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Aims Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. Methods and results We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4−/−) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4−/− mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P Conclusion Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.

Aims Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation. Methods and results General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis. Conclusion By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes.

Aims In heart failure (HF), enhanced persistent Na+ current (I-NaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. However, the underlying regulatory mechanisms remain unclear. Our aim was to potentially investigate the regulation and electrophysiological contribution of neuronal sodium channel Na(V)1.8 in failing human heart and eventually to reveal a novel anti-arrhythmic therapy Methods and results By western blot, we found that Na(V)1.8 protein expression is significantly up-regulated, while of the predominant cardiac isoform Na(V)1.5 is inversely reduced in human HF. Furthermore, to investigate the relation of Na(V)1.8 regulation with the cellular proarrhythmic events, we performed comprehensive electrophysiology recordings and explore the effect of Na(V)1.8 on I-NaL, action potential duration (APD), Ca2+ spark frequency, and arrhythmia induction in human failing cardiomyocytes. Na(V)1.8 inhibition with the specific blockers A-803467 and PF-01247324 decreased I-NaL, abbreviated APD and reduced cellular-spontaneous Ca2+-release and proarrhythmic events in human failing cardiomyocytes. Consistently, in mouse cardiomyocytes stressed with isoproterenol, pharmacologic inhibition and genetically knockout of Na(V)1.8 (SCN10A(-/-)), were associated with reduced I-NaL and abbreviated APD Conclusion We provide first evidence of differential regulation of Na(V)1.8 and Na(V)1.5 in the failing human myocardium and their contribution to arrhythmogenesis due to generation of I-NaL. We propose inhibition of Na(V)1.8 thus constitutes a promising novel approach for selective anti-arrhythmic therapy in HF.

Aims Cardiac angiogenesis is an important determinant of heart failure. We examined the hypothesis that protein tyrosine phosphatase (PTP)-1B, a negative regulator of vascular endothelial growth factor (VEGF) receptor-2 activation, is causally involved in the cardiac microvasculature rarefaction during hypertrophy and that deletion of PTP1B in endothelial cells prevents the development of heart failure. Methods and results Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in mice with endothelial-specific deletion of PTP1B (End.PTP1B-KO) and controls (End.PTP1B-WT). Survival up to 20 weeks after TAC was significantly improved in mice lacking endothelial PTP1B. Serial echocardiography revealed a better systolic pump function, less pronounced cardiac hypertrophy, and left ventricular dilation compared with End.PTP1B-WT controls. Histologically, banded hearts from End.PTP1B-KO mice exhibited increased numbers of PCNA-positive, proliferating endothelial cells resulting in preserved cardiac capillary density and improved perfusion as well as reduced hypoxia, apoptotic cell death, and fibrosis. Increased relative VEGFR2 and ERK1/2 phosphorylation and greater eNOS expression were present in the hearts of End.PTP1B-KO mice. The absence of PTP1B in endothelial cells also promoted neovascularization following peripheral ischaemia, and bone marrow transplantation excluded a major contribution of Tie2-positive haematopoietic cells to the improved angiogenesis in End.PTP1B-KO mice. Increased expression of caveolin-1 as well as reduced NADPH oxidase-4 expression, ROS generation and TGFβ signalling were observed and may have mediated the cardioprotective effects of endothelial PTP1B deletion. Conclusions Endothelial PTP1B deletion improves cardiac VEGF signalling and angiogenesis and protects against chronic afterload-induced heart failure. PTP1B may represent a useful target to preserve cardiac function during hypertrophy.

Aims Enhanced cardiac late Na current (late I Na) and increased sarcoplasmic reticulum (SR)-Ca2+-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late I Na and SR-Ca2+-leak in atrial cardiomyocytes (CMs). Methods and results In murine atrial CMs, SR-Ca2+-leak was increased by the late I Na enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca2+/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late I Na (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca2+-leak. The SR-Ca2+-leak induction by ATX-II was not detected when either the Na+/Ca2+ exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late I Na-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late I Na did not alter Ca2+-transient amplitude or SR-Ca2+-load. However, upon late I Na activation and simultaneous CaMKII inhibition, Ca2+-transient amplitude and SR-Ca2+-load were increased, whereas PKA inhibition reduced Ca2+-transient amplitude and load and additionally slowed Ca2+ elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late I Na, CaMKII, or PKA reduced the SR-Ca2+-leak. Conclusion Late I Na exerts distinct effects on Ca2+ homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late I Na represents a potential approach to attenuate CaMKII activation and decreases SR-Ca2+-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late I Na inhibition.

