Cardiac Cyclic AMP Research Articles

Abstract 19593: Sirtuin-3 is Essential for Glucagon-like Peptide-1-mediated Restoration of Diabetic Cardiomyopathy

Introduction: Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase and its expression is increased by fasting and exercise in skeletal muscle; however, the impact of anti-diabetic intervention on diabetic myocardium remained uncertain. Hypothesis: We hypothesized that the anti-diabetic remedy glucagon-like peptide-1 agonist (exendin-4; Ex4) may ameliorate mitochondrial remodeling observed in diabetic cardiomyopathy. Methods: Type 2 diabetic mice [T2DM; KKAy and dietary diabetes induced by high fat and high sucrose diet (HF/HS), male 14-16 w/o] and age- and gender-matched SIRT3 knockout mice with dietary diabetes (sirHF/HS) diet were allocated into Ex4 (24 nmole/kg/day for 40 days) and vehicle (CON) groups. Results: T2DM-CON and sirHF/HS-CON exhibited myocardial hypertrophy, increased fibrosis, decreased cardiac capillary density, and defragmented mitochondria, which were reversed by Ex4. Consistently with the changes in mitochondrial morphology, the mitochondrial regulatory proteins mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2) ratio was reduced in T2DM-CON and sirHF/HS-CON, which were ameliorated by Ex4 treatment exclusively to T2DM-CON. T2DM-CON exhibited decrease in cardiac SIRT3 level compared to nondiabetic counterpart, which was ameliorated by Ex4. Interestingly, cardiac remodeling of sirHF/HS-CON remained unchanged by Ex4 treatment, exclusively to cardiac capillary density. T2DM-CON and sirHF/HS-CON exhibited decrease in cardiac cyclic AMP level, which were attenuated by Ex4. The GLP-1 inactive analog (Ex9-39) failed to increase cardiac cyclic AMP concentration in mouse heart. In vitro analysis using cultured cardiomyocytes revealed that increase in intracellular cAMP levels by isoprotelenol (10microM) or 8-bromo-cyclic AMP (10 microM) enhanced SIRT3 mRNA, which was reversed by protein kinase A (PKA) inhibition. Conclusions: Our results indicate that Ex4 reversed abnormal suppression of SIRT3 in mitochondria of diabetic heart via restoration of cardiac cAMP.

Abstract 205: Mitochondrial Sirt3 Is Upregulated By Glucagon-like Peptide-1 Receptor Activation And Contributes To Reversal Of Cardiac Mitochondrial Remodeling Induced By Type 2 Diabetes.

RATIONALE: Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that maintains basal ATP yield and its expression level is increased by fasting, exercise, and some NAD+ intermediates. We recently reported that the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 (Ex-4) ameliorated cardiac mitochondrial remodeling in diabetic cardiomyopathy via increase in cardiac cyclic AMP (AJP2013). Because changes in cyclic AMP level is regulated by adenylyl cyclase which is one of the downstream target of Ex-4, we hypothesized that SIRT3 may involve in the Ex-4-mediated myocardial reverse remodeling of mitochondria in diabetic mice. METHODS: Type 2 diabetic Mice (16-week old male) were allocated into experimental groups as follows: Ex-4 (24 nmole/kg/day, subcutaneously administrated by osmotic pump for 40 days, DIO/Ex-4) and vehicle control (DIO/CON). Heart samples and cultured rat neonatal cardiomyocytes were subjected to mitochondrial fractionation using density gradient. RESULTS: Cardiac cyclic AMP concentration and phosphorylation of CREB were elevated in DIO-ex4, suggesting successful administration of Ex-4. Electron microscopic analysis revealed that Ex-4 reversed destroyed cristae structure and defragmented mitochondria of DIO/CON heart. The ratio of expression levels of Mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2) was consistently suppressed by Ex-4 treatment, suggesting normalization of mitochondrial morphology. Of note, DIO-CON exhibited marked decrease in cardiac SIRT3 level compared to lean/nondiabetic counterpart, which was reversed by exendin-4 treatment. Pharmacological production of intracellular cAMP levels (Isoprotelenol (10 microM) and 8-bromo-cyclic AMP (1 mM)) increased SIRT3 mRNA in cultured primary cardiomyocytes. The AMP-activated protein kinase (AMPK) inhibitition blocked the increase in SIRT3 mRNA, indicating that the SIRT3 expression was regulated by AMPK-dependent manner. CONCLUSIONS: Our results indicate that Ex-4 reversed abnormal suppression of SIRT3 in mitochondria via activating the PKA/AMPK-dependent pathway.

