CIC   05421
CENTRO DE INVESTIGACIONES CARDIOVASCULARES "DR. HORACIO EUGENIO CINGOLANI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
CaMKII: an apoptotic signal in ischemia-reperfusion injury
Autor/es:
MARGARITA A. SALAS
Lugar:
San Pablo, Brasil
Reunión:
Congreso; 62 Congreso Brasilero de CardiologĂ­a y XVI Meeting of the International Society for Heart Research, Latin American Section; 2007
Institución organizadora:
International Society for Heart Research, Latin American Section
Resumen:
CaMKII like PKA and PKC, is a Ser-Thr protein kinase which is highly enriched in the nervous system but is also present in the heart, and phosphorylates several proteins in response to calcium signals. The delta isoform is the predominant isoform in the heart, delta c localized in the cytosol and delta b in the nucleus. The enzyme is activated by the binding of the Ca-CaM complex. Then, by autophosphorylation of Thr287 on the autoinhibitory domain of an adjacent monomer, this enzyme can lock itself into the activated state CaM independent. The CaMKII is inactivated by several phosphatases including PP1, PP2A, PP2C. Among the different CaMKII inhibitors we have used KN-93 which competitively inhibits Ca-CaM binding at the regulatory domain. CaMKII is a kinase with several functions on excitation contraction coupling since it phosphorylates Ca2+  channels, RyR and PLB. It has also been demonstrated its role in the recovery from acidosis and the production of arrhythmias. In addition to the acute effects of CaMKII on ECC in the heart, this enzyme may be involved in long term regulation of gene expression During excitation–contraction coupling, Ca2+ entry, mainly via voltage dependent L-type Ca2+ channels , triggers sarcoplasmic reticulum (SR) Ca2+ release via (RyRs), by Ca2+ induced Ca2+ release. The resultant increase in intracellular [Ca2+], causes Ca2+ binding to troponin C, which activates myofilaments, leading to contraction. For relaxation to occur, Ca2+ must be removed from the cytoplasm by Na+/ Ca2+ exchanger (NCX) and the SR Ca2+ -ATPase (SERCA). PLB is the major inhibitor of SERCA2 and this inhibition can be relieved by phosphorylation of two residues: the PKA dependent Ser 16 and de CaMKII dependent Threo 17 . In previous experiments our group had demonstrated that Thr 17 of PLN could be phosphorylated by CaMKII,  in the absence of beta adrenergic stimulation, under conditions of high intracellular  calcium and inhibition of phosphatases, for instance by  acidosis. These two conditions are present during ischemia and reperfusion suggesting that Thr17 of PLN may be phosphorylated in that situation. It was further hypothesized that this phosphorylation might be relevant during reperfusion as a mechanism of the cell to cope with Ca2+ overload. We therefore studied the relationship between Thre17 PLB phosphorylation site and postischemic recovery The ischemia reperfusion injury can be reversible or irreversible depending on the absence or presence of cell death. The stunned heart is characterized by impairment of contractility and preserved Ca2+  transient. I will briefly show you the findings from our lab on the role of CaMKII-dependent Thr 17 phosphorylation in a protocol of reversible IRI We performed the experiments on Langendorf perfused rat hearts submitted to 20 min of ischemia followed  by different times of  reperfusion up to 30min in the presence or absence of 1uM of the CaMKII inhibitor KN-93 The inhibitor was perfused during the preischemic period and the first minute of reperfusion. We assessed : 1)      Contractile recovery 2)      PLB phosphorylation at  Ser 16 and Thr  17 sites   It was found a significant increase in the phosphorylation of Thr17 of PLN at the beginning of reperfusion, without significant changes in Ser16 phosphorylation. The increase in Thr17 phosphorylation was significantly diminished by inhibition of CaMKII with KN-93 and was also significantly decreased by inhibition of the NHE by EIPA and the reverse mode of NCX by KB-R7943. These last results suggest that the following cascade would be involved: The increase of Na/H exchanger activity due to the intracellular acidosis as a result of the ischemia, activates de reverse mode of the NCX increasing intracellular Ca2+. This in turn activates CaMKII leading to Thr 17 phosphorylation during reperfusion. Looking at the functional consequences of the inhibition of CaMKII with  KN-93 we found a significant impairment of the contractile and relaxation recovery, suggesting that  CaMKII-dependent phosphorylations play a role in the mechanical recovery. We did not know at that moment whether phosphorylation of Thr that occurs so early during reperfusion and was transient was the only one involved in the effects of CaMKII activation. To explore this point, we performed experiments in mice from Litsa Kranias lab, in which  Thr17 was mutated to Ala. The results of these experiments were very similar to those obtained in rats using the CaMKII-inhibitor KN-93. Substitution of Thr17 by Ala resulted in an impairment of contractility and relaxation during stunning. These results suggest that phosphorylation of Thr17 residue is essential for the contractile recovery during reperfusion. Taken together these results allowed us to described a cascade of events that occurs simultaneously with the one believed to be responsible for the mechanical alterations in contractility in the stunning, at least in rodents, but that is beneficial and ameliorates the detrimental effect of the reversible IR injury. Upon reperfusion the activity