Abstract
Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is increased in heart failure (HF), a syndrome characterized by markedly increased risk of arrhythmia. Activation of CaMKII increases peak L-type Ca(2+) current (I(Ca)) and slows I(Ca) inactivation. Whether these events are linked mechanistically is unknown. I(Ca) was recorded in acutely dissociated subepicardial and subendocardial murine left ventricular (LV) myocytes using the whole cell patch clamp method. Pressure overload heart failure was induced by surgical constriction of the thoracic aorta. I(Ca) density was significantly larger in subepicardial myocytes than in subendocardial/myocytes. Similar patterns were observed in the cell surface expression of alpha1c, the channel pore-forming subunit. In failing LV, I(Ca) density was increased proportionately in both cell types, and the time course of I(Ca) inactivation was slowed. This typical pattern of changes suggested a role of CaMKII. Consistent with this, measurements of CaMKII activity revealed a 2-3-fold increase (p < 0.05) in failing LV. To test for a causal link, we measured frequency-dependent I(Ca) facilitation. In HF myocytes, this CaMKII-dependent process could not be induced, suggesting already maximal activation. Internal application of active CaMKII in failing myocytes did not elicit changes in I(Ca). Finally, CaMKII inhibition by internal diffusion of a specific peptide inhibitor reduced I(Ca) density and inactivation time course to similar levels in control and HF myocytes. I(Ca) density manifests a significant transmural gradient, and this gradient is preserved in heart failure. Activation of CaMKII, a known pro-arrhythmic molecule, is a major contributor to I(Ca) remodeling in load-induced heart failure.
Highlights
Mortality associated with this disease [1]
We found that increases peak L-type Ca2ϩ current (ICa) density was significantly larger in SEP than in SEN myocytes (Fig. 1)
There were no significant differences in current decay kinetics or in the voltage dependence of ICa activation between cells isolated from these two regions of left ventricular (LV)
Summary
Severe Thoracic Aortic Banding—Increased pressure in the proximal aorta was induced by means of severe thoracic aortic banding (sTAB) [12] according to protocols approved by the institution’s Animal Care and Use Committee. Ca2ϩ/CaMKII Activity Measurement—Acutely isolated myocytes were lysed in cell extraction buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, Mini-Complete protease inhibitor mixture (Roche Applied Science), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich)). Cell lysates were incubated in reaction buffer (250 mM Tris-HCl, pH 7.5, 50 mM MgCl2, 2.5 mM dithiothreitol, 5 mM CaCl2, 5 M calmodulin, 0.1 mg/ml bovine serum albumin, 0.5 mM ATP) containing the biotinylated CaMKII peptide substrate and 0.5 Ci of [␥-32P]ATP. Surface-Protein Biotinylation Assay—To assay for cell surface protein expression, myocytes isolated from both SEP and SEN regions of sham and sTAB LVs were washed three times on ice with Kϩ-reversal Tyrode buffer (140 mM KCl, 8 mM KHCO3, 2 mM MgCl2, and 0.4 mM KH2PO4, pH 8.0). Mann-Whitney Rank Sum tests were performed if tests for normality or equal variance failed
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