Addition of 1 mmol/L tetracaine to intact ventricular myocytes reduced resting intracellular Ca2+ by 25% (126 vs 94 nmol/L; P<0.05), indicating a diastolic SR Ca2+ leak of 12 micromol/L per second.
The study quantifies diastolic SR Ca2+ leak in intact ventricular myocytes, demonstrating it is steeply dependent on SR Ca2+ load and accounts for 30% of SR diastolic efflux.
Absolute Event Rate: 94% vs 126%
p-value: p=<0.05
Increased diastolic SR Ca2+ 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 Ca2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na+, 0 Ca2+ solution +/-1 mmol/L tetracaine (to block resting leak). Total cell Ca2+ does not change under these conditions with Na+-Ca2+ exchange inhibited. Resting Ca2+i declined 25% after tetracaine addition (126+/-6 versus 94+/-6 nmol/L; P<0.05). At the same time, SR Ca2+ (Ca2+(SRT)) increased 20% (93+/-8 versus 108+/-6 micromol/L). From this Ca2+ shift, we calculate J(leak) to be 12 micromol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca2+ effluxes is counterbalanced by unidirectional forward Ca2+ pump flux. J(leak) also increased nonlinearly with Ca2+(SRT) with a steeper increase at higher load. We conclude that J(leak) is 4 to 15 micromol/L cytosol per second at physiological Ca2+(SRT). The data suggest that the leak is steeply Ca2+(SRT)-dependent, perhaps because of increased Ca2+i sensitivity of the ryanodine receptor at higher Ca2+(SRT). Key factors that determine Ca2+(SRT) in intact ventricular myocytes include (1) the thermodynamically limited Ca2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca2+ ATPase) and (2) diastolic SR Ca2+ leak (ryanodine receptor mediated).
Shannon et al. (2002) studied Normal, intact ventricular myocytes. Tetracaine in 0 Na+, 0 Ca2+ solution vs. Without tetracaine was evaluated on Resting [Ca2+]i (p=<0.05). Addition of 1 mmol/L tetracaine to intact ventricular myocytes reduced resting intracellular Ca2+ by 25% (126 vs 94 nmol/L; P<0.05), indicating a diastolic SR Ca2+ leak of 12 micromol/L per second.