Regulation of Sodium/Calcium Homeostasis by BacNav Gene Therapy Rescues Cardiac Dysfunction in Chronic Heart Failure
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE306122 | GEO | 2026/03/11
REPOSITORIES: GEO
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Project description:BACKGROUND: The global prevalence of heart failure is increasing significantly, and the prognosis for patients with heart failure remains poor, highlighting the urgent need for novel therapeutic strategies. Protein kinase C alpha (PKCα) plays a central role in heart failure progression. Phosphorylation of PRKCA at T497 triggers a series of phosphorylation cascades, leading to its activation. Ablation of T497 phosphorylation leads to reduced protein stability and activity. However, there is no therapeutic strategy for heart failure targeting PKCα T497. METHODS: We employed CRISPR-Cas9 adenine base editing to ablate the phosphorylation site T497 of PKCα. We generated mice harboring a PKCα phosphoresistant T497A mutation in the germline and administered adeno-associated virus 9 (AAV9) harboring base editor of PrkcaT497A to postnatal wild-type (WT) mice to explore its clinical feasibility. To model for heart failure, mice were subjected to transverse aortic constriction (TAC). Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson’s trichrome staining, and RNA sequencing, respectively. The editing efficiency of PrkcaT497A was assessed using Sanger sequencing and deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2–loaded human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) carrying the PRKCAT497A mutation under chronic angiotensin II (AngII) stimulation. RESULTS: The T497A mutation in PKCα prevented subsequent phosphorylation and led to PKCα degradation. Four weeks after TAC surgery, WT mice showed dramatically impaired cardiac function, cardiac remodeling, and increased lung weight, whereas PKCα phosphoresistant mice were protected from these effects. PKCα phosphoresistant mice also showed protection against heart failure-related aberrant changes in cardiac gene expression, cardiac hypertrophy, and fibrosis. Likewise, mice administrated with AAV9-base editors exhibited similar cardioprotective effects against TAC-induced heart failure. In vitro, PKCα-edited iPSC-CMs were protected from AngII-induced impairments in contractility and Ca2+ transients observed in WT iPSC-CMs. CONCLUSIONS: The editing of PRKCAT497A through adenine base editing may potentially provide a promising therapeutic approach for human cardiac diseases.
2026-03-04 | GSE294246 | GEO
Project description:BACKGROUND: The global prevalence of heart failure is increasing significantly, and the prognosis for patients with heart failure remains poor, highlighting the urgent need for novel therapeutic strategies. Protein kinase C alpha (PKCα) plays a central role in heart failure progression. Phosphorylation of PRKCA at T497 triggers a series of phosphorylation cascades, leading to its activation. Ablation of T497 phosphorylation leads to reduced protein stability and activity. However, there is no therapeutic strategy for heart failure targeting PKCα T497. METHODS: We employed CRISPR-Cas9 adenine base editing to ablate the phosphorylation site T497 of PKCα. We generated mice harboring a PKCα phosphoresistant T497A mutation in the germline and administered adeno-associated virus 9 (AAV9) harboring base editor of PrkcaT497A to postnatal wild-type (WT) mice to explore its clinical feasibility. To model for heart failure, mice were subjected to transverse aortic constriction (TAC). Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson’s trichrome staining, and RNA sequencing, respectively. The editing efficiency of PrkcaT497A was assessed using Sanger sequencing and deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2–loaded human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) carrying the PRKCAT497A mutation under chronic angiotensin II (AngII) stimulation. RESULTS: The T497A mutation in PKCα prevented subsequent phosphorylation and led to PKCα degradation. Four weeks after TAC surgery, WT mice showed dramatically impaired cardiac function, cardiac remodeling, and increased lung weight, whereas PKCα phosphoresistant mice were protected from these effects. PKCα phosphoresistant mice also showed protection against heart failure-related aberrant changes in cardiac gene expression, cardiac hypertrophy, and fibrosis. Likewise, mice administrated with AAV9-base editors exhibited similar cardioprotective effects against TAC-induced heart failure. In vitro, PKCα-edited iPSC-CMs were protected from AngII-induced impairments in contractility and Ca2+ transients observed in WT iPSC-CMs. CONCLUSIONS: The editing of PRKCAT497A through adenine base editing may potentially provide a promising therapeutic approach for human cardiac diseases.