Aims Methylation of CpG island promoters is a prototypical epigenetic mechanism to stably control gene expression. The aim of this study was to elucidate the contribution of aberrant promoter DNA methylation in pathological endothelial to mesenchymal transition (EndMT) and subsequent cardiac fibrosis. Methods and results In human coronary endothelial cells, TGFβ1 causes aberrant methylation of RASAL1 promoter, increased Ras-GTP activity, and EndMT. In end-stage failing vs. non-failing human myocardium, increased fibrosis was associated with significantly increased RASAL1 promoter methylation, decreased RASAL1 expression, increased Ras-GTP activity, and increased expression of markers of EndMT. In mice with pressure overload due to ascending aortic constriction, BMP7 significantly reduced RASAL1 promoter methylation, increased RASAL1 expression, and decreased EndMT markers as well as decreased cardiac fibrosis. The ten eleven translocation (TET) family enzyme TET3, which demethylates through hydroxymethylation, was significantly decreased in fibrotic mouse hearts, restored with BMP7, and BMP7 effects were absent in coronary endothelial cells with siRNA knockdown of TET3. Conclusion Our study provides proof-in-principle evidence that transcriptional suppression of RASAL1 through aberrant promoter methylation contributes to EndMT and ultimately to progression of cardiac fibrosis. Such aberrant methylation can be reversed through Tet3-mediated hydroxymethylation, which can be specifically induced by BMP7. This may reflect a new treatment strategy to stop cardiac fibrosis.

Aim Activation of the kinase RAF and its downstream targets leads to cardiomyocyte hypertrophy. It has been hypothesized that B-RAF might be the main activator of MEK in various cell types. Therefore, the aim of this study was to investigate the role of B-RAF and its modulating factors in cardiomyocyte hypertrophy. Methods and results Neonatal rat cardiomyocytes were pre-treated with and without the specific B-RAF inhibitor SB590885 and then stimulated with phenylephrine to induce hypertrophy. Inhibition of B-RAF completely impeded the hypertrophic response and led to a significant reduction of MEK1/2 phosphorylation. By applying a eukaryotic cDNA expression screen, based on a dual-luciferase reporter assay for B-RAF activity measurement, we identified RCN1 as a new negative modulator of B-RAF activity. Adenovirus-mediated overexpression of reticulocalbin 1 (RCN1) completely impeded phenylephrine-induced hypertrophy and led to significantly reduced MEK1/2 phosphorylation. Conversely, adenoviral knockdown of RCN1 with a specific synthetic miRNA induced cardiomyocyte hypertrophy and significantly increased MEK1/2 phosphorylation. Conclusions In summary, our results show that the inhibition of B-RAF abolishes cardiomyocyte hypertrophy and we identified RCN1 as novel negative modulator of cardiomyocyte hypertrophy by inhibition of the mitogen-activated protein kinase signalling cascade. Our results show that B-RAF kinase activity is essential for cardiac hypertrophy and RCN1, its newly identified negative regulator, abolishes hypertrophic response of cardiomyocytes in vitro .

Aims The calcineurin and nuclear factor of activated T cells (NFAT) pathway can mediate pro-hypertrophic signalling in the heart. Recently, it has been shown that dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) phosphorylates NFAT, which limits calcineurin/NFAT signal transduction in T cells and hypertrophy in cultured cardiomyocytes. The hypothesis tested in this study was that DYRK1A prevents calcineurin/NFAT-mediated cardiac hypertrophy in vivo . Methods and results In cultured rat cardiomyocytes, adenovirus-mediated overexpression of DYRK1A antagonized calcineurin-mediated nuclear NFAT translocation and the phenylephrine-induced hypertrophic growth response. To test the ability of DYRK1A to reduce hypertrophic cardiac growth in vivo , we created tetracycline-repressible Dyrk1a transgenic mice to avoid the cardiac developmental defects associated with embryonic DYRK1A expression. However, in the mouse model , histological determination of myocyte diameter, heart weight/body weight ratio, and echocardiographic measurements revealed that myocardial expression of DYRK1A failed to reduce hypertrophy induced via aortic banding or co-expression of calcineurin. This discrepancy is explained, at least in part, by insufficient long-term inhibition of NFAT and the activation of DYRK1A-resistant maladaptive genes in vivo . Conclusion Isolated augmentation of DYRK1A can be compensated for in vivo , and this may significantly limit anti-hypertrophic interventions aimed at enhancing DYRK1A activity.

In Western Europe, as in the USA, mortality of cardiovascular disease (CVD) has declined significantly in the last 30 years. Life expectancy for patients with coronary heart disease in the USA increased on average by 3 years between 1970 and 2000.1 Several epidemiological analyses support this positive evolution. An analysis of the underlying causes identifies improvements in quality of care and treatment as a major cause, accounting from 50 up to 75% of the success depending on the study samples, the remainder being accounted for by changes in lifestyle and prevention.2 Research and development in several areas of CVD have contributed to this success. In acute coronary events, the identification and development of efficient and safe thrombolytic agents followed by percutaneous coronary intervention with stent implantation were milestones in the reduction of acute mortality and salvage of myocardium.3–5 Statins have brought a major advance in preventing the progression of atherosclerotic disease and have been recognized for their wide mode of action.6 In heart failure (HF), beta-blockers and ACE-inhibitors have increased life expectancy by a leap.7 Some of these improvements have been the result of a classic bench-to-bedside development of a targeted treatment, such as the statins, others have known a more serendipitous development, such as beta-blockers. In other areas, progress in terms of treatment has been less spectacular and fraught with difficulties. Development of antiarrhythmic agents received a large boost from basic insights into cardiac ion channel structure and function, but translation into pharmacology unveiled unexpected pro-arrhythmia risks of some of the highly specific ion channel blockers in class III.8 Automatic defibrillators have taken an important role in the treatment of life-threatening arrhythmias and save lives, but are not without burden and come at a high cost.9 This latter example is …

