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1.
Am J Physiol Heart Circ Physiol ; 320(2): H881-H890, 2021 02 01.
Article in English | MEDLINE | ID: mdl-33337957

ABSTRACT

Morbidity and mortality associated with heart disease is a growing threat to the global population, and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca2+ sensitivity of contraction for both genotypes, but this reduction in pCa50 was nearly twice as large for WT-RLC versus N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed up myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca2+ sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM.NEW & NOTEWORTHY Mavacamten is a pharmaceutical that binds to myosin, and it is under investigation as a therapy for some forms of heart disease. We show that mavacamten reduces isometric tension and Ca2+ sensitivity of contraction in skinned myocardial strips from a mouse model of hypertrophic cardiomyopathy that expresses the N47K mutation in cardiac myosin regulatory light chain. Mavacamten reduces contractility by decreasing strong cross-bridge binding, partially due to faster cross-bridge nucleotide handling rates that speed up myosin detachment.


Subject(s)
Benzylamines/pharmacology , Calcium Signaling/drug effects , Cardiomyopathy, Hypertrophic/drug therapy , Enzyme Inhibitors/pharmacology , Myocardial Contraction/drug effects , Myosin Light Chains/metabolism , Papillary Muscles/drug effects , Uracil/analogs & derivatives , Ventricular Myosins/antagonists & inhibitors , Animals , Cardiomyopathy, Hypertrophic/enzymology , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/physiopathology , Disease Models, Animal , Humans , Kinetics , Male , Mice, Transgenic , Mutation , Myosin Light Chains/genetics , Papillary Muscles/enzymology , Papillary Muscles/physiopathology , Uracil/pharmacology , Ventricular Myosins/metabolism
2.
Eur J Pharmacol ; 891: 173724, 2021 Jan 15.
Article in English | MEDLINE | ID: mdl-33152335

ABSTRACT

Previously, we have shown that an increased cGMP-activated protein Kinase (PKG) activity after phosphodiesterase 5 (PDE5) inhibition by Sildenafil (SIL), leads to myocardial Na+/H+ exchanger (NHE1) inhibition preserving its basal homeostatic function. Since NHE1 is hyperactive in the hypertrophied myocardium of spontaneous hypertensive rats (SHR), while its inhibition was shown to prevent and revert this pathology, the current study was aimed to evaluate the potential antihypertrophic effect of SIL on adult SHR myocardium. We initially tested the inhibitory capability of SIL on NHE1 in isolated cardiomyocytes of SHR by comparing H+ efflux during the recovery from an acid load. After confirmed that effect, eight-month-old SHR were chronically treated for one month with SIL through drinking water. Compared to their littermate controls, SIL-treated rats presented a decreased NHE1 activity, which correlated with a reduction in its phosphorylation level assigned to activation of a PKG-p38 MAP kinase-PP2A signaling pathway. Moreover, treated animals showed a decreased oxidative stress that appears to be a consequence of a decreased mitochondrial NHE1 phosphorylation. Treated SHR showed a significant reduction in the pro-hypertrophic phosphatase calcineurin, despite slight tendency to decrease hypertrophy was detected. When SIL treatment was prolonged to three months, a significant decrease in myocardial hypertrophy and interstitial fibrosis that correlated with a lower myocardial stiffness was observed. In conclusion, the current study provides evidence concerning the ability of SIL to revert established cardiac hypertrophy in SHR, a clinically relevant animal model that resembles human essential hypertension.


Subject(s)
Cardiomegaly/prevention & control , Myocytes, Cardiac/drug effects , Papillary Muscles/drug effects , Phosphodiesterase 5 Inhibitors/pharmacology , Sildenafil Citrate/pharmacology , Sodium-Hydrogen Exchanger 1/metabolism , Animals , Cardiomegaly/enzymology , Cardiomegaly/etiology , Cardiomegaly/physiopathology , Cyclic GMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Down-Regulation , Fibrosis , Hypertension/complications , Male , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , Papillary Muscles/enzymology , Papillary Muscles/physiopathology , Phosphorylation , Protein Phosphatase 2/metabolism , Rats, Inbred SHR , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
3.
World J Gastroenterol ; 22(19): 4685-94, 2016 May 21.
Article in English | MEDLINE | ID: mdl-27217700

