β-Alanine supplementation ameliorates right ventricular remodeling caused by MCT-induced pulmonary hypertension

β-Alanine supplementation ameliorates right ventricular remodeling caused by MCT-induced pulmonary hypertension | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article β-Alanine supplementation ameliorates right ventricular remodeling caused by MCT-induced pulmonary hypertension Hongling Su, MD，Bo Li, MD，Zhaoxia Guo, MD，Fu Zhang, MD，Aqian Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7077018/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Pulmonary hypertension (PH) is a disease characterized by progressive pulmonary vascular resistance and right heart failure. Beta-alanine (β-Ala), a non-proteinogenic amino acid, exhibits potential cardiovascular benefits; however, its role in PH pathophysiology remains underexplored. Objective To assess the effects of β-Ala supplementation on right ventricular remodeling and dysfunction in a monocrotaline (MCT)-induced PH rat model. Methods Male Wistar rats were randomly divided into three groups: control, MCT-induced PH, and β-Ala treated PH. Right ventricular function was evaluated using RVSP and RVHI. Protein expression and mRNA levels of cardiac markers and signaling pathways were analyzed by Western blotting and qPCR. Histological analysis was performed to assess right ventricular hypertrophy and fibrosis. Results β-Ala supplementation significantly improved RVSP and RVHI in MCT-induced PH rats. Protein expression analysis showed reduced ERK and p38 MAPK, along with increased activation of AKT in the β-Ala group compared to the MCT group. Additionally, β-Ala reduced the expression of pro-apoptotic markers Bax and Caspase-3 while increasing levels of the anti-apoptotic protein Bcl-2. qPCR analysis revealed decreased expression of ANP, BNP, α-MHC, β-MHC, and TGF-β in the β-Ala group. Histological analysis confirmed that β-Ala treatment alleviated right ventricular hypertrophy and fibrosis. Conclusion β-Ala improves right ventricular remodeling and dysfunction in MCT-induced PH rats by modulating signaling pathways and apoptotic markers, indicating its potential as a therapeutic agent for PH-induced right heart dysfunction. Pulmonary hypertension right ventricular remodeling beta-alanine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pulmonary hypertension (PH) is a group of rapidly progressive diseases with a poor prognosis, caused by various factors such as hereditary, chronic thromboembolic, and hypoxic conditions. It is characterized by significant remodeling of the pulmonary vasculature and a progressive increase in pulmonary vascular resistance, ultimately leading to right heart failure and death[ 1 ]. Although current treatment regimens have significantly improved the overall quality of life, exercise capacity, and long-term outcomes for PH patients, the five-year survival rate remains below 60%[ 2 ]. In patients with PH, the deterioration of right ventricular function is a major determinant of clinical outcomes and survival. Right ventricular remodeling may be adaptive, showing concentric hypertrophy, retention of myocardial microcirculation, and mild fibrosis; or it may be maladaptive, exhibiting eccentric hypertrophy, reduced micro vessels (leading to an imbalance in oxygen supply and demand), and fibrosis[ 1 ]. Right heart failure caused by PH is a significant reason for the poor prognosis in these patients, and currently, there are few treatment options for right heart failure. Recent advancements in metabolomics have highlighted its significance in elucidating PH pathogenesis. Metabolomics systematically analyzes changes in metabolites within biological samples, providing new insights for identifying disease-related biomarkers and potential therapeutic targets[ 3 ]. In PH patients, metabolic abnormalities include disturbances in energy metabolism, alterations in lipid metabolism, and imbalances in amino acid metabolism, which are closely associated with pathophysiological processes such as pulmonary vascular remodeling, endothelial dysfunction, and right ventricular hypertrophy[ 4 ]. β-Alanine (β-Ala) is a known non-proteinogenic and non-essential amino acid, which acts as a degradation product of serine, dihydropyridine, L-carnitine, and coumarin in animals. It serves as the rate-limiting amino acid in the synthesis of L-carnitine. β-Ala serves as a precursor for L-carnitine synthesis via carnosine synthase, thereby exerting anti-proliferative effects[ 5 ]. In humans, decreased levels of β-Ala are positively correlated with the prognosis of hypertensive patients[ 6 ].Furthermore, increased β-Ala levels in the heart have been shown to provide protective effects against ischemia-reperfusion injury in mouse models[ 7 ]. Despite the important role of β-Ala in cardiac health, research regarding its involvement in right ventricular remodeling induced by PH remains relatively scarce. Metabolomic analyses of lung tissues from PH patients and various pulmonary hypertension animal models [ 8 – 10 ], have demonstrated a downward trend in β-Ala levels within the right ventricular tissues of rats with severe pulmonary hypertension leading to right ventricular failure[ 11 ]. The anti-proliferative properties of β-Ala, along with its cardioprotective role, suggest that it may have a potential intervention effect on right ventricular remodeling in PH patients. However, the current understanding of β-Ala in the context of PH is still limited, necessitating more research to elucidate its specific mechanisms and therapeutic potential in right ventricular remodeling caused by PH. Future studies could investigate the effects of β-Ala supplementation on right ventricular function in PH patients, alongside potential molecular mechanisms, to provide new strategies for the clinical treatment of PH. This study further highlights the potential role of β-Ala in mitigating the progression of right ventricular remodeling in PH. Materials and methods 1. Population data This study analyzed plasma β-alanine (β-Ala) levels in pulmonary hypertension (PH) patients and healthy controls from Gansu Provincial Hospital. Fasting venous blood was collected using EDTA tubes, centrifuged (3000 rpm, 15 min, 4°C), and stored at -80°C. Untargeted metabolomics via UPLC-MS/MS identified elevated β-Ala in PH patients, confirmed by targeted quantification. Ethical approval (protocol 2020-021) and informed consent were obtained. 2. Animal models and experimental protocols In this study, male Wistar rats weighing 180–220 grams and aged 6–8 weeks were selected as experimental animals. All rats were purchased from the Gansu Chinese Medicine University Experimental Animal Center (License Number: SCXK (Gansu) 2015—0001) and housed in a clean-grade animal room at the Gansu University of Chinese Medicine Animal Experiment Center. All animal experiments were conducted in accordance with the protocols approved by the Animal Protection and Utilization Committee of Gansu University of Chinese Medicine. The rats were randomly divided into three groups: the control group (CRL group, n=7), the MCT-induced pulmonary hypertension group (MCT group, n=7), and the β-Ala treatment MCT-induced pulmonary hypertension group (β-Ala group, n=7). Rats were housed under controlled conditions (23 ± 1°C, 60% humidity), under a 12-hour light/dark cycle, with access to sterilized food and water ad libitum. MCT group rats received a single subcutaneous injection of monocrotaline (60 mg/kg) to induce the pulmonary hypertension model, with observations conducted from the first week until the fifth week. Rats in the β-Ala group received daily intraperitoneal injections of β-Ala (300 mg/kg)[12] from day 22 to day 35 for two weeks. On day 35, the rats in each group were anesthetized with pentobarbital sodium (30 mg/kg) and subsequently euthanized. Right ventricular systolic pressure (RVSP) was measured through catheterization via the right external jugular vein into the right ventricle. Right ventricular hypertrophy index (RVHI) was assessed by calculating the ratio of right ventricle weight to the sum of left ventricle and septal weights. 3．Western blot Right ventricular myocardial tissues were homogenized in liquid nitrogen and a mortar or a tissue homogenizer, followed by lysis with a protein extraction buffer containing Tris.HCl, NaCl, glycerophosphate, NP-40, EDTA, EGTA, Na3VO4, NaF, PMSF, and Benzamidine. After centrifugation at 4°C for 10 minutes, the supernatant was collected for quantification. Subsequently, Western blot analysis was performed using non-phosphorylated and phosphorylated-specific antibodies (purchased from CST, Santa Cruz, and Abcam) to detect the expression of total and phosphorylated proteins. 4. qPCR Detection To extract RNA from the right ventricular myocardial tissue, we initially ground the tissue using liquid nitrogen and a mortar or a tissue homogenizer. The tissue was then dissolved in Trizol reagent, mixed with chloroform, and vortexed. After standing at room temperature for 15 minutes, the mixture was centrifuged at 12,000 g for 15 minutes at 4°C to separate the aqueous phase. The aqueous phase was transferred to a new tube, and isopropanol was added and mixed, followed by incubation at room temperature for 5-10 minutes. It was subsequently centrifuged again at 12,000 g for 10 minutes at 4°C, discarding the supernatant while retaining the RNA pellet. The pellet was then suspended in 75% ethanol and centrifuged at 8,000 g for 5 minutes at 4°C. After discarding the supernatant, the pellet was air-dried or vacuum-dried. Finally, RNA samples were dissolved in H2O, TE buffer, or 0.5% SDS, and quantified using UV absorbance at 260/280 nm. The extracted total RNA was reverse transcribed using the Promega RT kit, followed by qPCR amplification of specific genes. 