Regulation Of Cardiac Contraction Research Articles

Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol

Previous work has shown that the endogenous cystathionine γ-synthase (CSE)/hydrogen sulfide (H 2S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H 2S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H 2S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H 2S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H 2S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.

Stimulus-specific Differences in Protein Kinase Cδ Localization and Activation Mechanisms in Cardiomyocytes

Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC delta actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKC delta and PKC delta-pY(311), we demonstrate that PKC delta partitions between soluble and particulate fractions (with little Tyr(311) phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKC delta translocation to membranes and phosphorylation at Tyr(311). H(2)O(2) also increases PKC delta-pY(311) in association with its release from membranes. Both PMA- and H(2)O(2)-dependent increases in PKC delta-pY(311) are mediated by Src family kinases, but they occur via different mechanisms. The H(2)O(2)-dependent increase in PKC delta-pY(311) results from Src activation and increased Src-PKC delta complex formation. The PMA-dependent increase in PKC delta-pY(311) results from a lipid cofactor-induced conformational change that renders PKC delta a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKC delta-Y(311) phosphorylation does not grossly alter the kinetics of PMA-dependent PKC delta down-regulation. Rather, tyrosine phosphorylation regulates PKC delta kinase activity. PKC delta is recovered from the soluble fraction of H(2)O(2)-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC delta phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC delta substrate specificity. The stimulus-specific modes for PKC delta signaling identified in this study allow for distinct PKC delta-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.

Exercise training improves cardiac function-related gene levels through thyroid hormone receptor signaling in aged rats.

Exercise training improves the aging-induced downregulation of myosin heavy chain (MHC) and sarcoplasmic reticulum (SR) Ca(2+)-ATPase, which participate in the regulation of cardiac contraction and relaxation. Thyroid hormone receptor (TR), a transcriptional activator, affected the regulation of gene expression of MHC and SR Ca(2+)-ATPase. We hypothesized that myocardial TR signaling contributes to a molecular mechanism of exercise training-induced improvement of MHC and SR Ca(2+)-ATPase genes with cardiac function in old age. We investigated whether TR signaling and gene expression of MHC and SR Ca(2+)-ATPase in the aged heart are affected by exercise training, using the hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo old, swimming training for 8 wk). Trained aged rats showed improvement in cardiac function. Expression of TR-alpha1 and TR-beta1 proteins in the heart were significantly lower in sedentary aged rats than in sedentary young rats and were significantly higher in trained aged rats than in sedentary aged rats. The activity of TR DNA binding to the transcriptional regulatory region in the alpha-MHC and SR Ca(2+)-ATPase genes and the mRNA and protein expression of alpha-MHC and SR Ca(2+)-ATPase in the heart and plasma 3,3'-triiodothyronine and thyroxine levels were altered in association with changes in the myocardial TR protein levels. These findings suggest that exercise training improves the aging-induced downregulation of myocardial TR signaling-mediated transcription of MHC and SR Ca(2+)-ATPase genes, thereby contributing to the improvement of cardiac function in trained aged hearts.

Familial Hypertrophic Cardiomyopathy-linked Alterations in Ca2+ Binding of Human Cardiac Myosin Regulatory Light Chain Affect Cardiac Muscle Contraction

The ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca(2+) sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca(2+)-Mg(2+) binding site of HCRLC, which demonstrated no Ca(2+) binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca(2+) sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca(2+) binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC.

Regulation of cardiac muscle contraction by intracellular Ca2+ under physiological and pathophysiological conditions

Regulation of cardiac muscle contraction by intracellular Ca2+ under physiological and pathophysiological conditions

The special structure and function of troponin I in regulation of cardiac contraction and relaxation.

In this chapter I review evidence for a pivotal role of the sarcomeric thin filament protein, troponin I, in cardiac muscle activation and its modulation by covalent modifications, sarcomere length, and intracellular pH. This evidence demonstrates that the cardiac variant of troponin I (cTnI), which is the only isoform expressed in the adult myocardium, has unique structure and function that are specialized for extrinsic and intrinsic control of cardiac contraction and relaxation.

Effects of calmodulin and okadaic acid on myofibrillar Ca2+ sensitivity in cardiac myocytes.

