Improvement of cardiomyocyte function by a novel pyrimidine-based CaMKII-inhibitor.

OBJECTIVE: Pathologically increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the associated Ca2+-leak from the sarcoplasmic reticulum are recognized to be important novel pharmacotherapeutic targets in heart failure and cardiac arrhythmias. However, CaMKII-inhibitory compounds for therapeutic use are still lacking. We now report on the cellular and molecular effects of a novel pyrimidine-based CaMKII inhibitor developed towards clinical use. METHODS AND RESULTS: Our findings demonstrate that AS105 is a high-affinity ATP-competitive CaMKII-inhibitor that by its mode of action is also effective against autophosphorylated CaMKII (in contrast to the commonly used allosteric CaMKII-inhibitor KN-93). In isolated atrial cardiomyocytes from human donors and ventricular myocytes from CaMKIIδC-overexpressing mice with heart failure, AS105 effectively reduced diastolic SR Ca2+ leak by 38% to 65% as measured by Ca2+-sparks or tetracaine-sensitive shift in [Ca2+]i. Consistent with this, we found that AS105 suppressed arrhythmogenic spontaneous cardiomyocyte Ca2+-release (by 53%). Also, the ability of the SR to accumulate Ca2+ was enhanced by AS105, as indicated by improved post-rest potentiation of Ca2+-transient amplitudes and increased SR Ca2+-content in the murine cells. Accordingly, these cells had improved systolic Ca2+-transient amplitudes and contractility during basal stimulation. Importantly, CaMKII inhibition did not compromise systolic fractional Ca2+-release, diastolic SR Ca2+-reuptake via SERCA2a or Ca2+-extrusion via NCX. CONCLUSION: AS105 is a novel, highly potent ATP-competitive CaMKII inhibitor. In vitro, it effectively reduced SR Ca2+-leak, thus improving SR Ca2+-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.

CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation.

RATIONALE: Although research suggests that diastolic Ca(2+) levels might be increased in atrial fibrillation (AF), this hypothesis has never been tested. Diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR) might increase diastolic Ca(2+) levels and play a role in triggering or maintaining AF by transient inward currents through Na(+)/Ca(2+) exchange. In ventricular myocardium, ryanodine receptor type 2 (RyR2) phosphorylation by Ca(2+)/calmodulin-dependent protein kinase (CaMK)II is emerging as an important mechanism for SR Ca(2+) leak. OBJECTIVE: We tested the hypothesis that CaMKII-dependent diastolic SR Ca(2+) leak and elevated diastolic Ca(2+) levels occurs in atrial myocardium of patients with AF. METHODS AND RESULTS: We used isolated human right atrial myocytes from patients with AF versus sinus rhythm and found CaMKII expression to be increased by 40+/-14% (P<0.05), as well as CaMKII phosphorylation by 33+/-12% (P<0.05). This was accompanied by a significantly increased RyR2 phosphorylation at the CaMKII site (Ser2814) by 110+/-53%. Furthermore, cytosolic Ca(2+) levels were elevated during diastole (229+/-20 versus 164+/-8 nmol/L, P<0.05). Most likely, this resulted from an increased SR Ca(2+) leak in AF (P<0.05), which was not attributable to higher SR Ca(2+) load. Tetracaine experiments confirmed that SR Ca(2+) leak through RyR2 leads to the elevated diastolic Ca(2+) level. CaMKII inhibition normalized SR Ca(2+) leak and cytosolic Ca(2+) levels without changes in L-type Ca(2+) current. CONCLUSION: Increased CaMKII-dependent phosphorylation of RyR2 leads to increased SR Ca(2+) leak in human AF, causing elevated cytosolic Ca(2+) levels, thereby providing a potential arrhythmogenic substrate that could trigger or maintain AF.

Inhibition of elevated Ca2+/calmodulin-dependent protein kinase II improves contractility in human failing myocardium.

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 CaMKIIδ 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 CaMKIIδ, both cytosolic δ(C) and nuclear δ(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 CaMKIIδ expression is increased. The mechanism proposed consists of a reduced SR Ca(2+) leak and consequently increased SR Ca(2+) load. Thus, CaMKII inhibition appears to be a possible therapeutic option for patients with HF and merits further investigation.

Midterm results after treatment of gram-positive deep sternal wound infections with daptomycin for cardiac surgery patients.

Daptomycin in combination with surgical therapy has shown to be effective for treatment of deep sternal wound infection in cardiac surgery. However, till now midterm results in terms of re-infection or re-operation in patients who were successfully treated with daptomycin for gram-positive deep sternal wound infection are not published. Herein, we present midterm results in patients treated successfully with daptomycin after cardiac surgery.

Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging.

Phosphatase inhibitor-1 (I-1) is a distal amplifier element of beta-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 cardiomyocyte-restricted expression of I-1c (referred to herein as dTGI-1c mice) on an I-1-deficient background. Young adult dTGI-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 cardiomyocyte-restricted I-1c and reversed all abnormalities. Hearts from dTGI-1c mice showed hyperphosphorylation of phospholamban and the ryanodine receptor, and this was associated with an increased number of catecholamine-induced Ca2+ sparks in isolated myocytes. Aged dTGI-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-alpha (I-1S67A). In conclusion, conditional expression of I-1c or I-1S67A enhanced steady-state phosphorylation of 2 key Ca2+-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.