Abstract 19085: Cross-bridge Activation at Diastolic Calcium Levels in Human Myocardium

Abstract

Tropomyosin is positioned on actin in dynamic equilibrium as a function of free calcium concentration. However, it is estimated that ∼5% of the myosin binding sites on actin are exposed at low calcium levels (e.g. pCa 8) raising the possibility of myosin cross-bridge formation at diastolic calcium levels. We tested this hypothesis in detergent-treated (skinned) strips dissected from epicardial biopsies obtained from male patients during coronary bypass grafting. Patients (n=15) had normal left ventricular (LV) ejection fraction and wall motion. Eight control (C) patients had neither hypertension (HTN) nor diabetes mellitus. Seven patients had HTN and concentric remodeling with increased LV wall thickness/radius ratio. Western blot analysis of skinning supernatant revealed no loss of tropomyosin or troponin-I that would implicate loss of normal thin filament regulation during the skinning process. Myofilament calcium sensitivity at sarcomere length 2.2 μm was similar between C and HTN (pCa 50 6.07±0.07 vs 6.06±0.04). Tension at pCa 8 was similar between C and HTN (4.26±0.52 vs 4.30±0.51 mN.mm -2 ), but was significantly reduced by application the cross-bridge inhibitor 2,3-butanedione monoximine (BDM) (2.15±0.24 vs 3.01±0.40 mN.mm -2 , p<0.05). The higher tension in HTN with BDM reflects a greater inherent stiffness in HTN independent of cross-bridges. We then used sinusoidal length perturbations (0.125-250 Hz) to detect the detachment rate of cycling cross-bridges. In both C and HTN strips, frequency-dependent viscoelastic characteristics of cycling cross-bridges were apparent at pCa 8 and abolished with BDM. At pCa 8 cross-bridge detachment rate was significantly greater in C than HTN (106.1±12.5 vs 74.6±9.3 s -1 , p<0.05). These results indicate that there are cycling cross-bridges in normal and concentrically remodeled human myocardium at diastolic calcium levels that contribute to diastolic tension. Furthermore, the rate of cross-bridge detachment under relaxed conditions is slower in concentrically remodeled myocardium from HTN compared with C patients. The presence and lifetime of cross-bridges formed in addition to cross-bridge independent stiffness must be considered as significant contributors to diastolic dysfunction in HTN.