Mitochondrial SIRT3 is upregulated by Glucagon-like peptide-1 receptor activation and contributes to reversal of cardiac mitochondrial remodeling induced by type 2 diabetes

Rationale: Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates mitochondrial function and maintains basal ATP yield, whereas the role in myocardium remain uncertain. We reported that the GLP-1 receptor agonist exendin-4 (Ex-4) ameliorated cardiac mitochondrial remodeling in diabetic cardiomyopathy via cyclic AMP axis (ESC 2012, AHA 2012). Changes in intracellular ATP levels are associated with cyclic AMP, which is generated by adenylyl cyclase utilizing ATP, we thus hypothesized that SIRT3 may involve in Ex-4-mediated myocardial reverse remodeling in diabetes. Methods: Type 2 diabetic mice were allocated into Ex-4 (24 nmole/kg/day, DIO/Ex-4) and vehicle (DIO/CON). Heart samples and cultured cardiomyocytes were subjected to mitochondrial fractionation followed by mRNA and protein analyses. Results: Cardiac cyclic AMP concentration and phosphorylation of CREB were elevated in DIO/Ex-4, suggesting successful administration of Ex-4. Electron microscopy revealed that DIO/CON exhibited mitochondria with destroyed cristae structure and defragmented, which was reversed by Ex-4 (Fig. A). The ratio of Mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2), which modulates mitochondria morphology, was consistently suppressed by Ex-4 (Fig. B), suggesting normalization of mitochondrial morphology. DIO-CON exhibited decrease in cardiac SIRT3 level compared to nondiabetic counterpart, which was reversed by Ex-4 (Fig. C). Increased cAMP levels (Isoprotelenol and 8-bromo-cyclic AMP) enhanced SIRT3 mRNA and protein levels in cultured cardiomyocytesm which was blocked by AMP-activated protein kinase (AMPK) inhibitor (compound C). ![Figure][1] Conclusions: Our results indicate that Ex-4 reversed abnormal suppression of SIRT3 in mitochondria via activating the PKA/AMPK-dependent pathway. [1]: pending:yes

Delayed cardiac protection against harmful consequences of stress can be induced in experimental atherosclerosis in rabbits.

Multiple brief periods of rapid ventricular pacing confer both short- and long-term protection on the ischaemic heart. The duration of the short-term protection does not exceed 2 h, whereas the long-term protective effect appears several hours after the inducing insults, with maximal protection 24-48 h later. Up to now, delayed cardiac protection by preceding ischaemic insults against harmful consequences of stress has been produced in the normal, healthy animal only. The purpose of this study was, therefore, to test whether delayed cardiac protection can be induced in experimental atherosclerosis in rabbits produced by feeding cholesterol-rich diet over 2 months. Repeated brief periods of rapid ventricular pacing were used to induce delayed protection of the heart. Moderation of post-pacing right intracavitary ST segment elevation and that of the left ventricular end-diastolic pressure (both produced by ventricular overpacing: 500 beats/min for 15 min) were found in normal animals as well as in those fed cholesterol-enriched diet. The short-lived protection induced by a single 'preconditioning' pacing was reproducible only in normal animals. As measured by means of radioimmunoassay, the protective effect of either short- or long-term protection appeared in parallel with an attenuation of ischaemia-induced increase in cardiac cyclic AMP content, in both normal and atherosclerotic rabbits. An increase in cardiac cyclic GMP content was characteristic of the short- but not long-term protection. These results suggest that the delayed cardiac protection by preceding multiple brief rapid pacings operates even in experimental atherosclerosis, but the short-term protection induced by a single preconditioning stimulus is lost.

