h-89 and Heart-Failure

Heart failure (HF) is a disease with high mortality and morbidity rate. Previous studies have shown that microRNAs (miRNAs) may be implicated in the pathogenesis of HF, potentially being able to improve the cardiac function in an HF rat model. The present study was designed to define the role of miR-665 in the cardiac function of the HF rats. Following the establishm;ent of the rat models of HF, the functional role miR-665 in HF was determined using an ectopic expression and knockdown experiments. The cardiac function was evaluated with the determination of ventricular mass index and hemodynamic parameters. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was performed, with the apoptosis of cardiac cells detected in the process. The expression of miR-665, glucagon-like peptide 1 receptor (GLP1R), cyclic adenosine monophosphate (cAMP) signaling pathway-related, and apoptosis-related genes was examined. Enzyme-linked immunosorbent assay was conducted to determine the levels of inflammation-related genes. Initially, the upregulation of miR-665, downregulation of GLP1R, and inactivation of cAMP signaling pathway were observed in HF rats. GLP1R was a target of miR-665. Forced expression of miR-665 promoted cell apoptosis and inhibited GLP1R and the cAMP signaling pathway. In addition, miR-665 overexpression has been known to impair cardiac function, promote inflammatory response while elevating malondialdehyde and superoxide dismutase levels, and decreasing mitochondrial respiratory chain enzyme activities. Furthermore, we also observed that the effects of miR-665 inhibition had been reversed when the cAMP signaling pathway was also inhibited. This study demonstrates that miR-665 inhibition can stabilize the cardiac function of HF rats via the cAMP signaling pathway via upregulation of the GLP1R.

Topics: Animals; Cardiac Output; Cyclic AMP; Gene Expression Regulation; Gene Silencing; Glucagon-Like Peptide-1 Receptor; Heart Failure; Isoquinolines; Male; MicroRNAs; Mitochondria; Molecular Mimicry; Myocardial Infarction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Rats, Wistar; Signal Transduction; Sulfonamides

Heart failure (HF) is a prevalent disease that is considered the foremost reason for hospitalization in the United States. Most protein kinases (PK) are activated in heart disease and their inhibition has been shown to improve cardiac function in both animal and human studies. However, little is known about the direct impact of PKA and PKC inhibitors on human cardiac contractile function.. We investigated the ex vivo effect of such inhibitors on force as well as on kinetics of left ventricular (LV) trabeculae dissected from non-failing and failing human hearts. In these experiments, we applied 0.5 μM of H-89 and GF109203X, which are PKA and PKC inhibitors, respectively, in comparison to their vehicle DMSO (0.05%).. Statistical analyses revealed no significant effect for H-89 and GF109203X on either contractile force or kinetics parameters of both non-failing and failing muscles even though they were used at a concentration higher than the reported IC

Topics: Adult; Aged; Cyclic AMP-Dependent Protein Kinases; Female; Heart; Heart Failure; Heart Ventricles; Humans; Indoles; Inhibitory Concentration 50; Isoquinolines; Male; Maleimides; Middle Aged; Myocardial Contraction; Myocardium; Protein Kinase C; Protein Kinase Inhibitors; Sulfonamides; Young Adult

Cross-bridge cycling kinetics play an essential role in the heart's ability to contract and relax. The rate of tension redevelopment (ktr) slows down as a muscle length is increased in intact human myocardium. We set out to determine the effect of rapid length step changes and protein kinase A (PKA) and protein kinase C-βII (PKC-βII) inhibitors on the ktr in ultra-thin non-failing and failing human right ventricular trabeculae. After stabilizing the muscle either at L90 (90% of optimal length) or at Lopt (optimal length), we rapidly changed the length to either Lopt or L90 and measured ktr. We report that length-dependent changes in ktr occur very rapidly (in the order of seconds or faster) in both non-failing and failing muscles and that the length at which a muscle had been stabilized prior to the length change does not significantly affect ktr. In addition, at L90 and at Lopt, PKA and PKC-βII inhibitors did not significantly change ktr. Our results reveal that length-dependent regulation of cross-bridge cycling kinetics predominantly occurs rapidly and involves the intrinsic properties of the myofilament rather than post-translational modifications that are known to occur in the cardiac muscle as a result of a change in muscle/sarcomere length.

