Ca2+entry-independent effects of L-type Ca2+channel modulators on Ca2+sparks in ventricular myocytes

Abstract

During the cardiac action potential, Ca2+entry through dyhidropyridine receptor L-type Ca2+channels (DHPRs) activates ryanodine receptors (RyRs) Ca2+-release channels, resulting in massive Ca2+mobilization from the sarcoplasmic reticulum (SR). This global Ca2+release arises from spatiotemporal summation of many localized elementary Ca2+-release events, Ca2+sparks. We tested whether DHPRs modulate Ca2+sparks in a Ca2+entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca2+sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca2+(100 nM). Under these conditions, Ca2+sparks were detected with a stable frequency of 3–5 sparks·s−1·100 μm−1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca2+sparks. The DHPR modulators were also without effect on SR Ca2+load (caffeine-induced Ca2+transients) or sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity (Ca2+loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca2+entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca2+-release units in ventricular myocytes is modulated by Ca2+entry-independent conformational changes in neighboring DHPRs.