Background Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)δC mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 μm−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

AimsThe EMPA‐REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+‐dependent activation of Ca2+/calmodulin‐dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII‐ activity and improves Ca2+‐handling in human and murine ventricular myocytes.Methods and resultsMyocytes from wild‐type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 μmol/L) or vehicle. CaMKII activity was assessed by CaMKII–histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+]i was measured using Na+/Ca2+‐exchanger (NCX) currents (whole‐cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII‐dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 μm−1 s−1, P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0: 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+‐handling but significantly reduced [Na+]i.ConclusionsWe show for the first time that empagliflozin reduces CaMKII activity and CaMKII‐dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

Aims To determine the characteristics of the late Na current (INaL) and its arrhythmogenic potential in the progression of pressure-induced heart disease. Methods and Results Transverse aortic constriction (TAC) was used to induce pressure overload in mice. After one week the hearts developed isolated hypertrophy with preserved systolic contractility. In patch-clamp experiments both, INaL and the action potential duration (APD90) were unchanged. In contrast, after five weeks animals developed heart failure with prolonged APDs and the slowed INaL decay time which could be normalized by addition of the INaL inhibitor ranolazine (Ran) or by the Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor AIP. Accordingly the APD90 could be significantly abbreviated by Ran, tetrodotoxin and the CaMKII inhibitor AIP. Isoproterenol increased the number of delayed afterdepolarizations (DAD) in myocytes from failing but not sham hearts. Application of either Ran or AIP prevented the occurrence of DADs. Moreover, the incidence of triggered activity was significantly increased in TAC myocytes and was largely prevented by Ran and AIP. Western blot analyses indicate that increased CaMKII activity and a hyperphosphorylation of the Nav1.5 at the CaMKII phosphorylation site (Ser571) paralleled our functional observations five weeks after TAC surgery. Conclusion In pressure overload-induced heart failure a CaMKII-dependent augmentation of INaL plays a crucial role in the AP prolongation and generation of cellular arrhythmogenic triggers, which cannot yet be found in early and still compensated hypertrophy. Inhibition of INaL and CaMKII exert potent antiarrhythmic effects and might therefore be of potential therapeutic interest.

Objective Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). Rationale We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca]i) handling. Methods Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ−/−, KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 µmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS]i) and [Ca]i handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 µmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 µmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. Results DOX increased [ROS]i and led to significant diastolic [Ca]i overload in rat myocytes. This was associated with reduced [Ca]i transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca]i overload after DOX superfusion and led to partially restored [Ca]i transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca]i overload and Ca spark frequency. Conclusions DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca]i homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca]i. In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.

Conclusions:We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

It is increasingly evident that redox-dependent modifications in cellular proteins and signaling pathways (or redox signaling) play important roles in many aspects of cardiac hypertrophy. Indeed, these redox modifications may be intricately linked with the process of hypertrophy wherein there is not only a significant increase in myocardial O2 consumption but also important alterations in metabolic processes and in the local generation of O2-derived reactive species (ROS) that modulate and/or amplify cell signaling pathways. This article reviews our current knowledge of redox signaling pathways and their roles in cardiac hypertrophy. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

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