Topics: Adult; Aged; Cyclic AMP-Dependent Protein Kinases; Female; Heart; Heart Failure; Heart Ventricles; Humans; Isoquinolines; Kinetics; Male; Middle Aged; Myocardial Contraction; Myocardium; Myofibrils; Protein Kinase C beta; Sarcomeres; Signal Transduction; Sulfonamides

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~18weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation-contraction coupling observed in heart failure.

Topics: Animals; Calcium; Calcium Channels, L-Type; Cyclic AMP-Dependent Protein Kinases; Heart Failure; Heart Ventricles; Humans; Isoquinolines; Myocytes, Cardiac; Patch-Clamp Techniques; Rats; Sulfonamides

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca](SRT)) versus control. At similar [Ca](SRT) (121muM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2+/-0.28 vs. 1.1+/-0.28 and 1.29+/-0.39 SCaWs cell(-)(1), respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca](SRT). Inhibiting CaMKII reversed this effect (14%). At similar [Ca](SRT) (71microM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5+/-0.5; 1.6+/-0.7 vs. 0.36+/-0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

Topics: Animals; Arrhythmias, Cardiac; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cyclic AMP-Dependent Protein Kinases; Diastole; Heart Failure; Heart Ventricles; Isoproterenol; Isoquinolines; Muscle Cells; Myocytes, Cardiac; Rabbits; Sulfonamides

Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether beta-adrenergic receptor (beta-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca](i)) and SR Ca load ([Ca](SRT)) were assessed using fluo-4. beta-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca](i) decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2-related inhibitory peptide II or beta-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the beta-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca](i) decline rate. The results suggest that beta-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca](i).

Topics: Adrenergic beta-Agonists; Animals; Benzylamines; Calcium; Calcium Channels, L-Type; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cells, Cultured; Colforsin; Cyclic AMP-Dependent Protein Kinases; Diastole; Heart Failure; Isoproterenol; Isoquinolines; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Peptides; Phosphorylation; Protein Processing, Post-Translational; Rabbits; Receptors, Adrenergic, beta; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sulfonamides; Tetracaine

Heart failure is associated with alterations in contractile parameters and accompanied by abnormalities in intracellular calcium homeostasis. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and phospholamban (PLB) are important in intracellular calcium cycling. The aim of the present study was to examine mechanisms causing reductions in SERCA2 activity in the failing heart.. Myocardial infarction (MI) was induced in male Wistar rats, and animals with congestive heart failure were examined 6 weeks after the primary operation.. Serine(16) monomeric and pentameric phosphorylated PLB were significantly downregulated (50 and 55%, respectively), whereas threonine(17) phosphorylated PLB was unchanged in failing compared to sham hearts. Protein phosphatases 1 and 2A were significantly upregulated (26 and 42%, respectively) and phosphatase 2C significantly downregulated (29%), whereas the level of protein kinase A regulatory subunit II remained unchanged during heart failure. Increasing PLB phosphorylation by forskolin in isolated cardiomyocytes after inhibition of the Na(+)-Ca(2+) exchanger activity had significantly greater effect on SERCA2 activity in failing than in sham cells (49 and 20% faster transient decline, respectively). Decreasing PLB phosphorylation by the protein kinase A inhibitor H89 had significantly less effect on SERCA2 activity in failing compared to sham cardiomyocytes (20 and 75% slower transient decline, respectively).. The observed changes in SERCA2 activity after increasing and decreasing serine(16) PLB phosphorylation in cardiomyocytes from sham and failing hearts, suggest that the observed reduction in serine(16) PLB phosphorylation is one major factor determining the reduced SERCA2 activity in heart failure after MI.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Heart Failure; Homeostasis; Immunoblotting; Intracellular Fluid; Isoproterenol; Isoquinolines; Male; Myocardial Infarction; Myocardium; Phosphorylation; Rats; Rats, Wistar; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides