Mechanisms that define sarcoplasmic reticulum Ca release restitution in cardiac myocytes

CELY-ORTIZ, DIANA CATALINA ALEJANDRA; FELICE, JUAN IGNACIO; VALVERDE, CARLOS ALFREDO; FEDERICO, MARILÉN; SAPIA, LUCIANA; PALOMEQUE, JULIETA; LASCANO, ELENA CATALINA; NEGRONI, JORGE ANTONIO; MATTIAZZI, RAMONA ALICIA

Rosario

Encuentro; Reunión Anual de la Sociedad Argentina de Fisiología (SAFIS); 2019

Sociedad Argentina de Fisiología

During EC-coupling, Ca influx inducesthe release of Ca from the sarcoplasmic reticulum (SR). The release mechanismneeds time between stimuli to produce a second full Ca release (Ca releaserestitution, CRR). Dissection of the mechanisms determining CRR is importantsince its alteration is associated with arrhythmias. However these mechanisms areunclear with controversial results, mainly regarding the role of SR Ca uptakeon CRR.Aims. Todissect the mechanisms that define CRR.Methods. Experimentswere performed in hearts and myocytes isolated from wild type (WT) mice, micewith increased SR Ca uptake (due to phospholamban ablation, PLNKO), mice withincreased SR Ca channel (RyR2) activity (due to constitutive pseudo-phosphorilationof RyR2-Ser2814 CaMKII site, S2814D), and double-mutant mice (SDKO) obtained bycrossbreeding S2814D and PLNKO. Intracellular Ca was measured using fluorescentprobes. CRR curves were obtained by a two pulse protocol and the time constant(Tau) was used to evaluate CRR, assuming an exponential relationship betweenthe Ca released and the time between stimuli. Hearts from each strain werefrozen for Western blot analysis (WB). A previously validated human cardiac myocytemodel that represented each experimental condition was used to perform simulationsof the CRR protocol.Results. WBanalysis showed 33% decrease in RyR2 expression in PLNKO and SDKO hearts. Analysisof CRR curves indicated that CRR depends on SR Ca load (Increasing SR Ca by increasingextracellular 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 WTmice, in spite of the fact that SR Ca content was 20% lower). The rate of SR Careuptake had no influence on Tau. The simulations obtained with the model reproducedthe experimental results. However, when these simulations were carried out inPLNKO and SDKO representing only the increase in Ca uptake (i.e. without anydecrease in RyR2 expression), the results of the model showed that the rate of SRCa uptake is largely responsible of CRR.Conclusion. CRRdepends on SR Ca content, RyR2 sensitivity/activity and SR Ca uptake rate.