2026-03-04 | GSE308849 | GEO
Project description:<p>Background: Heart failure with reduced ejection fraction (HFrEF) is characterized by impaired contractility and high mortality. Dysregulation of intracellular ion cycling underlies the decline in cardiac contractility. Modulation of cardiac Na+/H+ and Ca2+ handling is considered a valuable approach for restoring cardiac function; thus, an in-depth understanding of the molecular regulation is timely for the discovery of new strategies to treat HFrEF.</p><p>Methods: Cardiac tissues from HFrEF patients and mice subjected to transverse aortic constriction (TAC) were analyzed for sterol regulatory element-binding protein 1 (SREBP1) transactivation of Sodium-hydrogen exchanger 3 (NHE3).Cardiomyocyte-specific SREBP1 transgenic (Srebp1a-Tg) and knockdown (Cre-Srebp1f/f) mice were generated. AAV9 vectors carrying Slc9a3 (encoding NHE3), Srebf1 or shRNA against Slc9a3, driven by the cardiomyocyte-specific cTnT promoter, were used to validate the role of the SREBP1-NHE3 in HFrEF. </p><p>Results: SREBP1 was activated in human hearts with HFrEF (dilated cardiomyopathy without diabetes or hyperlipidemia) and in mouse hearts with TAC-induced HFrEF. Srebp1a-Tg mice exhibited impaired cardiac contractilitywith dysregulated calcium handling in cardiomyocytes without apparent lipid accumulation. Transcriptomics analysis, ChIP-seq, ChIP assay, and promoter activity assessment showed that Slc9a3 (encoding NHE3) is a transcriptional target of SREBP1. Consistently, NHE3 was upregulated in Srebp1a-Tg mouse hearts, hearts under TAC surgery, and human failing hearts. Cardiomyocyte-specific knockdown of Srebp1 or Slc9a3 restored calcium handling and improved cardiac function in TAC hearts. In Srebp1a-Tg mice, NHE3 knockdown alleviated Na+ and Ca2+ overload and rescued cardiac systolic dysfunction. Conversely, NHE3 overexpression caused contractile impairment in both Cre-Srebp1f/f mice and controls, which offset the protective effect due to SREBP1 loss in the context of Na+ and Ca2+ overload. These resultsdemonstrate that SREBP1 upregulation of NHE3 caused cardiac dysfunction. Notably, empagliflozin downregulated NHE3 via AMP-activated protein kinase activation and inhibition of SREBP1, which ameliorated calcium overload and restored cardiac systolic function.</p><p>Conclusions: SREBP1 transactivates cardiac NHE3 expression during the progression of HFrEF, leading to dysregulatedcalcium handling and impaired contractility, suggesting a non-canonical role of SREBP1 in heart failure. Empagliflozin mitigates these detrimental effects by inhibiting the cardiac SREBP1-NHE3 axis, thus revealing a new pharmacological mechanism by which SGLT2i alleviates HF.</p>
2026-02-20 | MTBLS13696 | MetaboLights
Project description:<p>Background: Heart failure with reduced ejection fraction (HFrEF) is characterized by impaired contractility and high mortality. Dysregulation of intracellular ion cycling underlies the decline in cardiac contractility. Modulation of cardiac Na+/H+ and Ca2+ handling is considered a valuable approach for restoring cardiac function; thus, an in-depth understanding of the molecular regulation is timely for the discovery of new strategies to treat HFrEF.</p><p>Methods: Cardiac tissues from HFrEF patients and mice subjected to transverse aortic constriction (TAC) were analyzed for sterol regulatory element-binding protein 1 (SREBP1) transactivation of Sodium-hydrogen exchanger 3 (NHE3).Cardiomyocyte-specific SREBP1 transgenic (Srebp1a-Tg) and knockdown (Cre-Srebp1f/f) mice were generated. AAV9 vectors carrying Slc9a3 (encoding NHE3), Srebf1 or shRNA against Slc9a3, driven by the cardiomyocyte-specific cTnT promoter, were used to validate the role of the SREBP1-NHE3 in HFrEF. </p><p>Results: SREBP1 was activated in human hearts with HFrEF (dilated cardiomyopathy without diabetes or hyperlipidemia) and in mouse hearts with TAC-induced HFrEF. Srebp1a-Tg mice exhibited impaired cardiac contractilitywith dysregulated calcium handling in cardiomyocytes without apparent lipid accumulation. Transcriptomics analysis, ChIP-seq, ChIP assay, and promoter activity assessment showed that Slc9a3 (encoding NHE3) is a transcriptional target of SREBP1. Consistently, NHE3 was upregulated in Srebp1a-Tg mouse hearts, hearts under TAC surgery, and human failing hearts. Cardiomyocyte-specific knockdown of Srebp1 or Slc9a3 restored calcium handling and improved cardiac function in TAC hearts. In Srebp1a-Tg mice, NHE3 knockdown alleviated Na+ and Ca2+ overload and rescued cardiac systolic dysfunction. Conversely, NHE3 overexpression caused contractile impairment in both Cre-Srebp1f/f mice and controls, which offset the protective effect due to SREBP1 loss in the context of Na+ and Ca2+ overload. These resultsdemonstrate that SREBP1 upregulation of NHE3 caused cardiac dysfunction. Notably, empagliflozin downregulated NHE3 via AMP-activated protein kinase activation and inhibition of SREBP1, which ameliorated calcium overload and restored cardiac systolic function.