Pub Date : 2025-03-30DOI: 10.1016/j.yjmcc.2025.03.012
Emily A Chapman, Holden T Rogers, Zhan Gao, Hsin-Ju Chan, Francisco J Alvarado, Ying Ge
S-glutathionylation (SSG) is increasingly recognized as a critical signaling mechanism in the heart, yet SSG modifications in cardiac sarcomeric proteins remain understudied. Here we identified SSG of the ventricular isoform of myosin light chain 1 (MLC-1v) in human, swine, and mouse cardiac tissues using top-down mass spectrometry (MS)-based proteomics. Our results enabled the accurate identification, quantification, and site-specific localization of SSG in MLC-1v across different species. Notably, the endogenous SSG of MLC-1v was observed in human and swine cardiac tissues but not in mice. Treating non-reduced cardiac tissue lysates with GSSG elevated MLC-1v SSG levels across all three species.
{"title":"In-depth characterization of S-glutathionylation in ventricular myosin light chain 1 across species by top-down proteomics.","authors":"Emily A Chapman, Holden T Rogers, Zhan Gao, Hsin-Ju Chan, Francisco J Alvarado, Ying Ge","doi":"10.1016/j.yjmcc.2025.03.012","DOIUrl":"https://doi.org/10.1016/j.yjmcc.2025.03.012","url":null,"abstract":"<p><p>S-glutathionylation (SSG) is increasingly recognized as a critical signaling mechanism in the heart, yet SSG modifications in cardiac sarcomeric proteins remain understudied. Here we identified SSG of the ventricular isoform of myosin light chain 1 (MLC-1v) in human, swine, and mouse cardiac tissues using top-down mass spectrometry (MS)-based proteomics. Our results enabled the accurate identification, quantification, and site-specific localization of SSG in MLC-1v across different species. Notably, the endogenous SSG of MLC-1v was observed in human and swine cardiac tissues but not in mice. Treating non-reduced cardiac tissue lysates with GSSG elevated MLC-1v SSG levels across all three species.</p>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":" ","pages":""},"PeriodicalIF":4.9,"publicationDate":"2025-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-28DOI: 10.1016/j.yjmcc.2025.03.011
W. Glen Pyle
{"title":"Heartbreak and trauma: Unraveling sex-specific effects of post-traumatic stress disorder on cardiovascular disease","authors":"W. Glen Pyle","doi":"10.1016/j.yjmcc.2025.03.011","DOIUrl":"10.1016/j.yjmcc.2025.03.011","url":null,"abstract":"","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 153-154"},"PeriodicalIF":4.9,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143738998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-21DOI: 10.1016/j.yjmcc.2025.03.010
Kunzhe Dong , Zhixia Bai , Xiangqin He , Lu Zhang , Guoqing Hu , Yali Yao , Chen-Leng Cai , Jiliang Zhou
Dysfunction in either embryonic or postnatal smooth muscle cells (SMCs) significantly contributes to the progression of various cardiovascular and visceral diseases. Therefore, elucidating the molecular mechanisms governing SMC development and homeostasis is crucial. MYH11 is the most reliable lineage gene for SMCs and has been utilized to develop tamoxifen-inducible Cre driver lines for achieving SMC-specific gene manipulation by crossing with mice carrying the loxP-flanked gene, particularly in adult mice. For studies involving SMCs during embryogenesis, the commonly used constitutive Cre driver is controlled by the Tagln (also known as SM22α) promoter. However, this Cre driver exhibits activity in multiple non-SMC populations, including cardiomyocytes and skeletal muscle precursors, introducing confounding effects. Additionally, most existing SMC-specific Cre drivers are generated using a transgenic approach, raising concerns about random site integration and variable gene copy numbers. To address these limitations, we report a novel Cre mouse model generated by knock-in (KI) of a nuclear-localized Cre recombinase into the Myh11 gene locus using homologous recombination. We confirmed that the Cre activity precisely recapitulates endogenous Myh11 expression by crossing with Rosa26 mTmG or tdTomato reporter mice. Moreover, Myh11-driven Cre can efficiently delete the floxed allele of the transcription factor Tead1 specifically in SMCs. The Tead1 SMC-specific knockout mice did not exhibit an overt phenotype, thereby circumventing the embryonic lethal phenotype mediated by Tagln-driven Cre, as we previously reported. These findings establish this novel Cre driver line as a robust tool for tracing the Myh11-positive SMC lineage and manipulating gene function specifically in SMCs during embryonic development in both male and female mice.