5. HE Staining In this study, right ventricular myocardial tissues from rats were isolated, and the tissue samples were immediately fixed in 4% paraformaldehyde for 24 hours to preserve their structure and morphology. The tissues were then embedded in paraffin for sectioning. We used a microtome to cut the tissue into 5-micron thick sections for subsequent staining and analysis. To measure the cross-sectional area of cardiomyocytes, we employed the classic Hematoxylin and Eosin (H&E) staining method. The stained sections were observed under a microscope, with the cell nuclei stained blue by hematoxylin and the cytoplasm and muscle fibers stained red by eosin, allowing for clear measurement of cardiomyocyte cross-sectional areas. 6. Masson Staining In this study, we performed a series of staining procedures on frozen sections of rat myocardial tissue, including potassium dichromate, iron hematoxylin, alcoholic hydrochloric acid, aniline blue, phosphomolybdic acid, and acid fuchsin, to observe and assess fibrotic lesions within the tissue. Through these staining steps, we could clearly differentiate collagen fibers, mucins, cartilage (appearing blue), cytoplasm, muscle, fibrin, and red blood cells (appearing red), as well as nuclei (appearing bluish-black), allowing for both quantitative and qualitative analysis of the fibrotic extent in myocardial tissue. 7. Statistical Analysis Statistical analysis of the experimental results was performed using GraphPad Prism software (version 9). The results were expressed as means ± standard error (X̄ ± SE). Comparisons between two groups were conducted using the t-test. Mean comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), with further pairwise comparisons using Tukey's test. A p-value of <0.05 was considered statistically significant. Results 1. β-Ala Levels in Right Ventricular Remodeling Induced by PH There was a significant difference in β-Ala levels between healthy individuals and patients with PH (p < 0.0001) (Figure 1A). This indicates that pulmonary hypertension may alter β-Ala metabolism, suggesting its potential role in the pathophysiology of PH. Figure 1B shows that in cardiac samples from rats, there was also a significant difference in β-Ala levels between the CRL group (control group) and the MCT group (pulmonary hypertension model group) (p < 0.05). β-Ala levels were significantly reduced in the MCT group compared to the CRL group ( p < 0.05). This further supports the importance of β-Ala in right heart failure induced by PAH, potentially related to changes in cardiac function. 2. In Vivo Supplementation of β-Ala Improves Right Ventricular Remodeling in MCT-Induced PH Rats Based on the above results, we considered β-Ala to play an essential role in right ventricular remodeling caused by PH. Therefore, we supplemented β-Ala to observe changes in the right ventricle of PH rats. The results showed that the hypertrophy index in the MCT group was significantly higher than in the CRL group (p < 0.05), while the β-Ala group was significantly lower than the MCT group (p < 0.001) (Figure 2A and 2B). The RVSP in the MCT group was significantly higher than in the CRL group (p < 0.05), whereas the β-Ala group exhibited significantly lower RVSP than the MCT group (p < 0.001) (Figure 2C and 2D). Histopathological examination indicated that the cross-sectional area of muscle fibers in the MCT group was significantly increased (p < 0.001), while that of the β-Ala group was significantly decreased compared to the MCT group (p < 0.05) (Figure 2E and 2F). Similarly, the collagen area fraction in the MCT group was significantly higher than that in the CRL group (p < 0.05), while the β-Ala group showed a significantly lower collagen area fraction than the MCT group (p < 0.05) (Figure 2E and 2G). This figure illustrates the impact of MCT treatment on cardiac structure and function, indicating that MCT may lead to right ventricular hypertrophy, cardiac dysfunction, and fibrosis, whereas β-Ala treatment partially ameliorated these pathological changes. 3. Supplementation of β-Ala Reduces the Expression Levels of Heart Failure Markers in MCT-Induced PH Rats We further investigated the effects of β-Ala supplementation on the expression levels of heart failure markers in rats with right ventricular failure induced by pulmonary arterial hypertension (PAH). The results showed that the mRNA expression of ANP in the MCT group was significantly higher than that in the CRL group (p < 0.05). The β-Ala treatment group exhibited significantly lower expression compared to the MCT group (p < 0.05) (Figure 3A). The mRNA expression of BNP in the MCT group was also significantly higher than that in the CRL group (p < 0.05), while the β-Ala treatment group showed significantly lower levels than the MCT group (p < 0.05) (Figure 3B). The α-MHC mRNA expression in the MCT group was significantly lower than that in the CRL group (p < 0.05), whereas the β-Ala treatment group had significantly higher expression compared to the MCT group (p < 0.05) (Figure 3C). The β-MHC mRNA expression in the MCT group was significantly higher than that in the CRL group (p < 0.05), while the β-Ala treatment group exhibited significantly lower levels compared to the MCT group (p < 0.05) (Figure 3D). Moreover, the TGF-β mRNA expression in the MCT group was significantly higher than that in the CRL group (p < 0.05), and the β-Ala treatment group showed significantly lower levels compared to the MCT group (p < 0.05) (Figure 3E). Figure 4A presents the Western blot results for ANP, BNP, TGF-β, α-MHC, and β-MHC proteins in cardiac samples from different groups (CRL, MCT, and β-Ala treatment groups). The quantitative analysis of ANP protein levels is shown in Figure 4B. The ANP protein level in the MCT group was significantly higher than that in the CRL group (p < 0.01), while the β-Ala treatment group exhibited significantly lower ANP levels compared to the MCT group (p < 0.0001), suggesting that β-Ala may improve cardiac function by inhibiting ANP expression. Figure 4C illustrates the changes in β-MHC protein levels. The β-MHC protein level in the MCT group was significantly higher than that in the CRL group (p < 0.01), while the β-Ala treatment group demonstrated significantly lower levels compared to the MCT group (p < 0.01), indicating that β-Ala may have a protective effect on cardiac remodeling. The quantitative results of α-MHC protein levels are depicted in Figure 4D. The α-MHC protein level in the MCT group was significantly lower than that in the CRL group (p < 0.01), while the β-Ala treatment group exhibited significantly higher α-MHC levels compared to the MCT group (p < 0.0001), suggesting that β-Ala may promote normal cardiac function. Figure 4E displays the changes in BNP protein levels. The BNP protein level in the MCT group was significantly higher than that in the CRL group (p < 0.01), while the β-Ala treatment group showed significantly lower BNP levels compared to the MCT group (p < 0.0001), further supporting the potential role of β-Ala in improving cardiac function. Finally, Figure 4F presents the quantitative analysis of TGF-β protein levels. The TGF-β protein level in the MCT group was significantly higher than that in the CRL group (p < 0.01), and the β-Ala treatment group exhibited significantly lower levels compared to the MCT group (p < 0.001), suggesting that β-Ala may alleviate cardiac remodeling by inhibiting TGF-β expression. Collectively, these results indicate that β-alanine treatment significantly reduces the expression of ANP, BNP, TGF-β, α-MHC, and β-MHC in the right ventricular myocardium of the pulmonary arterial hypertension model, implying its important protective role in improving cardiac function and mitigating cardiac remodeling. 4. The Effect of β-Ala Supplementation on Apoptosis Indicators in MCT-Induced PH Rats In our study, we investigated the effects of β-Ala on cardiomyocyte apoptosis induced by PH, revealing its potential protective mechanisms. Figure 5A presents the expression levels of Bax, Bcl-2, Caspase-3, and its active form C-Caspase-3 proteins in cardiac samples from different experimental groups, as detected by Western blot analysis. The quantitative analysis shown in Figure 5B indicates that the Bax protein level was significantly elevated in the MCT-induced PH model compared to the CRL group (p < 0.01). However, after treatment with β-Ala, the Bax protein level significantly decreased compared to the MCT group (p < 0.0001), suggesting that β-Ala may inhibit apoptosis by downregulating Bax expression. Figure 5C demonstrates that the Caspase-3 protein level was significantly increased in the MCT group compared to the CRL group (p < 0.01), while the β-Ala treatment group showed a significant reduction in Caspase-3 levels compared to the MCT group (p < 0.01), further indicating that β-Ala may alleviate apoptosis by suppressing Caspase-3 expression. The quantitative results in Figure 5D reveal that the Bcl-2 protein level in the MCT group was significantly lower than that in the CRL group (p < 0.01), whereas the Bcl-2 level in the β-Ala treatment group significantly increased compared to the MCT group (p < 0.0001), suggesting that β-Ala may inhibit apoptosis by upregulating Bcl-2 expression. Finally, Figure 5E displays that the C-Caspase-3 protein level was significantly higher in the MCT group compared to the CRL group (p < 0.0001), while the β-Ala treatment group showed a significant decrease in C-Caspase-3 levels compared to the MCT group (p < 0.0001), further confirming the potential role of β-Ala in inhibiting apoptosis. These findings collectively support the significant role of β-Ala in regulating the expression of apoptosis-related proteins and inhibiting apoptosis. 