ABSTRACT

AIM: To explore the role of mammalian target of rapamycin (mTOR) in the pathogenesis of cirrhotic cardiomyopathy and the potential of rapamycin to improve this pathologic condition. METHODS: Male albino Wistar rats weighing 100-120 g were treated with tetrachloride carbon (CCl4) for 8 wk to induce cirrhosis. Subsequently, animals were administered rapamycin (2 mg/kg per day). The QTc intervals were calculated in a 5-min electrocardiogram. Then, the left ventricular papillary muscles were isolated to examine inotropic responsiveness to ß-adrenergic stimulation using a standard organ bath equipped by Powerlab system. Phosphorylated-mTOR localization in left ventricles was immunohistochemically assessed, and ventricular tumor necrosis factor (TNF)-α was measured. Western blot was used to measure levels of ventricular phosphorylated-mTOR protein. RESULTS: Cirrhosis was confirmed by hematoxylin and eosin staining of liver tissues, visual observation of lethargy, weight loss, jaundice, brown urine, ascites, liver stiffness, and a significant increase of spleen weight (P < 0.001). A significant prolongation in QTc intervals occurred in cirrhotic rats exposed to CCl4 (P < 0.001), while this prolongation was decreased with rapamycin treatment (P < 0.01). CCl4-induced cirrhosis caused a significant decrease of contractile responsiveness to isoproterenol stimulation and a significant increase in cardiac TNF-α. These findings were correlated with data from western blot and immunohistochemical studies on phosphorylated-mTOR expression in left ventricles. Phosphorylated-mTOR was significantly enhanced in cirrhotic rats, especially in the endothelium, compared to controls. Rapamycin treatment significantly increased contractile force and myocardial localization of phosphorylated-mTOR and decreased cardiac TNF-α concentration compared to cirrhotic rats with no treatment. CONCLUSION: In this study, we demonstrated a potential role for cardiac mTOR in the pathophysiology of cirrhotic cardiomyopathy. Rapamycin normalized the inotropic effect and altered phosphorylated-mTOR expression and myocardial localization in cirrhotic rats.


Subject(s)
Cardiomyopathies/etiology , Chemical and Drug Induced Liver Injury/complications , Liver Cirrhosis, Experimental/complications , Myocardium/enzymology , Papillary Muscles/enzymology , TOR Serine-Threonine Kinases/metabolism , Ventricular Function, Left , Action Potentials , Animals , Carbon Tetrachloride , Cardiomyopathies/enzymology , Cardiomyopathies/pathology , Cardiomyopathies/physiopathology , Cardiotonic Agents/pharmacology , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/pathology , Heart Rate , Isoproterenol/pharmacology , Liver Cirrhosis, Experimental/enzymology , Liver Cirrhosis, Experimental/pathology , Male , Myocardial Contraction , Myocardium/pathology , Papillary Muscles/drug effects , Papillary Muscles/physiopathology , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Rats, Wistar , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Ventricular Function, Left/drug effects
4.
Am J Physiol Heart Circ Physiol ; 305(2): H228-37, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23709596

ABSTRACT

Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of an increased Ca(2+) transient that follows the NHE1 Na(+)/H(+) exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme COOH terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3(-) buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 µM). SFR control was 120 ± 3% (n = 8) of the rapid initial phase and was fully blocked by ETZ (99 ± 4%, n = 6). The SFR corresponded to a maximal increase in pHi of 0.18 ± 0.02 pH units (n = 4), and pHi changes were blocked by ETZ (0.04 ± 0.04, n = 6), as monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with an anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58 ± 9% (n = 6) upon stretch of muscles, assessed by immunoblots. The p90(RSK) kinase inhibitor SL0101-1 (10 µM) blocked the SFR of heart muscles after stretch 102 ± 2% (n = 4) and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch, which potentiates NHE1-mediated H(+) transport in the myocardium.


Subject(s)
Carbonic Anhydrase II/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/pharmacology , Ethoxzolamide/pharmacology , Muscle Spindles/metabolism , Papillary Muscles/drug effects , Sodium-Hydrogen Exchangers/metabolism , Animals , Carbonic Anhydrase II/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Hydrogen-Ion Concentration , Immunoprecipitation , Luminescent Measurements , Male , Papillary Muscles/enzymology , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Wistar , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Sodium-Hydrogen Exchanger 1 , Time Factors
5.
Can J Physiol Pharmacol ; 90(10): 1386-93, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22966876

ABSTRACT

Natriuretic peptides and digitalis-like compounds serve as regulators of homeostasis, including control of volume expansion and blood pressure. The aim of the present study was to explore possible interactions between atrial natriuretic peptide (ANP) and ouabain in the heart. ANP (1 nmol/L) had no effect in papillary muscle preparations from guinea pigs. Ouabain (1 µmol/L) induced positive inotropic effect. The addition of ANP prior to ouabain resulted in a significant decrease in the ouabain-induced positive inotropic effect, manifested as an attenuated increase in twitch maximal upward force slope and resting muscular tension. In addition, ANP caused an increase in Na⁺-K⁺-ATPase activity in heart microsomal preparations. The effect of ouabain on Na⁺-K⁺-ATPase activity was shown in a biphasic manner. Ouabain (0.01-1 nmol/L) had a small but significant increase on pump activity, but higher doses of ouabain inhibited activity. ANP attenuated ouabain-induced Na⁺-K⁺-ATPase activity. Furthermore, ouabain (50 nmol/L) or ANP (10 nmol/L) alone induced Akt activation in cardiomyocytes. However, ANP blocked ouabain-induced Akt activation. These results point to the existence of interactions between ANP and ouabain on Na⁺-K⁺-ATPase signaling and function in the heart, which may be mediated by regulation of Na⁺-K⁺-ATPase activity and (or) signal transduction mechanisms.


