Mechanisms that define sarcoplasmic reticulum Ca release restitution in cardiac myocytes

CELY-ORTIZ, ALEJANDRA; FELICE J; VALVERDE, CARLOS A; FEDERICO, MARILÉN; SAPIA, LUCIANA; PALOMEQUE, JULIETA; LASCANO, ELENA; NEGRONI, JORGE; MATTIAZZI, ALICIA

Rosario

Congreso; Reunión Anual SAFIS 2019; 2019

Sociedad Argentina de Fisiología

During EC-coupling, Ca influx induces the release of Ca from the sarcoplasmic reticulum (SR). The release mechanism needs time between stimuli to produce a second full Ca release (Ca release restitution, CRR). Dissection of the mechanisms determining CRR is important since its alteration is associated with arrhythmias. However these mechanisms are unclear with controversial results, mainly regarding the role of SR Ca uptake on CRR.Aims. To dissect the mechanisms that define CRR.Methods. Experiments were performed in hearts and myocytes isolated from wild type (WT) mice, mice with increased SR Ca uptake (due to phospholamban ablation, PLNKO), mice with increased SR Ca channel (RyR2) activity (due to constitutive pseudo-phosphorilation of RyR2-Ser2814 CaMKII site, S2814D), and double-mutant mice (SDKO) obtained by crossbreeding S2814D and PLNKO. Intracellular Ca was measured using fluorescent probes. CRR curves were obtained by a two pulse protocol and the time constant (Tau) was used to evaluate CRR, assuming an exponential relationship between the Ca released and the time between stimuli. Hearts from each strain were frozen for Western blot analysis (WB). A previously validated human cardiac myocyte model that represented each experimental condition was used to perform simulations of the CRR protocol.Results. WB analysis showed 33% decrease in RyR2 expression in PLNKO and SDKO hearts. Analysis of CRR curves indicated that CRR depends on SR Ca load (Increasing SR Ca by increasing extracellular Ca from 2 to 4 mM in WT mice, decreased Tau by 52%, p < 0.05), and on RyR2 activity (Tau value was not statistically different in S2814D vs WT mice, in spite of the fact that SR Ca content was 20% lower). The rate of SR Ca reuptake had no influence on Tau. The simulations obtained with the model reproduced the experimental results. However, when these simulations were carried out in PLNKO and SDKO representing only the increase in Ca uptake (i.e. without any decrease in RyR2 expression), the results of the model showed that the rate of SR Ca uptake is largely responsible of CRR.Conclusion. CRR depends on SR Ca content, RyR2 sensitivity/activity and SR Ca uptake rate.