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

Abstract

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.