Subject(s)
Atrial Natriuretic Factor/metabolism , Cardiotonic Agents/pharmacology , Myocardial Contraction/drug effects , Ouabain/pharmacology , Papillary Muscles/drug effects , Animals , Cardiotonic Agents/antagonists & inhibitors , Cells, Cultured , Guinea Pigs , In Vitro Techniques , Male , Mice , Mice, Transgenic , Microsomes/drug effects , Microsomes/enzymology , Microsomes/metabolism , Muscle Tonus/drug effects , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Ouabain/antagonists & inhibitors , Papillary Muscles/enzymology , Papillary Muscles/metabolism , Proto-Oncogene Proteins c-akt/agonists , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Wistar , Signal Transduction/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Up-Regulation/drug effects
6.
Can J Cardiol ; 27(3): 339-45, 2011.
Article in English | MEDLINE | ID: mdl-21477968

ABSTRACT

BACKGROUND: Fabry disease results from deficiency of alpha-galactosidase A (AGA), causing lysosomal storage of globotriaosylceramide in heart and other tissues. Since 2003, enzymatic replacement therapy with recombinant AGA agalsidase alfa (R-AGA) was approved for clinical use. METHODS: We evaluated whether, in mice knocked out for AGA (FM, n = 31), the myocardium was altered with respect to the wild-type mice (WT, n = 25) and whether alterations were reversed in FM treated with intravenous R-AGA, 0.5 mg/kg every other week during 2 months (FM-AGA, n = 12). RESULTS: Left ventricular (LV) contractility was depressed in FM, evaluated by LV ΔP/Δt (FM = 2832 ± 85 mm Hg/s, WT = 3179 ± 119 mm Hg/s; P < 0.05), papillary muscle contraction (FM = 39.8 ± 17.3 mg, WT = 67.5 ± 15.7 mg; P < 0.05), or shortening fraction measured by M-mode echocardiography (FM = 30% ± 6%, WT = 47% ± 2%; P < 0.05). LV stiffness (arrested hearts) decreased in FM (FM = 35.57 ± 3.5 mm Hg/20 µl; WT = 68.86 ± 6.12 mm Hg/20 µl; P < 0.05). FM myocytes showed augmented size, disorganized architecture, and intracytoplasmic vacuolization. Alterations reverted in FM-AGA: LV ΔP/Δt = 3281 ± 456 mm Hg/s and LV stiffness = 58.83 ± 2.15 mm Hg/20 µl, with normalization of myocyte architecture. No reversion was detected with AGA solvent. CONCLUSIONS: The FM represent a mild, early stage of the disease, since myocardial alterations are not prominent and appear in nonhypertrophic hearts. Reversion of alterations in the FM-AGA suggests that enzymatic replacement therapy can be useful when administered in early stages of this disease.


Subject(s)
Enzyme Replacement Therapy/methods , Fabry Disease/drug therapy , Fabry Disease/pathology , Myocardium/pathology , Papillary Muscles/enzymology , alpha-Galactosidase/pharmacology , Analysis of Variance , Animals , Biopsy, Needle , Disease Models, Animal , Echocardiography/methods , Fabry Disease/diagnostic imaging , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Papillary Muscles/drug effects , Random Allocation , Reference Values
7.
Circ Res ; 105(12): 1232-9, 2009 Dec 04.
Article in English | MEDLINE | ID: mdl-19850940

ABSTRACT

RATIONALE: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction. OBJECTIVE: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI(PKC-P)) expressing mutant cTnI (S43E, S45E, T144E). METHODS AND RESULTS: Two-dimensional gel analysis showed 7.2+/-0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, cTnI(PKC-P) mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F(max)). Additionally, cTnI(PKC-P) mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI(PKC-P) mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI(PKC-P) mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state. CONCLUSIONS: A modest increase in PKC-induced cTnI phosphorylation ( approximately 7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).


Subject(s)
Myocardial Contraction , Myocardium/enzymology , Protein Kinase C/metabolism , Troponin I/metabolism , Ventricular Function, Left , Actin Cytoskeleton/enzymology , Animals , Calcium Signaling , Electrophoresis, Gel, Two-Dimensional , Mice , Mice, Transgenic , Models, Cardiovascular , Muscle Strength , Mutation , Myocardial Contraction/genetics , Papillary Muscles/enzymology , Phosphorylation , Troponin I/genetics , Ventricular Function, Left/genetics , Ventricular Pressure
8.
Exp Physiol ; 93(12): 1223-32, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18586857