{"title":"Generation of a novel constitutive smooth muscle cell-specific Myh11-driven Cre mouse model","authors":"Kunzhe Dong , Zhixia Bai , Xiangqin He , Lu Zhang , Guoqing Hu , Yali Yao , Chen-Leng Cai , Jiliang Zhou","doi":"10.1016/j.yjmcc.2025.03.010","DOIUrl":"10.1016/j.yjmcc.2025.03.010","url":null,"abstract":"<div><div>Dysfunction in either embryonic or postnatal smooth muscle cells (SMCs) significantly contributes to the progression of various cardiovascular and visceral diseases. Therefore, elucidating the molecular mechanisms governing SMC development and homeostasis is crucial. <em>MYH11</em> is the most reliable lineage gene for SMCs and has been utilized to develop tamoxifen-inducible Cre driver lines for achieving SMC-specific gene manipulation by crossing with mice carrying the lox<em>P</em>-flanked gene, particularly in adult mice. For studies involving SMCs during embryogenesis, the commonly used constitutive Cre driver is controlled by the <em>Tagln</em> (also known as <em>SM22α</em>) promoter. However, this Cre driver exhibits activity in multiple non-SMC populations, including cardiomyocytes and skeletal muscle precursors, introducing confounding effects. Additionally, most existing SMC-specific Cre drivers are generated using a transgenic approach, raising concerns about random site integration and variable gene copy numbers. To address these limitations, we report a novel Cre mouse model generated by knock-in (KI) of a nuclear-localized Cre recombinase into the <em>Myh11</em> gene locus using homologous recombination. We confirmed that the Cre activity precisely recapitulates endogenous <em>Myh11</em> expression by crossing with <em>Rosa26</em> mTmG or tdTomato reporter mice. Moreover, <em>Myh11</em>-driven Cre can efficiently delete the floxed allele of the transcription factor <em>Tead1</em> specifically in SMCs. The <em>Tead1</em> SMC-specific knockout mice did not exhibit an overt phenotype, thereby circumventing the embryonic lethal phenotype mediated by <em>Tagln</em>-driven Cre, as we previously reported. These findings establish this novel Cre driver line as a robust tool for tracing the <em>Myh11</em>-positive SMC lineage and manipulating gene function specifically in SMCs during embryonic development in both male and female mice.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 144-152"},"PeriodicalIF":4.9,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-21DOI: 10.1016/j.yjmcc.2025.03.009
Zujuan Wang , Yujie Ren , Dongmei Zhang , Gang She , Yan Wang , Guangyao Li , Xiaodong Sun , Dong Zheng , Zhongjuan Wang , Xiu-Ling Deng , Ying Zhao , Limei Zhao
Atrial fibrillation (AF) is a prevalent cardiac arrhythmia characterized by atrial fibrosis which involves excessed proliferation and increased activity of fibroblast and myofibroblast, as well as alterations in the extracellular matrix (ECM). The specific mechanism driving fibrosis in atrial fibroblasts and myofibroblsats remains incompletely understood. This study investigates the role of the intermediate-conductance Ca2+-activated K+ channel (KCa3.1) in Angiotensin II (Ang II)-induced atrial fibrosis and elucidates the underlying mechanisms. Primary rat atrial fibroblasts/myofibroblasts were treated with Ang II to evaluate KCa3.1 expression, cells proliferation and ECM production. The involvement of ERK/NF-κB signaling pathway was assessed using specific inhibitors. Ang II treatment increased KCa3.1 expression, stimulated the proliferation of fibroblasts/myofibroblasts, and enhanced ECM production, effects that were attenuated by the Ang II receptor antagonist Losartan and the KCa3.1 inhibitor TRAM-34. Knockdown of KCa3.1 using siRNA significantly reduced Ang II-induced collagen synthesis, confirming its critical role in fibrosis. The ERK/NF-κB pathway was found to mediate Ang II-induced upregulation of KCa3.1, as evidenced by inhibition with specific inhibitors. In vivo, Ang II infusion in rats increased KCa3.1 expression and atrial fibrosis, with atria showing greater susceptibility to fibrosis compared to ventricle. These effects were mitigated by losartan and TRAM-34. In conclusion, our findings demonstrate that Ang II-induced upregulation of KCa3.1 through ERK/NF-κB pathway activation in atrial fibroblasts/myofibroblasts promotes cellular proliferation and collagen deposition, ultimately contributing to atrial fibrosis. KCa3.1 represents a promising therapeutic target for the treatment of atrial fibrosis in AF.