Aims Phosphatase inhibitor-1 (I-1) is a conditional amplifier of b-adrenergic signalling downstream of protein kinase A by inhibiting type-1 phosphatases only in its PKA-phosphorylated form. I-1 is downregulated in failing hearts and thus contributes to b-adrenergic desensitization. It is unclear whether this should be viewed as a predominantly adverse or protective response. Methods and results We generated transgenic mice with cardiac-specific I-1 overexpression (I-1-TG) and evaluated cardiac function and responses to catecholamines in mice with targeted disruption of the I-1 gene (I-1-KO). Both groups were compared with their wild-type (WT) littermates. I-1-TG developed cardiac hypertrophy and mild dysfunction which was accompanied by a substantial compensatory increase in PP1 abundance and activity, confounding cause‐effect relationships. I-1-KO had normal heart structure with mildly reduced sensitivity, but unchanged maximal contractile responses to badrenergic stimulation, both in vitro and in vivo. Notably, I-1-KO were partially protected from lethal catecholamine-induced arrhythmias and from hypertrophy and dilation induced by a 7 day infusion with the b-adrenergic agonist isoprenaline. Moreover, I-1-KO exhibited a partially preserved acute badrenergic response after chronic isoprenaline, which was completely absent in similarly treated WT. At the molecular level, I-1-KO showed lower steady-state phosphorylation of the cardiac ryanodine receptor/Ca 2þ release channel and the sarcoplasmic reticulum (SR) Ca 2þ -ATPase-regulating protein phospholamban. These alterations may lower the propensity for diastolic Ca 2þ release and Ca 2þ uptake and thus stabilize the SR and account for the protection. Conclusion Taken together, loss of I-1 attenuates detrimental effects of catecholamines on the heart, suggesting I-1 downregulation in heart failure as a beneficial desensitization mechanism and I-1 inhibition as a potential novel strategy for heart failure treatment.

Sarcomeric cardiomyopathies represent a perfect example of the power of translation and clinic/research interactions: The question arises from the clinical phenotype of the patient (signs and symptoms), basic laboratory research identifies molecular and cellular aspects of the phenotype and develops diagnostics and treatment strategies for the patient. In clinical terms, 25 years ago patients with unexplained left ventricular hypertrophy suffering from dyspnoea and potentially life-threatening arrhythmias were observed, since then underlying genetic mutations and molecular mechanisms have been identified, which already have helped to understand the disease and diagnose the patients, and nowadays molecular treatment options are on the way. The cycle from bedside to bench and back is ongoing. What are sarcomeric cardiomyopathies? As implied by the name, these cardiomyopathies are caused by mutations in a number of sarcomeric genes. The prototype is hypertrophic cardiomyopathy (HCM), which is clinically defined by the presence of increased left ventricular wall thickness that is not solely explained by abnormal loading conditions.1 The frequency of unexplained left ventricular thickness ranges from 0.02 to 0.23% in adults or up to 1:500.2 Patterns …

Time for primary review 21 days. Sarcolemmal Na+–Ca2+ exchange plays a pivotal role in ion transport of the myocardium which is crucial for cardiac contractile performance. The driving force of the exchanger molecule depends on sodium and calcium concentrations at either side of the plasma membrane and on the membrane potential. Section 2 of this article aims to review structural and thermodynamic aspects of the Na+–Ca2+ exchanger and its function. Recently, it has been recognized that Ca2+ as well as Na+ homeostasis is impaired in the failing myocardium. Thus, it has been postulated by numerous authors that Na+–Ca2+ exchanger may be altered with respect to expression and function. However, the literature is controversial. Section 3 comments upon a number of recent publications that have been published on this topic. Section 4 summarizes functional consequences of altered expression and function of the exchanger with respect to excitation–contraction coupling which have to be considered with the concomitant changes of other important Ca2+ cycling proteins such as sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and ryanodine receptor. Finally, besides contractile dysfunction in hypertrophy and heart failure, cytoplasmic Ca2+ overload can induce spontaneous SR Ca2+ release. This Ca2+ is partly removed from the cytoplasm by Na+–Ca2+ exchange generating transient inward currents, which were blamed for provoking arrhythmogenic, delayed afterdepolarizations. We have dedicated section 5 to recent literature and clinical studies that may elucidate the role of Na+–Ca2+ exchange in arrhythmogenesis. ### 2.1 Isoforms, expression, structure and regulation Besides the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), the sarcolemmal Na+–Ca2+ exchanger (NCX) is the most important Ca2+ transport protein responsible for maintaining the Ca2+ balance of the myocyte. Molecular aspects of its function have been subject to … * Corresponding author. Tel.: +49-551-39-6351; fax: +49-551-39-6389. hasenfus{at}med.uni-goettingen.de

