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Chronic activation of Ca 2 + /calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca 2 + leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca 2 + leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation. [ABSTRACT FROM AUTHOR]

Abstract: The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) targets a number of Ca2+ homeostatic proteins and regulates gene transcription. Many of the substrates phosphorylated by CaMKII are also substrates for protein kinase A (PKA), the best known downstream effector of β-adrenergic receptor (β-AR) signaling. While PKA and CaMKII are conventionally considered to transduce signals through separate pathways, there is a body of evidence suggesting that CaMKII is activated in response to β-AR stimulation and that some of the downstream effects of β-AR stimulation are actually mediated by CaMKII. The signaling pathway through which β-AR stimulation activates CaMKII, in parallel with or downstream of PKA, is not well-defined. This review considers the evidence for and mechanisms by which CaMKII is activated in response to β-AR stimulation. In addition the potential role of CaMKII in β-AR regulation of cardiac function is considered. Notably, although many CaMKII targets (e.g., phospholamban or the ryanodine receptor) are central to the regulation of Ca2+ handling, and effects of CaMKII on Ca2+ handling are detectable, inhibition or gene deletion of CaMKII has relatively little effect on the acute physiological contractile response to β-AR. On the other hand CaMKII expression and activity are increased in heart failure, a pathophysiological condition characterized by chronic stimulation of cardiac β-ARs. Blockade of β-ARs is an accepted therapy for treatment of chronic heart failure although the rationale for its beneficial effects in cardiomyocytes is uncertain. There is growing evidence that inhibition or gene deletion of CaMKII also has a significant beneficial impact on the development of heart failure. The possibility that excessive β-AR stimulation is detrimental because of its effects on CaMKII mediated Ca2+ handling disturbances (e.g., ryanodine receptor phosphorylation and diastolic SR Ca2+ leak) is an intriguing hypothesis that merits future consideration. [Copyright &y& Elsevier]

Abstract: The sustained positive inotropic effect of α-adrenoceptor agonists in the heart is associated with a small increase in intracellular Ca2+ transients together with a larger sensitization of myofilaments to Ca2+. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) could contribute to this effect, either by affecting the Ca2+ release (ryanodine receptor) or by an uptake mechanism (via phospholamban [PLB] and SR Ca2+ ATPase). Here we examined the role of CaMKII in the positive inotropic effect of the α-adrenoceptor agonist phenylephrine in left atria isolated from a genetic mouse model of cardiac CaMKII inhibition (AC3-I). Compared to atria from wild-type (WT) or AC3-C (scrambled peptide), AC3-I atria showed the following abnormalities. PLB phosphorylation at Thr17, a known CaMKII target, was significantly lower (∼20%). Post-rest (30 s, 1 Hz, 37 °C) potentiation of force was absent (AC3-C, 190% of pre-rest amplitude). Basal force was ∼20% lower at 1.8 mM Ca2+, but normal at high Ca2+ concentration (>4.5 mM). The maximal positive inotropic effect of phenylephrine, which was more pronounced at low frequencies in WT and AC3-C atria, lost its frequency dependence (1 Hz to 8 Hz). Thus, the effect of phenylephrine was reduced by ∼50% at 1 Hz, but was normal at 8 Hz. All three groups showed a negative force–frequency relation, and did not differ in the frequency-dependent acceleration of relaxation. Our data indicate a role of CaMKII in post-rest potentiation and the positive inotropic effect of α-adrenergic stimulation at low frequencies. [Copyright &y& Elsevier]

