiation. \u003cem\u003eJ. Mol. Cell. Cardiol.\u003c/em\u003e \u003cb\u003e152\u003c/b\u003e, 80\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.yjmcc.2020.11.016\u003c/span\u003e\u003cspan address=\"10.1016/j.yjmcc.2020.11.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuran, A. T., Garber, C. E., Cornelius, T., Schwartz, J. E. \u0026amp; Diaz, K. M. Patterns of Sedentary Behavior in the First Month After Acute Coronary Syndrome. \u003cem\u003eJ. Am. Heart Assoc.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (15), e011585. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/jaha.118.011585\u003c/span\u003e\u003cspan address=\"10.1161/jaha.118.011585\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalal, H. M., Doherty, P. \u0026amp; Taylor, R. S. Cardiac rehabilitation. \u003cem\u003eBMJ\u003c/em\u003e \u003cb\u003e351\u003c/b\u003e, h5000. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/bmj.h5000\u003c/span\u003e\u003cspan address=\"10.1136/bmj.h5000\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDibben, G. et al. Exercise-based cardiac rehabilitation for coronary heart disease. \u003cem\u003eCochrane Database Syst. Rev.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (11). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/14651858.CD001800.pub4\u003c/span\u003e\u003cspan address=\"10.1002/14651858.CD001800.pub4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021). Cd001800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicin, L., Wagner, J. U. G., Lux\u0026aacute;n, G. \u0026amp; Dimmeler, S. Fibroblast-mediated intercellular crosstalk in the healthy and diseased heart. \u003cem\u003eFEBS Lett.\u003c/em\u003e \u003cb\u003e596\u003c/b\u003e (5), 638\u0026ndash;654. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/1873-3468.14234\u003c/span\u003e\u003cspan address=\"10.1002/1873-3468.14234\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall, C., Gehmlich, K., Denning, C. \u0026amp; Pavlovic, D. Complex Relationship Between Cardiac Fibroblasts and Cardiomyocytes in Health and Disease. \u003cem\u003eJ. Am. Heart Assoc.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (5), e019338. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/jaha.120.019338\u003c/span\u003e\u003cspan address=\"10.1161/jaha.120.019338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Coronary artery disease, metabolic syndrome, mitochondrial dynamics, mitochondrial dysfunction, physical inactivity","lastPublishedDoi":"10.21203/rs.3.rs-6508918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6508918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA sedentary lifestyle such as prolonged periods of sitting is one of the most important modifiable risk factors for morbidity and mortality in myocardial infarction (MI). Recently, mitochondrial dynamics (fusion and fission) have gained considerable attention as imbalanced dynamics may play central roles in various organ injuries, including MI. This study aimed to elucidate whether imbalanced mitochondrial dynamics of cardiomyocytes are involved in sedentary lifestyle-associated MI using mouse models. An MI model created by permanent coronary artery ligation showed a decreased survival rate, and the hearts of mice developed cardiac necrosis in the apex, cardiomyocyte hypertrophy, reduced ejection fraction, inflammation, and fibrosis. Ultrastructural analysis revealed increased mitochondrial fission, abnormal cardiac remodeling such as sarcomere disruption, and increased mRNA expression of cardiac injury and mitochondrial fission markers. Compared to the simple MI model, the combined sedentary lifestyle model created by narrow cage breeding and the MI model showed a further decrease in survival rate, cardiac hypertrophy, increased mitochondrial fission in cardiomyocytes, and myofibroblast activation with cardiac fibrosis. These findings suggest that mitochondrial fission could be involved in sedentary lifestyle-associated MI via abnormal crosstalk between cardiomyocytes and fibroblasts. Our study highlights the importance of developing novel mitochondrial-dynamics-rebalancing treatments in patients with MI.\u003c/p\u003e","manuscriptTitle":"Promotion of Pathological Cardiac Remodeling by Excessive Mitochondrial Fission in Sedentary Lifestyle-Associated Myocardial Infarction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 03:06:39","doi":"10.21203/rs.3.rs-6508918/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-26T18:42:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-25T08:18:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T09:01:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38205941338058342685059858508662218812","date":"2025-05-11T13:26:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65224466211298914256026738621179148272","date":"2025-05-09T12:31:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T12:25:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T12:10:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-06T10:52:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-05T09:57:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-23T04:56:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a412b835-35b4-4df9-9ceb-8aa6d266d4b5","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48378201,"name":"Health sciences/Cardiology"},{"id":48378202,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-07-03T06:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 03:06:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6508918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6508918","identity":"rs-6508918","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}