</p><p>Conclusions: SREBP1 transactivates cardiac NHE3 expression during the progression of HFrEF, leading to dysregulatedcalcium handling and impaired contractility, suggesting a non-canonical role of SREBP1 in heart failure. Empagliflozin mitigates these detrimental effects by inhibiting the cardiac SREBP1-NHE3 axis, thus revealing a new pharmacological mechanism by which SGLT2i alleviates HF.</p>
2026-02-20 | MTBLS13690 | MetaboLights
Project description:<p>Background: Heart failure with reduced ejection fraction (HFrEF) is characterized by impaired contractility and high mortality. Dysregulation of intracellular ion cycling underlies the decline in cardiac contractility. Modulation of cardiac Na+/H+ and Ca2+ handling is considered a valuable approach for restoring cardiac function; thus, an in-depth understanding of the molecular regulation is timely for the discovery of new strategies to treat HFrEF.</p><p>Methods: Cardiac tissues from HFrEF patients and mice subjected to transverse aortic constriction (TAC) were analyzed for sterol regulatory element-binding protein 1 (SREBP1) transactivation of Sodium-hydrogen exchanger 3 (NHE3).Cardiomyocyte-specific SREBP1 transgenic (Srebp1a-Tg) and knockdown (Cre-Srebp1f/f) mice were generated. AAV9 vectors carrying Slc9a3 (encoding NHE3), Srebf1 or shRNA against Slc9a3, driven by the cardiomyocyte-specific cTnT promoter, were used to validate the role of the SREBP1-NHE3 in HFrEF. </p><p>Results: SREBP1 was activated in human hearts with HFrEF (dilated cardiomyopathy without diabetes or hyperlipidemia) and in mouse hearts with TAC-induced HFrEF. Srebp1a-Tg mice exhibited impaired cardiac contractilitywith dysregulated calcium handling in cardiomyocytes without apparent lipid accumulation. Transcriptomics analysis, ChIP-seq, ChIP assay, and promoter activity assessment showed that Slc9a3 (encoding NHE3) is a transcriptional target of SREBP1. Consistently, NHE3 was upregulated in Srebp1a-Tg mouse hearts, hearts under TAC surgery, and human failing hearts. Cardiomyocyte-specific knockdown of Srebp1 or Slc9a3 restored calcium handling and improved cardiac function in TAC hearts. In Srebp1a-Tg mice, NHE3 knockdown alleviated Na+ and Ca2+ overload and rescued cardiac systolic dysfunction. Conversely, NHE3 overexpression caused contractile impairment in both Cre-Srebp1f/f mice and controls, which offset the protective effect due to SREBP1 loss in the context of Na+ and Ca2+ overload. These resultsdemonstrate that SREBP1 upregulation of NHE3 caused cardiac dysfunction. Notably, empagliflozin downregulated NHE3 via AMP-activated protein kinase activation and inhibition of SREBP1, which ameliorated calcium overload and restored cardiac systolic function.</p><p>Conclusions: SREBP1 transactivates cardiac NHE3 expression during the progression of HFrEF, leading to dysregulatedcalcium handling and impaired contractility, suggesting a non-canonical role of SREBP1 in heart failure. Empagliflozin mitigates these detrimental effects by inhibiting the cardiac SREBP1-NHE3 axis, thus revealing a new pharmacological mechanism by which SGLT2i alleviates HF.</p>
2026-02-20 | MTBLS13701 | MetaboLights
Project description:In this study, we used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after myocardial infarction (MI) preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP in the murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, we found that AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated cell cycle gene expression, enhanced TGFβ-signaling, and activated of components of the inflammatory response.Cardiac specific YAP activation after MI mitigated myocardial injury after MI, improved cardiac function and mouse survival. These findings suggest that therapeutic activation of hYAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI. Three groups were involved in this study: sham group, AAV9:Luci+MI group and AAV9-YAP+MI group. Each group contained three biological replicates. The sham group had neither myocardial infarction nor AAV injection. The AAV9:Luci +MI(L for brief) group had myocardial infarction and injected with AAV9:Luic. The AAV9:hYAP+MI(YAP for brief) group had myocardial infarction and injected with AAV9:hYAP. 5 days after MI and AAV injection, the heart apexes were collected and the total RNA were isolated for microarray analysis.
