Ca²⁺ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes

1. Shortening and intracellular Ca²⁺ (Ca(i)²⁺) transients were measured in isolated rabbit ventricular myocytes during paired contractures induced by rapid application of 10 mM caffeine 2. Caffeine-induced contractures relax despite maintained presence of caffeine. In control solution, a second phasic caffeine contracture failed to appear, unless the sarcoplasmic reticulum (SR) was refilled by a series of electrically stimulated twitches during the interval between caffeine exposures. 3. The relaxation of caffeine-induced contractures in 0 Na⁺-0 Ca²⁺ solution has previously been shown to rely on mitochondrial Ca²⁺ uptake and sarcolemmal Ca²⁺-ATPase. Thus, a second caffeine contracture (T₂) while still in O Na⁺-0 Ca²⁺ was greatly reduced compared to the first one (T₁). However, the amplitude of T₂ increased exponentially with the time interval, attaining a maximum of ~ 50% of T₁ for an interval of 180-300 s, with a time constant (Υ) of 41.2 s. Similar results were found for Ca(i)²⁺ transients (Υ = 45 S). 4. Inhibition of the mitochondrial Ca²⁺ uptake by the oxidative phosphorylation uncoupler, FCCP during T₁ dramatically depressed T₂. On the other hand, inhibition of the sarcolemmal Ca²⁺-ATPase (by increasing extracellular Ca²⁺ concentration, [Ca²⁺](o)) resulted in increase of T₂. Spermine inclusion during T₁ also increased T₂, possibly by an increase of mitochondrial Ca²⁺ uptake. 5. We conclude that Ca²⁺ taken up by mitochondria during the decline of T₁ moves back to the SR after caffeine is removed, with a τ ~40 s. 6. Partial intracellular :Na⁺ depletion by prolonged (3 min) perfusion with O Na⁺ Ca²⁺,solution before T₁ (a) accelerated relaxation and [Ca²⁺](i) decline during T₁, and (b) slowed, but did not abolish, the recovery of T₂ as the interval was increased. This effect was particularly pronounced when choline was used instead of Li⁺ as the Na⁺ substitute. 7. We further conclude that the mitochondrial Na⁺-Ca²⁺ antiporter influences the rate of net Ca²⁺ uptake by mitochondria and is also important in Ca²⁺ efflux from mitochondria during rest.