Abstract Physiologically, following myocardial infarction (MI), retinoid levels elevate locally in the infarcted area. Whereas therapeutic systemic application of retinoids was shown to reduce the progression of ventricular dilatation and the onset of heart failure, the role of acute physiologically increased retinoids in the infarction zone is unknown to date. To reveal the role of local retinoids in the MI zone is the central aim of this study. Using human cell culture and co-culture models for hypoxia as well as various assays systems, lentivirus-based transgene expression, in silico molecular docking studies, and an MI model in rats, we analysed the impact of the retinoid all-trans retinoic acid (ATRA) on cell signalling, cell viability, tissue survival, heart function, and MI-induced death in rats. Based on our results, ATRA-mediated signalling does aggravate the MI phenotype (e.g. 2.5-fold increased mortality compared to control), whereas 5′-methoxyleoligin (5ML), a new agent which interferes with ATRA-signalling rescues the ATRA-dependent phenotype. On the molecular level, ATRA signalling causes induction of TXNIP, a potent inhibitor of the physiological antioxidant thioredoxin (TRX1) and sensitizes cells to necrotic cell death upon hypoxia. 5ML-mediated prevention of ATRA effects were shown to be based on the inhibition of cellular ATRA uptake by interference with the cholesterol (and retinol) binding motif of the transmembrane protein STRA6. 5ML-mediated inhibition of ATRA uptake led to a strong reduction of ATRA-dependent gene expression, reduced ROS formation, and protection from necrotic cell death. As 5ML exerted a cardioprotective effect, also independent of its inhibition of cellular ATRA uptake, the agent likely has another cardioprotective property, which may rely on the induction of TRX1 activity. In summary, this is the first study to show i) that local retinoids in the early MI zone may worsen disease outcome, ii) that inhibition of endothelial retinoid uptake using 5ML may constitute a novel treatment strategy, and iii) that targeting endothelial and myocardial retinoid uptake (e.g. via STRA6 inhibition) may constitute a novel treatment target in acute MI. Highlights • Retinoids in the myocardial infraction zone have adverse effects on MI outcome. • Inhibition of cellular retinoid uptake reduces cell death under hypoxic conditions. • Endothelial cell death enhances cardiomyocyte death under hypoxic conditions. • 5′-Methoxyleoligin inhibits cellular retinoid uptake by binding to STRA6. • 5′-Methoxyleoligin protects from hypoxia and ATRA-enhanced cell death in MI. [ABSTRACT FROM AUTHOR]

The health benefit through the control of lipid levels in hyperlipidaemic individuals is evident from a large number of studies. The pharmacological options to achieve this goal shall be as specific and personalized as the reasons for and co-factors of hyperlipidaemia. It was the goal of this study to reveal the impact of leoligin on cholesterol levels and to define its mechanism of action. Oral application of leoligin in ApoE −/− mice led to significantly reduced total serum cholesterol levels and a reduction in postprandial blood glucose peak levels. In the absence of biochemical signs of toxicity, leoligin treatment resulted in reduced weight gain in mice. The effects of leoligin on serum cholesterol levels may be due to a direct inhibition of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) by a unique, non-statin-like binding mode. Postprandial serum glucose peaks may be reduced by a mild peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonistic activity of leoligin. No effect on atherosclerotic plaque size was observed. As a non-toxic, cholesterol-, peak glucose-, and weight gain-lowering compound, leoligin continues to fulfil characteristics of a potential agent for the treatment of cardiovascular disease (CVD). The counterregulatory overexpression of hepatic HMGCR in leoligin treated animals possibly explains the missing permanent anti-atherosclerotic effect. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the possible role of tubulin βII, a cytoskeletal protein, in regulation of mitochondrial oxidative phosphorylation and energy fluxes in heart cells. This isotype of tubulin is closely associated with mitochondria and co-expressed with mitochondrial creatine kinase (MtCK). It can be rapidly removed by mild proteolytic treatment of permeabilized cardiomyocytes in the absence of stimulatory effect of cytochrome c, that demonstrating the intactness of the outer mitochondrial membrane. Contrary to isolated mitochondria, in permeabilized cardiomyocytes (in situ mitochondria) the addition of pyruvate kinase (PK) and phosphoenolpyruvate (PEP) in the presence of creatine had no effect on the rate of respiration controlled by activated MtCK, showing limited permeability of voltage-dependent anion channel (VDAC) in mitochondrial outer membrane (MOM) for ADP regenerated by MtCK. Under normal conditions, this effect can be considered as one of the most sensitive tests of the intactness of cardiomyocytes and controlled permeability of MOM for adenine nucleotides. However, proteolytic treatment of permeabilized cardiomyocytes with trypsin, by removing mitochondrial βII tubulin, induces high sensitivity of MtCK-regulated respiration to PK–PEP, significantly changes its kinetics and the affinity to exogenous ADP. MtCK coupled to ATP synthasome and to VDAC controlled by tubulin βII provides functional compartmentation of ATP in mitochondria and energy channeling into cytoplasm via phosphotransfer network. Therefore, direct transfer of mitochondrially produced ATP to sites of its utilization is largely avoided under physiological conditions, but may occur in pathology when mitochondria are damaged. This article is part of a Special Issue entitled ‘‘Local Signaling in Myocytes’’. [Copyright &y& Elsevier]

Abstract: The Na+–Ca2+ exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. β-Adrenergic receptor (β-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca2+/calmodulin-dependent protein kinase II (CaMKIIδc) null mouse, we demonstrate that β-AR-stimulated Ncx1 upregulation is dependent on CaMKII. β-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that β-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically β-AR-stimulated heart. [Copyright &y& Elsevier]