Objectives: The functional consequences of Na+/Ca2+ exchanger (NCX) overexpression in heart failure have been controversially discussed. NCX function strongly depends on intracellular sodium which has been shown to be increased in heart failure. Methods and results: We investigated the Na+/K+-ATPase (NKA) inhibitor ouabain (0.5–16 μmol/l) in electrically stimulated, isotonically contracting adult rabbit cardiocytes overexpressing NCX after adenoviral gene transfer (Ad-NCX-GFP, 48 h culture time). Myocytes transfected with adenovirus encoding for green fluorescent protein (Ad-GFP) served as a control. Contractions were analyzed by video-edge detection. In the Ad-NCX-GFP group, the maximum inotropic response was significantly reduced by 50.7% ( P

Objective: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1±2.0%) compared to donor (31.9±4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. Methods: Cooperative effects in isometric force and rate of force redevelopment (Ktr) were studied in single Triton-skinned human cardiomyocytes at various [Ca2+] before and after protein phosphatase-1 (PP-1) incubation. Results: Maximum force and Ktr values did not differ between failing and donor hearts, but Ca2+-sensitivity of force (pCa50) was significantly higher in failing myocardium (ΔpCa50=0.17). Ktr decreased with decreasing [Ca2+], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa50 to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute Ktr values was found after PP-1 in failing and donor myocytes, while the shape of the Ktr-Ca2+ relationships remained unaltered. Conclusions: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca2+-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca2+-sensitivity and impaired Ca2+-handling on diastolic function in human heart failure.

Increased levels of inhibitory G proteins have been observed in heart failure, but their physiological relevance in mediating the reduced beta-adrenergic response is largely unknown.To evaluate the functional consequences of Galpha(i2) overexpression, we studied myocardial contraction in intact isometric contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of Galpha(i2).Neither Galpha(i2) nor lacZ (control) overexpression altered baseline contractile force. After 72 h of continuous contractions, developed force (F(dev)) increased after addition of 1 microM isoproterenol by 28.5+/-9.7 mN/mm(2) in the control group, which was unchanged from the initial response at t=0 h (23.7+/-3.8 mN/mm(2)). In sharp contrast, in preparations transfected with AdGalpha(i2), the response to isoproterenol was significantly attenuated (5.9+/-2.0 vs. 27.6+/-4.2 mN/mm(2), t=72 vs. 0 h, respectively, P0.01). In a primary culture of transfected isolated myocytes from a nearly identical baseline, isoproterenol increased cell shortening by 3.1+/-0.6% in the lacZ transfected myocytes, but only by 1.3+/-0.5% in Galpha(i2) transfected myocytes (t=72 h, P0.01). In Galpha(i2) transfected myocytes, pertussis toxin restored beta-adrenergic responsiveness, indicating specificity of attenuation by the transgene.Overexpression of Galpha(i2) attenuates the positive inotropic effects of beta-adrenergic stimulation in myocardium. In addition, the method we developed allows investigation of a causal link between altered protein expression and subsequent alterations in contractile function in a physiological relevant in vitro manner.

Background: Pyruvate has been shown to increase contractile function in isolated myocardium and to improve hemodynamics in patients with congestive heart failure. We tested the hypothesis that pyruvate potentiates the inotropic response to β-adrenergic stimulation and to elevated extracellular calcium, since this may be of potential therapeutic value in the clinical setting of acute heart failure in order to circumvent deleterious effects on energy demand as can occur during catecholamine therapy. Methods and Results: We investigated isometrically contracting isolated multicellular muscle preparations from terminal failing human hearts at 37°C, pH 7.4, and a stimulation frequency of 1 Hz. At an extracellular calcium concentration of 1.25 mM, pyruvate (10 mM) alone increased developed force ( F dev) from 9.0±2.3 to 21.1±4.3 mN/mm2 ( n =9, P

Protein kinase C (PKC) is thought to be involved in the regulation of the mammalian cardiac excitation-contraction coupling process by vasoactive peptides like endothelin-1 (ET-1). However, the demonstration of a causal link between activation of specific PKC isoforms and the increase in contractility mediated by ET-1 is still inferential.By means of adenovirus-mediated gene transfer, we specifically overexpressed PKC epsilon in cultured adult rabbit ventricular myocytes (Ad-PKC epsilon). Myocyte shortening and [Ca2+]i transients under basal and ET-1-stimulated conditions were measured in Ad-PKC epsilon and Ad-LacZ control transfected cells.Infection with Ad-PKC epsilon resulted in a strong, virus dose-dependent increase in PKC epsilon protein levels, whereas protein expression of other PKC isoforms remained unchanged. Using a multiplicity of infection of 100 plaque-forming units/myocyte, basal and cofactor-dependent PKC epsilon kinase activity was increased 28- and 90-fold, respectively, when compared to control. Myocyte basal fractional shortening and [Ca2+]i transient amplitude were both increased by 21% (P0.05 each) in Ad-PKC epsilon transfected myocytes when compared to Ad-LacZ transfected control myocytes. The positive inotropic effect of ET-1 in control myocytes was markedly blunted in PKC epsilon-overexpressing myocytes.Specific overexpression of PKC epsilon in rabbit ventricular myocytes increases basal myocyte contractility and [Ca2+]i transients, and modifies their responsiveness to ET-1.