{"title":"Elevated KCa3.1 expression by angiotensin II via the ERK/NF-κB pathway contributes to atrial fibrosis","authors":"Zujuan Wang , Yujie Ren , Dongmei Zhang , Gang She , Yan Wang , Guangyao Li , Xiaodong Sun , Dong Zheng , Zhongjuan Wang , Xiu-Ling Deng , Ying Zhao , Limei Zhao","doi":"10.1016/j.yjmcc.2025.03.009","DOIUrl":"10.1016/j.yjmcc.2025.03.009","url":null,"abstract":"<div><div>Atrial fibrillation (AF) is a prevalent cardiac arrhythmia characterized by atrial fibrosis which involves excessed proliferation and increased activity of fibroblast and myofibroblast, as <strong>well as alterations</strong> in the extracellular matrix (ECM). The specific mechanism driving fibrosis in atrial fibroblasts and myofibroblsats remains incompletely understood. This study investigates the role of the intermediate-conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channel (K<sub>Ca</sub>3.1) in Angiotensin II (Ang II)-induced atrial fibrosis and elucidates the underlying mechanisms. Primary rat atrial fibroblasts/myofibroblasts were treated with Ang II to evaluate K<sub>Ca</sub>3.1 expression, cells proliferation and ECM production. The involvement of ERK/NF-κB signaling pathway was assessed using specific inhibitors. Ang II treatment increased K<sub>Ca</sub>3.1 expression, stimulated the proliferation of fibroblasts/myofibroblasts, and enhanced ECM production, effects that were attenuated by the Ang II receptor antagonist Losartan and the K<sub>Ca</sub>3.1 inhibitor TRAM-34. Knockdown of K<sub>Ca</sub>3.1 using siRNA significantly reduced Ang II-induced collagen synthesis, confirming its critical role in fibrosis. The ERK/NF-κB pathway was found to mediate Ang II-induced upregulation of K<sub>Ca</sub>3.1, as evidenced by inhibition with specific inhibitors. In vivo, Ang II infusion in rats increased K<sub>Ca</sub>3.1 expression and atrial fibrosis, with atria showing greater susceptibility to fibrosis compared to ventricle. These effects were mitigated by losartan and TRAM-34. In conclusion, our findings demonstrate that Ang II-induced upregulation of K<sub>Ca</sub>3.1 through ERK/NF-κB pathway activation in atrial fibroblasts/myofibroblasts promotes cellular proliferation and collagen deposition, ultimately contributing to atrial fibrosis. K<sub>Ca</sub>3.1 represents a promising therapeutic target for the treatment of atrial fibrosis in AF.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 133-143"},"PeriodicalIF":4.9,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-17DOI: 10.1016/j.yjmcc.2025.03.008
Laetitia S. Gaspar , Santoshi Pyakurel , Na Xu , Shane P. D'Souza , Bala S.C. Koritala
A dysregulated circadian system is independently associated with both Obstructive Sleep Apnea (OSA) and cardiovascular disease (CVD). OSA and CVD coexistence is often seen in patients with prolonged untreated OSA. However, the role of circadian dysregulation in their relationship is unclear. Half of the human genes, associated biological pathways, and physiological functions exhibit circadian rhythms, including blood pressure and heart rate regulation. Mechanisms related to circadian dysregulation and heart function are potentially involved in the coexistence of OSA and CVD. In this article, we provide a comprehensive overview of circadian dysregulation in OSA and associated CVD. We also discuss feasible animal models and new avenues for future research to understand their relationship. Oxygen-sensing pathways, inflammation, dysregulation of cardiovascular processes, oxidative stress, metabolic regulation, hormone signaling, and epigenetics are potential clock-regulated mechanisms connecting OSA and CVD.
