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

Abstract

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.

Figure 1. Left ventricular developed pressure (LVDP) and epicardial monophasic action potentials (MAPs) during ischemia/reperfusion protocol

A: Representative recordings showing the appearance of reperfusion arrhythmias triggered bya delay after depolarization (DAD). B: Representative example in which the PBs are associated with an early afterdepolarization (EAD). The inset of the Figure indicates in an expanded time scale, the occurrence of EAD before the completion of the action potential.

Figure 2. CaMKII-dependence of reperfusion arrhythmias

Typical example (A) depicting that CaMKII-inhibition by 2.5 μM KN-93 significantly decreased the number of PBs. The inactive analogue KN-92 (B) failed to affect the reperfusion arrhythmic pattern. C: Overall results comparing the number of PBs in the pre-ischemic period (Pre-I) and in the first 3 min of reperfusion in the absence of drugs (ND) and in the presence of KN-92 and KN-93. D: MAP duration at 90 % repolarization (MAPD₉₀) measured at Pre-I during the first 3 min of reperfusion in the absence and the presence of KN-93. n = 4–7. * indicates P < 0.05 with respect to Pre-I. ^ indicates P < 0.05 with respect to reperfusion with ND.

Figure 3. CaMKII is active at the onset of reperfusion

Typical blots and overall results of experiments showing an increase in CaMKII autophosphorylation (A) and in the phosphorylation of two typical substrates of CaMKII: Thr17 site of PLN (B) and Ser2814 of RyR2 (C). Treatment with KN-93 prevented the CaMKII-dependent protein-phosphorylations detected during reperfusion. n = 3–6. * indicates P < 0.05 with respect to control (Ctrl). ^ indicates p < 0.05 with respect to reperfusion with ND.

Figure 4. CaMKII inhibition targeted to SR completely suppresses reperfusion arrhythmias

Typical examples (A, B, D and E) and overall results (C and F) of experiments performed in WT mice and mice with targeted inhibition of CaMKII at the SR level (SR-AIP mice). A and B are MAP recordings, D and E are optical AP recordings obtained with the fluorescent dye Di-8-ANEPPS. Reperfusion arrhythmias were virtually completely inhibited in SR-AIP mice. n = 3–6. * P < 0.05 with respect to Pre-I. ^ indicates P < 0.05 with respect to reperfusion in WT mice.

Figure 5. CaMKII-dependent phosphorylation of RyR2-Ser2814 site is critical for generation of reperfusion arrhythmias

Typical examples (A and B) and overall results (C) of reperfusion arrhythmias in WT mice and mice with genetic mutation of Ser2814 site of RyR2 (RyR2-S2814A mice). There was a significant decrease in PBs in RyR2-S2814A mice relative to WT. n = 3–6. * P < 0.05 with respect to Pre-I. ^ indicates P < 0.05 with respect to reperfusion in WT mice.

Figure 6. Effect of genetic mutation of PLN-phosphorylation sites (PLN-DM mice) on reperfusion arrhythmias

Typical examples of MAP recordings (A and B) and overall results (C), indicating that the number of PBs upon reperfusion was similar in transgenic mice with non-phosphorylatable PLN than in WT mice with intact PLN. The percentage of PBs triggered by EADs was increased in PLN-DM mice vs. WT mice (D). Half relaxation time at 1 min reperfusion was prolonged in PLN-DM mice with respect to WT mice (E). n = 4–7. * P < 0.05 with respect to Pre-I.