AimsIn late-stage chronic heart failure (CHF), elevated cytokines and cachexia are often observed. Several studies have shown that exercise training exerts beneficial effects on skeletal muscle in this setting. Furthermore, it has been shown that the expression of myostatin, a key regulator of skeletal muscle mass, is increased in a variety of cachectic states. This study aimed to investigate the expression of myostatin in CHF, the influence of exercise training on myostatin levels, and regulation of myostatin by tumour necrosis factor-a (TNF-a). Methods and resultsIn an animal model of CHF (LAD-ligation model), protein expression of myostatin was elevated 2.4-fold in the skeletal muscle and more than four-times in the myocardium, compared with control (Co). Exercise training on a treadmill over 4 weeks led to a significant reduction in myostatin protein expression in the skeletal muscle and the myocardium of CHF animals, with values returning to baseline levels. In differentiated C2C12 cells, TNF-a induced the expression of myostatin through a p38MAPK-dependent pathway involving nuclear factor kappa-B (NF-kB). The increased TNF-a mRNA levels in the skeletal muscle of CHF animals correlated significantly with myostatin expression. ConclusionThese alterations in myostatin expression in the skeletal and heart muscle following exercise training could help to explain the beneficial anti-catabolic effects of exercise training in CHF.--

Cardiac cachexia is a serious complication of chronic heart failure with a prevalence of 10–16% and poor prognosis. There are no current therapy options for cardiac cachexia. Ghrelin is the natural ligand for the GHS-1a-receptor and a potential target for conditions associated with cachexia. Ghrelin has been shown to increase weight in several species. The GHS-1a-receptor is not only found in the brain, but also in other tissues, including the myocardium. Human clinical trials with native ghrelin in cardiac cachexia demonstrated increases in appetite, weight and cardiac output.MethodsHuman ghrelin or one of two analogues BIM-28125 and BIM-28131 (also known as RM-131) were tested at 50 nmole/kg/d and 500 nmole/kg/d versus placebo in a rat model of heart failure (myocardial infarction). Animals (SD-rats, approx. 225 g at surgery) received diuretics from day 14 and compounds from day 28 for 4 weeks using osmotic pumps. Weight was monitored and body composition analysed (NMR-scanning). Cardiac function was assessed by echocardiography and hemodynamics.ResultsAnimals with MI gained less weight compared to sham rats until start of the therapy (311 g vs 324 g, p = 0.0129). Animals treated with BIM-28131 at 50 nmole/kg/d or all compounds at 500 nmole/kg/d displayed stronger weight gain compared to placebo and sham (all p<0.001). Before treatment, body composition was similar in all groups (average: 36 g fat, 248 g lean). Placebo-treated rats gained no fat, but only lean mass. The active compounds induced both fat and lean mass gain, but to a different extent. The fat-to-muscle-ratio of tissue gain was 0.9±0.07 for BIM-28131 at 50 nmole/kg/d, whereas at 500 nmole/kg/d it was 0.76±0.07 for BIM-28131, 0.68±0.12 for BIM-28125, and 0.48±0.05 for ghrelin. MuRF-1 and MAFbx were differentially regulated by treatment.ConclusionGhrelin is a very promising treatment option for cardiac cachexia, with the analogue BIM-28131 (RM-131) being the most effective compound.

BackgroundIn chronic heart failure (CHF), cachexia is a hallmark of the terminal stage of this disease and is associated with a severely reduced quality of life and poor prognosis. Therapeutic options are currently not available. Ghrelin and its analogues BIM-28125 and BIM-28131 (now known as RM-131) have been shown to increase weight in a rat model of CHF. It has been further demonstrated that the expression of myostatin, a negative regulator of skeletal muscle mass, is increased in CHF. The aim of the study was to investigate the influence of ghrelin or its analogues on myostatin in CHF.MethodsIn an animal model of CHF, Sprague–Dawley rats received either ghrelin or two ghrelin analogues BIM-28125 and BIM-28131 in two different concentrations (50 and 500 nmol/kg/day) compared to placebo. The compounds were delivered using osmotic mini pumps. The expression of myostatin was analyzed in skeletal muscle by RT-PCR and Western blot, and muscle mass of gastrocnemius muscle was measured. The plasma levels of tumor necrosis factor alpha (TNF-α) were measured.ResultsThe relative weight of the gastrocnemius muscle of the sham-operated group was significantly increased compared to placebo-treated CHF rats. The application of ghrelin analogue BIM-28125 and BIM-28131 in their higher concentrations led to a significant reduction in myostatin mRNA expression in comparison to placebo. Myostatin protein expression was significantly reduced in both concentrations of ghrelin and BIM-28131 and in the lower concentration of BIM-28125. The increase of TNF-α plasma concentration in the CHF-animals could be abolished by all used substances.ConclusionsIn an animal model of CHF, the expression of myostatin is significantly reduced in the skeletal muscle after application of ghrelin and most concentrations of its analogues BIM-28125 and BIM-28131 possibly due to anti-inflammatory effects.

