Abstract
Overexpression of the sarcoplasmic reticulum Ca ATPase (SERCA2a) produces positive inotropism and it has been proposed as a promising strategy to counteract defective excitation–contraction coupling in the failing heart. However, the effects of overexpressing SERCA2a on action potential duration (APD), which can affect diastolic parameters in the heart, is unknown. We, therefore, investigated the relationship between SERCA2a overexpression and APD in adult rabbit ventricular myocytes which were cultured for 48 h. Overexpression of SERCA2a was achieved by infection with an adenovirus carrying both SERCA2a and GFP independently driven by CMV promoters, Ad.SERCA2a. Myocytes infected with Ad.GFP only and/or non-infected myocytes were used as controls. Electrophysiological measurements were taken using switch clamping with 15–25 MΩ resistance microelectrodes. In Ad.SERCA2a infected myocytes, APD was significantly reduced compared with both groups of control cells at 0.5 Hz (APD50 (ms) non-infected: 481±98, n=12; Ad.GFP: 464±85, n=11; Ad.SERCA2a: 285±69, n=13 (mean±S.E.M.) and at 1 Hz (APD50 (ms) non-infected: 375±64, n=22; Ad.GFP: 363±47, n=18; Ad.SERCA2a: 231±54, n=24). Using AP voltage-clamping, we recorded a 0.2 mM Cd-sensitive current which can be ascribed to Ca current flowing during the AP. The integral of this current was reduced in Ad.SERCA2a myocytes compared with control (non-infected charge (pC): 27.5±4.2, n=8; Ad.SERCA2a: 15.5±4.1, n=11; P<0.01). Using AP clamping during the loading protocol, to take into account changes in APD, SR Ca content (assessed by integrating a 20 mM caffeine-induced inward current) was significantly larger in Ad.SERCA2a compared with both controls (SR Ca content (μM/l non-mitochondrial volume): non-infected: 25.5±7, n=8; Ad.GFP: 25.7±11, n=6; Ad.SERCA2a: 80.5±19, n=8). In conclusion, this study shows that SR Ca content is increased despite decreased Ca entry after overexpression of SERCA2a, and this can lead to positive inotropism. This effect coupled with shorter APD may be a useful therapeutic modality in heart failure.
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