Activation of Ca2+ Sparks during β-Adrenergic Stimulation in Resting Cardiomyocytes May Involve CAMKII and No, But Not ROS

Abstract

During β-adrenergic stimulation of cardiac myocytes, phosphorylation of the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors, RyRs) by PKA and CaMKII has been linked to the observed diastolic Ca2+ leak (as Ca2+ sparks) from the SR. Using confocal Ca2+ imaging, we previously showed that β-adrenergic stimulation with 1 μM isoproterenol (ISO) increases the spark frequency in quiescent voltage-clamped guinea-pig cardiomyocytes, without altering the SR Ca2+ content. Protein kinase inhibitors (KN-93 and H89) indicated an involvement of CaMKII in the change of spark frequency, but not PKA. Additional experiments showed that the change in sensitivity of the RyRs upon β-adrenergic stimulation may involve reactive nitrogen intermediates (RNI), as incubation with the NOSs inhibitor L-NAME (500 μM) and specific nNOS inhibitor AAAN (100 μM) prevented the increase in spark frequency without changes in SR Ca2+ content. This observation was confirmed by using the NO scavenger PTIO (130 μM). Furthermore, recent evidence suggests that redox activation of CaMKII or redox modifications of RyRs may contribute to the SR Ca2+ leakage. Therefore, we examined the effect of reactive oxygen species (ROS) on the spark frequency upon ISO application. Incubation with the SOD mimetic (and peroxynitrite decomposition catalyst) Mn-TBAP (100 μM) did not prevent the increase in spark frequency in cells with constant SR Ca2+ content. Similarly, acute application of the ROS scavenger TIRON (200 μM) could not suppress the progressive increase in spark frequency upon ISO application. Our observations suggest that CaMKII and RNI, but not ROS, are involved in the upregulation of spark frequency during β-adrenergic stimulation.