2014-05-26 | E-GEOD-54612 | biostudies-arrayexpress
Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.
2021-04-12 | PXD020175 | Pride
Project description:Calpains are calcium (Ca2+) activated neutral proteases that are involved in several essential signaling pathways. One Calpain-specific proteolytic target is Junctophilin-2 (JP2), an important structural protein of Ca2+ release units required for the excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in mouse models of heart failure has been reported previously, the precise molecular identity of the Calpain cleavage sites and the (patho-) physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565, but subsequently also at three additional secondary Calpain cleavage sites. We identified the Calpain-specific primary cleavage products not only in mouse but also in human iPSC-derived cardiomyocytes (hiPSC-CMs). Knockout of RyR2 in hiPSC-CMs destabilized JP2 resulting in an increase of the Calpain-specifc cleavage fragments. The primary N-terminal cleavage product (NT1) accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+ dependent manner, closely associated with DNA-rich chromosomal regions, where NT1 is proposed to function as a cardioprotective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.
2022-08-12 | PXD031424 | Pride
Project description:Calpains are calcium (Ca2+) activated neutral proteases that are involved in several essential signaling pathways. One Calpain-specific proteolytic target is Junctophilin-2 (JP2), an important structural protein of Ca2+ release units required for the excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in mouse models of heart failure has been reported previously, the precise molecular identity of the Calpain cleavage sites and the (patho-) physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565 but subsequently also at three additional secondary Calpain cleavage sites. We identified the Calpain-specific primary cleavage products not only in mouse but also in human iPSC-derived cardiomyocytes (hiPSC-CMs). Knockout of RyR2 in hiPSC-CMs destabilized JP2 resulting in an increase of the Calpain-specifc cleavage fragments. The primary N-terminal cleavage product (NT1) accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+ dependent manner, closely associated with DNA-rich chromosomal regions, where NT1 is proposed to function as a cardioprotective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.
2022-08-12 | PXD031321 | Pride
Project description:This a model from the article:
Simulation study of cellular electric properties in heart failure
Priebe L, Beuckelmann DJ. Circ Res.
1998; 82(11):1206-23 9633920
,
Abstract:
Patients with severe heart failure are at high risk of sudden cardiac death. In the majority of these patients, sudden cardiac death is thought to be due to ventricular tachyarrhythmias. Alterations of the electric properties of single myocytes in heart failure may favor the occurrence of ventricular arrhythmias in these patients by inducing early or delayed afterdepolarizations. Mathematical models of the cellular action potential and its underlying ionic currents could help to elucidate possible arrhythmogenic mechanisms on a cellular level. In the present study, selected ionic currents based on human data are incorporated into a model of the ventricular action potential for the purpose of studying the cellular electrophysiological consequences of heart failure. Ionic currents that are not yet sufficiently characterized in human ventricular myocytes are adopted from the action potential model developed by Luo and Rudy (LR model). The main results obtained from this model are as follows: The action potential in ventricular myocytes from failing hearts is longer than in nonfailing control hearts. The major underlying mechanisms for this prolongation are the enhanced activity of the Na+-Ca2+ exchanger, the slowed diastolic decay of the [Ca2+]i transient, and the reduction of the inwardly rectifying K+ current and the Na+-K+ pump current in myocytes of failing hearts. Furthermore, the fast and slow components of the delayed rectifier K+ current (I(Kr) and I(Ks), respectively) are of utmost importance in determining repolarization of the human ventricular action potential. In contrast, the influence of the transient outward K+ current on APD is only small in both cell groups. Inhibition of I(Kr) promotes the development of early afterdepolarizations in failing, but not nonfailing, myocytes. Furthermore, spontaneous Ca2+ release from the sarcoplasmic reticulum triggers a premature action potential only in failing myocytes. This model of the ventricular action potential and its alterations in heart failure is intended to serve as a tool for investigating the effects of therapeutic interventions on the electric excitability of the human ventricular myocardium.
This model was taken from the CellML repository
and automatically converted to SBML.
The original model was:
Priebe, Beuckelmann. (1998) - version02
The original CellML model was created by:
Lloyd, Catherine, May
c.lloyd@auckland.ac.nz
The University of Auckland
Auckland Bioengineering Institute
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2005-01-01 | MODEL8683876463 | BioModels