oxiCaMKII-dependent RyR2 phosphorylation mediates contractile dysfunction associated with sepsis.

SEPÚLVEDA MARISA; GONANO LUIS; LÓPEZ ALARCÓN MICAELA; RAMOS ISALIRA; VILA PETROFF MARTÍN; VIOTTI MANUEL; MEDEI EMILIANO

Buenos Aires

Congreso; ISHR; 2016

Centro de Investigaciones Cardiovasculares "Dr. Horacio E Cingolani"

In sepsis, there is a recognized association between cardiac dysfunction and mortality. Contractile dysfunction associated with sepsis has been attributed to a decrease in the amplitude of the intracellular Ca2+ transient and recent studies have proposed that altered ryanodine receptor (RyR2) function is responsible for sarcoplasmic reticulum (SR) Ca2+ loss and reduced Ca2+ transients.We examined the subcellular mechanisms involved in SR Ca2+ loss and contractile dysfunction associated with sepsis.Using a colon ascendens stent peritonitis mouse model of sepsis (CASP) and Sham controls, we observed that after 24hs CASP mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction and fractional shortening (EF%54,76±0,67; FS%27,53±0,5) compared to sham (EF%73,57±0,2; FS%46,75±0,38). At the cardiac myocyte level, CASP cells showed reduced cell shortening, Ca2+ transient amplitude and SR Ca2+ content compared to Sham cardiomyocytes. CASP hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II (CaMKII) activity (CASP 0.92 ± 0.1 AU, Sham 0.56 ± 0.05 AU) which could be prevented by pretreating animals with the antioxidant Tempol (1mM for 7 days in drinking water).Pharmacological inhibition of CaMKII with 2.5 µM KN93 prevented the decrease in cell shortening, Ca2+ transient amplitude and SR Ca2+ content in CASP myocytes. Contractile function was also preserved in CASP myocytes isolated from transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) and in CASP myocytes isolated from mutant mice that have the RyR2 CaMKII-dependent phosphorylation site (Ser 2814) mutated to alanine (2814A). Furthermore, CASP 2814A mice showed preserved EF and FS (EF%59,54±3,42; FS%35,88±5,58) compared to sham 2814A mice EF%65,89±6,95; FS%33,33±2,38) . Results indicate that oxidation and subsequent activation of CaMKII has a causal role in the contractile dysfunction associated with sepsis. CaMKII, through phosphorylation of RyR2 would lead to Ca2+ leak from the SR, reducing SR Ca2+ content, Ca2+ transient and contractility.