S24-3 - PDE isozyme specific regulation of cardiac cyclic AMP and contractility in mammalian myocardium: clinical relevance

S24-3 - PDE isozyme specific regulation of cardiac cyclic AMP and contractility in mammalian myocardium: clinical relevance

Anion Manipulation, a Novel Antiarrhythmic Approach: Mechanism of Action

We recently reported that modulation of anion homeostasis by substitution of extracellular chloride by nitrate prevents ischaemia- and reperfusion-induced ventricular fibrillation (VF) in rat and rabbit in vitro by an unknown mechanism independent of haemodynamic changes but related to widening of QT interval (Ridley and Curtis 1991). In the present study we have examined three possible explanations for the mechanism: modification of membrane anion permeability, alteration of cyclic nucleotide homeostasis and alteration of intracellular pH. In isolated Langendorff-perfused rat heart ( n = 12/group), substitution of chloride in modified Krebs perfusion solution by anion surrogates (methylsulphate, bromide, nitrate or iodide) inhibited left regional ischaemia-and reperfusion-induced arrhythmias only when the membrane permeability of the surrogate was greater than that of chloride (e.g., nitrate, bromide, iodide); the least permeant anion, methylsulphate, was proarrhythmic during ischaemia. Rank order of arrhythmia susceptibility correlated with the relative permeability of each anion, with near abolition of both ischaemia- and reperfusion-induced VF ( P < 0.05) by the most permeant anions (iodide and nitrate). Arrhythmia suppression occurring in the iodide and nitrate groups was accompanied by significant widening of QT interval at 90% repolarization, with effects substantially more marked during ischaemia than before ischaemia. In separate studies using the same model we determined cardiac cyclic (c) AMP cGMP content and their molar ratios by radioimmunoassay of biopsies before, during and after ischaemia. There was no meaningful relation between cyclic nucleotide content and rank order of arrhythmia susceptibility ruling out changes in the former as a contributory mechanism to the latter. In further studies we measured intracellular pH in the isolated perfused rat heart by phosphorus NMR spectroscopy. Nitrate caused a slight intracellular acidosis which was exacerbated when hearts were made globally ischaemic, indicating that its antiarrhythmic activity was not a consequence of alkalinisation (e.g., inhibition of chloride-bicarbonate exchange). To test for inherent adverse effects on cardiac contractile function we analysed Starling curves in isolated rat hearts perfused under conditions equivalent to those used for arrhythmia studies. There was no relationship between perfusion anion composition and systolic (developed pressure at constant intraventricular volume, and pressure-volume slope) or diastolic function (end-diastolic pressure at constant intraventricular volume). In conclusion, alteration of membrane permeability is a mechanism which may be sufficient to explain modulation of arrhythmias by manipulation of extracellular anion content, appears to be devoid of deleterious effects on contractile function, and may represent a focus for future antiarrhythmic drug development.

Cardiac inotropic responses to calcium and forskolin are not altered by prolonged isoproterenol infusion

Effects of prolonged isoproterenol infusion upon the density of cardiac calcium channels, calcium-mediated contractile responses, and the ability of forskolin to enhance tension development and cyclic AMP accumulation were studied in ventricular muscle preparations from Sprague-Dawley rats. Isoproterenol infusion (400 μg/kg per h s.c., 4 days) significantly decreased calcium channel density (B max) in cardiac microsomal membranes as quantified by a 32% decrease in specific [ 3H]nitrendipine binding sites; binding affinity (K D) was unchanged. A 57% decrease of β-adrenoceptors confirmed homologous down regulation. To examine functional effects of decreased [ 3H]nitrendipine binding sites, responses to calcium, BAY K8644 and nifedipine were determined in isolated right ventricular strips. Significant decreases in basal developed tension were observed in muscles from isoproterenal-infused rats. However, concentration-dependent increases in contractility in response to CaCl 2 or BAY K8644 were comparable, and the negative inotropic effect of nifedipine was unchanged. Whereas isoproterenol infusion was associated with significantly decreased basal cardiac cyclic AMP concentrations, exposure of ventricular strips from either vehicle- or isoproterenol-infused rats to 10 μM forskolin resulted in comparable increases in cyclic AMP and in developed tension. Cumulative, submaximal concentrations of forskolin also produced similar increases in contraclity with maximum responses in ventricular strips from vehicle-infused animals attained at 4.4 μM forskolin. Higher concentrations resulted in automaticity. By contrast, ventricle from isoproterenol-infused animals responded to 14.4 μM forskoklin with maximal increases in force of contraction.