5. The Effect of β-Ala Supplementation on the Expression Levels of Relevant Proteins in the Right Ventricular Myocardium of MCT-Induced PH Rats Additionally, we assessed the expression levels of several relevant proteins. Figure 6 displays the Western blot results and quantitative analysis of ERK, p38 MAPK, and AKT protein expression levels across different groups. Figures 6A, C, and E show the Western blot images, while Figures 6B, D, and F present the corresponding quantitative analysis results. These figures indicate the expression levels of the target proteins relative to GAPDH. The expression levels of ERK, p38 MAPK, and AKT proteins in the MCT group were significantly higher than those in the CRL group. In contrast, the β-Ala treatment group significantly reduced the expression levels of ERK, p38 MAPK, and AKT proteins. These results suggest that β-Ala may improve MCT-induced right ventricular remodeling by inhibiting the expression of ERK, p38 MAPK, and AKT. Discussion This study utilized the observation that β-alanine levels in patients with PH are significantly lower than those in healthy individuals. By simulating PH in rats, we found results consistent with those observed in human PH cases, thus confirming our findings through animal experiments. We investigated the effects of β-Ala on right ventricular remodeling in experimental PH rats using the classic MCT-induced PH rat model to explore the pharmacological efficacy of β-Ala. The pathological development of MCT-induced PH is characterized by initial endothelial dysfunction in pulmonary arteries, interstitial edema, vasoconstriction, inflammatory infiltration around blood vessels, and the secretion of cytokines, followed by the gradual emergence of intimal hypertrophy and fibrosis[ 13 ].This classic small animal model of PH plays a crucial role in studying the pathological mechanisms of human diseases at the animal level. Our research demonstrates that β-Ala can improve cardiac function and hemodynamics in PH rats, inhibit the apoptosis of myocardial tissues, inhibit ventricular remodeling, and delay the progression of right heart dysfunction caused by PH. β-Ala is an isomer of alanine that exhibits anti-proliferative effects primarily through the synthesis of carnosine[ 5 ]. Carnosine plays a protective role against myocardial damage and oxidative stress, and it also helps regulate cardiac rhythm and delay aging[ 14 – 16 ]. Research indicates that endothelial β-Ala metabolism is essential for maintaining the homeostasis of vascular endothelial cells. β-Ala has a significant role in modulating endothelial function and phenotype. Disruption of β-Ala biosynthesis can lead to increased phosphorylation of Smad2/3 and activation of TGF-β signaling, ultimately resulting in endothelial neoplasia and atherosclerosis[ 17 ]. Studies have found that supplementation with β-Ala further improves the therapeutic effects on exercise capacity in rats with congestive heart failure, leading to additional benefits[ 12 ]. In preliminary studies, we observed that β-Ala levels in the blood of PH patients and in the right ventricular tissue of PH rats were significantly lower than those in healthy individuals. Supplementation with β-Ala resulted in the alleviation of right heart failure in PH rats, indicating that β-Ala metabolism plays a crucial role in the process of cardiomyocyte remodeling. In fact, in the context of heart failure due to myocardial infarction, β-Ala supplementation has been shown to reduce the size of myocardial infarction and provide certain protective effects on the damaged heart[ 18 ]. The levels of apoptosis are an important pathological characteristic reflecting ventricular remodeling. Detection of cell apoptosis is not only useful in the diagnosis and risk stratification of patients with ischemic heart disease in clinical settings, but interventions targeting apoptosis have also proven effective in preventing and treating post-infarction remodeling and heart failure[ 19 ]. Induction of increased Bcl-2 expression and inhibition of Bax activity are effective triggers for the death and apoptosis of ischemic cardiac myocytes[ 20 ]. As a classic anti-apoptotic factor [ 21 ], high levels of Bcl-2 expression can protect against oxidative stress-induced apoptosis and safeguard cardiomyocyte viability and left ventricular function in the context of ischemia[ 22 ].The Bcl-2/Bax ratio serves as a "rheostat" regulating cell death, dependent on the balance between Bcl-2 and Bax in the cell. A reduction in apoptosis is often associated with a significant increase in the Bcl-2/Bax ratio[ 23 ]. Factors influencing cardiotoxicity may enhance cardiomyocyte apoptosis by affecting the Bcl-2/Bax apoptotic pathway and altering the Bcl-2/Bax ratio[ 24 ]。In this study, the Bcl-2/Bax ratio was significantly decreased in rats with right ventricular remodeling. Following β-Ala supplementation, the Bcl-2/Bax ratio increased significantly. This suggests that a reduction in apoptosis during right ventricular remodeling may help to mitigate the occurrence and progression of right heart remodeling by promoting cardiomyocyte survival. The ERK signaling pathway is typically associated with cell proliferation and differentiation. In PH, sustained activation of ERK may lead to aberrant proliferation and remodeling of cardiac myocytes[ 25 ]. Our study found that the expression levels of ERK protein were significantly upregulated in the MCT-induced PH model compared to the CRL group, while β-Ala treatment significantly reversed this upregulation, indicating that β-Ala may alleviate the abnormal proliferation and remodeling of cardiac myocytes by inhibiting the ERK signaling pathway. The p38 MAPK signaling pathway is closely related to cellular stress responses and apoptosis. In PAH, activation of p38 MAPK may promote cardiomyocyte apoptosis[ 26 ]. Our findings demonstrated that p38 MAPK expression levels were significantly increased in the MCT group compared to the CRL group, and β-Ala treatment significantly reduced its expression, suggesting that β-Ala may decrease apoptosis of cardiac myocytes by inhibiting the p38 MAPK signaling pathway. The AKT signaling pathway plays a central role in cell survival and apoptosis inhibition[ 27 ]. In PAH, activation of AKT may be suppressed, leading to increased cardiomyocyte apoptosis[ 28 ]. Our results indicate that compared to the CRL group, the MCT group showed a significant decrease in AKT protein expression, whereas β-Ala treatment significantly increased its expression levels. This suggests that β-Ala may inhibit cardiomyocyte apoptosis by activating the AKT signaling pathway. Limitations Although this study revealed the improvement of right ventricular remodeling caused by PH through β-Ala, there are still several limitations. First, we did not thoroughly investigate the specific molecular mechanisms by which β-Ala improves right ventricular remodeling due to PH. While existing research has suggested that β-Ala may exert its effects by modulating the expression of key signaling molecules such as ERK, p38 MAPK, and AKT, our study lacks direct evidence of alterations in these signaling pathways. Furthermore, it is generally believed that β-Ala acts by increasing the concentration of carnosine in cardiomyocytes; however, we have not validated the changes in carnosine levels in this study, which may be an avenue for further exploration in future research. Although our study achieved preliminary results in exploring the beneficial effects of β-Ala on right ventricular remodeling due to PH, further in-depth research is needed to investigate its potential mechanisms of action, particularly in regulating cardiomyocyte apoptosis and vascular remodeling. Future studies could focus on the effects of β-Ala on specific signaling pathways, such as ERK, p38 MAPK, and AKT, and verify its biological effects by measuring carnosine concentration, thereby providing more scientific evidence for the treatment of PH. Conclusion In conclusion, this study is the first to reveal that β-Ala has a significant ameliorative effect on right ventricular remodeling caused by PH. These findings identify β-Ala as a novel therapeutic target for PH-induced right ventricular dysfunction and highlight its translational potential. Our findings indicate that β-Ala may regulate the expression of key signaling molecules such as ERK, p38 MAPK, and AKT to suppress myocardial remodeling, fibrosis, and apoptosis induced by PAH, thereby exerting its potential therapeutic effects. By modulating critical cell signaling pathways, including ERK, p38 MAPK, and AKT, β-Ala demonstrates positive effects on the processes of cardiomyocyte apoptosis, fibrosis, and remodeling. Our results highlight the important role of β-Ala as a potential therapeutic agent in alleviating PH-related cardiac damage and improving right heart function, laying a scientific foundation for further drug development and clinical application. Declarations Author Contributions H.S. contributed to writing the original draft. B.L., Z.G. and F.Z. contributed to reviewing and editing the manuscript. A.W., K.J. and H.Z. contributed to data collection. Y.Y., J.Z. and W.L. contributed to reviewing the manuscript. J.L. contributed to conceptualization, resources, and supervision. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the Lanzhou Science and Technology Program of Gansu Province of China awarded to Hongling Su (LX-62000001-2022-090), the Natural Science Foundation of Gansu Province of China to Bo Li (24JRRA1049) and The Intramural Fund of Gansu Provincial Hospital of China to Hongling Su (22GSSYB-2). Clinical trial number Not applicable. Data availability statement All the authors confirm that all the data that support the findings of this study are available upon request from the corresponding author. Ethics approval and consent to participate All animal experiments were conducted in accordance with the protocols approved by the Animal Protection and Utilization Committee of Gansu University of Chinese Medicine. Consent for publication Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. Conflicts of interest The authors declare no conflicts of interest. Author details 1 The First Clinical Medical College of Lanzhou University, Lanzhou 730000, P. R. China. 2 Department of Cardiology, Pulmonary Vascular Disease Center (PVDC), Gansu Provincial Hospital, Lanzhou 730000, P. R. China. 3 Department of laboratory, Gansu Wuwei Tumor Hospital, Wuwei 733000, P. R. China. 4 Heart, Lung and Vessels Center, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610072, P. R. China. 5 Department of Intensive Care Unit, Gansu Provincial Maternity and Child Health Hospital, Gansu Provincial General Hospital, Lan Zhou, Gansu Province, 730050, China. References Hassoun PM. Pulmonary Arterial Hypertension. 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Theranostics. 2023;13(13):4392-4411.https://doi.org/10.7150/thno.84427 Allo SN, Bagby L, Schaffer SW. Taurine depletion, a novel mechanism for cardioprotection from regional ischemia. The American journal of physiology. 1997;273(4):H1956-1961.https://doi.org/10.1152/ajpheart.1997.273.4.H1956 Abbate A, Bussani R, Amin MS, Vetrovec GW, Baldi A. Acute myocardial infarction and heart failure: role of apoptosis. Int J Biochem Cell Biol. 2006;38(11):1834-1840.https://doi.org/10.1016/j.biocel.2006.04.010 Ahmad F, Lal H, Zhou J, Vagnozzi RJ, Yu JE, Shang X, et al. Cardiomyocyte-specific deletion of Gsk3α mitigates post-myocardial infarction remodeling, contractile dysfunction, and heart failure. J Am Coll Cardiol. 2014;64(7):696-706.https://doi.org/10.1016/j.jacc.2014.04.068 Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993;75(2):241-251.https://doi.org/10.1016/0092-8674(93)80066-n Chatterjee S, Stewart AS, Bish LT, Jayasankar V, Kim EM, Pirolli T, et al. Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation. 2002;106(12 Suppl 1):I212-217 Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE, Oltvai ZN. Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death. Semin Cancer Biol. 1993;4(6):327-332 Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol. 2013;65(2):157-170.https://doi.org/10.1111/j.2042-7158.2012.01567.x Feng WA-O, Wang J, Yan XA-O, Zhang Q, Chai L, Wang Q, et al. ERK/Drp1-dependent mitochondrial fission contributes to HMGB1-induced autophagy in pulmonary arterial hypertension. (1365-2184 (Electronic)) Romero-Becerra R, Santamans AM, Folgueira C, Sabio G. p38 MAPK Pathway in the Heart: New Insights in Health and Disease. International journal of molecular sciences. 2020;21(19).https://doi.org/10.3390/ijms21197412 Wang AW, Song L, Miao J, Wang HX, Tian C, Jiang X, et al. Baicalein attenuates angiotensin II-induced cardiac remodeling via inhibition of AKT/mTOR, ERK1/2, NF-κB, and calcineurin signaling pathways in mice. American journal of hypertension. 2015;28(4):518-526.https://doi.org/10.1093/ajh/hpu194 Cao YY, Ba HX, Li Y, Tang SY, Luo ZQ, Li XH. Regulatory effects of Prohibitin 1 on proliferation and apoptosis of pulmonary arterial smooth muscle cells in monocrotaline-induced PAH rats. Life sciences. 2020;250:117548.https://doi.org/10.1016/j.lfs.2020.117548 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7077018","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502516444,"identity":"acdaac32-41b1-44d0-b731-8b8dcadd4433","order_by":0,"name":"Hongling Su","email":"","orcid":"","institution":"The First Clinical Medical College of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Hongling","middleName":"","lastName":"Su","suffix":""},{"id":502516445,"identity":"e7bb8179-8630-4065-bea7-a04e0918a3ee","order_by":1,"name":"MD，Bo Li","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"MD，Bo","middleName":"","lastName":"Li","suffix":""},{"id":502516446,"identity":"c3811b96-2ce6-495e-b52f-206b74790207","order_by":2,"name":"MD，Zhaoxia Guo","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"MD，Zhaoxia","middleName":"","lastName":"Guo","suffix":""},{"id":502516447,"identity":"41275a40-ccb0-4b10-b3cd-587f8a79d78e","order_by":3,"name":"MD，Fu Zhang","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"MD，Fu","middleName":"","lastName":"Zhang","suffix":""},{"id":502516448,"identity":"75ee3c64-2e6d-48c4-a479-9f4e57e89638","order_by":4,"name":"MD，Aqian Wang","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"MD，Aqian","middleName":"","lastName":"Wang","suffix":""},{"id":502516449,"identity":"c597323d-9d7d-47ff-92a8-4902aa7b3d78","order_by":5,"name":"MD，Kaiyu Jiang","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"MD，Kaiyu","middleName":"","lastName":"Jiang","suffix":""},{"id":502516450,"identity":"6c091b02-7f51-4bf1-bdb2-c9a33c4191fc","order_by":6,"name":"Hai Zhu","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Zhu","suffix":""},{"id":502516451,"identity":"ead72e2b-1581-4f01-8beb-42726893ce56","order_by":7,"name":"Yanxia Yang","email":"","orcid":"","institution":"Gansu Wuwei Tumor Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanxia","middleName":"","lastName":"Yang","suffix":""},{"id":502516452,"identity":"72b14300-8aa3-48f1-a670-46c9d8c3dbe2","order_by":8,"name":"Jing Zeng","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zeng","suffix":""},{"id":502516453,"identity":"14d8dcde-18cb-4d6e-8847-790e9fc925a3","order_by":9,"name":"Wen Li","email":"","orcid":"","institution":"Gansu Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Li","suffix":""},{"id":502516454,"identity":"ce6cf1b5-bfb2-4a97-a8cf-7acfe2d3f2c3","order_by":10,"name":"Yunshan Cao","email":"","orcid":"","institution":"University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Yunshan","middleName":"","lastName":"Cao","suffix":""},{"id":502516455,"identity":"cabc35d1-b5a7-4ad4-b51e-c81db3f3f17f","order_by":11,"name":"Jian Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYHACAxCSYWNgSDjAwGDDw8/fQJwWHqiWNBnJGQeI0cLAwAPlHLYxaEjAr163vXnjgx8Fd3j4JBIeHvi54zyPAcMBxg8fc3BrMTtzrNiwx+AZD5tEQsLB3jO3ecyZG5glZ27Do+VGjpk0g8FhsJYDvG23eSwbDrAx8+LTcv+N+W+YloN/287xGBxIIKDlBo8ZM0zLYd62A0RoOZNWLNkD0sLzIOGwbFsyj+SMg834/XL88MYPP/4clpNvz0n++LbNzp6fv/ngh494tCABngQog7GBKPVAwH6AWJWjYBSMglEwwgAA9etTm+bWtgYAAAAASUVORK5CYII=","orcid":"","institution":"The First Clinical Medical College of Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-07-08 17:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7077018/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7077018/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89486647,"identity":"b7a3eee1-09b3-45ff-8209-d3f7068ccebe","added_by":"auto","created_at":"2025-08-20 12:57:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Ala Content in Human and Rat Heart Samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Comparison of β-Ala content (ng/mg) in healthy individuals (Health) versus patients with PH. Data are presented as mean ± SEM, with statistical significance indicated by ****p \u0026lt; 0.0001. B. β-Ala content (ng/mg) in rat heart tissues from the control group (CRL) and the MCT-induced PAH group (MCT). Data are presented as mean ± SEM, with statistical significance indicated by *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/e7151cfb735bb290f5899099.jpg"},{"id":89486867,"identity":"cdfd65ca-d96e-43bb-9efd-1cd3c4d50de5","added_by":"auto","created_at":"2025-08-20 13:05:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":435982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of β-Ala on Right Ventricular Remodeling in MCT-Induced PAH Rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative images of the right ventricles from the CRL group, MCT group, and β-Ala group. B. RVHI in the three groups. C. RVSP waveforms recorded from the CRL, MCT, and β-Ala groups. D. RVSP measurements in the three groups. E. Histological analysis of right ventricular myocardium stained with H\u0026amp;E, showing differences in morphology among the groups. F. Myocyte cross-sectional area (μm²) in the three groups. G. Right ventricular collagen area fraction (%) in the three groups. Data are presented as mean ± SEM. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant. Scale bars in (E) represent 50 μm.\u003c/p\u003e","description":"","filename":"Figure02.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/c07470ba3bcf7ded0b8b5e4a.jpg"},{"id":89486651,"identity":"4eef8167-150d-460b-a8fe-d6c84a2d5926","added_by":"auto","created_at":"2025-08-20 12:57:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of β-Ala on mRNA Expression of Cardiac Markers in MCT-Induced PAH Rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Relative mRNA expression of ANP normalized to GAPDH in the control group (CRL), MCT group, and β-Ala group. B. Relative mRNA expression of BNP normalized to GAPDH in the three groups. C. Relative mRNA expression of α-MHC normalized to GAPDH. D. Relative mRNA expression of β-MHC normalized to GAPDH. E. Relative mRNA expression of TGF-β normalized to GAPDH. Data are presented as mean ± SEM. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns = not significant.\u003c/p\u003e","description":"","filename":"Figure03.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/0b2cf759a50b13942f65fd83.jpg"},{"id":89486866,"identity":"61facdee-caeb-49b4-8c27-33cf71d6a697","added_by":"auto","created_at":"2025-08-20 13:05:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein Expression Levels of Cardiac Markers in MCT-Induced PAH Rats Treated with β-Ala.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative Western blot images showing the expression of ANP, BNP, TGF-β, α-MHC, and β-MHC in heart tissues from the CRL group, MCT group, and β-Ala treated group. B. Quantification of ANP protein levels relative to GAPDH. C. Quantification of BNP protein levels relative to GAPDH. D. Quantification of TGF-β protein levels relative to GAPDH. E. Quantification of α-MHC protein levels relative to GAPDH. F. Quantification of β-MHC protein levels relative to GAPDH. Data are presented as mean ± SEM. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure04.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/8247826f0781b62a4045d1dd.jpg"},{"id":89486653,"identity":"1f8de6ab-e12d-4c8f-a37a-ffd871a85522","added_by":"auto","created_at":"2025-08-20 12:57:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":106716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of β-Ala on Apoptotic Protein Expression in MCT-Induced PAH Rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative Western blot images showing the expression of Bax, Bcl-2, Caspase 3, and cleaved Caspase 3 in heart tissues from the CRL group, MCT group, and β-Ala treated group. B. Quantification of Bax protein levels relative to GAPDH in the three groups. C. Quantification of Bcl-2 protein levels relative to GAPDH. D. Ratio of Bcl-2 to Bax protein levels in the three groups. E. Quantification of total Caspase 3 protein levels relative to GAPDH. F. Quantification of cleaved Caspase 3 protein levels relative to GAPDH. Data are presented as mean ± SEM. Statistical significance is indicated as follows: **p \u0026lt; 0.01, ****p \u0026lt; 0.0001, ns = not significant.\u003c/p\u003e","description":"","filename":"Figure05.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/e1390ab02ed7c09529763f88.jpg"},{"id":89489418,"identity":"b3c1c136-a37f-411a-b158-15a37b25493f","added_by":"auto","created_at":"2025-08-20 13:29:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of β-Ala on Key Signaling Proteins in MCT-Induced PAH Rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative Western blot images showing the expression of ERK and GAPDH in heart tissues from the CRL group, MCT group, and β-Ala treated group. B. Quantification of ERK protein levels relative to GAPDH in the three groups. C. Representative Western blot images showing the expression of p38 MAPK and GAPDH. D. Quantification of p38 MAPK protein levels relative to GAPDH in the three groups. E. Representative Western blot images showing the expression of AKT and GAPDH. F. Quantification of AKT protein levels relative to GAPDH in the three groups. Data are presented as mean ± SEM. Statistical significance is indicated as follows: **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns = not significant.\u003c/p\u003e","description":"","filename":"Figure06.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/f69adbebae10415f98b1f39d.jpg"},{"id":91851348,"identity":"2fc187ea-6b39-4f69-a68c-ddb5e7de8af5","added_by":"auto","created_at":"2025-09-22 11:24:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1727025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7077018/v1/f1512fbf-2b86-4531-9558-8d4b67b29dbc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"β-Alanine supplementation ameliorates right ventricular remodeling caused by MCT-induced pulmonary hypertension","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePulmonary hypertension (PH) is a group of rapidly progressive diseases with a poor prognosis, caused by various factors such as hereditary, chronic thromboembolic, and hypoxic conditions. It is characterized by significant remodeling of the pulmonary vasculature and a progressive increase in pulmonary vascular resistance, ultimately leading to right heart failure and death[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although current treatment regimens have significantly improved the overall quality of life, exercise capacity, and long-term outcomes for PH patients, the five-year survival rate remains below 60%[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In patients with PH, the deterioration of right ventricular function is a major determinant of clinical outcomes and survival. Right ventricular remodeling may be adaptive, showing concentric hypertrophy, retention of myocardial microcirculation, and mild fibrosis; or it may be maladaptive, exhibiting eccentric hypertrophy, reduced micro vessels (leading to an imbalance in oxygen supply and demand), and fibrosis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Right heart failure caused by PH is a significant reason for the poor prognosis in these patients, and currently, there are few treatment options for right heart failure.\u003c/p\u003e\u003cp\u003eRecent advancements in metabolomics have highlighted its significance in elucidating PH pathogenesis. Metabolomics systematically analyzes changes in metabolites within biological samples, providing new insights for identifying disease-related biomarkers and potential therapeutic targets[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In PH patients, metabolic abnormalities include disturbances in energy metabolism, alterations in lipid metabolism, and imbalances in amino acid metabolism, which are closely associated with pathophysiological processes such as pulmonary vascular remodeling, endothelial dysfunction, and right ventricular hypertrophy[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eβ-Alanine (β-Ala) is a known non-proteinogenic and non-essential amino acid, which acts as a degradation product of serine, dihydropyridine, L-carnitine, and coumarin in animals. It serves as the rate-limiting amino acid in the synthesis of L-carnitine. β-Ala serves as a precursor for L-carnitine synthesis via carnosine synthase, thereby exerting anti-proliferative effects[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In humans, decreased levels of β-Ala are positively correlated with the prognosis of hypertensive patients[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].Furthermore, increased β-Ala levels in the heart have been shown to provide protective effects against ischemia-reperfusion injury in mouse models[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite the important role of β-Ala in cardiac health, research regarding its involvement in right ventricular remodeling induced by PH remains relatively scarce. Metabolomic analyses of lung tissues from PH patients and various pulmonary hypertension animal models [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], have demonstrated a downward trend in β-Ala levels within the right ventricular tissues of rats with severe pulmonary hypertension leading to right ventricular failure[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The anti-proliferative properties of β-Ala, along with its cardioprotective role, suggest that it may have a potential intervention effect on right ventricular remodeling in PH patients. However, the current understanding of β-Ala in the context of PH is still limited, necessitating more research to elucidate its specific mechanisms and therapeutic potential in right ventricular remodeling caused by PH. Future studies could investigate the effects of β-Ala supplementation on right ventricular function in PH patients, alongside potential molecular mechanisms, to provide new strategies for the clinical treatment of PH. This study further highlights the potential role of β-Ala in mitigating the progression of right ventricular remodeling in PH.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e1.\u0026nbsp;Population data\u003c/p\u003e\n\u003cp\u003eThis study analyzed plasma \u0026beta;-alanine (\u0026beta;-Ala) levels in pulmonary hypertension (PH) patients and healthy controls from Gansu Provincial Hospital. Fasting venous blood was collected using EDTA tubes, centrifuged (3000 rpm, 15 min, 4\u0026deg;C), and stored at -80\u0026deg;C. Untargeted metabolomics via UPLC-MS/MS identified elevated \u0026beta;-Ala in PH patients, confirmed by targeted quantification.\u0026nbsp;Ethical approval (protocol 2020-021) and informed consent were obtained.\u003c/p\u003e\n\u003cp\u003e2. Animal models and experimental protocols\u003c/p\u003e\n\u003cp\u003eIn this study, male Wistar rats weighing 180\u0026ndash;220 grams and aged 6\u0026ndash;8 weeks were selected as experimental animals. All rats were purchased from the Gansu Chinese Medicine University Experimental Animal Center (License Number: SCXK (Gansu) 2015\u0026mdash;0001) and housed in a clean-grade animal room at the Gansu University of Chinese Medicine Animal Experiment Center. All animal experiments were conducted in accordance with the protocols approved by the Animal Protection and Utilization Committee of Gansu University of Chinese Medicine. The rats were randomly divided into three groups: the control group (CRL group, n=7), the MCT-induced pulmonary hypertension group (MCT group, n=7), and the \u0026beta;-Ala treatment MCT-induced pulmonary hypertension group (\u0026beta;-Ala group, n=7).