Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of ß-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a ß-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

Genistein and exercise treatment reduced NASH related HDAC3, IL-13 and MMP-12 expressions in ovariectomized rats fed with high fat high fructose diet.

BACKGROUND AND AIM: Genistein (GEN) and exercise (Ex) may be regarded as an alternative treatment for non-alcoholic steatohepatitis (NASH). However, the mechanisms behind their therapeutic effects in NASH are not well-understood. EXPERIMENTAL PROCEDURE: This study investigated the roles of histone deacetylase (HDAC)3 and interleukin-(IL-)13 in the NASH model of ovariectomized (OVX) rats fed with high fat high fructose (HFHF) diet. RESULTS AND CONCLUSION: Nine weeks after being fed with HFHF diet, severe NASH pathology with mild fibrosis were seen along with an increase in HDAC3, IL-13 and matrix metalloelastase (MMP-12) expressions in OVX rats. Five weeks of either GEN or Ex treatments abrogated the increase in both HDAC3 and IL-13 expressions in OVX rats fed with HFHF diet and ameliorated NASH features, liver fibrosis and MMP-12 expression. The combination of Gen and Ex, however, did not provide additional benefits on NASH features in OVX rats fed with HFHF diet. These results suggested that GEN and Ex treatments improved HFHF diet induced NASH in OVX rats through the suppression of HDAC3, IL-13 and MMP-12 expression.

Genistein and exercise modulated lipid peroxidation and improved steatohepatitis in ovariectomized rats.

BACKGROUND: The prevalence of nonalcoholic steatohepatitis (NASH) in menopausal women is increasing, but current treatments have not been proven effective. The objective of this study was to investigate the treatment effects of genistein and running exercise in ovariectomized (OVX) rats with NASH. METHODS: Thirty-six female Sprague-Dawley rats were divided into 6 groups, control; OVX with standard diet; OVX with high fat and high fructose (HFHF) diet for 4 weeks; OVX with HFHF and genistein treatment (16 mg/kg BW/day) for 5 weeks (OVX + HFHF+GEN); OVX with HFHF and moderate intensity exercise for 5 weeks (OVX + HFHF+EX); OVX with HFHF and combined treatments (OVX + HFHF+GEN + EX). Serum interleukin-6 (IL-6) levels, hepatic free fatty acid (FFA), hepatic glutathione (GSH), and hepatic malondialdehyde (MDA) levels were measured. Liver histology was examined to determine NASH severity. RESULTS: OVX + HFHF group had the highest levels of hepatic FFA compared with OVX and control groups (5.92 ± 0.84 vs. 0.37 ± 0.01 vs. 0.42 ± 0.04 nmol/mg protein, respectively, p < 0.01). Serum IL-6 levels were significantly elevated in both OVX and OVX + HFHF groups as compared with controls (112.13 ± 6.50 vs. 121.47 ± 3.96 vs. 86.13 ± 2.40 pg/mL, respectively, p < 0.01). In OVX + HFHF group, hepatic MDA levels were higher, while GSH levels were lower than in OVX and control groups (MDA; 0.98 ± 0.04 vs. 0.82 ± 0.02 vs. 0.78 ± 0.03 nmol/mg protein, and GSH; 46.01 ± 0.91 vs. 55.21 ± 1.40 vs. 57.94 ± 0.32, respectively; p < 0.01 for both). Comparing with OVX + HFHF group, rats that received genistein, exercise and combined treatments demonstrated an improvement in liver histopathology, decreased levels of hepatic FFA (1.44 ± 0.21 vs. 0.45 ± 0.04 vs. 0.49 ± 0.05 nmol/mg protein, respectively, p < 0.01), serum IL-6 (82.80 ± 2.07 vs. 83.47 ± 2.81 vs. 94.13 ± 1.61 pg/mL, respectively, p < 0.01), and hepatic MDA (0.80 ± 0.03 vs. 0.76 ± 0.02 vs. 0.76 ± 0.03 nmol/mg protein, respectively, p < 0.01). CONCLUSIONS: Genistein and moderate intensity exercise were effective in reducing the severity of NASH in OVX rats through the reduction in liver inflammation, oxidative stress and liver fat contents.

