Rationale: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors. Objective: Establishment of mouse models, which allow the quantification of the glutathione redox potential ( E GSH ) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2). Methods and Results: We generated transgenic mice with cardiac myocyte–restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward H 2 O 2 , diamide, and dithiothreitol was titrated and used to determine the E GSH in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct E GSH were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction. Conclusions: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of E GSH in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.

Background Considerable evidence suggests that CaMKII overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive β-adrenoceptor (β-AR) stimulation. Recent studies indicate a significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in HF. In this study we investigated the effect of chronic β-AR blocker treatment on CaMKII activity in human and experimental HF. Methods and Results Immunoblot analysis of myocardium from end stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with β-AR blockers revealed no difference in CaMKII activity when compared to non-β-AR-blocker-treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKIIδC transgenic mice were treated with the β1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29, p<0.001), attenuated the development of cardiac hypertrophy (−14±6% heart weight/tibia length, p<0.05) and strongly reduced ventricular arrhythmias (−70±22% PVCs, p<0.05). On a molecular level, metoprolol expectedly decreased PKA dependent phospholamban (PLN) and ryanodine receptor 2 (RyR2) phosphorylation (−42±9% for P-PLN-S16 and −22±7% for P-RyR2-S2808, p<0.05). However, this was neither paralleled by a reduction in CaMKII autophosphorylation, oxidation and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by β-AR blocker treatment was confirmed in healthy wildtype mice receiving metoprolol. Conclusions Chronic β-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggests that the molecular effects of β-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may therefore further improve HF therapy in addition to β-AR blockade.