ABSTRACT

High extracellular Mg(2+) concentrations ([Mg(2+)](o)) caused a remarkable concentration-dependent and reversible increase in intracellular Mg(2+) concentrations ([Mg(2+)](i)) in beating and quiescent guinea-pig papillary muscles, accompanied by a definite decrease in intracellular Na(+) concentrations ([Na(+)](i)). A change in 1 mm [Mg(2+)](o) evoked a direct change in 0.0161 mm [Mg(2+)](i) and an inverse change in 0.0263 mm [Na(+)](i). Imipramine completely abolished the high [Mg(2+)](o)-induced decrease in [Na(+)](i) and remarkably diminished the high [Mg(2+)](o)-induced increase in [Mg(2+)](i) in papillary muscles. High [Mg(2+)](o) also produced a significant activation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-related kinase 2 (ERK2) that was inhibited by pretreatment with imipramine. These results suggest that the high [Mg(2+)](o)-induced increase in [Mg(2+)](i) could be coupled with the decrease in [Na(+)](i), which might involve activation of the reverse mode of Na(+)-Mg(2+) exchange, accompanied by activation of p38 MAP kinase and ERK2 in the guinea-pig heart.


Subject(s)
Extracellular Fluid/metabolism , Intracellular Fluid/metabolism , Magnesium/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Myocardial Contraction , Papillary Muscles/enzymology , Sodium/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Action Potentials , Animals , Antiporters/antagonists & inhibitors , Antiporters/metabolism , Enzyme Activation , Guinea Pigs , Heart Ventricles/enzymology , Imipramine/pharmacology , In Vitro Techniques , Ion-Selective Electrodes , Kinetics , Muscle Strength , Myocardial Contraction/drug effects , Papillary Muscles/drug effects
9.
J Appl Physiol (1985) ; 105(3): 951-7, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18566187

ABSTRACT

During acclimatization to sustained hypobaric hypoxia, retardation of age-associated decline in left ventricle mechanical activity and improved posthypoxic recovery were accompanied by upregulation of mitochondrial nitric oxide synthase (mtNOS). To evaluate the time course of regression of these effects on deacclimatization, rats exposed to 53.8 kPa in a hypopressure chamber for 5 mo were returned to 101.3 kPa, whereas controls remained at 101.3 kPa throughout the study. At three time points, contractile function in response to calcium and to hypoxia-reoxygenation (H/R) were determined in papillary muscle, and NOS activity and expression were determined in mitochondria isolated from left ventricle. Developed tension was, before H/R, 65, 58, and 40%, and, after H/R, 129, 107, and 71% higher than in controls at 0.4, 2, and 5 mo of normoxia, respectively. Maximal rates of contraction and relaxation followed a similar pattern. All three parameters showed a linear decline during deacclimatization, with mean half-time (t(1/2)) of 5.9 mo for basal mechanical activity and 5.3 mo for posthypoxic recovery. Left ventricle mtNOS activity was 42, 27, and 20% higher than in controls at 0.4, 2, and 5 mo, respectively (t(1/2) = 5.0 mo). The expression of mtNOS showed similar behavior. The correlation of mtNOS activity with muscle contractility sustained a biphasic modulation, suggesting an optimal mtNOS activity. This experimental model would provide the most persistent effect known at present on preservation of myocardial mechanical activity and improved tolerance to O(2) deprivation. Results support the putative role of mtNOS in the mechanism involved.


Subject(s)
Acclimatization , Altitude , Hypoxia/enzymology , Mitochondria, Heart/enzymology , Myocardium/enzymology , Nitric Oxide Synthase/biosynthesis , Ventricular Function, Left , Aging/metabolism , Animals , Atmospheric Pressure , Calcium/metabolism , Disease Models, Animal , Enzyme Induction , Hypoxia/physiopathology , Male , Myocardial Contraction , Nitric Oxide/metabolism , Papillary Muscles/enzymology , Papillary Muscles/physiopathology , Rats , Rats, Wistar , Time Factors
10.
Basic Res Cardiol ; 103(3): 232-43, 2008 May.
Article in English | MEDLINE | ID: mdl-18274801

ABSTRACT

Chronic hemodynamic overload on the heart results in pathological myocardial hypertrophy, eventually followed by heart failure. Phosphatase calcineurin is a crucial mediator of this response. Little is known, however, about the role of calcineurin in response to acute alterations in loading conditions of the heart, where it could be mediating beneficial adaptational processes. We therefore analyzed proteome changes following a short-term increase in preload in rabbit myocardium in the absence or presence of the calcineurin inhibitor cyclosporine A. Rabbit right ventricular isolated papillary muscles were cultivated in a muscle chamber system under physiological conditions and remained either completely unloaded or were stretched to a preload of 3 mN/mm(2), while performing isotonic contractions (zero afterload). After 6 h, proteome changes were detected by two-dimensional gel electrophoresis and ESI-MS/MS. We identified 28 proteins that were upregulated by preload compared to the unloaded group (at least 1.75-fold regulation, all P < 0.05). Specifically, mechanical load upregulated a variety of enzymes involved in energy metabolism (i.e., aconitase, pyruvate kinase, fructose bisphosphate aldolase, ATP synthase alpha chain, acetyl-CoA acetyltransferase, NADH ubiquinone oxidoreductase, ubiquinol cytochrome c reductase, hydroxyacyl-CoA dehydrogenase). Cyclosporine A treatment (1 micromol/l) abolished the preload-induced upregulation of these proteins. We demonstrate for the first time that an acute increase in the myocardial preload causes upregulation of metabolic enzymes, thereby increasing the capacity of the myocardium to generate ATP production. This short-term adaptation to enhanced mechanical load appears to critically depend on calcineurin phosphatase activity.


