Abstract
Introduction: Small conductance calcium (Ca)-activated K (SK) channels present in the plasmalemma of ventricular cardiomyocytes (VCMs) are dormant in health and become functional in cardiac disease. We recently showed that SK channels contribute to VCM repolarization in a rat model of hypertrophy induced by thoracic aortic banding (TAB), despite reduced expression levels, suggestive of increased activity of the channels. However, the mechanisms responsible for disease-associated functional upregulation of the channels are yet to be defined. Hypothesis: Functional recruitment of SK channels in cardiac hypertrophy is caused by enhancement of adrenergic signaling cascades. Methods: The effects of α1 adrenergic stimulation by phenylephrine (10 μM) + propranolol (100 μM) were studied in patch-clamped rat VCMs overexpressing rat SK2 after 48 hrs in culture. Calcium transients were recorded simultaneously using confocal microscopy and were used as a ramp [Ca] to assess possible changes in Ca sensitivity of the channel. Phosphorylation of SKs by Ser/Thr and tyrosine kinases, phosphorylation of Calmodulin at Thr-79, expression levels and phosphorylation of CaMKII and tyrosine kinase Pyk2 were assessed in cultured rat VCMs and freshly isolated VCMs from TABs and Shams using Western blot analysis. Results: WB analysis showed increased Ser/Thr and Tyr phosphorylation of SK2 in TABs. Alpha1 stimulation of rat VCMs overexpressing rat SK2 mimicked this pattern, increasing I SK at high voltages in parallel with the increase in Ca transient amplitude and reduction in Ca sensitivity of the channels. Overexpression of Pyk2 inhibitor CRNK did not restore I SK despite reduction in Ca transient amplitude because of attenuating α1-agonist mediated decrease in Ca sensitivity of SKs. Application of CaMKII inhibitor KN93 fully reversed α1-mediated I SK increase at high voltages without affecting Ca sensitivity of the channel. Conclusion: Upregulation of SKs in hypertrophic hearts is likely caused by CaMKII-dependent phosphorylation which reduces voltage-dependent block of the channels. Pyk2 inhibition may present therapeutic potential by attenuating negative effects on SK Ca sensitivity thereby increasing repolarization reserve diminished in cardiac disease.
Published Version
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