Connexin 43 (Cx43), a product of the GJA1 gene, is a gap junction protein facilitating intercellular communication between cardiomyocytes. Cx43 protects the heart from ischemic injury by mechanisms that are not well understood. GJA1 mRNA can undergo alternative translation, generating smaller isoforms in the heart, with GJA1-20k being the most abundant. Here, we report that ischemic and ischemia/reperfusion (I/R) injuries upregulate endogenous GJA1-20k protein in the heart, which targets to cardiac mitochondria and associates with the outer mitochondrial membrane. Exploring the functional consequence of increased GJA1-20k, we found that AAV9-mediated gene transfer of GJA1-20k in mouse hearts increases mitochondrial biogenesis while reducing mitochondrial membrane potential, respiration, and ROS production. By doing so, GJA1-20k promotes a protective mitochondrial phenotype, as seen with ischemic preconditioning (IPC), which also increases endogenous GJA1-20k in heart lysates and mitochondrial fractions. As a result, AAV9-GJA1-20k pretreatment reduces myocardial infarct size in mouse hearts subjected to in vivo ischemic injury or ex vivo I/R injury, similar to an IPC-induced cardioprotective effect. In conclusion, GJA1-20k is an endogenous stress response protein that induces mitochondrial biogenesis and metabolic hibernation, preconditioning the heart against I/R insults. Introduction of exogenous GJA1-20k is a putative therapeutic strategy for patients undergoing anticipated ischemic injury.

Background—: The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting of ryanodine receptors (RyRs) at sarcoplasmic reticulum apposing Ca _V 1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through β-adrenergic receptor (β-AR)–mediated phosphorylation of dyadic proteins. Phosphorylated RyRs (P-RyR) have increased calcium sensitivity and open probability, amplifying calcium transient at a cost of receptor instability. Given that bridging integrator 1 (BIN1) organizes t-tubule microfolds and facilitates Ca _V 1.2 delivery, we explored whether β-AR–regulated RyRs are also affected by BIN1. Methods and Results—: Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the β-AR agonist isoproterenol (1 µmol/L) or the blockers CGP+ICI (baseline). Using biochemistry and superresolution fluorescent imaging, we identified that BIN1 clusters P-RyR and Ca _V 1.2. Acute β-AR activation increases coimmunoprecipitation between P-RyR and cardiac spliced BIN1+13+17 (with exons 13 and 17). Isoproterenol redistributes BIN1 to t-tubules, recruiting P-RyRs and improving the calcium transient. In cardiac-specific Bin1 heterozygote mice, isoproterenol fails to concentrate BIN1 to t-tubules, impairing P-RyR recruitment. The resultant accumulation of uncoupled P-RyRs increases the incidence of spontaneous calcium release. In human hearts with end-stage ischemic cardiomyopathy, we find that BIN1 is also 50% reduced, with diminished P-RyR association with BIN1. Conclusions—: On β-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electric stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.

Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1^M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1^M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.

Heart failure with preserved ejection fraction (HFpEF) is a growing epidemic worldwide with a high burden of morbidity and mortality.¹ Its incompletely understood pathophysiology has limited development of diagnostic tools to define and follow the disease progression.² Quiz Ref ID Cardiac bridging integrator 1 (cBIN1) is a membrane-scaffolding protein in cardiomyocytes that organizes the dyad-containing microdomains at the transverse tubules that are responsible for the initiation and regulation of systolic and diastolic calcium transients.^3-7 Quiz Ref ID The level of cBIN1 is reduced in animal models of heart failure, as well as in human biopsy samples from patients with end-stage cardiomyopathy.^3,8-11 Its blood availability makes it an attractive biomarker of cardiomyocyte remodeling.¹² In this study, we demonstrate the performance of the cBIN1 score (CS), a dimensionless index based on the normalized reciprocal of cBIN1 plasma levels, in a population of patients with HFpEF who are stable and ambulatory.

Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood. Microparticles are small vesicles generated from the cell surface membrane and externally released for communication with other cells. We now show that heart ventricular muscle cells, which form the main pumping chambers of the heart, release microparticles in both mouse and human. Ventricular microparticles arise from surface membrane microdomains organized by cardiac bridging integrator 1 (cBIN1), a membrane deformation protein that has been shown to be reduced during human heart failure. Here we identify microparticles containing cBIN1 in blood, which were reduced in mutant mice lacking heart cBIN1 expression. Furthermore, the process leading to microparticle release involves the recruitment of CHMP4B protein to snip the cBIN1 membrane. In humans, cBIN1 is present in blood and within microparticles. Upon osmotic shock, human microparticles burst, allowing for the quantification of cBIN1 in plasma by enzyme-linked immunosorbent assay (ELISA). The measured cBIN1 level was greatly reduced in patients with heart failure. Thus, we introduce the biology for a new blood-based diagnostic tool that can assess cardiac muscle health and identify failing heart in human patients.