Adiponectin (APN) is a multifunctional adipocytokine that inhibits myocardial fibrosis, dilatation, and left ventricular (LV) dysfunction after myocardial infarction (MI). Coxsackievirus B3 (CVB3) myocarditis is associated with intense extracellular matrix (ECM) remodeling which might progress to dilated cardiomyopathy. Here, we investigated in experimental CVB3 myocarditis whether APN inhibits adverse ECM remodeling following cardiac injury by affecting matrix metalloproteinase (MMP) expression. Cardiac injury was induced by CVB3 infection in APN knockout (APN‐KO) and wild‐type (WT) mice. Expression and activity of MMPs was quantified by qRT‐PCR and zymography, respectively. Activation of protein kinases was assessed by immunoblot. In cardiac myocytes and fibroblasts APN up‐regulates MMP‐9 expression via activation of 5′ adenosine monophosphate‐activated protein kinase (AMPK) and extracellular signal‐regulated kinase (ERK)1/2 which function as master regulators of inflammation‐induced MMP‐9 expression. Correspondingly, APN further increased up‐regulation of MMP‐9 expression triggered by tumor necrosis factor (TNF)α, lipopolysaccharide (LPS) and R‐848 in cardiac fibroblasts. In vivo, compared to WT mice cardiac MMP‐9 activity and serum levels of carboxy‐terminal telopeptide of type I collagen (ICTP) were attenuated in APN‐KO mice in subacute (day 7 p.i.) CVB3 myocarditis. Moreover, on day 3 and day 7 post CVB3 infection splenic MMP‐9 expression was diminished in APN‐KO mice correlating with attenuated myocardial immune cell infiltration in subacute CVB3 myocarditis. These results indicate that APN attenuates adverse cardiac remodeling following cardiac injury by up‐regulating MMP‐9 expression in cardiac and immune cells. Thus, APN mediates intensified collagen cleavage that might explain inhibition of LV fibrosis and dysfunction.

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Introduction: Transdifferentiation of cardiac fibroblasts into myofibroblasts regulated by TGFβ/SMAD3 signaling is a major mechanism of scar formation and adverse remodeling following myocardial infarction. Hypothesis: We hypothesized that the transcription factor FOXO3a, a key regulator of cell differentiation, cycle and size, might inhibit this process. Methods: Acute myocardial infarction was induced in FOXO3-/- and WT mice (FVB) by permanent LAD ligation and myofibroblast transdifferentiation markers were assessed. FOXO3a-/- and WT cardiac fibroblasts were investigated in transdifferentiation assays ex vivo. FOXO3a gene transfer was performed with gain of function adenoviral vectors. IP/IF and Western blotting were used to test for a direct interaction between FOXO3a and SMAD3. Results: FOXO3a-/- mice had significantly higher survival rates compared to WT mice due to reduced rates of ventricle perforation. Myocardial expression of alpha smooth muscle actin (ASMA) and Collagen1A1 (Col1A1) was significantly enhanced in FOXO3a-/- mice 3 and 14 days post infarction. Moreover, Foxo3a-/- mice showed larger fibrotic areas following MI. In line with these results, cardiac fibroblasts isolated from FOXO3a-/- mice showed significantly enhanced expression levels of Col1A1 and ASMA 24 hours following stretch or TGF-β stimulation when compared to WT cells in vitro. Furthermore supernatants of FOXO3a -/- fibroblasts showed significantly higher protein expression of Col1A1 in (p<0.01) while FOXO3a gene-transfer dose-dependently downregulated Col1A1. Immunfluorescence staining for ASMA protein was significantly attenuated following FOXO3a gene transfer in cardiac fibroblasts. Mechanistically, immunoprecipitation showed direct interaction of FOXO3a with SMAD3a that was enhanced following activation of the transcription factor leading to diminished SMAD3 downstream gene expression. Conclusions: Our results identify FOXO3a as a direct inhibitor of TGF- β regulated matrix remodeling via FOXO3a-SMAD3 interaction. FOXO3a regulates fibrosis and scar formation following myocardial infarction and thus targeting the FOXO3a-SMAD3 axis might be of therapeutic interest.