To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 x 10(-15)) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10(-5) to 10(-20)). SCN10A encodes Na(V)1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a(-/-) mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.

Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte-nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart.

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Prolonged mechanical unloading (UN) of the heart is associated with detrimental changes to the structure and function of cardiomyocytes. The mechanisms underlying these changes are unknown. In this study, we report the influence of UN on excitationcontraction coupling, Ca 2؉ -induced Ca 2؉ release (CICR) in particular, and transverse (t)-tubule structure. UN was induced in male Lewis rat hearts by heterotopic abdominal heart transplantation. Left ventricular cardiomyocytes were isolated from the transplanted hearts after 4 wk and studied using whole-cell patch clamping, confocal microscopy, and scanning ion conductance microscopy (SICM). Recipient hearts were used as control (C). UN reduced the volume of cardiomyocytes by 56.5% compared with C (UN, n‫;09؍‬ C, n‫;95؍‬ P<0.001). The variance of time-to-peak of the Ca 2؉ transients was significantly increased in unloaded cardiomyocytes (UN 227.4؎24.9 ms 2 , n‫24؍‬ vs.

Aims Ca2+‐induced Ca2+ release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)‐tubule system guarantees the proximity of the triggers for Ca2+ release [L‐type Ca2+ channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca2+ release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t‐tubule structure and improves CICR. Methods and results Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham‐operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF‐UN). HF reduced the t‐tubule density measured by di‐8‐ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t‐tubule system in HF was also reversed in HF‐UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF‐UN. Electron microscopy revealed recovery of normal t‐tubule microarchitecture in HF‐UN. L‐type Ca2+ current density, measured using whole‐cell patch clamping, was reduced in HF but unaffected by unloading. The variance of the time‐to‐peak of the Ca2+ transient, an index of CICR dyssynchrony, was increased in HF and normalized by unloading. The increased Ca2+ spark frequency observed in HF was reduced in HF‐UN. These results could be explained by the recoupling of orphaned RyRs in HF, as indicated by immunofluorescence. Conclusions Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t‐tubule system and improves CICR.

AimsCombined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a β2-adrenoceptor (β2-AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF.Methods and resultsLeft coronary artery ligation or sham operation was performed in male Lewis rats. After 4–6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF + UN + Sal or HF + UN + Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham + Sal, HF + Sal) or clenbuterol (HF + Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Clenbuterol treatment improved in vivo LV function measured with echocardiography (LVEF (%): HF 35.9 ± 2 [16], HF + Clen 52.1 ± 1.4 [16]; P < 0.001; mean ± SEM [n]). In combination with unloading, clenbuterol increased sarcomere shortening (amplitude (µm): HF + UN + Clen 0.1 ± 0.01 [50], HF + UN + Sal 0.07 ± 0.01 [38]; P < 0.001) by normalizing the depressed myofilament sensitivity to Ca2+ (slope of the linear relationship between Ca2+ transient and sarcomere shortening hysteresis loop during relaxation (μm/ratio unit): HF + UN + Clen 2.13 ± 0.2 [52], HF + UN + Sal 1.42 ± 0.13 [38]; P < 0.05).ConclusionClenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation–contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function.

Abstract-The 4.1 proteins are a family of multifunctional adaptor proteins. They promote the mechanical stability of plasma membranes by interaction with the cytoskeletal proteins spectrin and actin and are required for the cell surface expression of a number of transmembrane proteins. Protein 4.1R is expressed in heart and upregulated in deteriorating human heart failure, but its functional role in myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient (knockout) mice (4.1R KO). ECG analysis revealed reduced heart rate with prolonged Q-T interval in 4.1R KO. No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration in isolated ventricular myocytes was prolonged in 4.1R KO. Ca 2ϩ transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca 2ϩ content and Ca 2ϩ sparks frequency were increased. The Na ϩ /Ca 2ϩ exchanger current density was reduced in 4.1R KO. The transient inward current inactivation was faster and the persistent Na ϩ current density was increased in the 4.1R KO group, with possible effects on action potential duration. Although no major morphological changes were noted, 4.1R KO hearts showed reduced expression of NaV1.5␣ and increased expression of protein 4.1G. Our data indicate an unexpected and novel role for the cytoskeletal protein 4.1R in modulating the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease. Key Words: cardiac cytoskeleton Ⅲ ion transporter regulation Ⅲ EC coupling T he cardiac cytoskeleton is important in conferring stability to the myocardium, in sensing the mechanical stretch, and in coordinating the assembly of cellular structures and intercellular signaling. 1 One group of cytoskeletal proteins, the spectrin-and ankyrin-associated system, is involved in the complex interplay between actin, spectrin, and various ion transporters in relation to the regulation of intracellular [Ca 2ϩ ]. 2 Beta II spectrin, muscle LIM-only protein, and ankyrin B and G have all been associated with cardiac function and regulation of the electrophysiological properties of the myocardium. 3 The 4.1 protein family is also part of the spectrin-associated cytoskeleton. It promotes the interaction between spectrin and F-actin and, thus, membrane stability. 2 In mammals, the 4 genes, EPB41, EPB41L1, EPB41L2, and EPB41L3, encode proteins 4.1R, 4.1G, 4.1N, and 4.1B. mRNA transcripts from all 4 genes are found in mouse myocardium. 4 All 4.1 proteins have a FERM membranebinding domain, a spectrin-actin binding (SAB) domain, and a C-terminal domain. 2 A FERM-adjacent domain regulates the activities of both FERM and SAB domains. 5 The FERM and C-terminal domains bind to membrane proteins, 6 whereas the SAB domain binds the spectrin-actin cytoskeleton. 7 Multiple ion channels, pumps, and exc...

The transverse (t)-tubule system plays an essential role in healthy and diseased heart muscle, particularly in Ca(2+)-induced Ca(2+) release (CICR), and its structural disruption is an early event in heart failure. Both mechanical overload and unloading alter t-tubule structure, but the mechanisms mediating the normally tight regulation of the t-tubules in response to load variation are poorly understood. Telethonin (Tcap) is a stretch-sensitive Z-disc protein that binds to proteins in the t-tubule membrane. To assess its role in regulating t-tubule structure and function, we used Tcap knockout (KO) mice and investigated cardiomyocyte t-tubule and cell structure and CICR over time and following mechanical overload. In cardiomyocytes from 3-month-old KO (3mKO), there were isolated t-tubule defects and Ca(2+) transient dysynchrony without whole heart and cellular dysfunction. Ca(2+) spark frequency more than doubled in 3mKO. At 8 months of age (8mKO), cardiomyocytes showed progressive loss of t-tubules and remodelling of the cell surface, with prolonged and dysynchronous Ca(2+) transients. Ca(2+) spark frequency was elevated and the L-type Ca(2+) channel was depressed at 8 months only. After mechanical overload obtained by aortic banding constriction, the Ca(2+) transient was prolonged in both wild type and KO. Mechanical overload increased the Ca(2+) spark frequency in KO alone, where there was also significantly more t-tubule loss, with a greater deterioration in t-tubule regularity. In conjunction, Tcap KO showed severe loss of cell surface ultrastructure. These data suggest that Tcap is a critical, load-sensitive regulator of t-tubule structure and function.