Quantitative assessment of the SR Ca 2+ leak-load relationship

Abstract

Increased diastolic SR Ca 2+ leak (J leak) could depress contractility in heart failure, but there are conflicting reports regarding the J leak magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca 2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na +, 0 Ca 2+ solution ± 1 mmol/L tetracaine (to block resting leak). Total cell [Ca 2+] does not change under these conditions with Na +-Ca 2+ exchange inhibited. Resting [Ca 2+]i declined 25% after tetracaine addition (126±6 versus 94±6 nmol/L; P<0.05). At the same time, SR [Ca 2+] ([Ca 2+] SRT) increased 20% (93±8 versus 108±6 μmol/L). From this Ca 2+ shift, we calculate J leak to be 12 μmol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca 2+ effluxes is counterbalanced by unidirectional forward Ca 2+ pump flux. J leakalso increased nonlinearly with [Ca 2+] SRT with a steeper increase at higher load. We conclude that J leak is 4 to 15 μmol/L cytosol per second at physiological [Ca 2+] SRT. The data suggest that the leak is steeply [Ca 2+] SRT-dependent, perhaps because of increased [Ca 2+] i sensitivity of the ryanodine receptor at higher [Ca 2+] SRT. Key factors that determine [Ca 2+] SRT in intact ventricular myocytes include (1) the thermodynamically limited Ca 2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca 2+ ATPase) and (2) diastolic SR Ca 2+ leak (ryanodine receptor mediated).