{"title":"Circadian Biology in Obstructive Sleep Apnea-Associated Cardiovascular Disease","authors":"Laetitia S. Gaspar , Santoshi Pyakurel , Na Xu , Shane P. D'Souza , Bala S.C. Koritala","doi":"10.1016/j.yjmcc.2025.03.008","DOIUrl":"10.1016/j.yjmcc.2025.03.008","url":null,"abstract":"<div><div>A dysregulated circadian system is independently associated with both Obstructive Sleep Apnea (OSA) and cardiovascular disease (CVD). OSA and CVD coexistence is often seen in patients with prolonged untreated OSA. However, the role of circadian dysregulation in their relationship is unclear. Half of the human genes, associated biological pathways, and physiological functions exhibit circadian rhythms, including blood pressure and heart rate regulation. Mechanisms related to circadian dysregulation and heart function are potentially involved in the coexistence of OSA and CVD. In this article, we provide a comprehensive overview of circadian dysregulation in OSA and associated CVD. We also discuss feasible animal models and new avenues for future research to understand their relationship. Oxygen-sensing pathways, inflammation, dysregulation of cardiovascular processes, oxidative stress, metabolic regulation, hormone signaling, and epigenetics are potential clock-regulated mechanisms connecting OSA and CVD.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 116-132"},"PeriodicalIF":4.9,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-12DOI: 10.1016/j.yjmcc.2025.03.007
Timothy D. Roberts , Dana S. Hutchinson , Denise Wootten , Miles J. De Blasio , Rebecca H. Ritchie
The global prevalence of obesity is skyrocketing at an alarming rate, with recent data estimating that one-in-eight people are now living with the disease. Obesity is a chronic metabolic disorder that shares underlying pathophysiology with other metabolically-linked diseases such as type 2 diabetes mellitus, cardiovascular disease and diabetic cardiomyopathy. There is a distinct correlation between type 2 diabetes status and the likelihood of heart failure. Of note, there is an apparent sexual dimorphism, with women disproportionately affected with respect to the degree of severity of the cardiac phenotype of diabetic cardiomyopathy that results from diabetes. The current pharmacotherapies available for the attenuation of hyperglycaemia in type 2 diabetes are not always effective, and have varying degrees of efficacy in the setting of heart failure. Insulin can worsen heart failure prognosis whereas metformin, sodium-glucose cotransporter 2 inhibitors (SGLT2i) and more recently, glucagon-like peptide-1 receptor agonists (GLP-1RAs), have demonstrated cardioprotection with their administration. This review will highlight the advancement of incretin therapies for individuals with diabetes and heart failure and explore newly-reported evidence of the clinical usefulness of GLP-1R agonists in this distinct phenotype of heart failure.
{"title":"Advances in incretin therapies for targeting cardiovascular disease in diabetes","authors":"Timothy D. Roberts , Dana S. Hutchinson , Denise Wootten , Miles J. De Blasio , Rebecca H. Ritchie","doi":"10.1016/j.yjmcc.2025.03.007","DOIUrl":"10.1016/j.yjmcc.2025.03.007","url":null,"abstract":"<div><div>The global prevalence of obesity is skyrocketing at an alarming rate, with recent data estimating that one-in-eight people are now living with the disease. Obesity is a chronic metabolic disorder that shares underlying pathophysiology with other metabolically-linked diseases such as type 2 diabetes mellitus, cardiovascular disease and diabetic cardiomyopathy. There is a distinct correlation between type 2 diabetes status and the likelihood of heart failure. Of note, there is an apparent sexual dimorphism, with women disproportionately affected with respect to the degree of severity of the cardiac phenotype of diabetic cardiomyopathy that results from diabetes. The current pharmacotherapies available for the attenuation of hyperglycaemia in type 2 diabetes are not always effective, and have varying degrees of efficacy in the setting of heart failure. Insulin can worsen heart failure prognosis whereas metformin, sodium-glucose cotransporter 2 inhibitors (SGLT2i) and more recently, glucagon-like peptide-1 receptor agonists (GLP-1RAs), have demonstrated cardioprotection with their administration. This review will highlight the advancement of incretin therapies for individuals with diabetes and heart failure and explore newly-reported evidence of the clinical usefulness of GLP-1R agonists in this distinct phenotype of heart failure.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 102-115"},"PeriodicalIF":4.9,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-10DOI: 10.1016/j.yjmcc.2025.03.003
Xutu Wang , R. John Solaro , Wen-Ji Dong