Objective: The immunosuppressive drug Cyclosporine A (CsA) is a key substance in pharmacological therapy following solid organ transplantation and has been suggested to prevent cardiac hypertrophy. We investigated the direct effects of CsA on myocardial function, because these are largely unknown. Methods: In multicellular cardiac muscle preparations from end-stage failing and non-failing human hearts as well as from non-failing rabbit hearts we investigated the effects of CsA on contractile performance, sarcoplasmic reticulum (SR) Ca2+-load, cytosolic calcium transients, calcium sensitivity of the myofilaments, and myocardial oxygen consumption. Results: In failing human muscle preparations there was a concentration dependent decrease in contractile force; the maximal effect amounted to 55.6±6.4% of control while EC50 was reached at 1.0±0.3 nM ( n =6). These concentrations are at and even below the therapeutic plasma levels. CsA decreased the aequorin light signal in human failing trabeculae to 71.5±5.9% ( n =5), indicating decreased calcium transients. Estimation of the SR calcium load via measurement of rapid cooling contractures revealed a decrease to 84.4±6.5% in failing human preparations ( n =6). Measurements of both decreased SR calcium load and force development in presence of CsA were also observed in four non-failing human muscle preparations. In rabbit muscle preparations ( n =8), developed force decreased to 50.2±7.7% ( n =8, EC50: 1.9±0.4 nM) and rapid cooling contractures to 74.0±7.4% of control at 100 nmol/l CsA. No direct effects were observed on myofilament calcium sensitivity nor on maximal force development of permeabilized preparations from the rabbit ( n =7). Oxygen consumption measurements showed that CsA decreased the economy of contraction to 76.4±7.9% in rabbit preparations ( n =8). Conclusions: CsA causes a direct cardio-depressive effect at clinically relevant concentrations, most likely due to altered handling of Ca2+ by the SR.

Aims Apolipoprotein E (apoE) may act as a vasculoprotective factor by promoting plasma lipid clearance and cholesterol efflux. Moreover, apoE accumulates at sites of vascular injury and modulates the effect of growth factors on smooth muscle cells (SMCs). Experimental data suggested that hypothalamic apoE expression is reduced in obesity and associated with leptin resistance. In this study, we examined the role of apoE in mediating the effects of leptin on vascular lesion formation. Methods and results Leptin was administered to apoE knockout (apoE−/−) mice via osmotic pumps to increase its circulating levels. Morphometric analysis revealed that leptin did not alter neointima formation and failed to increase α-actin- or PCNA-immunopositive SMCs after vascular injury. Similar findings were obtained after analysis of atherosclerotic lesions. Comparison of apoE−/−, wild-type, or LDL receptor−/− mice and functional analyses in aortic SMCs from WT or apoE−/− mice or human arterial SMCs after treatment with small interfering (si)RNA or heparinase revealed that leptin requires the presence of apoE, expressed, secreted and bound to the cell surface, to fully activate leptin receptor signalling and to promote SMC proliferation and neointima formation. Mechanistically, leptin induced the phosphorylation and membrane translocation of caveolin (cav)-1, and apoE down-regulation or caveolae disruption inhibited the leptin-induced p47phox activation, ROS formation and SMC proliferation. Finally, leptin failed to increase neointima formation in mice lacking cav-1. Conclusion Our findings suggest that apoE mediates the effects of leptin on vascular lesion formation by stabilizing cav-1-enriched cell membrane microdomains in SMCs, thus allowing NADPH oxidase assembly and ROS-mediated mitogenic signalling.

Objective: The aim was to study the effect of three positive inotropic interventions on myocardial force development and heat production in guinea pig papillary muscles in order to investigate the energetic consequences. Methods: The positive inotropic agents used were epinine (β adrenoceptor stimulation), E-1020 (phosphodiesterase inhibition), and ouabain (sodium-potassium ATPase inhibition). Heat measurements were accomplished using antimony-bismuth thermopiles, and initial heat was separated into tension dependent and tension independent heat using the butanedione-monoxime (BDM) and the shortening methods. Results: Optimal concentrations of epinine, E-1020, and ouabain increased peak developed force from 20.0(SD 6.6) to 55.5(9.3) (n = 5; p < 0.01), from 20.9(9.1) to 27.2(7.2) (n = 6; p < 0.05), and from 23.4(9.2) to 44.9(18.0) mN·mm−2(n = 6; p < 0.01). respectively. Epinine and E-1020 decreased the tension-time integral per unit initial heat, ie, the economy of isometric contraction, from 5.5(1.4) to 3.6(0.5) (p < 0.01) and from 5.5(1.4) to 3.1(0.9) N·m·s·J−1 (p < 0.01). respectively: no significant change was observed with ouabain [6.7(1.4) to 8.3(0.5) N·m·s·J−1]. The tension independent heat (calcium turnover) was measured in two different ways using BDM or shortening to abolish force production. It was increased significantly by epinine (by 141-243%), E-1020 (by 77-114%), and ouabain (by 23-38%). The first measurement in brackets is the BDM estimate, the second is the shortening estimate. From the tension-time integral and the tension dependent heat the crossbridge force-time integral was analysed: epinine and E-1020 decreased the crossbridge force-time integral from 0.46(0.16) to 0.31(0.06) pN·s (p < 0.01) and from 0.50(0.19) to 0.31 (0.08) pN·s (p < 0.01), respectively, while ouabain left the force-time integral unchanged [0.59(0.27) to 0.63(0.20) pN·s]. Conclusions: (1) The inotropic effect of ouabain results from an increase in muscle activation with no change in crossbridge kinetics; (2) epinine and E-1020 increase the tension independent heat and decrease the crossbridge force-time integral, both effects reducing the overall economy; and (3) the shortening and BDM methods for measuring the tension independent heat give qualitatively similar but quantitatively different results. Cardiovascular Research 1994; 28: 994-1002

