Wassim A. Basheer

Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)–Organized Dyads

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 CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through &bgr;-adrenergic receptor (&bgr;-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 CaV1.2 delivery, we explored whether &bgr;-AR–regulated RyRs are also affected by BIN1. Methods and Results— Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the &bgr;-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 CaV1.2. Acute &bgr;-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 &bgr;-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.

Isoproterenol Promotes Rapid Ryanodine Receptor Movement to BIN1 Organized Dyads

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 CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through &bgr;-adrenergic receptor (&bgr;-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 CaV1.2 delivery, we explored whether &bgr;-AR–regulated RyRs are also affected by BIN1. Methods and Results— Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the &bgr;-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 CaV1.2. Acute &bgr;-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 &bgr;-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.

The “tail” of Connexin43: An unexpected journey from alternative translation to trafficking☆

With each heartbeat, Connexin43 (Cx43) cell-cell communication gap junctions are needed to rapidly spread and coordinate excitation signals for an effective heart contraction. The correct formation and delivery of channels to their respective membrane subdomain is referred to as protein trafficking. Altered Cx43 trafficking is a dangerous complication of diseased myocardium which contributes to the arrhythmias of sudden cardiac death. Cx43 has also been found to regulate many other cellular processes that cannot be explained by cell-cell communication. We recently identified the existence of up to six endogenous internally translated Cx43 N-terminal truncated isoforms from the same full-length mRNA molecule. This is the first evidence that alternative translation is possible for human ion channels and in human heart. Interestingly, we found that these internally translated isoforms, more specifically the 20 kDa isoform (GJA1-20k), is important for delivery of Cx43 to its respective membrane subdomain. This review covers recent advances in Cx43 trafficking and potential importance of alternatively translated Cx43 truncated isoforms. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated DiscsNovelty and Significance

Rationale: Delivery of Cx43 (connexin 43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking and that Cx43 has a 20 kDa internally translated small C terminus isoform, GJA1-20k (Gap Junction Protein Alpha 1- 20 kDa), which is required for full-length Cx43 trafficking, but by an unknown mechanism. Objective: We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs. Methods and Results: Using an in vivo Adeno-associated virus serotype 9–mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin without affecting actin protein expression and that GJA1-20k complexes with both actin and tubulin. We also found that filamentous actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A disrupts the targeting of microtubules to cell–cell junctions. GJA1-20k protects actin filament from latrunculin A disruption, preserving microtubule trajectory to the cell–cell border. For therapeutic implications, we found that prior in vivo Adeno-associated virus serotype 9–mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury. Conclusions: The internally translated GJA1-20k isoform stabilizes actin filaments, which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell–cell coupling in instances of anticipated myocardial ischemia.

Connexin 43 and CaV1.2 Ion Channel Trafficking in Healthy and Diseased Myocardium.

A remarkable aspect of cardiac ion channel biology is that individual ion channels have half-lives on the order of hours. For example, Connexin 43 (Cx43) gap junction proteins have a half-life of 1 to 3 hours,1,2 whereas potassium channels, calcium channels, and the sodium–calcium exchanger have half-lives that are reported in the 2 to 8 hours range.3–6 The short life span of ion channels suggests that there needs to be efficiency in their life cycle and movements which follow the order of: formation, delivery to the correct subdomain on plasma membrane, behavior once in membrane, and internalization back into the cell. To maintain this efficiency in ion trafficking, thousands of individual proteins contribute to a functional equilibrium. Mutations in a single protein can disrupt this equilibrium and over time manifest as arrhythmogenic substrate or cardiomyopathy. Cardiomyocytes use common intracellular organelles and machinery to produce and shuttle ion channel proteins to their specific organelles and functional subdomains at the cell membrane. After gene transcription in the nuclei, proteins are translated and subjected to post-translational modification in the endoplasmic reticulum (ER) and then further modified in the Golgi apparatus. For ion channels, sorting and delivery to their subcellular destination begins in the Golgi apparatus. The Golgi complex is usually found adjacent to the lateral side of each nucleus in mammalian ventricular cardiomyocytes. Co-localized with each Golgi is the centrosome at which microtubules are nucleated and extend throughout the cell.7 Sorting of proteins mainly takes place at the trans-Golgi network (TGN).8 Cargo proteins are sorted into post-Golgi carriers, which are docked onto molecular motors and delivered to the cell periphery along microtubules.9 These extending microtubules form an intricate and dynamic outgoing network capable of shuttling ion channel–containing vesicles to their destinations. In the context …

Cx43 Isoform GJA1-20k Promotes Microtubule Dependent Mitochondrial Transport

Connexin 43 (Cx43, encoded by GJA1) is a cell-cell communication gap junction protein expressed in all organ systems. It was recently found that GJA1 mRNA undergoes alternative translation to generate N-terminal truncated isoforms, of which GJA1-20k is the most abundant. Here we report a surprising finding that, unlike full length GJA1-43k, GJA1-20k has a strong tropism for mitochondria. Exploring function, we found that GJA1-20k appears to be an organelle chaperone and that overexpression of GJA1-20k is sufficient to rescue mitochondrial localization to the cell periphery upon exposure to hydrogen peroxide, which effectively limits the network fragmentation that occurs with oxidative stress. By high-resolution fluorescent imaging and electron microscopy, we determined that GJA1-20k is enriched at the interface between mitochondria and microtubules, appearing to load organelles for transport. Mutagenesis experiments revealed that although the microtubule-binding domain (MTBD) in GJA1-20k is not necessary for protein localization to mitochondria, the MTBD is essential for GJA1-20k to facilitate mitochondrial transport and maintain mitochondrial localization at the periphery. These results reveal an unexpected role for the alternatively translated isoform of the Cx43 gap junction protein, GJA1-20k, which is to facilitate microtubule-based mitochondrial transport and to maintain mitochondrial network integrity during cellular stress.

Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury

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.