The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.