Posttranslational modifications of calcium/calmodulin-dependent protein kinase IIdelta and its downstream signaling in human failing hearts

BACKGROUND: In human failing hearts (HF) of different origin (coronary artery disease-CAD, dilated-DCM, restrictive and hypertrophic cardiomyopathy-OTHER), we investigated the active forms of Ca2+/calmodulin-dependent protein kinase IIdelta (p-Thr287-CaMKIIdelta, oxMet281/282-CaMKIIdelta) and their role in phenotypes of the disease. METHODS AND RESULTS: Although basic diagnostic and clinical markers indicating the attenuated cardiac contractility and remodeling were comparable in HF groups, CaMKIIdelta-mediated axis was different. P-Thr287-CaMKIIdelta was unaltered in CAD group, whereas it was upregulated in non-ischemic cardiomyopathic groups. No correlation between the upregulated p-Thr287-CaMKIIdelta and QT interval prolongation was detected. Unlike in DCM, oxMet281/282-CaMKIIdelta did not differ among HF groups. Independently of CaMKIIdelta phosphorylation/oxidation, activation of its downstreams-phospholamban and cardiac myosin binding protein-C was significantly downregulated supporting both diminished cardiac lusitropy and inotropy in all hearts. Content of sarcoplasmic reticulum Ca2+-ATPase 2a in all HF was unchanged. Protein phosphatase1beta was upregulated in CAD and DCM only, while 2A did not differ among groups. CONCLUSION: This is the first demonstration that the posttranslational activation of CaMKIIdelta differs in HF depending on etiology. Lower levels of downstream molecular targets of CaMKIIdelta do not correlate with either activation of CaMKIIdelta or the expression of major protein phosphatases in the HF. Thus, it is unlikely that these mechanisms exclusively underlie failing of the heart

Hearts from Mice Fed a Non-Obesogenic High-Fat Diet Exhibit Changes in Their Oxidative State, Calcium and Mitochondria in Parallel with Increased Susceptibility to Reperfusion Injury

High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown.To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury.Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet.This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Novel, non-conventional pathways of necroptosis in the heart and other organs: Molecular mechanisms, regulation and inter-organelle interplay

Necroptosis, a cell death modality that is defined as a necrosis-like cell death depending on the receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL), has been found to underlie the injury of various organs. Nevertheless, the molecular background of this cell loss seems to also involve, at least under certain circumstances, some novel axes, such as RIPK3–PGAM5–Drp1 (mitochondrial protein phosphatase 5–dynamin-related protein 1), RIPK3–CaMKII (Ca2+/calmodulin-dependent protein kinase II) and RIPK3–JNK–BNIP3 (c-Jun N-terminal kinase–BCL2 Interacting Protein 3). In addition, endoplasmic reticulum stress and oxidative stress via the higher production of reactive oxygen species produced by the mitochondrial enzymes and the enzymes of the plasma membrane have been implicated in necroptosis, thereby depicting an inter-organelle interplay in the mechanisms of this cell death. However, the role and relationship between these novel non-conventional signalling and the well-accepted canonical pathway in terms of tissue- and/or disease-specific prioritisation is completely unknown. In this review, we provide current knowledge on some necroptotic pathways being not directly associated with RIPK3–MLKL execution and report studies showing the role of respective microRNAs in the regulation of necroptotic injury in the heart and in some other tissues having a high expression of the pro-necroptotic proteins

Oxidative activation of CaMKIIdelta in acute myocardial ischemia/reperfusion injury: A role of angiotensin AT receptor-NOX2 signaling axis

During ischemia/reperfusion (IR), increased activation of angiotensin AT1 receptors recruits NADPH oxidase 2 (NOX2) which contributes to oxidative stress. It is unknown whether this stimulus can induce oxidative activation of Ca2+/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta) leading into the aggravation of cardiac function and whether these effects can be prevented by angiotensin AT1 receptors blockade. Losartan, a selective AT1 blocker, was used. Its effects were compared with effects of KN-93, an inhibitor of CaMKIIdelta. Global IR was induced in Langendorff-perfused rat hearts. Protein expression was evaluated by immunoblotting and lipoperoxidation was measured by TBARS assay. Losartan improved LVDP recovery by 25%; however, it didn't reduce reperfusion arrhythmias. Oxidized CaMKIIdelta (oxCaMKIIdelta) was downregulated at the end of reperfusion compared to before ischemia and losartan did not change these levels. Phosphorylation of CaMKIIdelta mirrored the pattern of changes in oxCaMKIIdelta levels. Losartan did not prevent the higher lipoperoxidation due to IR and did not influence NOX2 expression. Inhibition of CaMKII ameliorated cardiac IR injury; however, this was not accompanied with changes in the levels of either active form of CaMKIIdelta in comparison to the angiotensin AT1 receptor blockade. In spite of no changes of oxCaMKIIdelta, increased cardiac recovery of either therapy was abolished when combined together. This study showed that oxidative activation of CaMKIIdelta is not elevated at the end of R phase. NOX2-oxCAMKIIdelta signaling is unlikely to be involved in cardioprotective action of angiotensin AT1 receptor blockade which is partially abolished by concomitant CaMKII inhibition

Effect of Isoproterenol on Tissue Defense Enzymes, Hemodynamic and Left Ventricular Contractile Function in Rats

Present study investigated the effects of isoproterenol-induced oxidative stress on hemodynamic and ventricular functions in rats. Subcutaneous injections of isoproterenol (85 mg/kg for two consecutive days at 24 h interval) significantly decreased myocardial antioxidant enzymes; superoxide dismutase, catalase and glutathione peroxidase in heart. Isoproterenol-induced oxidative stress was also evidenced by significant depletion of reduced glutathione and increased formation of lipid peroxidation product, thiobarbituric acid reactive substances along with depletion of myocyte injury specific marker enzymes; creatine phosphokinase isoenzyme and lactate dehydrogenase. The deleterious outcome of oxidative stress on hemodyanmic parameters and ventricular function were further evidenced by decreased systolic, diastolic and mean arterial blood pressure, heart rate, ventricular contractility; [(+)LVdP/dt] and relaxation; [(−)LVdP/dt], along with an increased left ventricular end diastolic pressure (LVEDP). Subsequent to changes in heart rate and arterial pressure, isoproterenol also decreased rate pressure product. Present study findings clearly demonstrate the detrimental outcome of isoproterenol induced-oxidative stress on cardiac function and tissue antioxidant defense and substantiate its suitability as an animal model for the evaluation of cardioprotective agents