Introduction: Nitric oxide can regulate cardiac function by targeting calcium-handling protein kinases such as calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ). Recent evidence has shown that during stress response, there is an increase in nitric oxide production which can activate CaMKII through S-nitrosylation. Chronic CaMKII activation, as a result of S-nitrosylation, has been implicated in the progression of arrhythmias. However, CaMKII S-nitrosylation can have protective or detrimental effects on cardiac function depending on the S-nitrosylation site that is modified. In the heart, the Cys 273 site inhibits CaMKII activation while the Cys 290 site enhances autonomous CaMKII activity. The mechanism underlying the role of CaMKII S-nitrosylation sites in the regulation of cardiac function during acute ischemia/reperfusion (I/R) injury has not been fully elucidated.  

Aim: To determine if CaMKII S-nitrosylation site was detrimental to the heart during I/R injury.

Methods: Male mouse hearts were isolated and perfused with modified Krebs-Henseleit buffer via the Langendorff perfusion system. Hearts from wild-type C57BL/6 (WT) (n = 5) and C273S knock-in mice (C273S-KI, that lack the Cys 273 site; n = 5) were acutely exposed to 100 nM isoproterenol (ISO), a β-adrenergic receptor agonist before or after 150 µM S-nitrosoglutathione (GSNO), a nitric oxide donor for 10 min. Arrhythmias were counted per heart. For ischemia studies, WT (n = 4 – 6) and C273S-KI (n = 3) hearts were exposed to 100 nM ISO for 5 min prior to 20 min global ischemia, followed by 90 min reperfusion. Duration of arrhythmias was measured for 10 minutes post-ischemia. Data is presented as mean ± standard error of mean and ANOVA was used determine the variance in means.

Results: Treatment of isolated WT hearts with GSNO before ISO reduced arrhythmias compared to control (15.2 ± 4.2 vs 7.6 ± 2.4; p<0.05) while treating isolated hearts with ISO before GSNO promoted arrhythmias compared to control (38.0 ± 14.5 vs 4.6 ± 1.4; p < 0.05). When S-nitrosylation at C273 was not available in C273S-KI hearts, ISO before GSNO significantly increased arrhythmic events and GSNO before ISO had the similar effects (46.2 ± 23.2 vs 5.5 ± 2.9; 46.5 ± 8.9 vs 4.0 ± 1.7; p<0.05). The ischemic WT hearts and C273S-KI hearts had similar % LVDP recovery after ISO treatment. In addition, we did not detect a difference in the duration of arrhythmic events early during reperfusion in WT and C273S-KI hearts before (175.0 ± 41.3 vs 196.7 ± 38.4 secs) and after (277.5 ± 96.0 vs 336.7 ± 132.5 secs) treatment with ISO.

Conclusion: Our findings suggest that CaMKII S-nitrosylation at Cys 273 may provide some protection against ISO-induced arrhythmias. Our preliminary ischemia experiments showed no significant benefit of Cys 273 presence on post-ischemic LVDP recovery or early arrhythmias, without prior GSNO treatment. We are still testing the hypothesis that pre-treatment with GSNO before ischemia will provide protection in WT, but not in the C273S knock-in mouse. If that hypothesis is supported, S-nitrosylation of CaMKIIδ at Cvs 273 may be a more generally protective event under β-adrenergic and ischemia-reperfusion stress.