\u003c/p\u003e\n\u003cp\u003eRats were housed under controlled conditions (23 \u0026plusmn; 1\u0026deg;C, 60% humidity), under a 12-hour light/dark cycle, with access to sterilized food and water ad libitum. MCT group rats received a single subcutaneous injection of monocrotaline (60 mg/kg) to induce the pulmonary hypertension model, with observations conducted from the first week until the fifth week. Rats in the \u0026beta;-Ala group received daily intraperitoneal injections of \u0026beta;-Ala (300 mg/kg)[12] from day 22 to day 35 for two weeks. On day 35, the rats in each group were anesthetized with pentobarbital sodium (30 mg/kg) and subsequently euthanized. Right ventricular systolic pressure (RVSP) was measured through catheterization via the right external jugular vein into the right ventricle. Right ventricular hypertrophy index (RVHI) was assessed by calculating the ratio of right ventricle weight to the sum of left ventricle and septal weights.\u003c/p\u003e\n\u003cp\u003e3．Western blot\u003c/p\u003e\n\u003cp\u003eRight ventricular myocardial tissues were homogenized in liquid nitrogen and a mortar or a tissue homogenizer, followed by lysis with a protein extraction buffer containing Tris.HCl, NaCl, glycerophosphate, NP-40, EDTA, EGTA, Na3VO4, NaF, PMSF, and Benzamidine. After centrifugation at 4\u0026deg;C for 10 minutes, the supernatant was collected for quantification. Subsequently, Western blot analysis was performed using non-phosphorylated and phosphorylated-specific antibodies (purchased from CST, Santa Cruz, and Abcam) to detect the expression of total and phosphorylated proteins.\u003c/p\u003e\n\u003cp\u003e4. qPCR Detection\u003c/p\u003e\n\u003cp\u003eTo extract RNA from the right ventricular myocardial tissue, we initially ground the tissue using liquid nitrogen and a mortar or a tissue homogenizer. The tissue was then dissolved in Trizol reagent, mixed with chloroform, and vortexed. After standing at room temperature for 15 minutes, the mixture was centrifuged at 12,000 g for 15 minutes at 4\u0026deg;C to separate the aqueous phase. The aqueous phase was transferred to a new tube, and isopropanol was added and mixed, followed by incubation at room temperature for 5-10 minutes. It was subsequently centrifuged again at 12,000 g for 10 minutes at 4\u0026deg;C, discarding the supernatant while retaining the RNA pellet. The pellet was then suspended in 75% ethanol and centrifuged at 8,000 g for 5 minutes at 4\u0026deg;C. After discarding the supernatant, the pellet was air-dried or vacuum-dried. Finally, RNA samples were dissolved in H2O, TE buffer, or 0.5% SDS, and quantified using UV absorbance at 260/280 nm. The extracted total RNA was reverse transcribed using the Promega RT kit, followed by qPCR amplification of specific genes.\u003c/p\u003e\n\u003cp\u003e5. HE Staining\u003c/p\u003e\n\u003cp\u003eIn this study, right ventricular myocardial tissues from rats were isolated, and the tissue samples were immediately fixed in 4% paraformaldehyde for 24 hours to preserve their structure and morphology. The tissues were then embedded in paraffin for sectioning. We used a microtome to cut the tissue into 5-micron thick sections for subsequent staining and analysis. To measure the cross-sectional area of cardiomyocytes, we employed the classic Hematoxylin and Eosin (H\u0026amp;E) staining method. The stained sections were observed under a microscope, with the cell nuclei stained blue by hematoxylin and the cytoplasm and muscle fibers stained red by eosin, allowing for clear measurement of cardiomyocyte cross-sectional areas.\u003c/p\u003e\n\u003cp\u003e6. Masson Staining\u003c/p\u003e\n\u003cp\u003eIn this study, we performed a series of staining procedures on frozen sections of rat myocardial tissue, including potassium dichromate, iron hematoxylin, alcoholic hydrochloric acid, aniline blue, phosphomolybdic acid, and acid fuchsin, to observe and assess fibrotic lesions within the tissue. Through these staining steps, we could clearly differentiate collagen fibers, mucins, cartilage (appearing blue), cytoplasm, muscle, fibrin, and red blood cells (appearing red), as well as nuclei (appearing bluish-black), allowing for both quantitative and qualitative analysis of the fibrotic extent in myocardial tissue.\u003c/p\u003e\n\u003cp\u003e7. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eStatistical analysis of the experimental results was performed using GraphPad Prism software (version 9). The results were expressed as means \u0026plusmn; standard error (X̄ \u0026plusmn; SE). Comparisons between two groups were conducted using the t-test. Mean comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), with further pairwise comparisons using Tukey\u0026apos;s test. A p-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. \u0026beta;-Ala Levels in Right Ventricular Remodeling Induced by PH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant difference in \u0026beta;-Ala levels between healthy individuals and patients with PH (p \u0026lt; 0.0001) (Figure 1A). This indicates that pulmonary hypertension may alter \u0026beta;-Ala metabolism, suggesting its potential role in the pathophysiology of PH. Figure 1B shows that in cardiac samples from rats, there was also a significant difference in \u0026beta;-Ala levels between the CRL group (control group) and the MCT group (pulmonary hypertension model group) (p \u0026lt; 0.05). \u0026beta;-Ala levels were significantly reduced in the MCT group compared to the CRL group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). This further supports the importance of \u0026beta;-Ala in right heart failure induced by PAH, potentially related to changes in cardiac function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. In Vivo Supplementation of \u0026beta;-Ala Improves Right Ventricular Remodeling in MCT-Induced PH Rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the above results, we considered \u0026beta;-Ala to play an essential role in right ventricular remodeling caused by PH. Therefore, we supplemented \u0026beta;-Ala to observe changes in the right ventricle of PH rats. The results showed that the hypertrophy index in the MCT group was significantly higher than in the CRL group (p \u0026lt; 0.05), while the \u0026beta;-Ala group was significantly lower than the MCT group (p \u0026lt; 0.001) (Figure 2A and 2B). The RVSP in the MCT group was significantly higher than in the CRL group (p \u0026lt; 0.05), whereas the \u0026beta;-Ala group exhibited significantly lower RVSP than the MCT group (p \u0026lt; 0.001) (Figure 2C and 2D). Histopathological examination indicated that the cross-sectional area of muscle fibers in the MCT group was significantly increased (p \u0026lt; 0.001), while that of the \u0026beta;-Ala group was significantly decreased compared to the MCT group (p \u0026lt; 0.05) (Figure 2E and 2F). Similarly, the collagen area fraction in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.05), while the \u0026beta;-Ala group showed a significantly lower collagen area fraction than the MCT group (p \u0026lt; 0.05) (Figure 2E and 2G). This figure illustrates the impact of MCT treatment on cardiac structure and function, indicating that MCT may lead to right ventricular hypertrophy, cardiac dysfunction, and fibrosis, whereas \u0026beta;-Ala treatment partially ameliorated these pathological changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Supplementation of \u0026beta;-Ala Reduces the Expression Levels of Heart Failure Markers in MCT-Induced PH Rats\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further investigated the effects of\u0026nbsp;\u0026beta;-Ala\u0026nbsp;supplementation on the expression levels of heart failure markers in rats with right ventricular failure induced by pulmonary arterial hypertension (PAH). The results showed that the mRNA expression of ANP in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.05). The\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group exhibited significantly lower expression compared to the MCT group (p \u0026lt; 0.05) (Figure 3A). The mRNA expression of BNP in the MCT group was also significantly higher than that in the CRL group (p \u0026lt; 0.05), while the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group showed significantly lower levels than the MCT group (p \u0026lt; 0.05) (Figure 3B). The \u0026alpha;-MHC mRNA expression in the MCT group was significantly lower than that in the CRL group (p \u0026lt; 0.05), whereas the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group had significantly higher expression compared to the MCT group (p \u0026lt; 0.05) (Figure 3C). The \u0026beta;-MHC mRNA expression in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.05), while the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group exhibited significantly lower levels compared to the MCT group (p \u0026lt; 0.05) (Figure 3D). Moreover, the TGF-\u0026beta; mRNA expression in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.05), and the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group showed significantly lower levels compared to the MCT group (p \u0026lt; 0.05) (Figure 3E). Figure 4A presents the Western blot results for ANP, BNP, TGF-\u0026beta;, \u0026alpha;-MHC, and \u0026beta;-MHC proteins in cardiac samples from different groups (CRL, MCT, and\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment groups). The quantitative analysis of ANP protein levels is shown in Figure 4B. The ANP protein level in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.01), while