Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

Widespread changes in cardiac gene expression occur during heart failure, yet the mechanisms responsible for coordinating these changes remain poorly understood. The Mediator complex represents a nodal point for modulating transcription by bridging chromatin-bound transcription factors with RNA polymerase II activity; it is reversibly regulated by its cyclin-dependent kinase 8 (Cdk8) kinase submodule. Here, we identified increased Cdk8 protein expression in human failing heart explants and determined the consequence of this increase in cardiac-specific Cdk8-expressing mice. Transgenic Cdk8 overexpression resulted in progressive dilated cardiomyopathy, heart failure, and premature lethality. Prior to functional decline, left ventricular cardiomyocytes were dramatically elongated, with disorganized transverse tubules and dysfunctional calcium handling. RNA sequencing results showed that myofilament gene isoforms not typically expressed in adult cardiomyocytes were enriched, while oxidative phosphorylation and fatty acid biosynthesis genes were downregulated. Interestingly, candidate upstream transcription factor expression levels and MAPK signaling pathways thought to determine cardiomyocyte size remained relatively unaffected, suggesting that Cdk8 functions within a novel growth regulatory pathway. Our findings show that manipulating cardiac gene expression through increased Cdk8 levels is detrimental to the heart by establishing a transcriptional program that induces pathological remodeling and eccentric hypertrophy culminating in heart failure.

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.

Mitochondrial oxidative stress has been implicated in aging and several cardiovascular diseases, including heart failure and cardiomyopathy, ventricular tachycardia, and atrial fibrillation. The role of mitochondrial oxidative stress in bradyarrhythmia is less clear. Mice with a germline deletion of Ndufs4 subunit respiratory complex I develop severe mitochondrial encephalomyopathy resembling Leigh Syndrome (LS). Several types of cardiac bradyarrhythmia are present in LS mice, including a frequent sinus node dysfunction and episodic atrioventricular (AV) block. Treatment with the mitochondrial antioxidant Mitotempo or mitochondrial protective peptide SS31 significantly ameliorated the bradyarrhythmia and extended the lifespan of LS mice. Using an ex vivo Langendorff perfused heart with live confocal imaging of mitochondrial and total cellular reactive oxygen species (ROS), we showed increased ROS in the LS heart, which was potentiated by ischemia-reperfusion. A simultaneous ECG recording showed a sinus node dysfunction and AV block concurrent with the severity of the oxidative stress. Treatment with Mitotempo abolished ROS and restored the sinus rhythm. Our study reveals robust evidence of the direct mechanistic roles of mitochondrial and total ROS in bradyarrhythmia in the setting of LS mitochondrial cardiomyopathy. Our study also supports the potential clinical application of mitochondrial-targeted antioxidants or SS31 for the treatment of LS patients.