Whereas it has been established that the phosphorylation of 20 kD regulatory myosin light chain (MLC20) is a key regulator of contraction in smooth muscle, troponin complex has been thought to be that of myofibrillar Ca2+ sensitivity in cardiac muscle. To elucidate the role of the phosphorylation of cardiac regulatory myosin light chain (MLC2) in the regulation of cardiac muscle contraction, we observed effects of calmodulin and okadaic acid, a protein phosphatase inhibitor, on myofibrillar Ca2+ sensitivity as estimated by pCa50 values obtained from pCa-tension relationships using beta-escin-skinned cardiomyocytes from Wistar rat hearts, in relation to changes in the phosphorylation of myofibrillar regulatory proteins. Whereas myofibrillar Ca2+ sensitivity tended to be progressively decreased by repeated Ca2+-activation in the absence of calmodulin (pCa50; from 5.91 to 5.86, n = 5), calmodulin (2.5 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.92 to 6.03, n = 5, p < 0.05). Okadaic acid over 3 microM enhanced Ca2+-activated force, which was inhibited by 50 microM trifluoperazine, a calmodulin antagonist. Okadaic acid (3 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.96 to 6.11, n = 6, p < 0.05). Whereas the phosphorylation level of troponin I was not changed by 3 microM okadaic acid, that of MLC2 was significantly increased by the same dose of okadaic acid (from 12 to 31%, n = 4, p < 0.05). These results suggest that MLC2 phosphorylation plays a partial role in the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle.

Site-Specific Antibody of (Na+ + K+)-ATPase Augments Cardiac Myocyte Contraction without Inactivating Enzyme Activity

(Na+ + K+)-ATPase regulates both excitability and contractility of the heart. Little is known about the molecular basis of the enzyme that underlies its cardiac regulatory functions. Here we demonstrate that the 833KRQPRNPKTDKLVNE847 region, which resides in the α-subunit of rat (Na+ + K+)-ATPase, directly participates in the regulation of cardiac contraction. A site-specific antibody (SSA95) against this peptide sequence markedly increased intracellular Ca2+ transients and contraction (EC50 = 11.4 nM) in intact rat heart cells without inactivating the (Na+ + K+)-ATPase. These novel findings establish the first link between a precise structural region of the (Na+ + K+)-ATPase and cardiac positive inotropy.

A pH-Sensitive Interaction of Troponin I with Troponin C Coupled with Strongly Binding Cross-Bridges in Cardiac Myofilament Activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca2+ regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca2+ regulation.

Regulation of Cardiac Contraction and Relaxation

Regulation of Cardiac Contraction and Relaxation

Altered Regulation of Cardiac Muscle Contraction by Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.

Molecular Cloning of a Novel P2 Purinoceptor from Human Erythroleukemia Cells

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

Thin Filament-Mediated Regulation of Cardiac Contraction

Thin Filament-Mediated Regulation of Cardiac Contraction

Regulation of Cardiac Muscle Contraction by Intracellular Ca2+.

In this short review, some of the factors which influence the intracellular Ca2+ transients and contraction in mammalian cardiac muscles which we have been examining are discussed. The processes concerning the change in [Ca2+]i and contraction are divided into three steps following the scheme proposed by Blinks [6]: Upstream, Central, and Downstream. However, these processes are interrelated. Although the Ca2+ transient is an important step for triggering contraction, it is influenced by the following Central and Downstream mechanisms. The evaluation of Ca2+ sensitivity of the contractile elements (Central mechanism) in intact preparations is discussed; measurement of the relation between [Ca2+]i and tension in tetanic contraction is one of the possible methods. A change in the Ca2+ binding to troponin-C is an important factor to determine the Ca2+ sensitivity but other mechanisms are also considered to be involved. In cardiac muscles, the feedback mechanism from the Downstream, in particular the cross-bridge attachment and detachment, to the Central processes is a physiologically important mechanism in the determination of contractile properties. This feedback mechanism alternatively influences the intracellular Ca2+ transient by changing the affinity of troponin-C for Ca2+. Thus, the interpretation of the measured Ca2+ transients is not simple and the numerous factors involved must be considered. Further study is necessary to elucidate the molecular mechanisms which are responsible for the feedback from the Downstream mechanism to the Central and Upstream mechanisms.