Subject(s)
Calcineurin/metabolism , Cardiomyopathies/metabolism , Energy Metabolism , Myocardial Contraction , Papillary Muscles/metabolism , Proteomics , Adaptation, Physiological , Adenosine Triphosphate/metabolism , Animals , Calcineurin Inhibitors , Cardiomyopathies/physiopathology , Cyclosporine/pharmacology , Electrophoresis, Gel, Two-Dimensional , Energy Metabolism/drug effects , Enzyme Induction , Female , Papillary Muscles/enzymology , Phosphoric Monoester Hydrolases/metabolism , Proteomics/methods , Rabbits , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Time Factors , Tissue Culture Techniques
11.
Am J Physiol Cell Physiol ; 294(1): C106-17, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17942637

ABSTRACT

Although beta(2)-adrenoceptors represent 15-25% of beta-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of beta(2)-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5% at 60 nM) followed by a moderate positive inotropic effect (+36% at 6 microM) due to activation of beta(1)-adrenoceptors. In the presence of 4 microM atenolol, the concentration-dependent NIE (-12% at 6 microM) was biphasic, best described by a double logistic equation with respective EC(50) values of 3 and approximately 420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/cytosolic phospholipase A(2) (cPLA(2)) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by cPLA(2). The possibility that these effects are due to an equilibrium between different affinity states of the receptor (G(s)/G(i) coupled and G(i) independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that beta(2)-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state.


Subject(s)
Adrenergic beta-2 Receptor Agonists , Adrenergic beta-Agonists/pharmacology , Albuterol/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Group IV Phospholipases A2/metabolism , Myocardial Contraction/drug effects , Nitric Oxide/metabolism , Papillary Muscles/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Adrenergic beta-1 Receptor Agonists , Adrenergic beta-Antagonists/pharmacology , Animals , Arachidonic Acid/metabolism , Atenolol/pharmacology , Catecholamines/metabolism , Depression, Chemical , Dose-Response Relationship, Drug , Drug Partial Agonism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guinea Pigs , In Vitro Techniques , Male , Papillary Muscles/enzymology , Papillary Muscles/metabolism , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Propanolamines/pharmacology , Prostaglandin-Endoperoxide Synthases/metabolism , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/drug effects
12.
Can J Physiol Pharmacol ; 85(3-4): 326-31, 2007.
Article in English | MEDLINE | ID: mdl-17612641

ABSTRACT

To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P<0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2=0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity.


Subject(s)
Heart Ventricles/enzymology , Myosins/metabolism , Papillary Muscles/enzymology , Animals , Blood Pressure , Calcium Chloride/pharmacology , Cardiac Output , Cats , Deer , Dogs , Electrophoresis, Polyacrylamide Gel , Hares , Humans , Middle Aged , Mitral Valve Insufficiency/metabolism , Mitral Valve Insufficiency/physiopathology , Mitral Valve Stenosis/metabolism , Mitral Valve Stenosis/physiopathology , Myosins/chemistry , Rabbits , Rats , Rats, Inbred SHR , Rats, Wistar , Sus scrofa , Ventricular Function
13.
Scand Cardiovasc J ; 40(1): 37-42, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16448996

ABSTRACT

OBJECTIVE: Anti-ss-adrenergic actions of several substances influence heart function significantly. The anti-ss-adrenergic effect of melatonin was investigated, with special attention to protein kinase C (PKC) and nitric oxide (NO). DESIGN: Guinea pig papillary muscles were exposed to melatonin (500 pM) for 15 min and 20 min washout. Contractile force was measured during a bolus of isoproterenol (300 nM) given before melatonin, at the end of melatonin-exposure and after washout. In separate experiments blockers of PKC, NO-synthase (NOS) and melatonin receptors were added, or forskolin (10 microM) substituted for isoproterenol. RESULTS: Melatonin significantly reduced the increase in contractile force in response to isoproterenol, both when present and after melatonin-washout. The reduction was unaffected by inhibition of PKC, while inhibition of melatonin receptors or NOS seemed to abolish the effect. Melatonin induced a sustained but not acute reduction of contractile force response with forskolin stimulation. This was abolished by NOS-inhibition. CONCLUSION: Receptor-mediated immediate and sustained anti-ss-adrenergic effects of melatonin were demonstrated in contractile function. A role for NO in the response was indicated, while a role for PKC was not verified.