<div><div>Sarcomere length-dependent activation (LDA) is essential to engaging the Frank-Starling mechanism in the beat-to-beat regulation of cardiac output. Through LDA, the heart increases the Ca<sup>2+</sup> sensitivity of myocardial contraction at a longer sarcomere length, leading to an enhanced maximal force at the same level of Ca<sup>2+</sup>. Despite its importance in both normal and pathological states, the molecular mechanism underlying LDA, especially the origin of the sarcomere length (SL) induced increase in myofilament Ca<sup>2+</sup>sensitivity, remains elusive. The aim of this study is to interrogate the role of changes in the state of myosin heads during diastole as well as effects of strong force-generating cross-bridges (XB) as determinants of SL-induced Ca<sup>2+</sup> sensitivity of troponin in membrane-free (skinned) rat myocardial fibers. Skinned myocardial fibers were reconstituted with troponin complex containing a fluorophore-modified cardiac troponin C, cTnC(13C/51C)<sub>AEDANS-DDPM</sub>, and recombinant cardiac troponin I (cTnI) mutant, ΔSP-cTnI, in which the switch peptide (Sp) of cTnI was replaced by a non-functional peptide link to partially block the force-generating reaction of myosin with actin. We used the reconstituted myocardial fibers as a platform to investigate how Ca<sup>2+</sup> sensitivity of troponin within skinned myocardial fibers responds to sarcomere stretch with variations in the status of myosin-actin XBs. Muscle mechanics and fluorescence measurements clearly showed similar SL-induced increases in troponin Ca<sup>2+</sup> sensitivity in either the presence or the absence of strong XBs, suggesting that the SL-induced Ca<sup>2+</sup> sensitivity change is independent of reactions of force generating XB with the thin filament. The presence of mavacamten, a selective myosin-motor inhibitor known to promote transition of myosin heads from the weakly actin-bound state (ON or disordered relaxed (DRX) state) to the ordered off state (OFF or super-relaxed (SRX) state), blunted the observed SL-induced increases in Ca<sup>2+</sup> sensitivity of troponin regardless of the presence of XBs, suggesting that the presence of the myosin heads in the weakly actin bound state, is essential for Ca<sup>2+</sup>-troponin to sense the sarcomere stretch. Results from skinned myocardial fibers reconstituted with troponin containing engineered TEV digestible mutant cTnI and cTnT suggest that the observed SL effect on Ca<sup>2+</sup> sensitivity may involve potential interactions of weakly bound myosin heads with troponin in the actin/Tm cluster region interacting with cTnT-T1 and residues 182–229 of cTnT-T2. The mechanical stretch effects may then be subsequently transmitted to the N-cTnC via the IT arm of troponin and the N-terminus of cTnI. Our findings strongly indicate that SL-induced potential myosin-troponin interaction in diastole, rather than strong myosin-actin XBs, may be an essential molecular mechanism under
{"title":"Myosin-actin crossbridge independent sarcomere length induced Ca2+ sensitivity changes in skinned myocardial fibers: Role of myosin heads","authors":"Xutu Wang , R. John Solaro , Wen-Ji Dong","doi":"10.1016/j.yjmcc.2025.03.003","DOIUrl":"10.1016/j.yjmcc.2025.03.003","url":null,"abstract":"<div><div>Sarcomere length-dependent activation (LDA) is essential to engaging the Frank-Starling mechanism in the beat-to-beat regulation of cardiac output. Through LDA, the heart increases the Ca<sup>2+</sup> sensitivity of myocardial contraction at a longer sarcomere length, leading to an enhanced maximal force at the same level of Ca<sup>2+</sup>. Despite its importance in both normal and pathological states, the molecular mechanism underlying LDA, especially the origin of the sarcomere length (SL) induced increase in myofilament Ca<sup>2+</sup>sensitivity, remains elusive. The aim of this study is to interrogate the role of changes in the state of myosin heads during diastole as well as effects of strong force-generating cross-bridges (XB) as determinants of SL-induced Ca<sup>2+</sup> sensitivity of troponin in membrane-free (skinned) rat myocardial fibers. Skinned myocardial fibers were reconstituted with troponin complex containing a fluorophore-modified cardiac troponin C, cTnC(13C/51C)<sub>AEDANS-DDPM</sub>, and recombinant cardiac troponin I (cTnI) mutant, ΔSP-cTnI, in which the switch peptide (Sp) of cTnI was replaced by a non-functional peptide link to partially block the force-generating reaction of myosin with actin. We used the reconstituted myocardial fibers as a platform to investigate how Ca<sup>2+</sup> sensitivity of troponin within skinned myocardial fibers responds to sarcomere stretch with variations in the status of myosin-actin XBs. Muscle mechanics and fluorescence measurements clearly showed similar SL-induced increases in troponin Ca<sup>2+</sup> sensitivity in either the presence or the absence of strong XBs, suggesting that the SL-induced Ca<sup>2+</sup> sensitivity change is independent of reactions of force generating XB with the thin filament. The presence of mavacamten, a selective myosin-motor inhibitor known to promote transition of myosin heads from the weakly actin-bound state (ON or disordered relaxed (DRX) state) to the ordered off state (OFF or super-relaxed (SRX) state), blunted the observed SL-induced increases in Ca<sup>2+</sup> sensitivity of troponin regardless of the presence of XBs, suggesting that the presence of the myosin heads in the weakly actin bound state, is essential for Ca<sup>2+</sup>-troponin to sense the sarcomere stretch. Results from skinned myocardial fibers reconstituted with troponin containing engineered TEV digestible mutant cTnI and cTnT suggest that the observed SL effect on Ca<sup>2+</sup> sensitivity may involve potential interactions of weakly bound myosin heads with troponin in the actin/Tm cluster region interacting with cTnT-T1 and residues 182–229 of cTnT-T2. The mechanical stretch effects may then be subsequently transmitted to the N-cTnC via the IT arm of troponin and the N-terminus of cTnI. Our findings strongly indicate that SL-induced potential myosin-troponin interaction in diastole, rather than strong myosin-actin XBs, may be an essential molecular mechanism under","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 90-101"},"PeriodicalIF":4.9,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-09DOI: 10.1016/j.yjmcc.2025.03.002
