Carlos Alfredo Valverde

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr(17) site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na(+)/Ca(2+)exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca(2+) loading and release (thapsigargin/dantrolene), mitochondrial Ca(2+) uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr(17) of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.

Calcium-calmodulin dependent protein kinase II (CaMKII): a main signal responsible for early reperfusion arrhythmias.

To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca(2+)-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31±6 to 5±1 beats/3min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0±14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca(2+) channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca(2+) channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca(2+) channels might contribute to reperfusion-induced PBs, by increasing SR Ca(2+) content and Ca(2+) influx.

Calcium-Calmodulin Kinase II Mediates Digitalis-Induced Arrhythmias

Background —Digitalis-induced Na + accumulation results in an increase in Ca 2+ i via the Na + /Ca 2+ exchanger leading to enhanced SR Ca 2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. Digitalis-induced increase in Ca 2+ i could also activate CaMKII which has been shown to have proarrhythmic effects. Here we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results —In paced rat ventricular myocytes (0.5 Hz), 50 μmol/l ouabain increased contraction amplitude by 160 ± 5%. In the absence of electrical stimulation, ouabain promoted spontaneous contractile activity and Ca 2+ waves. Ouabain activated CaMKII (P-CaMKII) which phosphorylated its downstream targets, phospholamban (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/l KN93 but not by 2.5 μmol/l of the inactive analogue KN92. Similar results were obtained using the CaMKII inhibitor, AIP (1-2.5 μmol/l) and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2+ content and Ca 2+ spark frequency, indicative of enhanced SR Ca 2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca 2+ content. Similar results were obtained with digoxin. In vivo , ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions —These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2+ leak from the SR is the underlying mechanism involved.Background— Digitalis-induced Na+ accumulation results in an increase in Ca2+ i via the Na+/Ca2+ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca2+ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results— In paced rat ventricular myocytes (0.5 Hz), 50 &mgr;mol/L ouabain increased contraction amplitude by 160±5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca2+ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 &mgr;mol/L KN93 but not by 2.5 &mgr;mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 &mgr;mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca2+ content and Ca2+ spark frequency, indicative of enhanced SR Ca2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca2+ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions— These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca2+ leak from the SR, is the underlying mechanism involved.

CaMKII Mediates Digitalis-Induced Arrhythmias

Background —Digitalis-induced Na + accumulation results in an increase in Ca 2+ i via the Na + /Ca 2+ exchanger leading to enhanced SR Ca 2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. Digitalis-induced increase in Ca 2+ i could also activate CaMKII which has been shown to have proarrhythmic effects. Here we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results —In paced rat ventricular myocytes (0.5 Hz), 50 μmol/l ouabain increased contraction amplitude by 160 ± 5%. In the absence of electrical stimulation, ouabain promoted spontaneous contractile activity and Ca 2+ waves. Ouabain activated CaMKII (P-CaMKII) which phosphorylated its downstream targets, phospholamban (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/l KN93 but not by 2.5 μmol/l of the inactive analogue KN92. Similar results were obtained using the CaMKII inhibitor, AIP (1-2.5 μmol/l) and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2+ content and Ca 2+ spark frequency, indicative of enhanced SR Ca 2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca 2+ content. Similar results were obtained with digoxin. In vivo , ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions —These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2+ leak from the SR is the underlying mechanism involved.Background— Digitalis-induced Na+ accumulation results in an increase in Ca2+ i via the Na+/Ca2+ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca2+ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results— In paced rat ventricular myocytes (0.5 Hz), 50 &mgr;mol/L ouabain increased contraction amplitude by 160±5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca2+ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 &mgr;mol/L KN93 but not by 2.5 &mgr;mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 &mgr;mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca2+ content and Ca2+ spark frequency, indicative of enhanced SR Ca2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca2+ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions— These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca2+ leak from the SR, is the underlying mechanism involved.

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.