Chronic high-dose testosterone treatment: impact on rat cardiac contractile biology.

Androgen therapy provides cardiovascular benefits for hypogonadism. However, myocardial hypertrophy, fibrosis, and infarction have been reported in testosterone or androgenic anabolic steroid abuse. Therefore, better understanding of the factors leading to adverse results of androgen abuse is needed. The aim of the present study was to examine the impact of high dose of androgen treatment on cardiac biology, and whether exposure duration modulates this response. Male rats were treated with 10 mg/kg testosterone, three times a week, for either 4 or 12 weeks; vehicle injections served as controls. Four weeks of testosterone treatment induced an increase in ventricular wall thickness, indicative of concentric hypertrophy, as well as increased ejection fraction; in contrast, both parameters were blunted following 12 weeks of high-dose testosterone treatment. Cardiac myocyte contractile parameters were assessed in isolated electrically stimulated myocytes (sarcomere and intracellular calcium dynamics), and in chemically permeabilized isolated myocardium (myofilament force development and tension-cost). High-dose testosterone treatment for 4 weeks was associated with increased myocyte contractile parameters, while 12 weeks treatment induced significant depression of these parameters, mirroring the cardiac pump function results. In conclusion, chronic administration of high-dose testosterone initially induces increased cardiac function. However, this initial beneficial impact is followed by significant depression of cardiac pump function, myocyte contractility, and cardiac myofilament function. Our results indicate that chronic high-testosterone usage is of limited use and may, instead, induce significant cardiac dysfunction.

Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium.

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca(2+) transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 µm) and long (2.3 µm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca(2+) sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca(2+) sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3ß.

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HF(dys)) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HF(dys); however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HF(dys) cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3ß (GSK-3ß). We found that GSK-3ß was deactivated in HF(dys) and reactivated by CRT. Mass spectrometry of myofilament proteins from HF(dys) animals incubated with GSK-3ß confirmed GSK-3ß­dependent phosphorylation at many of the same sites observed with CRT. GSK-3ß restored calcium sensitivity in HF(dys), but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HF(dys) through GSK-3ß reactivation, identifying a therapeutic approach to enhancing contractile function

Testosterone regulates cardiac contractile activation by modulating SERCA but not NCX activity.

Alterations in intracellular Ca(2+) transients of cardiomyocytes in orchidectomized (ORX) rats could be a cause of cardiac dysfunction in the hypogonadal condition. To investigate the role of male sex hormones in intracellular Ca(2+) homeostasis during relaxation, Ca(2+)-handling activities by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and the Na(+)/Ca(2+) exchanger (NCX) were evaluated in the ventricular muscle of 10-wk-old ORX rats with and without testosterone supplementation (2.5 mg/kg testosterone propionate, 2 times/wk). ORX induced a 50% decrease in contraction force accompanied by a prolonged time to achieve 50% relaxation (T(50)) in isolated intact ventricular trabeculae, which was partially corrected by testosterone administration. Maximum active tension was also suppressed in ORX rats without changes in myofilament Ca(2+) sensitivity and passive stiffness of the heart. Using a sarcoplasmic reticulum-enriched membrane preparation, the maximum thapsigargin-sensitive SERCA activity of the ORX rat was 27% lower with an increased Ca(2+) sensitivity, which was prevented by testosterone treatment. However, neither changes in SERCA content nor its modulating components, sarcolipin and heat shock protein 20, were detected in the ORX rat, but there was a significant decrease in the phosphorylated Thr(17) form of phospholamban. Despite a lower level of NCX protein in the heart of ORX rats, prolonged T(50) disappeared after an incubation with thapsigargin (10 µM), implying a lack of effect of male sex hormone deficiency on NCX function. These findings indicate that male sex hormones can regulate cardiac relaxation by acting mainly through SERCA. However, a detailed mechanism of SERCA modulation under male sex hormone deficiency status remains to be explored.