M.A. Rodríguez , J.B. Torres , E.C. Lascano , A. Mattiazzi , C. Mundiña-Weilenmann , M. Said
Spontaneously hypertensive rats (SHR) are more susceptible to cardiac alternans, a precursor to arrhythmias. Ca2+ alternans is a beat-to-beat oscillation in Ca2+ transient amplitude at constant stimulation frequency. We previously found that the early onset of alternans in SHR hearts is associated with prolonged sarcoplasmic reticulum (SR) Ca2+ release refractoriness, primarily influenced by SR Ca2+ load and RyR2 sensitivity. The Na+/Ca2+ exchanger (NCX) is critical for regulating intracellular Ca2+. In SHR myocytes, elevated intracellular Na+ and Ca2+ levels and prolonged action potential duration along with structural changes in T-tubules, where NCX is primarily located, could alter NCX function. The effect of NCX on Ca2+ alternans is complex: enhanced NCX activity may hasten Ca2+ decay, offering protection, but also reduce SR Ca2+ content, potentially promoting alternans. This study aimed to investigate NCX's role in alternans in SHR hearts using pharmacological and computational approaches. ORM-10962, a selective NCX inhibitor, increased Ca2+ transient amplitude and SR Ca2+ content in SHR myocytes, but had no effect on normotensive myocytes, suggesting preferential forward mode activation in SHR. The inhibitor delayed alternans onset and normalized SR Ca2+ release refractoriness. These findings were confirmed by the computational model. Further experiments showed that blocking of NCX's reverse mode had no impact on Ca2+ alternans in SHR myocytes. The results suggest that NCX hyperactivity in SHR myocytes prevents the necessary increase in SR Ca2+ load to overcome the prolonged refractoriness. The findings highlight NCX inhibition as a potential therapeutic strategy to prevent Ca2+ alternans and reduce arrhythmic risk in hypertensive conditions.
{"title":"Inhibiting NCX delays the early onset of Ca2+ alternans in myocytes from spontaneously hypertensive rats (SHR)","authors":"M.A. Rodríguez , J.B. Torres , E.C. Lascano , A. Mattiazzi , C. Mundiña-Weilenmann , M. Said","doi":"10.1016/j.yjmcc.2025.03.002","DOIUrl":"10.1016/j.yjmcc.2025.03.002","url":null,"abstract":"<div><div>Spontaneously hypertensive rats (SHR) are more susceptible to cardiac alternans, a precursor to arrhythmias. Ca<sup>2+</sup> alternans is a beat-to-beat oscillation in Ca<sup>2+</sup> transient amplitude at constant stimulation frequency. We previously found that the early onset of alternans in SHR hearts is associated with prolonged sarcoplasmic reticulum (SR) Ca<sup>2+</sup> release refractoriness, primarily influenced by SR Ca<sup>2+</sup> load and RyR2 sensitivity. The Na<sup>+</sup>/Ca<sup>2+</sup> exchanger (NCX) is critical for regulating intracellular Ca<sup>2+</sup>. In SHR myocytes, elevated intracellular Na<sup>+</sup> and Ca<sup>2+</sup> levels and prolonged action potential duration along with structural changes in T-tubules, where NCX is primarily located, could alter NCX function. The effect of NCX on Ca<sup>2+</sup> alternans is complex: enhanced NCX activity may hasten Ca<sup>2+</sup> decay, offering protection, but also reduce SR Ca<sup>2+</sup> content, potentially promoting alternans. This study aimed to investigate NCX's role in alternans in SHR hearts using pharmacological and computational approaches. ORM-10962, a selective NCX inhibitor, increased Ca<sup>2+</sup> transient amplitude and SR Ca<sup>2+</sup> content in SHR myocytes, but had no effect on normotensive myocytes, suggesting preferential forward mode activation in SHR. The inhibitor delayed alternans onset and normalized SR Ca<sup>2+</sup> release refractoriness. These findings were confirmed by the computational model. Further experiments showed that blocking of NCX's reverse mode had no impact on Ca<sup>2+</sup> alternans in SHR myocytes. The results suggest that NCX hyperactivity in SHR myocytes prevents the necessary increase in SR Ca<sup>2+</sup> load to overcome the prolonged refractoriness. The findings highlight NCX inhibition as a potential therapeutic strategy to prevent Ca<sup>2+</sup> alternans and reduce arrhythmic risk in hypertensive conditions.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 81-89"},"PeriodicalIF":4.9,"publicationDate":"2025-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597232","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Critical limb ischemia is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes. Diabetes-induced premature senescence of endothelial cells (EC) has been proposed as a mechanism leading to impaired ischemia-driven angiogenesis. We showed that hyperglycemia induced expression of the protein tyrosine phosphatase SHP-1, which reduced angiogenic factor activity in ischemic muscle of diabetic mice. Here, we evaluate the impact of SHP-1 deletion on EC function and senescence.
