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

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).