Subject(s)
Adrenergic beta-Antagonists/pharmacology , Melatonin/pharmacology , Papillary Muscles/drug effects , Animals , Enzyme Inhibitors/pharmacology , Guinea Pigs , In Vitro Techniques , Myocardial Contraction , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Papillary Muscles/enzymology , Receptors, Melatonin/drug effects , Receptors, Melatonin/metabolism , Tryptamines/pharmacology
14.
Basic Clin Pharmacol Toxicol ; 98(1): 74-8, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16433895

ABSTRACT

In order to clarify the mechanisms of the positive inotropic actions of levosimendan and its optical isomer, dextrosimendan, we compared their concentration-dependent effects in intact papillary muscles, permeabilized cardiomyocytes and in purified phosphodiesterase enzyme preparations of guinea-pig hearts. In papillary muscles twitch tension increased with EC50 values of 60 nM and 2.8 microM for levosimendan and dextrosimendan, respectively. Hence, the two enantiomers exhibited a 47 times potency difference in their positive inotropic effects in a preparation where theoretically Ca2+-sensitization and phosphodiesterase inhibition could both contribute to the positive inotropic effects. In guinea-pig cardiomyocytes, levosimendan and dextrosimendan increased isometric force production (at pCa 6.2) due to Ca2+-sensitization with EC50 values of 8.4 nM and 0.64 microM, respectively, with a similar relative potency difference of 76. A major difference appeared in their relative pharmacological potencies, however, when the inhibitory effects of the two enantiomers were assayed on phosphodiesterase III, purified from guinea pig left ventricle (i.e. the phosphodiesterase isoenzyme which is dominant in that tissue). Levosimendan was a 427 times more potent phosphodiesterase inhibitor than dextrosimendan, with IC50 values of 7.5 nM, and 3.2 microM, respectively. Taken together, our data support the hypothesis that levosimendan and dextrosimendan exert their positive inotropic effects via a stereoselective Ca2+-sensitizing mechanism and not via stereoselective inhibition of phosphodiesterase III in the myocardium.


Subject(s)
Calcium/metabolism , Cardiotonic Agents/pharmacology , Hydrazones/pharmacology , Pyridazines/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Animals , Cardiotonic Agents/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3 , Dose-Response Relationship, Drug , Guinea Pigs , Heart Ventricles/cytology , Heart Ventricles/drug effects , Heart Ventricles/enzymology , Hydrazones/chemistry , In Vitro Techniques , Isomerism , Myocardial Contraction , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Papillary Muscles/cytology , Papillary Muscles/drug effects , Papillary Muscles/enzymology , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Pyridazines/chemistry , Simendan
15.
Basic Res Cardiol ; 100(3): 224-30, 2005 May.
Article in English | MEDLINE | ID: mdl-15630521

ABSTRACT

UNLABELLED: Heme oxygenase-1 (HO-1) is the inducible isoform of heme oxygenase and plays a role in defense against cellular stress. The effects of HO-1 on cardiac muscle contractility, however, are unknown. METHODS: HO-1 was induced by intraperitoneal injection of hemin in rabbits 24 and 48 h before isolating right ventricular papillary muscles for mechanical in vitro analysis at baseline and during stimulation with isoprenalin. Western blotting and activity measurement con.rmed upregulation of HO-1 in ventricular tissue, and immunohistochemical stainings showed localization in the cardiac endothelium. RESULTS: Baseline mechanical performance of papillary muscles and maximal inotropic response to ISO was not significantly affected by HO-1 induction. Also, the log(EC50) of the ISO concentration-response curve was not affected by HO-1 induction. Inhibition of heme oxygenase with stanneous mesoporphyrin or chromium mesoporphyrin in muscles with induced HO-1, however, shifted the log(EC50) of the ISO concentration-response curve from -6.9 +/- 0.2 to -6.0 +/- 0.2 (p = 0.008). CONCLUSION: These results indicate that induction of cardiac HO-1 has no direct effect on baseline contractility. Pharmacological inhibition of HO-1 upon induction, however, diminishes cardiac muscle sensitivity to beta-adrenergic stimulation. These results caution against pharmacologically targeting HO-1 when an activated adrenergic system is important for hemodynamic stability.