of NHE increased leading to an increase in intracellular Na and in intracellular Ca2+ through the reverse mode of the NCX. The increase in Ca2+ would activate calpains, in turn responsible for the protein proteolysis and the decrease in contractility of the stunned heart (detrimental effect). Simultaneously the increase in Ca2+ would also activate CaMKII, and phosphorylate PLN favoring Ca2+ handling thus allowing the recovery of intracellular Ca2+ transient typical of stunning and partially ameliorating the contractile dysfunction. Being CaMKII playing a beneficial role in the RIRI while recent reports have pointed to CamKII as an important signal in the apoptotic cascade we decided to investigate its role in a model of irreversible IRI. Since the irreversible IRI is characterized by death of myocardial cells, the two major patterns of cell death were evaluated: apoptosis and necrosis. To produce irreversible I/R injury, we prolonged the ischemic period to 45 min and the reperfusion period up to 2 hours, The CaMKII inhibitor was perfused during the preischemic period and the first 10 min of reperfusion. We assessed: 1)       Contractile recovery 2)      PLN phosphorylation 3)       Infarct size with Trifenil tetrazolium staining 4)      Levels of lacticodeshidrogenase , in the perfusion effluent of the first 10 min of reflow 5)       Apoptosis: by TUNEL assay, levels of activated caspase 3 and the ratio of Bcl2/Bax proteins   Reperfusion was again associated with an increase in the phosphorylation of Thr17, which indicates that CaMKII was activated at least at the beginning of reperfusion. Contractile recovery was very poor after the long ischemic period but in contrast to what happened in the stunned heart,  inhibition of CaMKII produced a slight but significant enhancement of the contractile recovery.  Inhibition of CaMKII significantly decreased the size of the infarct produced by I/R injury, also decreased the release of LDH used to estimate necrosis, as well as the number of tunel positive cells, indicating amelioration of apoptotic death. Another parameter of apoptosis, the levels of activated caspase -3 was diminished by CaMKII inhibition The results showed that CaMKII activation during irreversible IRI was mediating myocytes death by both necrosis and apoptosis processes. We are currently studying the apoptotic pathway triggered by CaMKII   There are two major mechanisms that conduct to apoptosis: one named extrinsic pathway, activated by interaction of death receptors with their ligands. The second one, the intrinsic pathway where prodeath factors are released from the mitochondria via the action of proapoptotic proteins members of the Bcl2 family.   We hipotetized that CaMKII is involved in the intrinsic apoptotic pathway. We propose a cascade where CaMKII is activated during reperfusion by Ca2+ influx through the NCX acting in reverse mode inducing SR Ca overload. This situation of SR stress could lead  to apoptosis by two ways: by activating caspase 12 or/and by releasing Ca2+  through the RyR2, provoking mitochondrial Ca2+overload, rupture of outer membrane and releasing cytochrome c with the subsequent caspase activation We used different inhibitors of this pathway in order to confirm this. KBR to inhibit NCX, Thapsigarguin to inhibit SERCA, Dantrolene to block Ca2+ leak through  IP3 and RyR and Rutheniun Red to block mitochondrial Ca uniporter We were able to diminish infarct size and the number of TUNEL positive cells by inhibiting Ca2+entrance through the reverse mode of the NCX with KBR, leak of Ca2+  through the RyR with dantrolene and mitochondrial Ca2+ overload with the mitochondrial Ca2+  uniporter inhibitor, ruthenium red. These results indicated an apoptotic pathway which involves the reverse mode of the NCX, the SR and the mitochondria. This apoptotic pathway is not new and has been previously described. Now the question is: does CaMKII participate in this typical pathway of apoptosis?  The participation of CaMKII is supported by different types of evidence. In the first place, in experiments done on isolated myocytes subjected to simulated I/R it was found that reperfusion produced in most of the cells multiple and large contractions and intracellular calcium oscillations that culminate in cell membrane disruption These oscillations that are an indication of SR Ca2+  overload,  were prevented by inhibition of CaMKII with 1 uM KN-93. In contrast the cells treated with KN-93 preserved the contractile activity after the ischemic/reperfusion insult. Similar results were obtained with Thapsigargin. The role of CaMKII in this pathway that includes the SR and the mitochondria, was also supported by the effect of CaMKII inactivation increasing the rate of antiapoptotic to proapoptotic proteins, which as you may remember act at the mitochondria level in the release of citochrome C. Thus, our results suggest a dual effect of the activation of this kinase in the I/R injury. In the stunned heart the beneficial cascade would involve the phosphorylation of Thr17 site of PLN, improving Ca2+ handling by the SR and ameliorating the depressed contractility. The detrimental cascade would involve the Ca2+ overload of SR and possibly the mitochondria, and the consequent cell alterations conducting to apoptosis and necrosis. We still do not know if Thr 17 is involved in the detrimental effect or if its phosphorylation is beneficial but not sufficient to counteract other deleterious effects due to others substrates of CaMKII.  
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