Methods
Ligation of the femoral artery was performed in nondiabetic (NDM) and 3 months diabetic (DM) mice with EC-specific deletion of SHP-1. Cell migration, proliferation and protein expression were evaluated in EC exposed to normal (NG) or high glucose (HG) concentrations. Gastrocnemius and tibial artery of patients with diabetes were collected and analyzed.
Results
Blood flow reperfusion and limb function were reduced by 43 % and 82 %, respectively in DM mice as compared to NDM mice. EC-specific deletion of SHP-1 in DM mice restored blood flow reperfusion by 60 %, and limb function by 86 %, while capillary density was similar to NDM mice. Moreover, ablation of SHP-1 in EC prevented diabetes-induced expression of the senescence markers p53 and p21 and counteracted Nrf2 downregulation. In EC, elevated expression of beta-galactosidase, p21 and p53, and suppression of Nrf2 and VEGF actions were observed in EC exposed to HG levels and human muscle and artery of patients with diabetes, effects that were reversed by overexpression of dominant negative SHP-1.
Conclusion
SHP-1 in EC is a central effector of diabetes-induced senescence and induces aberrant collateral vessel formation and blood flow reperfusion. Reduced SHP-1 expression counteracts these pathologic features.
{"title":"Endothelial SHP-1 regulates diabetes-induced abnormal collateral vessel formation and endothelial cell senescence","authors":"Alexandre Nadeau , Marike Ouellet , Raphaël Béland , Clément Mercier , Stéphanie Robillard , Farah Lizotte , Marc-Antoine Despatis , C. Florian Bentzinger , Pedro Geraldes","doi":"10.1016/j.yjmcc.2025.03.005","DOIUrl":"10.1016/j.yjmcc.2025.03.005","url":null,"abstract":"<div><h3>Background</h3><div>Critical limb ischemia is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes. Diabetes-induced premature senescence of endothelial cells (EC) has been proposed as a mechanism leading to impaired ischemia-driven angiogenesis. We showed that hyperglycemia induced expression of the protein tyrosine phosphatase SHP-1, which reduced angiogenic factor activity in ischemic muscle of diabetic mice. Here, we evaluate the impact of SHP-1 deletion on EC function and senescence.</div></div><div><h3>Methods</h3><div>Ligation of the femoral artery was performed in nondiabetic (NDM) and 3 months diabetic (DM) mice with EC-specific deletion of SHP-1. Cell migration, proliferation and protein expression were evaluated in EC exposed to normal (NG) or high glucose (HG) concentrations. Gastrocnemius and tibial artery of patients with diabetes were collected and analyzed.</div></div><div><h3>Results</h3><div>Blood flow reperfusion and limb function were reduced by 43 % and 82 %, respectively in DM mice as compared to NDM mice. EC-specific deletion of SHP-1 in DM mice restored blood flow reperfusion by 60 %, and limb function by 86 %, while capillary density was similar to NDM mice. Moreover, ablation of SHP-1 in EC prevented diabetes-induced expression of the senescence markers p53 and p21 and counteracted Nrf2 downregulation. In EC, elevated expression of beta-galactosidase, p21 and p53, and suppression of Nrf2 and VEGF actions were observed in EC exposed to HG levels and human muscle and artery of patients with diabetes, effects that were reversed by overexpression of dominant negative SHP-1.</div></div><div><h3>Conclusion</h3><div>SHP-1 in EC is a central effector of diabetes-induced senescence and induces aberrant collateral vessel formation and blood flow reperfusion. Reduced SHP-1 expression counteracts these pathologic features.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 50-63"},"PeriodicalIF":4.9,"publicationDate":"2025-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605203","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-09DOI: 10.1016/j.yjmcc.2025.03.004
Xutu Wang , Nathan Kallish , R. John Solaro , Wen-Ji Dong
Sarcomere length-dependent activation (LDA) is the key cellular mechanism underlying the Frank-Starling law of the heart, in which sarcomere stretch leads to increased Ca2+ sensitivity of myofilament and force of contraction. Despite its key role in both normal and pathological states, the precise mechanisms underlying LDA remain unclear but are thought to involve multiple interactions among sarcomere proteins, including troponin of the thin filament, myosin, titin and myosin binding protein C (MyBP-C). Our previous study with permeabilized rat cardiac fibers demonstrated that the mechanism underlying the increase in Ca2+ sensitivity of thin filament induced by sarcomere stretch may involve sarcomere length (SL)-induced interactions between troponin