Subject(s)
Adrenergic beta-Agonists/pharmacology , Isoproterenol/pharmacology , Myocardial Contraction/drug effects , Papillary Muscles/enzymology , Animals , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Mesoporphyrins/pharmacology , Myocardial Contraction/physiology , Myocardium/enzymology , Papillary Muscles/drug effects , Rabbits
16.
Am J Physiol Endocrinol Metab ; 287(5): E834-41, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15265762

ABSTRACT

AMP-activated protein kinase (AMPK) is a serine-threonine kinase that regulates cellular metabolism and has an essential role in activating glucose transport during hypoxia and ischemia. The mechanisms responsible for AMPK stimulation of glucose transport are uncertain, but may involve interaction with other signaling pathways or direct effects on GLUT vesicular trafficking. One potential downstream mediator of AMPK signaling is the nitric oxide pathway. The aim of this study was to examine the extent to which AMPK mediates glucose transport through activation of the nitric oxide (NO)-signaling pathway in isolated heart muscles. Incubation with 1 mM 5-amino-4-imidazole-1-beta-carboxamide ribofuranoside (AICAR) activated AMPK (P < 0.01) and stimulated glucose uptake (P < 0.05) and translocation of the cardiomyocyte glucose transporter GLUT4 to the cell surface (P < 0.05). AICAR treatment increased phosphorylation of endothelial NO synthase (eNOS) approximately 1.8-fold (P < 0.05). eNOS, but not neuronal NOS, coimmunoprecipitated with both the alpha(2) and alpha(1) AMPK catalytic subunits in heart muscle. NO donors also increased glucose uptake and GLUT4 translocation (P < 0.05). Inhibition of NOS with N(omega)-nitro-l-arginine and N(omega)-methyl-l-arginine reduced AICAR-stimulated glucose uptake by 21 +/- 3% (P < 0.05) and 25 +/- 4% (P < 0.05), respectively. Inhibition of guanylate cyclase with ODQ and LY-83583 reduced AICAR-stimulated glucose uptake by 31 +/- 4% (P < 0.05) and 22 +/- 3% (P < 0.05), respectively, as well as GLUT4 translocation to the cell surface (P < 0.05). Taken together, these results indicate that activation of the NO-guanylate cyclase pathway contributes to, but is not the sole mediator of, AMPK stimulation of glucose uptake and GLUT4 translocation in heart muscle.


Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Glucose/metabolism , Monosaccharide Transport Proteins/metabolism , Multienzyme Complexes/metabolism , Muscle Proteins/metabolism , Nitric Oxide Synthase/metabolism , Papillary Muscles/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases , Aminoimidazole Carboxamide/pharmacology , Animals , Biological Transport/drug effects , Enzyme Activation , Glucose Transporter Type 4 , Hypoglycemic Agents/pharmacology , In Vitro Techniques , Male , Multienzyme Complexes/drug effects , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III , Papillary Muscles/drug effects , Papillary Muscles/enzymology , Protein Serine-Threonine Kinases/drug effects , Protein Transport/physiology , Rats , Rats, Sprague-Dawley , Ribonucleotides/pharmacology
17.
Acta Anaesthesiol Belg ; 54(1): 25-31, 2003.
Article in English | MEDLINE | ID: mdl-12703343

ABSTRACT

Sevoflurane has dose-dependent negative inotropic effects on myocardial contractility. The current study investigated whether the nitric oxide pathway is involved in these effects. A laboratory, ex-vivo experiment was performed on 66 isolated papillary muscles. Effects of increasing concentrations of sevoflurane (1, 2 and 3 MAC) were assessed in control conditions, in the presence of Nw-nitro-L-arginine (L-NOARG) and in beta-adrenergic stimulated rat papillary muscles. Contractility was assessed by total developed tension. In baseline conditions, the administration of increasing concentrations of sevoflurane caused a dose-dependent reduction in contractility of respectively 8.6 +/- 1.7%, 14.4 +/- 4.8% and 23.6 +/- 3.9%. This negative inotropic effect was not significantly altered by the administration of the NO-synthase inhibitor L-NOARG (p = 0.09). Under continuous administration of 3 MAC sevoflurane, 4 consecutive concentrations of isoproterenol induced a mean increase of contractility of respectively 43.0 +/- 13.7%, 65.9 +/- 22.6%, 131.2 +/- 25.6% and 122.3 +/- 31.2%. After administration of L-NOARG, the 4 consecutive concentrations of isoproterenol induced a mean increase in contractility of respectively 36.0 +/- 8.5%, 75.0 +/- 17.8%, 143.0 +/- 42.8% and 120.0 +/- 51.4% (p = 0.85). These data indicated that the negative inotropic effects of sevoflurane in rat papillary muscles, both in basic as in beta-adrenergic stimulated conditions, were not altered by blocking the NO-cGMP-system.


