Cardiomyocyte T-tubules are important for regulating ionic flux. Bridging Integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is down-regulated in failing hearts. Here we find that cardiac T-tubules normally contain dense protective inner membrane folds that are formed by a cardiac spliced isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased which does not change overall cardiomyocyte morphology, but frees diffusion of local extracellular calcium and potassium ions, prolonging action potential duration, and increasing susceptibility to ventricular arrhythmias. We also find that T-tubule inner folds are rescued only by the BIN1 isoform BIN1+13+17, which promotes N-WASP dependent actin polymerization to stabilize T-tubule membrane at cardiac Z-discs. In conclusion, BIN1+13+17 recruits actin to fold T-tubule membrane, creating a fuzzy space that protectively restricts ionic flux. When BIN1+13+17 is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered and arrhythmias can result.

Background A variety of studies carried out using either human subjects or laboratory animals suggest that vitamin D and its analogues possess important beneficial activity in the cardiovascular system. Using Cre-Lox technology we have selectively deleted the vitamin D receptor (VDR) gene in the cardiac myocyte in an effort to better understand the role of vitamin D in regulating myocyte structure and function. Methods and Results Targeted deletion of exon 4 coding sequence in the VDR gene resulted in an increase in myocyte size and left ventricular weight/body weight versus controls both at baseline and following a 7-day infusion of isoproterenol. There was no increase in interstitial fibrosis. These knockout mice demonstrated a reduction in end diastolic and end systolic volume by echocardiography, activation of the fetal gene program (i.e. increased atrial natriuretic peptide and alpha skeletal actin gene expression) and increased expression of MCIP 1, a direct downstream target of calcineurin/NFAT signaling. Treatment of neonatal cardiomyocytes with 1,25- dihydroxyvitamin D partially reduced isoproterenol-induced MCIP 1 mRNA and protein levels and MCIP 1 gene promoter activity. Conclusions Collectively, these studies demonstrate that the vitamin D-VDR signaling system possesses direct, anti-hypertrophic activity in the heart. This appears to involve, at least in part, suppression of the pro-hypertrophic calcineurin/NFAT/MCIP 1 pathway. These studies identify a potential mechanism to account for the reported beneficial effects of vitamin D in the cardiovascular system.

Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organelles that consist of sarcolemma penetrating into the myocyte interior, forming a highly branched and interconnected network. Mature t-tubule networks are found in mammalian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomyocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the current knowledge in gross morphology and structure, membrane and protein composition, and function of the cardiac t-tubule network. We also review in detail current knowledge on the formation of functional membrane subdomains within t-tubules, with a particular focus on the cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts. Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and therapeutic opportunities to better treat patients with failing hearts.

Rationale The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown. Objective We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking. Methods and Results High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 μm/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell–cell borders. In Langendorff-perfused mouse heart preparations, Cx43/β-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs. Conclusions Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.

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Rationale Delivery of connexin 43 (Cx43) 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 20kDa internally translated small C-terminus isoform (GJA1-20k) 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 AAV9-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 (F-actin) without affecting actin protein expression, and that GJA1-20k complexes with both actin and tubulin. We also found that F-actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A (LatA) disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from LatA disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo AAV9-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.

Background The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting ryanodine receptors (RyR) at sarcoplasmic reticulum apposing CaV1.2 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 BIN1 (Bridging Integrator 1) organizes t-tubule microfolds and facilitates CaV1.2 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 min with β-AR agonist isoproterenol (1µmol/L) or blockers CGP+ICI (baseline). Using biochemistry and super-resolution fluorescent imaging, we identified that BIN1 clusters P-RyR and CaV1.2. 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 heterozygotes (Bin1 HT) 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 Upon β-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electrical stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14-3-3 proteins can regulate protein trafficking, and a 14-3-3 mode-1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C-terminus. We hypothesized that Cx43Ser373 phosphorylation is important to pathologic gap junction remodeling. Immunofluorescence in human heart reveals enrichment of 14-3-3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14-3-3τ in cell lines increases gap junction plaque size at cell-cell borders. Cx43S373A mutation prevents Cx43/14-3-3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff-perfused mouse hearts we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 minutes of no-flow ischemia. Phosphorylation of Cx43 at Ser368 by PKC and Ser255 by MAPK has previously been implicated in Cx43 internalization. The Cx43S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gate-keeper of a post-translational modification cascade necessary for Cx43 internalization. Cx43Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.