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Rationale: Heart failure (HF) is known to be associated with increased Ca 2؉ /calmodulin-dependent protein kinase (CaMK)II expression and activity. There is still controversial discussion about the functional role of CaMKII in HF. Moreover, CaMKII inhibition has never been investigated in human myocardium.Objective: We sought to investigate detailed CaMKII␦ expression in end-stage failing human hearts (dilated and ischemic cardiomyopathy) and the functional effects of CaMKII inhibition on contractility. Methods and Results:Expression analysis revealed that CaMKII␦, both cytosolic ␦ C and nuclear ␦ B splice variants, were significantly increased in both right and left ventricles from patients with dilated or ischemic cardiomyopathy versus nonfailing. Experiments with isometrically twitching trabeculae revealed significantly improved force frequency relationships in the presence of CaMKII inhibitors (KN-93 and AIP). Increased postrest twitches after CaMKII inhibition indicated an improved sarcoplasmic reticulum (SR) Ca 2؉ loading. This was confirmed in isolated myocytes by a reduced SR Ca 2؉ spark frequency and hence SR Ca 2؉ leak, resulting in increased SR Ca 2؉ load when inhibiting CaMKII. Ryanodine receptor type 2 phosphorylation at Ser2815, which is known to be phosphorylated by CaMKII thereby contributing to SR Ca 2؉ leak, was found to be markedly reduced in KN-93-treated trabeculae. Interestingly, CaMKII inhibition did not influence contractility in nonfailing sheep trabeculae.Conclusions: The present study shows for the first time that CaMKII inhibition acutely improves contractility in human HF where CaMKII␦ expression is increased. The mechanism proposed consists of a reduced SR Ca Key Words: Ca 2ϩ /calmodulin-dependent kinase II Ⅲ heart failure Ⅲ contractility Ⅲ calcium Ⅲ sarcoplasmic reticulum Ca 2ϩ leak Ⅲ ryanodine receptor H eart failure (HF) is accompanied by systolic and diastolic contractile dysfunction caused by abnormalities in intracellular Ca 2ϩ handling and structural remodeling. Several targets associated with the remodeling processes have been identified. The sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (SERCA) protein levels have been reported to be downregulated and paralleled by a reduced SR Ca 2ϩ uptake capacity in the human failing heart. [1][2][3] In contrast, the sarcolemmal Na ϩ /Ca 2ϩ exchanger protein expression and activity were found to be increased thereby even more effectively competing with the reduced SERCA activity for cytosolic Ca 2ϩ -removal. 3,4 The net effect is an impaired SR Ca 2ϩ loading, which leads to smaller intracellular Ca 2ϩ transients and elevated diastolic Ca 2ϩ levels in HF. 5 Thus, impaired contractility with reduced contractile force and diastolic dysfunction are well-accepted determinants in HF. 6 Intracellular Ca 2ϩ homeostasis of cardiac myocytes is also regulated by phosphorylation of several key Ca 2ϩ -handling proteins. An important regulatory kinase is the Ca 2ϩ /calmodulin-dependent protein kinase (CaMK)II. 7 It is a serine/ threonine protein kinase t...

Phosphatase inhibitor-1 (I-1) is a distal amplifier element of β-adrenergic signaling that functions by preventing dephosphorylation of downstream targets. I-1 is downregulated in human failing hearts, while overexpression of a constitutively active mutant form (I-1c) reverses contractile dysfunction in mouse failing hearts, suggesting that I-1c may be a candidate for gene therapy. We generated mice with conditional cardiomyocyterestricted expression of I-1c (referred to herein as dTG I-1c mice) on an I-1-deficient background. Young adult dTG I-1c mice exhibited enhanced cardiac contractility but exaggerated contractile dysfunction and ventricular dilation upon catecholamine infusion. Telemetric ECG recordings revealed typical catecholamine-induced ventricular tachycardia and sudden death. Doxycycline feeding switched off expression of cardiomyocyterestricted I-1c and reversed all abnormalities. Hearts from dTG I-1c mice showed hyperphosphorylation of phospholamban and the ryanodine receptor, and this was associated with an increased number of catecholamine-induced Ca 2+ sparks in isolated myocytes. Aged dTG I-1c mice spontaneously developed a cardiomyopathic phenotype. These data were confirmed in a second independent transgenic mouse line, expressing a full-length I-1 mutant that could not be phosphorylated and thereby inactivated by PKC-α (I-1 S67A ). In conclusion, conditional expression of I-1c or I-1 S67A enhanced steady-state phosphorylation of 2 key Ca 2+ -regulating sarcoplasmic reticulum enzymes. This was associated with increased contractile function in young animals but also with arrhythmias and cardiomyopathy after adrenergic stress and with aging. These data should be considered in the development of novel therapies for heart failure. IntroductionHeart failure is among the most frequent causes of morbidity and mortality worldwide and is, despite improved treatment options, associated with poor prognosis. Current treatment with angiotensin-converting enzyme inhibitors, aldosterone receptor antagonists, and beta blockers is suboptimal, with the 5-year survival rate being less than 50%. New drug principles targeting neurohumoral activation mechanisms, such as antagonists of endothelin receptors, TNF-α or IL-6, and statins, failed to improve survival in clinical studies. Thus, new approaches are needed, and an attractive one is to target the abnormal function of cardiomyocytes in failing hearts directly (as opposed to the more indirect affection by neurohumoral blockade).Two of the best studied alterations of failing myocyte function are (a) desensitization of the β-adrenergic signaling system (1, 2) and (b) alterations of intracellular Ca 2+ handling (3, 4). The latter include decreased diastolic sarcoplasmic reticulum (SR) Ca 2+ uptake via the SR Ca 2+ ATPase (SERCA2a) and relatively increased

AS105 is a novel, highly potent ATP-competitive CaMKII inhibitor. In vitro, it effectively reduced SR Ca-leak, thus improving SR Ca-accumulation and reducing cellular arrhythmogenic correlates, without negatively influencing excitation-contraction coupling. These findings further validate CaMKII as a key target in cardiovascular disease, implicated by genetic, allosteric inhibitors, and pseudo-substrate inhibitors.