Aims Bone marrow (BM) progenitors participate in new vessel formation and endothelial repair. The leptin receptor (ObR) is expressed on hematopoietic cells; however, the effects of leptin on BM progenitor cells and their angiogenic potential are unknown. Methods and results In the present study, we show that the short-term administration of leptin (over five consecutive days) into wild-type mice increased the number of circulating, BM-derived sca-1+, flk-1+ vascular progenitors, 95 ± 1.7% of which also expressed ObR. Ex vivo stimulation of BM cells with leptin enhanced the expression of NADPH oxidase isoform 2 (NOX2), and the leptin-induced increase in reactive oxygen species production, matrix metalloproteinase-9 (MMP9) expression and circulating soluble KitL levels was absent in mice lacking NOX2. Furthermore, intraperitoneal injections of leptin improved perfusion and increased the number of BM-derived, CD31-positive endothelial cells in ischaemic hindlimbs after femoral artery ligation. The effects of leptin on the mobilization of sca-1+, flk-1+ cells and neovascularization were abolished in mice transplanted with BM from ObR-deficient and in NOX2−/− mice. Conclusion Our findings suggest that the angiogenic effects of leptin involve sca-1+, flk-1+ vascular progenitor cells mobilized from the BM in response to ObR-mediated activation of NOX2, increased MMP9 expression, and sKitL release.

In heart failure (HF), the generation of reactive oxygen species (ROS) is enhanced. It was shown that failing cardiac myocytes are more susceptible to ROS-induced damage, possibly due to increased expression of the sarcolemmal Na-Ca exchanger (NCX).We investigated the consequences of increased expression levels of NCX in adult rabbit ventricular cardiomyocytes (via adenovirus-mediated gene transfer, Ad-NCX1-GFP) with respect to tolerance towards ROS. After 48-h incubation, cells were monitored for morphological changes on an inverted microscope. ROS were generated via hydrogen peroxide (H(2)O(2)) (100 micromol/l) and Fe(3+)/nitrilotriacetate (Fe(3+)/NTA, 100/200 micromol/l) for 4 min and cell morphology was followed over 30 min. [Na(+)](i) and [Ca(2+)](i) in native cells were measured using SBFI-AM and Indo1-AM, respectively.In native myocytes, exposure to ROS induced hypercontracture. This was accompanied by a 1.3-fold increase in diastolic Indo1 fluorescence ratio (P0.05). Overexpression of NCX significantly enhanced development of hypercontracture. After 15 min, the percentage of cells that had undergone hypercontracture (F(hyper)) was 85+/-4% vs. only 44+/-10% in control cells (P0.05). Inhibition of NCX-mediated Ca(2+) entry with KB-R7943 (5 micromol/l) reduced F(hyper) to 33+/-11% (P0.05). [Na(+)](i) was increased 2.9-fold 1 min prior to hypercontracture (P0.05).ROS-induced hypercontracture is due to Ca(2+) entry via NCX which could be triggered by a concomitant substantial increase in [Na(+)](i). Elevated NCX levels predispose to ROS-induced injury, a mechanism likely contributing to myocyte dysfunction and death in heart failure.

We would like to thank da Costa Martins and De Windt for giving us the opportunity to discuss our recent paper in more detail.1 They state that ‘… a quantitative analysis of endogenous or transgenic Dyrk1a expression was not provided …’ and ‘… it remains unclear what level of overexpression over endogenous myocardial Dyrk1a was actually achieved by the transgenic system …’. The authors may have overlooked that we provide exactly this information in detail in our article: see Fig. 2A displaying the protein overexpression time course, see page 523 line 101 where we state that the level of overexpression was 5.1-fold, and see the quantitative real-time RT–PCR data on the level of Dyrk1a mRNA expression (see Supplementary material online, Figure S2B ). Furthermore, da Costa Martins and De Windt state that ‘… endogenous Rcan1 protein expression was not measured in the study by Grebe et al. ’ However, endogenous Rcan1–4 mRNA expression was indeed quantified in both the TAC and calcineurin transgenic disease models and is displayed in Fig. 4B . da Costa Martins and De Windt ask whether we observed alterations in endogenous DYRK1A expression following aortic banding or calcineurin co-expression. Although the data were not fully included in the paper (but see Supplementary material online, Figure S2C …