The Role of Cyclic AMP and the Dihydropyridine-Sensitive Channels on the Mechanism of Action of Milrinone (Corotrope)

Milrinone (Corotrope) increased cyclic AMP levels in dog guinea pig and rat cardiac muscle. A correlation between the increase in contractile force and cyclic AMP levels in dog ventricular trabeculae was obtained when measurements were made 60-70 s after the addition of milrinone. When cyclic AMP levels were determined at the time of maximal contractile response, only concentrations of milrinone 200-300 times the inotropic dose had any effects in elevating cyclic nucleotide levels. In dog Purkinje tissue and rat cardiac muscle, milrinone had minimal or no effects on contractile force but increased cardiac cyclic AMP levels. Sequential doses of milrinone to perfused guinea pig hearts resulted in severe tachyphylaxis to the inotropic activity of milrinone. However, under these conditions, milrinone was found to elevate cardiac cyclic AMP upon each administration. Furthermore, in this preparation, cross-tachyphylaxis between Bay K 8644 and milrinone was demonstrated. The mechanism of action of Bay K 8644, which acts on sarcolemmal Ca2+, is not mediated by increases in cyclic AMP. Following development of tachyphylaxis to Bay K 8644, in the guinea pig hearts, addition of milrinone results in no increases in contractile force but a significant increase in cyclic AMP levels. In all of the instances of tachyphylaxis, isoproterenol increased both contractile force and cyclic AMP. The data are discussed and we put forth the hypothesis that increased cardiac force due to milrinone is in part due to a direct or indirect action on sarcolemmal Ca2+ channels.

Effect of exercise on cardiac cyclic AMP

The effect of one bout of intense swimming caused significant increases in the cyclic AMP content of fast-twitch white skeletal muscle, liver, and heart. Further investigation of the exercise-induced increase in myocardial cyclic AMP indicates that the nucleotide content remained elevated long after (24 h) termination of exercise. This increase in cyclic AMP was time dependent, with the level increasing gradually throughout the work bout. The increase in cardiac cyclic AMP seemed to be independent of work intensity, provided that work time was of sufficient duration (greater than or equal to 30 min). Increases in cardiac cyclic AMP were also seen when rats were exposed to 2 degrees C for 1-7 d. The increases in cyclic AMP seen following exercise and cold exposure were accompanied by an increase in cardiac cyclic AMP phosphodiesterase (PDE) activity. Our working hypothesis was that prolonged elevations in cyclic AMP produced an induction and/or activation of one or more of the PDE isozymes. When we administered dibutyryl cyclic AMP to rats, cardiac PDE activity was increased. This increase was inhibited by cycloheximide, suggesting that the elevated enzyme activity is mediated by the synthesis of new protein. These data support the concept that cyclic AMP is involved in the regulation of its own metabolism during physiological stress.

Pharmacological Actions of DPN 205–734, a Novel Cardiotonic Agent

DPN 205-734 [5-(4-cyanophenyl)-1,2-dihydro-6-methyl-2-oxopyridin-3-carbo nitrile] was investigated in vitro in Langendorff rabbit hearts, guinea pig and rabbit papillary muscles, and rat myocardium and in vivo in anesthetized and unanesthetized dogs, pithed open-chest cats, anesthetized rats, and cardiomyopathic hamsters. In vitro, this substance caused a concentration-dependent positive inotropic effect. Left ventricular dP/dtmax was increased in anesthetized dogs after intravenous injection of 0.02 and 0.2 mg/kg (35 +/- 10 and 130 +/- 13%, respectively) and in unanesthetized dogs after oral doses of 0.05-0.5 mg/kg (15 +/- 2 to 71 +/- 14%). DPN 205-734 lowered blood pressure and total peripheral resistance in several experimental models, indicating an afterload-reducing effect. It induced moderate tachycardia. The positive inotropic effect is not explainable by beta-stimulation as shown in pithed open-chest cats pretreated with propranolol. A phosphodiesterase-inhibiting activity (IC50 = 35.5 microM), measured in rat myocardium, may be primarily responsible for the positive inotropic action. In guinea pig papillary muscles partially depolarized with 22 mM K+, DPN 205-734 in a concentration of 1 microM restored slow action potentials, which were then blocked by carbachol. These actions can be explained by the increase in cardiac cyclic AMP level due to a phosphodiesterase-inhibiting effect. In rabbit papillary muscles the positive inotropic effect of DPN 205-734 (100 microM) was only moderately inhibited by carbachol (3 microM), suggesting an additional mechanism.

