Control of Maximum Sarcoplasmic Reticulum Ca Load in Intact Ferret Ventricular Myocytes : Effects of Thapsigargin and Isoproterenol

In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively with known amounts of Ca via L-type Ca channels (I_Ca), using Na-free solutions to prevent Na/Ca exchange. We then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when SR Ca pumping was accelerated by isoproterenol (1 μM) or slowed by thapsigargin (0.2–0.4 μM). Under control conditions, SR Ca content reached a limit of 137 μmol·liter cytosol⁻¹ (nonmitochondrial volume) when measured by integrating caffeine-induced Na/Ca exchange currents (∫I_NaCaXdt) and of 119 μmol·liter cytosol⁻¹ when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]_i). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (∫I_NaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (∫I_NaCaXdt method). The relative stability of the SR Ca load suggests that dissipative losses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gradient approaching the thermodynamic limit.