and weakly bound, disordered relaxed state (DRX) myosin heads in diastole, rather than strong myosin–actin crossbridge interactions. In this study we investigated the role of the N-domains of MyBP-C in this newly discovered mechanism. To examine the potential role of the N-domain of MyBP-C in SL-induced myosin-troponin interactions, skinned myocardial fibers from a transgenic ΔN-MyBP-C rat with deleted N-terminal C0-C2 domains and a non-transgenic rat were reconstituted with troponin containing wild-type cTnT, cTnC(13C/51C)AEDANS-DDPM and mutant ΔSP-cTnI or wild-type cTnI. Because the switching peptide (SP) of ΔS-cTnI is replaced by a nonfunctional peptide linker, force-generating actin-myosin crossbridge interactions of the reconstituted skinned fibers with mutant ΔSP-cTnI are inhibited regardless of the presence of Ca2+. This approach allowed us to examine the sensitivity of troponin/thin filament to Ca2+ binding in response to sarcomere stretch by monitoring Ca2+-induced changes in fluorescence resonance energy transfer (FRET) between AEDANS and DDPM attached to the N-domain of cTnC in the presence/absence of myosin–actin crossbridge interaction with or without deletion of C0-C2 domains of MyBP-C. Our measurements of SL-induced changes in muscle fiber mechanics and FRET Ca2+ sensitivities provide strong evidence that both the weakly bound myosin heads and the N-terminus of MyBP-C are critical for SL to activate troponin in the diastolic state. A model based on the results is proposed for the mechanism underlying LDA of myofilament.
{"title":"Ca2+ sensitivity changes in skinned myocardial fibers induced by myosin–actin crossbridge-independent sarcomere stretch: Role of N-domain of MyBP-C","authors":"Xutu Wang , Nathan Kallish , R. John Solaro , Wen-Ji Dong","doi":"10.1016/j.yjmcc.2025.03.004","DOIUrl":"10.1016/j.yjmcc.2025.03.004","url":null,"abstract":"<div><div>Sarcomere length-dependent activation (LDA) is the key cellular mechanism underlying the Frank-Starling law of the heart, in which sarcomere stretch leads to increased Ca<sup>2+</sup> sensitivity of myofilament and force of contraction. Despite its key role in both normal and pathological states, the precise mechanisms underlying LDA remain unclear but are thought to involve multiple interactions among sarcomere proteins, including troponin of the thin filament, myosin, titin and myosin binding protein C (MyBP-C). Our previous study with permeabilized rat cardiac fibers demonstrated that the mechanism underlying the increase in Ca<sup>2+</sup> sensitivity of thin filament induced by sarcomere stretch may involve sarcomere length (SL)-induced interactions between troponin and weakly bound, disordered relaxed state (DRX) myosin heads in diastole, rather than strong myosin–actin crossbridge interactions. In this study we investigated the role of the N-domains of MyBP-C in this newly discovered mechanism. To examine the potential role of the N-domain of MyBP-C in SL-induced myosin-troponin interactions, skinned myocardial fibers from a transgenic ΔN-MyBP-C rat with deleted N-terminal C0-C2 domains and a non-transgenic rat were reconstituted with troponin containing wild-type cTnT, cTnC(13C/51C)<sub>AEDANS-DDPM</sub> and mutant ΔSP-cTnI or wild-type cTnI. Because the switching peptide (SP) of ΔS-cTnI is replaced by a nonfunctional peptide linker, force-generating actin-myosin crossbridge interactions of the reconstituted skinned fibers with mutant ΔSP-cTnI are inhibited regardless of the presence of Ca<sup>2+</sup>. This approach allowed us to examine the sensitivity of troponin/thin filament to Ca<sup>2+</sup> binding in response to sarcomere stretch by monitoring Ca<sup>2+</sup>-induced changes in fluorescence resonance energy transfer (FRET) between AEDANS and DDPM attached to the N-domain of cTnC in the presence/absence of myosin–actin crossbridge interaction with or without deletion of C0-C2 domains of MyBP-C. Our measurements of SL-induced changes in muscle fiber mechanics and FRET Ca<sup>2+</sup> sensitivities provide strong evidence that both the weakly bound myosin heads and the N-terminus of MyBP-C are critical for SL to activate troponin in the diastolic state. A model based on the results is proposed for the mechanism underlying LDA of myofilament.</div></div>","PeriodicalId":16402,"journal":{"name":"Journal of molecular and cellular cardiology","volume":"202 ","pages":"Pages 24-34"},"PeriodicalIF":4.9,"publicationDate":"2025-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143592457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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