the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group exhibited significantly lower ANP levels compared to the MCT group (p \u0026lt; 0.0001), suggesting that\u0026nbsp;\u0026beta;-Ala\u0026nbsp;may improve cardiac function by inhibiting ANP expression. Figure 4C illustrates the changes in \u0026beta;-MHC protein levels. The \u0026beta;-MHC protein level in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.01), while the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group demonstrated significantly lower levels compared to the MCT group (p \u0026lt; 0.01), indicating that\u0026nbsp;\u0026beta;-Ala\u0026nbsp;may have a protective effect on cardiac remodeling. The quantitative results of \u0026alpha;-MHC protein levels are depicted in Figure 4D. The \u0026alpha;-MHC protein level in the MCT group was significantly lower than that in the CRL group (p \u0026lt; 0.01), while the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group exhibited significantly higher \u0026alpha;-MHC levels compared to the MCT group (p \u0026lt; 0.0001), suggesting that\u0026nbsp;\u0026beta;-Ala\u0026nbsp;may promote normal cardiac function. Figure 4E displays the changes in BNP protein levels. The BNP protein level in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.01), while the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group showed significantly lower BNP levels compared to the MCT group (p \u0026lt; 0.0001), further supporting the potential role of\u0026nbsp;\u0026beta;-Ala\u0026nbsp;in improving cardiac function. Finally, Figure 4F presents the quantitative analysis of TGF-\u0026beta; protein levels. The TGF-\u0026beta; protein level in the MCT group was significantly higher than that in the CRL group (p \u0026lt; 0.01), and the\u0026nbsp;\u0026beta;-Ala\u0026nbsp;treatment group exhibited significantly lower levels compared to the MCT group (p \u0026lt; 0.001), suggesting that\u0026nbsp;\u0026beta;-Ala\u0026nbsp;may alleviate cardiac remodeling by inhibiting TGF-\u0026beta; expression. Collectively, these results indicate that \u0026beta;-alanine treatment significantly reduces the expression of ANP, BNP, TGF-\u0026beta;, \u0026alpha;-MHC, and \u0026beta;-MHC in the right ventricular myocardium of the pulmonary arterial hypertension model, implying its important protective role in improving cardiac function and mitigating cardiac remodeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. The Effect of \u0026beta;-Ala Supplementation on Apoptosis Indicators in MCT-Induced PH Rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our study, we investigated the effects of \u0026beta;-Ala on cardiomyocyte apoptosis induced by PH, revealing its potential protective mechanisms. Figure 5A presents the expression levels of Bax, Bcl-2, Caspase-3, and its active form C-Caspase-3 proteins in cardiac samples from different experimental groups, as detected by Western blot analysis. The quantitative analysis shown in Figure 5B indicates that the Bax protein level was significantly elevated in the MCT-induced PH model compared to the CRL group (p \u0026lt; 0.01). However, after treatment with \u0026beta;-Ala, the Bax protein level significantly decreased compared to the MCT group (p \u0026lt; 0.0001), suggesting that \u0026beta;-Ala may inhibit apoptosis by downregulating Bax expression. Figure 5C demonstrates that the Caspase-3 protein level was significantly increased in the MCT group compared to the CRL group (p \u0026lt; 0.01), while the \u0026beta;-Ala treatment group showed a significant reduction in Caspase-3 levels compared to the MCT group (p \u0026lt; 0.01), further indicating that \u0026beta;-Ala may alleviate apoptosis by suppressing Caspase-3 expression. The quantitative results in Figure 5D reveal that the Bcl-2 protein level in the MCT group was significantly lower than that in the CRL group (p \u0026lt; 0.01), whereas the Bcl-2 level in the \u0026beta;-Ala treatment group significantly increased compared to the MCT group (p \u0026lt; 0.0001), suggesting that \u0026beta;-Ala may inhibit apoptosis by upregulating Bcl-2 expression. Finally, Figure 5E displays that the C-Caspase-3 protein level was significantly higher in the MCT group compared to the CRL group (p \u0026lt; 0.0001), while the \u0026beta;-Ala treatment group showed a significant decrease in C-Caspase-3 levels compared to the MCT group (p \u0026lt; 0.0001), further confirming the potential role of \u0026beta;-Ala in inhibiting apoptosis. These findings collectively support the significant role of \u0026beta;-Ala in regulating the expression of apoptosis-related proteins and inhibiting apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. The Effect of \u0026beta;-Ala Supplementation on the Expression Levels of Relevant Proteins in the Right Ventricular Myocardium of MCT-Induced PH Rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditionally, we assessed the expression levels of several relevant proteins. Figure 6 displays the Western blot results and quantitative analysis of ERK, p38 MAPK, and AKT protein expression levels across different groups. Figures 6A, C, and E show the Western blot images, while Figures 6B, D, and F present the corresponding quantitative analysis results. These figures indicate the expression levels of the target proteins relative to GAPDH. The expression levels of ERK, p38 MAPK, and AKT proteins in the MCT group were significantly higher than those in the CRL group. In contrast, the \u0026beta;-Ala treatment group significantly reduced the expression levels of ERK, p38 MAPK, and AKT proteins. These results suggest that \u0026beta;-Ala may improve MCT-induced right ventricular remodeling by inhibiting the expression of ERK, p38 MAPK, and AKT.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study utilized the observation that β-alanine levels in patients with PH are significantly lower than those in healthy individuals. By simulating PH in rats, we found results consistent with those observed in human PH cases, thus confirming our findings through animal experiments. We investigated the effects of β-Ala on right ventricular remodeling in experimental PH rats using the classic MCT-induced PH rat model to explore the pharmacological efficacy of β-Ala. The pathological development of MCT-induced PH is characterized by initial endothelial dysfunction in pulmonary arteries, interstitial edema, vasoconstriction, inflammatory infiltration around blood vessels, and the secretion of cytokines, followed by the gradual emergence of intimal hypertrophy and fibrosis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].This classic small animal model of PH plays a crucial role in studying the pathological mechanisms of human diseases at the animal level. Our research demonstrates that β-Ala can improve cardiac function and hemodynamics in PH rats, inhibit the apoptosis of myocardial tissues, inhibit ventricular remodeling, and delay the progression of right heart dysfunction caused by PH.\u003c/p\u003e\u003cp\u003eβ-Ala is an isomer of alanine that exhibits anti-proliferative effects primarily through the synthesis of carnosine[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Carnosine plays a protective role against myocardial damage and oxidative stress, and it also helps regulate cardiac rhythm and delay aging[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Research indicates that endothelial β-Ala metabolism is essential for maintaining the homeostasis of vascular endothelial cells. β-Ala has a significant role in modulating endothelial function and phenotype. Disruption of β-Ala biosynthesis can lead to increased phosphorylation of Smad2/3 and activation of TGF-β signaling, ultimately resulting in endothelial neoplasia and atherosclerosis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Studies have found that supplementation with β-Ala further improves the therapeutic effects on exercise capacity in rats with congestive heart failure, leading to additional benefits[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In preliminary studies, we observed that β-Ala levels in the blood of PH patients and in the right ventricular tissue of PH rats were significantly lower than those in healthy individuals. Supplementation with β-Ala resulted in the alleviation of right heart failure in PH rats, indicating that β-Ala metabolism plays a crucial role in the process of cardiomyocyte remodeling. In fact, in the context of heart failure due to myocardial infarction, β-Ala supplementation has been shown to reduce the size of myocardial infarction and provide certain protective effects on the damaged heart[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe levels of apoptosis are an important pathological characteristic reflecting ventricular remodeling. Detection of cell apoptosis is not only useful in the diagnosis and risk stratification of patients with ischemic heart disease in clinical settings, but interventions targeting apoptosis have also proven effective in preventing and treating post-infarction remodeling and heart failure[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Induction of increased Bcl-2 expression and inhibition of Bax activity are effective triggers for the death and apoptosis of ischemic cardiac myocytes[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As a classic anti-apoptotic factor [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], high levels of Bcl-2 expression can protect against oxidative stress-induced apoptosis and safeguard cardiomyocyte viability and left ventricular function in the context of ischemia[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].The Bcl-2/Bax ratio serves as a \"rheostat\" regulating cell death, dependent on the balance between Bcl-2 and Bax in the cell. A reduction in apoptosis is often associated with a significant increase in the Bcl-2/Bax ratio[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Factors influencing cardiotoxicity may enhance cardiomyocyte apoptosis by affecting the Bcl-2/Bax apoptotic pathway and altering the Bcl-2/Bax ratio[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]。