Purpose: Transverse tubules (T-Tubules, TTs) form a complex network of continuous membrane invaginations which are essential to ensure effective EC coupling in ventricular myocytes (VMs). Resolution limited microscopic studies suggested a loss of TTs as a leading mechanism in late stages of heart failure (HF). However, TTs have dimensions below the resolution limit of conventional light microscopy. Therefore, we employed superresolution microscopy and novel quantitative image analysis strategies to directly quantify TT remodeling in different stages during HF development. Methods: TTs were investigated in VMs of two different murine HF models. Analyses were performed at an early (1 week), an intermediate (4 wks) and a late (8 wks) stage of HF development both after myocardial infarction (MI) and transverse aortic constriction (TAC). Stimulated emission depletion (STED) microscopy was applied to visualize di-8-ANEPPS stained TTs in living VMs. STED live cell imaging was complemented by imaging of immunostained fixed VMs, by protein biochemical analyses and by multimodal imaging of TTs and Ca2+ transients. Results: Already 1 week after MI and TAC, analysis of STED images revealed significant changes on the level of the TT network. A strong increase of TT density was accompanied by significantly more axial TT elements and a higher branching complexity compared to Sham control groups. 4 wks after MI and TAC these changes were observed to almost the same extent, whereas a tendency towards a decreasing TT density occured 8 wks after the interventions. In addition, the size of individual TT cross-sections increased significantly after both interventions, starting early and progressing during HF development. Analysis of Caveolin-3 (Cav3) immunostains revealed a strong increase of Cav3 positive axial structures after MI and TAC suggesting a potential role in the development of the increasing amount of axial TT structures. This was supported by an increased Cav3 protein expression determined by Western blot analysis. Simultaneous imaging of TTs and Ca2+ transients suggested that TT remodeling might directly lead to a delayed and heterogeneous intracellular Ca2+ release. Conclusions: We identified significant proliferative TT remodeling mechanisms in two independent mouse models (MI and TAC), already in early stages of HF development. Our data suggest that TT remodeling might directly lead to defective Ca2+ release units and accordingly to dyssynchronous intracellular Ca2+ release. This might in turn increase the risk of delayed afterdepolarizations, lead to action potential prolongation and arrhythmias.

The epigenetic signature for transcription regulation is poorly understood in cardiac hypertrophy. Here, we investigate the genome-wide distribution of several histone modifications and the correlated transcriptome in adult mouse cardiomyocytes subjected to a pro-hypertrophy stimulus in vivo. We demonstrate that the epigenetic profile dynamic of cardiac genes changes in response to the stimulus, and that distinct epigenetic signatures at promoters regulate the gene re-programming occurring with cardiac hypertrophy. Moreover, we identify 9,207 enhancers that impact transcription in normal and hypertrophic cardiomyocytes and whose activity is governed by unique epigenetic signatures. In fact, combinations of H3K27ac, H3K9ac, and H3K4me3 marks define new subclasses of highly active and dynamic enhancers in cardiac myocytes. We also analyzed the transcriptional network within which these genetic elements act to orchestrate hypertrophy gene expression, finding a role for myocyte enhancer factor (MEF)2C and MEF2A in regulating enhancers. The association of cardiac hypertrophy with a specific epigenetic profile provides a new basis for understanding the molecular mechanisms underlying hear failure, and opens up the possibility of developing new therapies based on the control of the epigenetic profile of cardiomyocytes.

Purpose: The ability to generate patient-specific human induced pluripotent stem cells (ps-iPSCs) provides a unique opportunity for modeling heart disease. Dilated cardiomyopathy (DCM) is due to a progressive enlargement of the heart leading finally to heart failure. Mutations in many genes have been implicated in the pathogenesis of DCM including the RNA-binding motif 20 (RBM20). We aimed to generate iPSCs from DCM patients with a RBM20 mutations, and to analyze the functionality and cell biology of cardiomyocytes derived from these ps-iPSCs (ps-iPSC-CM) with regard to the cardiac DCM phenotype. Methods and Results: Dermal fibroblasts from two patients (34y; 75y) with DCM harboring different RBM20 mutations were reprogrammed to ps-iPSCs via lentiviral infection using the STEMCCA system. The ps-iPSCs show pluripotent characteristics by expressing pluripotency markers on mRNA and protein level similar to human embryonic stem cells. Furthermore, bisulfite sequencing analysis showed that the OCT4 and NANOG promoters were significantly demethylated in ps-iPSCs compared to their parental cells. In addition, the generated RBM20-iPSCs are able to differentiate in vitro and in vivo into derivatives of all three germ layers. The cardiac in vitro differentiation efficiency into beating cardiomyocytes of the RBM20-iPSCs is similar to control iPSCs. Electrophysiological studies showed that under basal conditions ps-iPSC-CM exhibited spontaneous action potentials characteristic for pacemaker-, atrial-, ventricular- and Purkinje-like cells in a similar manner as control iPSC-CM. Using Fast Fourier Transformation the sarcomere-associated periodic signal amplitude was analyzed for α-actinin. A significant higher percentage of RBM20-iPSC-CM showed abnormal sarcomeric α-actinin distribution compared to control CM, suggesting disorganized myofilament structure. But no significant difference was observed in cell size between RBM20-iPSC-CMs and control cells. Further, we found a different isoform expression pattern of the cardiac titin in the mutant ps-iPSC-CM compared to the control cells. This demonstrates a RBM20-dependent regulation of organized myofilament structure and titin splicing in an in vitro ps-DCM-iPSC-model. Conclusion: We demonstrate that iPSC-derived cardiomyocytes from young and old DCM patients harboring a RBM20 mutation recapitulate the abnormalities that were found in individuals with DCM caused by the same mutation. The differentiated cardiomyocytes may be used for individual drug testing and development of novel treatments for this inherited disorder.

