BACKGROUND: Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca ²⁺ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca ²⁺ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. METHODS: Calpain-resistant JP2 knock-in mice (JP2 ^CR ) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2 ^CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2 ^CR , JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. RESULTS: JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2 ^CR cardiomyocytes. JP2 ^CR hearts are more resistant to pressure-overload stress, having significantly improved Ca ²⁺ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2 ^CR preserves the integrity of calpain-sensitive E-C coupling–related proteins, including ryanodine receptor 2, Ca _V 1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction–induced increases in calpain activity. Furthermore, JP2 ^CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. CONCLUSIONS: The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

BACKGROUND: Calcium (Ca ²⁺ ) homeostasis in cardiac fibroblasts (CFs) plays a critical role in myocardial repair and remodeling after injury. JPH2 (junctophilin-2; human JPH2 or mouse Jph2) is a structural protein known to regulate intracellular Ca ²⁺ signaling and excitation-contraction coupling in cardiomyocytes. However, the role of JPH2 in CF biology remains unexplored. METHODS: Junctophilin expression was assayed in human and mouse CFs using reverse transcription quantitative polymerase chain reaction, Western blotting, and immunofluorescence. To investigate the functional role of Jph2 in CFs, we assessed Ca ²⁺ handling with live-cell confocal imaging, conducted RNA sequencing analysis, and assayed TGFβ (transforming growth factor β) responses after adenovirus-mediated gene silencing of Jph2 and fibroblast-specific Jph2 knockout. Jph2 interactions in CFs were identified using immunoprecipitation and proximity biotin ligation assays and confirmed by coimmunoprecipitation and rescue experiments. The in vivo role of Jph2 in CFs was investigated in fibroblast-specific Jph2 knockout mice (Col1a2 ^CreERT /Jph2 ^flox/flox , Jph2 ^fKO ) at baseline and after myocardial infarction and evaluated for changes in cardiac function, fibrosis, angiogenesis, CF activation, differentiation, and proliferation. RESULTS: Jph2 was identified as the only junctophilin expressed in CFs and acutely upregulated in response to myocardial infarction. Cellular and RNA sequencing analyses revealed that isolated Jph2-deficient CFs exhibited impaired fibroblast activation, reduced extracellular matrix production, and diminished expression of VEGFA (vascular endothelial growth factor A), VEGFB, and VEGFC after TGFβ treatment. In vivo, Jph2 ^fKO mice displayed exacerbated adverse cardiac remodeling after myocardial infarction, characterized by decreased CF activation/extracellular matrix production, enhanced CF proliferation, worsened systolic dysfunction, increased left ventricular dilation, impaired scar maturation, and decreased angiogenesis. Mechanistically, biochemical assays demonstrated that Jph2 interacts directly with the coiled-coil 2 domain of Stim1 (stromal interaction molecule 1) via its joining region. Jph2 knockdown led to Stim1 protein destabilization, defective store-operated Ca ²⁺ entry, and a reduction in both canonical and noncanonical TGFβ signaling pathways. Stim1 overexpression partially rescued the sensitivity of Jph2-deficient CFs to TGFβ, as evidenced by increased expression of periostin and fibronectin-1, as well as enhanced Smad3 phosphorylation. CONCLUSIONS: Our results demonstrate that Jph2 is required in CFs to orchestrate Ca ²⁺ homeostasis, CF activation, and extracellular matrix production and promotes angiogenesis in the infarcted heart, positioning it as a central regulator of cardiac repair after injury.

Cardiovascular disease (CVD) remains the leading cause of mortality worldwide and is associated with profound pathological remodeling at both the cellular and molecular levels. Beyond transcriptional changes, post-transcriptional regulation—including RNA splicing, modification, nuclear export, and degradation—significantly expands the complexity of the eukaryotic transcriptome. Among these processes, RNA degradation remains comparatively understudied in cardiovascular biology. Traditionally viewed primarily as a quality-control mechanism that removes aberrant transcripts, including those containing premature termination codons, RNA degradation is now increasingly recognized as an active regulatory process that controls RNA quality, transcript abundance, and ultimately gene expression under both physiological and pathological conditions. Multiple mechanisms regulate RNA stability and degradation, including nonsense-mediated mRNA decay, RNA modifications, RNA-binding proteins, and noncoding RNAs, particularly microRNAs. These pathways have been extensively implicated in biological and disease processes including cancer and inflammation, and emerging evidence highlights their importance in cardiovascular homeostasis and disease. In the heart, altered RNA stability and degradation contribute to the regulation of cardiac contractility, ion-channel homeostasis, mitochondrial function, and inflammatory response. Recent advances in reporter-based RNA stability assays and metabolic RNA-labeling approaches have further enabled quantitative measurement of RNA degradation both in vitro and in vivo. In this review, we summarize the molecular mechanisms regulating RNA stability and degradation, discuss their emerging roles in cardiovascular physiology and disease, highlight recent technological advances for studying RNA turnover, and examine therapeutic strategies targeting RNA stability. Together, these advances establish RNA degradation as an important and potentially druggable target in CVD.