Contractile regulation by overexpressed ET_Arequires intact T tubules in adult rat ventricular myocytes

Abstract

Chung KY, Kang M, Walker JW. Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes. Am J Physiol Heart Circ Physiol 294: H2391-H2399, 2008. First published March 7, 2008 doi:10.1152/ajpheart.00011.2008.-Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ETA)/ET type B (ETB) receptor family. ETA, the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ETA in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ETA protein levels. In these T tubule-compromised myocytes, an overexpression of ETA using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ETA downregulation. The rescue of a normal PIE in 3-to 4-day cultured myocytes required both an increased expression of ETA and intact T tubules (preserved with CD). Therefore, the activation of ETA localized in T tubules was associated with a strong PIE, whereas the activation of ET A in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET A is upregulated and T-tubule morphology is altered. cytochalasin D; inotropism; endothelin type A receptor; heart failure THE POTENT vasoconstrictor peptide endothelin (ET)-1 is produced not only by vascular endothelial cells (46) but also by endocardial endothelial cells and cardiac myocytes (14). In the heart, ET-1 produces inotropic, chronotropic, and growthpromoting actions (30,38). Among these physiological effects of ET-1, the regulation of contractility has received considerable attention, with a positive inotropic effect (PIE) reported in rat, mouse, rabbit, ferret, and human but not in guinea pig or dog myocardium (18,22,(43)(44)(45). Biological functions of ET-1 are mediated by two G protein-coupled receptor (GPCR) subtypes, ET type A receptor (ET A ) and ET type B receptor

Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation–contraction coupling and inotropic stimulation

“…These data indicate that T-tubules are the primary sites of ET-1 signal transduction in ventricular myocytes, consistent with the immunolocalisation of ET A receptors in tubule membranes (Fig. 6) [40][41][42]. In contrast, ET A receptors in atrial myocytes are primarily located at the periphery (Fig.…”

Endothelin Receptor Overexpression Alters Diastolic Function in Cultured Rat Ventricular Myocytes

“…1B). In earlier studies, we presented evidence that Ttubule localized ET A was primarily responsible for regulation of myoycte contractility by ET-1 (Chung et al ., 2008). Here we examined the functional consequences of ET-1 stimulation in myocytes overexpressing ET receptors by measuring additional twitch and Ca 2+ handling parameters as described below.…”

“…After 48 hours of adenoviral infection, ET A -CFP was expressed to a level of 12-fold over endogenous ET A , and ET B -YFP was expressed to a level of 10-fold. Physiological responses of myocytes were evaluated within this 40-48 hour window of infection to minimize the impact of structural remodeling that can occur in cultured adult rat myocytes (Chung et al ., 2008).…”

“…Isolated myocytes were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, 1 mM CaCl 2 , 5 mM taurine, 5 mM carnitine, and 5 mM creatine, plated on laminine-coated cover-slip, and infected with adenoviruses as previously described (Chung et al ., 2008).…”

“…Quantitative analyses of Perinuclear regions, sarcolemma, and T tubules were performed with National Institutes of Health ImageJ software. The overexpression levels of ET A and ET B were analyzed as described previously (Chung et al ., 2008). Briefly, the radiance confocal microscope was calibrated by the use of a purified bacterial expressed CFP or YFP construct provided by Dr. Dapankar Battacharaya.…”