Subject(s)
Anesthetics, Inhalation/pharmacology , Anti-Arrhythmia Agents , Heart/drug effects , Methyl Ethers/pharmacology , Nitric Oxide Synthase/physiology , Adrenergic beta-Agonists/pharmacology , Animals , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Isoproterenol/pharmacology , Male , Myocardial Contraction/drug effects , Nitroarginine/pharmacology , Papillary Muscles/drug effects , Papillary Muscles/enzymology , Rats , Rats, Wistar , Sevoflurane
18.
J Appl Physiol (1985) ; 94(2): 555-60, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12531911

ABSTRACT

The present study was designed to examine the acute and chronic effects of endurance treadmill training on citrate synthase (CS) gene expression and enzymatic activity in rat skeletal and cardiac muscles. Adult rats were endurance trained for 8 wk on a treadmill. They were killed 1 h (T(1), n = 8) or 48 h (T(48), n = 8) after their last bout of exercise training. Eight rats were sedentary controls (C) during the training period. CS mRNA levels and enzymatic activities of the soleus and ventricle muscles were determined. Training resulted in higher CS mRNA levels in both the soleus muscles (21% increase in T(1); 18% increase in T(48), P < 0.05) and ventricle muscles (23% increase in T(1); 17% increase in T(48), P < 0.05) when compared with the C group. The CS enzyme activities were 42 (P < 0.01) and 25% (P < 0.01) greater in the soleus muscles of T(1) and T(48) groups, respectively, when compared with that of the C group. Soleus CS enzyme activity was significantly greater in the T(1) vs. T(48) groups (P < 0.05). However, no appreciable alterations in CS enzyme activities were observed in the ventricle muscles in both training groups. These findings suggest differential responses of skeletal and cardiac muscles in CS enzymatic activity but similar responses in CS gene expression at 1 and 48 h after the last session of endurance training. Moreover, our data support the existence of an acute effect of exercise on the training-induced elevation in CS activity in rat soleus but not ventricle muscles.


Subject(s)
Citrate (si)-Synthase/metabolism , Muscle, Skeletal/enzymology , Papillary Muscles/enzymology , Physical Education and Training , Physical Endurance , Animals , Citrate (si)-Synthase/genetics , Gene Expression , Male , Rats , Rats, Sprague-Dawley
19.
Biol Trace Elem Res ; 96(1-3): 203-8, 2003.
Article in English | MEDLINE | ID: mdl-14716099

ABSTRACT

This study was performed with the objective of assessing the mechanical response of the myocardium to different levels of cerium and delineation of the mechanism underlying the mediation of the functional changes. Rat ventricular papillary muscle was used as the experimental model. Isolated papillary muscles were exposed to different concentrations of CeCl3 and the force of contraction was measured using a force transducer. Experiments have revealed that the negative inotropic response to CeCl3 was proportional to its concentration. The inotropic changes were found to be completely reversible at concentrations < or =5 microM, and partially reversible at higher concentrations. Neutralization of cerium-induced inotropic changes by the superoxide anion scavenger superoxide dismutase (SOD) at concentrations < or =5 microM indicates that the mechanical changes are mediated by reactive oxygen species. At higher concentrations of Ce3+, SOD partially reversed the contractile changes. The beneficial effect of SOD was seen only if the muscles were pretreated with the scavenger prior to the addition of cerium chloride.


Subject(s)
Cerium/pharmacology , Papillary Muscles/drug effects , Reactive Oxygen Species/metabolism , Animals , In Vitro Techniques , Muscle Contraction/drug effects , Papillary Muscles/enzymology , Papillary Muscles/metabolism , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolism
20.
Am J Physiol Heart Circ Physiol ; 283(4): H1471-80, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12234799

ABSTRACT

The possible involvement of different kinases in the alpha(1)-adrenoreceptor (AR)-mediated positive inotropic effect (PIE) was investigated in rat papillary muscle and compared with beta-AR-, endothelin receptor- and phorbol ester-induced changes in contractility. The alpha(1)-AR-induced PIE was not reduced by the inhibitors of protein kinase C (PKC), MAPK (ERK and p38), phosphatidyl inositol 3-kinase, or calmodulin kinase II. However, PKC inhibition attenuated the effect of phorbol 12-myristate 13-acetate (PMA) on contractility. alpha(1)-AR-induced PIE was reduced by approximately 90% during inhibition of myosin light chain kinase (MLCK) by 1-(5-chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine (ML-9). Endothelin-induced PIE was also reduced by ML-9, but ML-9 had no effect on beta-AR-induced PIE. The Rho kinase inhibitor Y-27632 also reduced the alpha(1)-AR-induced PIE. The alpha(1)-AR-induced PIE in muscle strips from explanted failing human hearts was also sensitive to MLCK inhibition. alpha(1)-AR induced a modest increase in (32)P incorporation into myosin light chain in isolated rat cardiomyocytes. This effect was eliminated by ML-9. The PIE of alpha(1)-AR stimulation seems to be dependent on MLCK phosphorylation.


Subject(s)
Cardiotonic Agents/pharmacology , Isoproterenol/pharmacology , Myosin Light Chains/metabolism , Papillary Muscles/enzymology , Receptors, Adrenergic, alpha-1/metabolism , Animals , Azepines/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carcinogens/pharmacology , Endothelin-1/pharmacology , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Myosin Light Chains/antagonists & inhibitors , Papillary Muscles/drug effects , Phenylephrine/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Rats , Receptors, Endothelin/metabolism , Tetradecanoylphorbol Acetate/pharmacology , rho-Associated Kinases
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