Cardiac muscle ultrastructure and cyclic AMP reactions to altered gravity conditions.

Morphological and biochemical analyses of heart muscle of rats subjected to microgravity on Spacelab 3 (SL-3) flight and rats born and reared under increased gravity (1.7 G) conditions were compared with 1-G controls. Electronmicroscopic studies showed an increase in the number of lipid droplets and in areas of glycogen storage. Distribution changes of microtubules and cytoskeletal elements from both SL-3 and 1.7-G groups were observed. The high Km cyclic AMP phosphodiesterase activity was lower (P less than 0.05) in SL-3 heart muscle, and low Km activity was lower in 1.7-G males but was unaltered in females. Cyclic AMP-dependent protein kinase (cA-PK) activity was decreased in subcellular fractions of heart muscle of SL-3 animals. Recompartmentalization of cA-PK activity occurred in particulate tissue fraction of 1.7-G animals (70.3% of total for 1.7 G vs. 35.9% for controls). Phosphorylation of endogenous low-mobility proteins increased in SL-3 heart-soluble fractions. Photoaffinity labeling (18 h, 4 degrees C) decreased in type II cA-PK regulatory (R) subunits in both SL-3 and in 1.7-G male heart tissue particulate fractions. The 1.7-G female heart R subunit distribution did not differ from controls. These findings indicate that in heart muscle altered gravity conditions influenced physiological reactions similar to catecholamine-induced receptor-mediated hormonal responses.

Effects of several newer cardiotonic drugs on cardiac cyclic AMP metabolism

The purpose of this study was to investigate the possible roles of selective inhibition of cyclic nucleotide phosphodiesterase (PDE) isozymes, adenylate cyclase activation, and tissue cyclic 3',5'-adenosine monophosphate (cyclic AMP) elevation in the positive inotropic action of five NEW cardiotonic drugs. Three PDE isozymes (PDE I, II and III), homogenates, and slices of guinea pig ventricles were used. The inotropics amrinone, milrinone, AR-L 115BS, MDL 17,043, and RMI 82,249 all inhibited cyclic AMP hydrolysis by PDE III in a concentration-dependent manner, as did the PDE inhibitors aminophylline and 1-methyl-3-isobutylxanthine (MIX). All drugs except for AR-L 115BS inhibited PDE III at concentrations lower than those producing a standard inotropic response. A significant correlation ( r = 0.80, P < 0.05) was observed between PDE III inhibition and inotropic activity for six of the drugs. Only aminophylline and MIX, but none of the cardiotonic drugs, inhibited cyclic AMP hydrolysis by PDE I and II and cyclic 3',5'-guanosine monophosphate (cyclic GMP) hydrolysis (amrinone not tested) by PDE I. Further, none of the cardiotonic drugs inhibited the calmodulin-stimulated cyclic AMP hydrolysis by PDE I, indicating their lack of calmodulin antagonist activity. These drugs also did not stimulate adenylate cyclase activity but all increased net cyclic AMP formation from ATP in guinea pig ventricular homogenates through inhibition of cyclic AMP breakdown. Amrinone, milrinone, MDL 17,043 and RMI 82,249, but not AR-L 115BS, raised cyclic AMP levels significantly (P < 0.05) in guinea pig ventricular slices. Also, amrinone, MDL 17,043 and RMI 82,249, but not AR-L 115BS, potentiated forskolin-induced cyclic AMP increase. These data taken together suggest that the specific inhibition of cyclic AMP PDE III isozyme and the consequent elevation of tissue cyclic AMP levels in cardiac tissue are an important mechanism of action of amrinone, milrinone, MDL 17,043 and RMI 82,249. Because AR-L 115BS did not increase cyclic AMP levels, it is likely that another mechanism may participate in the inotropic response to AR-L 115BS.

Positive Inotropic and Electrophysiological Effects of APP 201-533 Can Be Explained by an Increase of Cardiac Cyclic AMP

The possible mechanism of action of the positive inotropic agent APP 201-533 [3-amino-6-methyl-5-phenyl-2(1H)-pyridinone] was investigated. In guinea pig papillary muscles, APP 201-533 increased force of contraction concentration dependently at 10(-4)-10(-3) M. The effect was associated with an abbreviation of contraction and relaxation time. In guinea pig papillary muscles partially depolarized with 22 mM K+, APP 201-533 in concentrations of 10(-4) and 10(-5) M restored slow action potentials, which were not influenced by cimetidine, propranolol, and prazosin but were blocked by the Ca2+ antagonist PY 108-068 and by carbachol in an atropine-sensitive manner. The concentration-effect curve of histamine was shifted to the left in the presence of APP 201-533. These actions can be explained by the increase in cardiac cyclic AMP level that was found in rabbit papillary muscles and guinea pig left atria treated with APP 201-533 due to the known phosphodiesterase inhibitory effect of pyridinones. APP 201-533 increased the inotropic potency of dihydroouabain in guinea pig papillary muscles. It seems possible that this effect is based on an increased Ca2+ sensitivity of myocardial contractile structures as described previously for APP 201-533.

Correlation between haemodynamic and metabolic changes in three models of experimental cardiac hypertrophy.

Three models of cardiac hypertrophy (aortic constriction, application of isoproterenol, daily injections of triiodothyronine) were characterized in haemodynamic and in metabolic terms. Heart function was evaluated in closed-chest rats after catheterization of the left ventricle with an ultraminiature catheter pressure transducer. Heart rate (HR), left ventricular systolic pressure (LVSP), and the maximal rate of rise of left ventricular pressure (LV dP/dtmax) were measured. The metabolic parameters determined included the levels of cardiac cyclic AMP and the rates of the biosynthesis of myocardial adenine nucleotides. During the first 72 h after aortic constriction, HR was decreased, whereas LVSP and LV dP/dtmax were both elevated. Within 12 h after administration of isoproterenol, HR and LV dP/dtmax were markedly increased, while LVSP was depressed. In triiodothyronine-treated animals, all haemodynamic parameters were elevated during the first 72 h. Comparison with the time course of changes in metabolic parameters revealed that the rise in cardiac contractility measured as LV dP/dtmax occurred at about the same time as the increase in the content of myocardial cyclic AMP in the isoproterenol- and triiodothyronine-models. In hypertrophy due to aortic constriction, cardiac cyclic AMP was elevated only moderately, and this elevation did not correlate with the enhancement of contractility. Beta-receptor-blockade with propranolol prevented entirely the increase of myocardial adenine nucleotide biosynthesis in the isoproterenol- and triiodothyronine- but not in pressure-induced hypertrophy.

Activation of the cyclic nucleotide phosphodiesterase from rat heart cytosol by phospholipase C

Phospholipase C (clostridium perfringens) significantly increased the cyclic nucleotide phosphodiesterase activity of a crude 105 000 g supernatant from rat heart. This activation only concerned the basal activity of cyclic GMP phosphodiesterase determined with 0.25 μM cyclic GMP as substrate, in the presence of EGTA, whereas stimulation was found to be independent of EGTA when phosphodiesterase activity was measured with 0.25 μM cyclic AMP. Similar qualitative results were found for the three cytosolic forms of phosphodiesterase separated from rat heart supernatant by isoelectric focusing. Supplementary experiments provided evidence that the activation of the cyclic AMP-specific phosphodiesterase was attributable to Phospholipase C activity and not to contaminating protease(s). In contrast, the stimulation of the cyclic GMP phosphodiesterase activity appeared to be largely dependent on the proteolytic activity of commercial Phospholipase C. Phosphatidic acid also significantly increased the cyclic AMP phosphodiesterase activity of the rat heart cytosol. These results suggest that the activation of cardiac cyclic AMP phosphodiesterase may be related to changes in phospholipid metabolism, notably the accumulation of phosphatidate, and relevant to physiological regulatory processes.

The influence of calcium utilization on cardiac cyclic AMP.

The influence of calcium utilization on cardiac cyclic AMP.

Increased cardiac myosin ATPase activity as a biochemical adaptation to running training: Enhanced response to catecholamines and a role for myosin phosphorylation

The effects were studied of prior running training on protein phosphorylation and adenosine triphosphatase (ATPase) activities of natural actomyosin isolated from perfused rat hearts. Myosin Ca 2+-ATPase activities were significantly higher in running-trained hearts than in controls, whereas the Ca 2+-stimulated, Mg 2+-dependent ATPase activities of natural actomyosin were not changed. After treatment of isolated perfused hearts with the β-agonist isoproterenol, both troponin-I and myosin P light chains became phosphorylated. Troponin-I phosphorylation (1 mol/mol) was the same in both sets of hearts and was accompanied by similar changes in cardiac cyclic AMP contents. The V max values for myosin Ca 2+-ATPase activity were increased after isoproterenol treatment in all the perfused hearts, but to a significantly greater extent in the hearts of running trained animals; this was correlated with enhancement of both the rate and extent of myosin P light chain phosphorylation. Enhanced Ca 2+-dependent myosin P light chain phosphorylation, further enhanced by β-adrenergic stimulation, represents, at the molecular level, a biochemical response to running training.

Exercise-induced increases in myocardial adenosine 3′,5′-cyclic monophosphate and phosphodiesterase activity

Numerous cellular biochemical events caused by hormones are mediated throught cyclic AMP. Although many changes occur in the cell during exercise that could be attributed to this nucleotide, little evidence is available implicating it as an important regulator of exercise metabolism. In this investigation it was found that a 60 min bout of treadmill exercise caused a 2.4-fold increase in myocardial cyclic AMP immediately following the work. Rather than the imemediate nucleotide hydrolysis that was expected, it was found that the elevated cyclic AMP level remained for approx. 24 h before returning to control levels. Cardiac glycogen fell to 30% of control after work but supercompensated 60% above control within 1 h following exercise. Therefore, cardiac cyclic AMP was elevated at a time when glycogen was being synthesized. Study of the temporal relationship between the exercise-induced increase in cyclic AMP and cyclic nucleotide phosphodiesterase indicated that the work caused an increase in the hearts' capacity to hydrolyze cyclic AMP. Measurement of heart phosphodiesterase at substrate concentrations of 1.0 and 100 μM produced significant increased in enzyme activity immediately following exercise which remained elevated for 48 h and was back to control activity 96 h following work. These data present a potentially fascinating model for the study of the dissociation between cyclic AMP, glycogenesis and elevations in phosphodiesterase activity in the heart.

Role of Chemosensitivity during Exercise in Normal Subjects and Patients with Pulmonary Emphysema

In 11 normal subjects (mean age = 22.8 years) and 8 patients with pulmonary emphysema (mean age = 70.4 years), the role of chemosensitivity in determining ventilation, cardiac output, lactic acid, and cyclic AMP and GMP was evaluated quantitatively during 150 or 30 W exercise and simulated exercise. Simulated exercise was done while the subjects took a rest by regulating arterial blood gases at exercise levels in patients and at PaO2 = 65 mm Hg and PaCO2 = 48 mm Hg in normal subjects. In normal subjects, the role of arterial blood gases in determining exercise ventilation, cardiac output, cyclic AMP and GMP are large, while those contributed much less to lactic acid. In patients, PaO2 contributed only half of the exercise ventilation. It accounted for a negligibly small portion of exercise cardiac output, lactic acid, and cyclic GMP. These results indicate, by deduction, that either augmentation of chemosensitivity, pH, or humoral factors is responsible for about half of the changes of exercise ventilation in patients with pulmonary emphysema. These factors seem to influence cardiac output, lactic acid, and cyclic AMP and GMP more strongly than PaO2 alone in exercising patients.

Dissociation between Cardiac Cyclic AMP and Myocardial Contractility Induced by Verapamil, Calcium and Magnesium Ions

Isolated, perfused rat hearts were used to study the effects of verapamil, excess calcium ions or excess magnesium ions on changes in heart cyclic AMP and myocardial force of contraction. Verapamil caused a dose-dependent decrease in force of contraction and a nondose-related reduction in cyclic AMP. Perfusion of hearts with medium containing 5 mM calcium produced a significant rise in cyclic AMP, but no change in contractile force. The depressant effects of verapamil on contractility and cyclic AMP were reversed by 5mM calcium; excess calcium in the perfusion fluid also containing verapamil prevented the myocardial depressant effects of verapamil. Acutely elevating the magnesium concentration in the perfusion medium decreased force of contraction, accentuated the negative inotropic effect of verapamil, but did not decrease cyclic AMP or enhance the verapamil-induced reduction in cyclic AMP.