Analysis of Dihydropyridine and Ryanodine Receptor Clusters in Healthy and Failing Human Cardiac Myocytes

Abstract

Excitation contraction coupling in cardiac ventricular muscle is regulated through calcium-induced calcium release (CICR). This process is where the action potential triggers the opening of voltage sensitive calcium channel or dyhyropyridine receptor (DHPR) on the plasma or t-tubular membrane of cardiac myocytes. This allows a small influx of Ca2+ (L-type current) into the cardiac cells which then induces the opening of the sarcoplasmic reticulum ryanodine receptor causing a much larger release of Ca2+ in the form of a calcium spark. Propagation of these sparks throughout the cell allows cytoplasmic build up of Ca2+ that in turn triggers muscle contraction. The close association of DHPR and RYR2 in a structure termed the dyad is critical to successful CICR. Several animal models of heart failure have demonstrated dyssynchronous Ca2+ release is associated with disrupted t-tubular network including a decrease in DHPR/RYR colocalisation. We hypothesised that similar disorganisation in myocyte excitation–contraction-coupling mechanisms in insulinresistant cardiomyopathy. Cardiac specific GLUT4 knock-out (GLUT4-KO)mice and their littermate controls which show a global GLUT4 knock-down (GLUT4-KD) were used for this study. We have previously reported that GLUT4-KO and GLUT4KD exhibit >95% and 85% reduction in GLUT4 expression respectively, relative to wild-type C57Bl6. Cardiomyocytes were isolated enzymatically from hearts of 15 to 35-week-old male mice. The GLUT4-KO mice exhibited significantly greater heart weight (380.5± 16.8 vs. 217± 6.0mg, p< 0.001) and cardiacweight index (14.2± 0.6 vs. 7.8± 0.2mg/g, p< 0.001) compared toGLUT4-KDmice. Myocyte Ca2+ currents and Na+/Ca2+ exchanger currents were measured using whole cell patch clamp recording techniques, and normalized for myocyte size determined by capacitance measurement (pA/pF). Mean capacitance of GLUT4-KO myocytes was significantly greater than GLUT4-KD(437± 28vs. 269± 15pF,p< 0.0001). InGLUT4KO, peak voltage-activated (holding potential −90mV) Ca2+ channel current density was significantly reduced compared with GLUT4-KD (−2.82± 0.3 vs. −5.33± 0.7 pA/pF, p< 0.05). The Ni-sensitive Na+/Ca2+ exchange currents were significantly larger in the GLUT4-KO in both inward (−1.56± 0.29 vs. −0.83± 0.16 pA/pF, p< 0.05) and outward modes (1.82± 0.25 vs. 1.09± 0.1 pA/pF, p< 0.02). Thus, cardiac-specific knockout of the GLUT4 trans-