Objective: Human heart failure is associated with a disturbed intracellular calcium (Ca2+) homeostasis. In this regard, ventricular wall stress is considered to be a determinant for expression of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). In the present study, we analyzed the transmural protein and/or mRNA levels of SERCA2a, other Ca2+-handling proteins, and of atrial and brain natriuretic peptides (ANP and BNP) in the human heart. Methods: Subepicardial (epi) , midmyocardial (mid) , and subendocardial (endo) sections of the left ventricular free wall from end-stage failing ( n =17) and nonfailing ( n =5) human hearts were analyzed by Western blot for immunoreactive protein levels of SERCA2a, phospholamban (PLN), and calsequestrin (CS). Subepi- and subendocardial sections were analyzed by Northern blot for steady-state mRNA levels of SERCA2a, Na+-Ca2+ exchanger (NCX1), ANP, and BNP. Results: SERCA2a protein and mRNA levels were reduced by 40±5% ( P

The progress made in our understanding of the pathophysiology and treatment of congestive heart failure (CHF) would not have been possible without a number of animal models of heart failure and hypertrophy, each one having unique advantages as well as disadvantages. The species and interventions used to create CHF depends on the scientific question as well as on factors such as ethical and economical considerations, accessibility and reproducibility or the model. How closely the model should mimic the human syndrome of CHF depends on the scientific question under investigation. If the goal is to study pathophysiological processes like remodeling or the function of subcellular systems such as excitation contraction-coupling processes, contractile protein function or energetics, the model of heart failure should mimic the clinical setting as closely as possible. However, if defined causal connections are under investigation such as structure–function analyses or regulation of gene expression, exact reflection of the clinical setting by the animal model may be less important. In this review, animal models of heart failure are discussed with particular focus on similarities between the animal model and the failing human heart regarding myocardial function as well as molecular and subcellular mechanisms. In addition, new models of heart failure and hypertrophy, and finally some recent animal models of myocarditis are reviewed.

Time for primary review 27 days. During the last decade there was accumulating evidence that alterations of excitation-contraction (EC) coupling may play a critical role in the pathophysiology of myocardial failure. EC-coupling comprises processes involved in calcium activation of contractile proteins and the subsequent removal of calcium facilitating relaxation (for review see [1]). The initial event is depolarisation of the membrane which opens voltage-gated, dihydropyridine-sensitive sarcolemmal calcium channels (dihydropyridine receptors) allowing an influx of calcium into the myocyte. There is a close proximity between one or a few sarcolemmal calcium channels and one or a few calcium channels of the sarcoplasmic reticulum (ryanodine receptors). This may enable calcium influx through one opening of a single sarcolemmal calcium channel to increase a local pool of calcium sufficiently to open adjacent ryanodine receptors to release calcium. This local, punctate, increase of calcium is termed calcium spark according to its visualization by confocal microscopy [2]. It is the sum of these local calcium releases which generate a global cytoplasmic increase in calcium which causes activation of contractile proteins. The global increase in calcium is immediately followed by calcium removal resulting in subsequent deactivation of the contractile machinery. Calcium removal from the cytosol occurs by activity of the sarcoplasmic reticulum calcium pump and by exchange of calcium for sodium by the sarcolemmal Na+-Ca2+-exchanger [1]. Defective EC-coupling in heart failure may result from altered density or function of proteins relevant for calcium homeostasis. Knowledge of these alterations is a prerequisite for understanding the pathophysiology of myocardial failure and for the development of new strategies to treat patients with heart failure. This review focuses on expression and function of calcium-regulatory proteins in failing myocardium from animal models of heart failure and from failing human hearts. Calcium entry through voltage-gated L-type … * Corresponding author. Tel. (+49-761) 270 3591; Fax (+49-761) 270 3788; E-mail: hasenfus@ruf.uni-freiburg.de

Myocyte Na+ homeostasis is crucially involved in a number of vital cell functions, such as excitability, excitation–contraction coupling, energy metabolism, pH regulation, as well as cardiac development and growth. However, consideration of Na+ regulation is often relegated to a secondary position in the discussion of cardiac (patho-)physiology, where the focus is typically on contractile proteins, Ca2+ regulation and pH regulation which appear more directly related to contractile function. However, myocyte Na+ homeostasis is as complex as Ca2+ or pH homeostasis and [Na+]i very directly influences intracellular [Ca2+] and pH via powerful cardiac Na/Ca exchange, Na/H exchange and Na-bicarbonate cotransport systems. Na+ flux my even be central in mediating effects of mechanical loading of the heart on excitation–contraction coupling. Moreover, [Na+]i homeostasis is regulated by a delicate balance of Na+ channels and transporters in the surface and mitochondrial membrane that maintain a large [Na+] gradient across the sarcolemmal membrane. Given this fundamental but often overlooked contribution of Na … * Corresponding author. Tel.: +49-551-3989-25; fax: +49-551-3919-127.