In this study, the Bcl-2/Bax ratio was significantly decreased in rats with right ventricular remodeling. Following β-Ala supplementation, the Bcl-2/Bax ratio increased significantly. This suggests that a reduction in apoptosis during right ventricular remodeling may help to mitigate the occurrence and progression of right heart remodeling by promoting cardiomyocyte survival.\u003c/p\u003e\u003cp\u003eThe ERK signaling pathway is typically associated with cell proliferation and differentiation. In PH, sustained activation of ERK may lead to aberrant proliferation and remodeling of cardiac myocytes[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our study found that the expression levels of ERK protein were significantly upregulated in the MCT-induced PH model compared to the CRL group, while β-Ala treatment significantly reversed this upregulation, indicating that β-Ala may alleviate the abnormal proliferation and remodeling of cardiac myocytes by inhibiting the ERK signaling pathway. The p38 MAPK signaling pathway is closely related to cellular stress responses and apoptosis. In PAH, activation of p38 MAPK may promote cardiomyocyte apoptosis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our findings demonstrated that p38 MAPK expression levels were significantly increased in the MCT group compared to the CRL group, and β-Ala treatment significantly reduced its expression, suggesting that β-Ala may decrease apoptosis of cardiac myocytes by inhibiting the p38 MAPK signaling pathway. The AKT signaling pathway plays a central role in cell survival and apoptosis inhibition[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In PAH, activation of AKT may be suppressed, leading to increased cardiomyocyte apoptosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our results indicate that compared to the CRL group, the MCT group showed a significant decrease in AKT protein expression, whereas β-Ala treatment significantly increased its expression levels. This suggests that β-Ala may inhibit cardiomyocyte apoptosis by activating the AKT signaling pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough this study revealed the improvement of right ventricular remodeling caused by PH through β-Ala, there are still several limitations. First, we did not thoroughly investigate the specific molecular mechanisms by which β-Ala improves right ventricular remodeling due to PH. While existing research has suggested that β-Ala may exert its effects by modulating the expression of key signaling molecules such as ERK, p38 MAPK, and AKT, our study lacks direct evidence of alterations in these signaling pathways. Furthermore, it is generally believed that β-Ala acts by increasing the concentration of carnosine in cardiomyocytes; however, we have not validated the changes in carnosine levels in this study, which may be an avenue for further exploration in future research. Although our study achieved preliminary results in exploring the beneficial effects of β-Ala on right ventricular remodeling due to PH, further in-depth research is needed to investigate its potential mechanisms of action, particularly in regulating cardiomyocyte apoptosis and vascular remodeling. Future studies could focus on the effects of β-Ala on specific signaling pathways, such as ERK, p38 MAPK, and AKT, and verify its biological effects by measuring carnosine concentration, thereby providing more scientific evidence for the treatment of PH.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study is the first to reveal that β-Ala has a significant ameliorative effect on right ventricular remodeling caused by PH. These findings identify β-Ala as a novel therapeutic target for PH-induced right ventricular dysfunction and highlight its translational potential. Our findings indicate that β-Ala may regulate the expression of key signaling molecules such as ERK, p38 MAPK, and AKT to suppress myocardial remodeling, fibrosis, and apoptosis induced by PAH, thereby exerting its potential therapeutic effects. By modulating critical cell signaling pathways, including ERK, p38 MAPK, and AKT, β-Ala demonstrates positive effects on the processes of cardiomyocyte apoptosis, fibrosis, and remodeling. Our results highlight the important role of β-Ala as a potential therapeutic agent in alleviating PH-related cardiac damage and improving right heart function, laying a scientific foundation for further drug development and clinical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.S. contributed to writing the original draft. B.L., Z.G. and F.Z. contributed to reviewing and editing the manuscript. A.W., K.J. and H.Z. contributed to data collection. Y.Y., J.Z. and W.L. contributed to reviewing the manuscript. J.L. contributed to conceptualization, resources, and supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Lanzhou Science and Technology Program of Gansu Province of China awarded to Hongling Su (LX-62000001-2022-090), the Natural Science Foundation of Gansu Province of China to Bo Li (24JRRA1049) and The Intramural Fund of Gansu Provincial Hospital of China to Hongling Su (22GSSYB-2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors confirm that all the data that support the findings of this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the protocols approved by the Animal Protection and Utilization Committee of Gansu University of Chinese Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eThe First Clinical Medical College of Lanzhou University, Lanzhou 730000, P. R. China.\u003csup\u003e2\u003c/sup\u003eDepartment of Cardiology, Pulmonary Vascular Disease Center (PVDC), Gansu Provincial Hospital, Lanzhou 730000, P. R. China.\u003csup\u003e3\u003c/sup\u003eDepartment of laboratory, Gansu Wuwei Tumor Hospital, Wuwei 733000, P. R. China.\u003csup\u003e4\u003c/sup\u003eHeart, Lung and Vessels Center, Sichuan Provincial People\u0026apos;s Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 610072, P. R. China.\u003csup\u003e5\u003c/sup\u003eDepartment of Intensive Care Unit, Gansu Provincial Maternity and Child Health Hospital, Gansu Provincial General Hospital, Lan Zhou, Gansu Province, 730050, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHassoun PM. Pulmonary Arterial Hypertension. 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Life sciences. 2020;250:117548.https://doi.org/10.1016/j.lfs.2020.117548\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary hypertension, right ventricular remodeling, beta-alanine","lastPublishedDoi":"10.21203/rs.3.rs-7077018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7077018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePulmonary hypertension (PH) is a disease characterized by progressive pulmonary vascular resistance and right heart failure. Beta-alanine (β-Ala), a non-proteinogenic amino acid, exhibits potential cardiovascular benefits; however, its role in PH pathophysiology remains underexplored.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo assess the effects of β-Ala supplementation on right ventricular remodeling and dysfunction in a monocrotaline (MCT)-induced PH rat model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMale Wistar rats were randomly divided into three groups: control, MCT-induced PH, and β-Ala treated PH. Right ventricular function was evaluated using RVSP and RVHI. Protein expression and mRNA levels of cardiac markers and signaling pathways were analyzed by Western blotting and qPCR. Histological analysis was performed to assess right ventricular hypertrophy and fibrosis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eβ-Ala supplementation significantly improved RVSP and RVHI in MCT-induced PH rats. Protein expression analysis showed reduced ERK and p38 MAPK, along with increased activation of AKT in the β-Ala group compared to the MCT group. Additionally, β-Ala reduced the expression of pro-apoptotic markers Bax and Caspase-3 while increasing levels of the anti-apoptotic protein Bcl-2. qPCR analysis revealed decreased expression of ANP, BNP, α-MHC, β-MHC, and TGF-β in the β-Ala group. Histological analysis confirmed that β-Ala treatment alleviated right ventricular hypertrophy and fibrosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eβ-Ala improves right ventricular remodeling and dysfunction in MCT-induced PH rats by modulating signaling pathways and apoptotic markers, indicating its potential as a therapeutic agent for PH-induced right heart dysfunction.\u003c/p\u003e","manuscriptTitle":"β-Alanine supplementation ameliorates right ventricular remodeling caused by MCT-induced pulmonary hypertension","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 12:56:59","doi":"10.21203/rs.3.rs-7077018/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25a94dfa-6b66-4709-9f7b-31bd1a6a1629","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T11:23:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 12:56:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7077018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7077018","identity":"rs-7077018","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}