Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death

Abstract

While Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5 μmol/dm(3)) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIIδ and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function.

Analysis of necroptotic proteins in failing human hearts

“…It can be mentioned that this axis of RIP3-dependent necroptosis was found in both I/R and doxorubicin-induced cardiotoxicity [13], animal model analogues to types of heart failure referred to in our study. The involvement of this protein kinase, a key regulator of Ca 2+ homeostasis in cardiomyocytes in myocardial I/R-induced necroptosis, has also been proposed in our recent study showing that the inhibition of CaMKII reversed certain changes in pronecroptotic markers what was accompanied by improved contractile function [10]. It should be noted, however, that the pronecroptotic axis involving RIP3-CaMKII resulting in cell death via effects on mitochondria [13] is contradictory to studies showing that necroptosis occurs due to plasma membrane disruption as a consequence of recruitment of MLKL following RIP1-RIP3 activation [7, 12, 20].…”

“…In fact, cleavage of RIP1 and RIP3 by caspase-8 prevents necroptotic signaling while simultaneously promoting apoptosis [6]. The importance of necroptosis has been shown mainly in non-cardiac pathologies [9] and recently, it has also been demonstrated in rodent reperfused hearts subjected to previous acute global and regional ischemia [10, 11] as well as in a model of cardiomyopathy [12, 13]. Relevance and proposed mechanisms of necroptotic cell death in heart failure have been reviewed elsewhere [14].…”

Cardiomyocyte apoptosis contributes to pathogenesis of cirrhotic cardiomyopathy in bile duct-ligated mice

“…More important is the ratio of pro-to anti-apoptotic proteins in a cell [23]. In this regard, Bcl-2 is an anti-apoptotic factor, whereas Bax is a pro-apoptotic factor, and the Bcl-2/Bax ratio is recognized as an index of intrinsic pathway activation [24,25]. We believe that the increased Bcl-2/Bax ratio in the BDL-mice reflects an anti-apoptotic compensatory reaction in the intrinsic pathway in reaction to the net pro-apoptotic imbalance mediated by the extrinsic pathway.…”

Effects of Necrostatin-1, an Inhibitor of Necroptosis, and its Inactive Analogue Nec-1i on Basal Cardiovascular Function

“…In line, a recent study has suggested that RIP3, being a downstream target of RIP1, activates CaMKII (Zhang et al 2016) which is known to phosphorylate and thus regulate excitation-contraction coupling of the heart (Luo et al 2013). The link between CaMKII, RIPs, necroptotic cell death and the force of contractile function has been proposed for the first time by our group (Szobi et al 2014). In fact, we have found that inhibition of CaMKII normalizes the upregulation of RIP1 levels and that these changes are linked with the improved contraction cycle in I/R heart.…”