Cytosolic and membrane-bound cyclic nucleotide phosphodiesterases from guinea pig cardiac ventricles

Cyclic nucleotide phosphodiesterase (PDE) activities were characterized in the cytosolic and post-nuclear membrane preparations of guinea pig cardiac ventricles. The cytosolic PDE activities were stimulated 5-fold by calmodulin (CaM) on both substrates (1 μM) and 1.2-fold by cGMP (5 μM) on cAMP hydrolysis. Conversely, in the membrane preparation, CaM only stimulated PDE activities 1.2- to 1.4-fold, but cGMP induced a 3-fold increase of the hydrolysis of cAMP. In both the cytosolic and the membrane preparations, the hydrolysis of cAMP was inhibited by 100 μM of either the PDE III inhibitor SK & F 94120 (27% and 31% respectively) or the PDE IV inhibitor rolipram (14% and 23% respectively). Four peaks were resolved from the cytosolic preparation by chromatography. Peak A and peak B hydrolyzed both cAMP and cGMP and were stimulated respectively by CaM and cGMP. Peak C and peak D selectively hydrolyzed cAMP. Peak C had an apparent K m value for cAMP of 3.3 μM and was inhibited by PDE IV inhibitors. Peak D showed an apparent K m value for cAMP of 0.43 μM and was inhibited by cGMP and by cardiotonic inhibitors of PDE III. Similar potencies of these inhibitors were observed in the membrane preparation. These results suggest that in guinea pig cardiac ventricles: (1) PDE I (CaM-activated) is almost exclusively cytosolic; (2) PDE II (cGMP-stimulated), PDE III (cGMP-inhibited and cardiotonic-sensitive) and PDE IV (rolipram-sensitive) are present in cytosolic and membrane preparations; (3) PDE III and PDE IV differ in their apparent K m values for cAMP. The latter observation could explain the differential effects of PDE III and PDE IV inhibitors in the regulation of cardiac contraction.

Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes.

Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

Involvement of Rolipram-Sensitive Cyclic AMP Phosphodiesterase in the Regulation of Cardiac Contraction

The involvement of rolipram-sensitive phosphodiesterase (PDE IV) in regulation of cardiac contraction was investigated by studying the effect of selective inhibitors (rolipram, denbufylline, Ro 20-1724) on guinea pig left atria contraction. In contrast to milrinone and SK&F 94120 (inhibitors of the cyclic GMP-inhibited PDE, PDE III), (+/-)-rolipram and denbufylline (0.1-30 microM) did not produce any positive inotropic effect in normal (2.5 mM) or elevated (3-3.2 mM) external CaCl2 concentration. In these conditions, Ro 20-1724 produced only a slight but significant increase of contraction over control levels. In the presence of forskolin (an adenylate cyclase activator) or SK&F 94120 (a PDE III inhibitor), which produced an increase of the response to electrical stimulation of approximately 10%, (+/-)-rolipram, denbufylline, and Ro 20-1724 all exerted concentration-dependent positive inotropic effects (mean EC50 values were 20, 25, and 125 nM, respectively, in the presence of forskolin). Rolipram exhibited stereospecificity: the (-)-enantiomer was 10 times more potent than the (+)-enantiomer. Neither preincubation of the atria with atenolol nor pretreatment of the guinea pigs with reserpine significantly modified the effect of PDE IV inhibitors obtained in the presence of forskolin. These data show that in the presence of cyclic AMP-dependent positive inotropic agents, PDE IV inhibitors exert a positive inotropic effect which probably does not involve enhanced catecholamine release from sympathetic nerve endings. This suggests that PDE IV may play a role in regulation of cardiac contraction in physiologic conditions in which the sympathetic outflow produces a stimulation of adenylate cyclase in cardiac cells.

Comparative Pharmacology of Cardiac and Vascular Tissues in Heart Failure

The purpose of this paper is to review various factors of the control of cardiac contractility and of vascular tone that may be altered in heart failure and that are targets for pharmacotherapeutic interventions. This paper is divided into three main sections: pathophysiological aspects of heart failure, differences in the regulation of cardiac contraction and of vascular tone, and pharmacological principles in the management of heart failure.

Ca-pools involved in the regulation of cardiac contraction under positive inotropy. X-ray microanalysis on rapidly-frozen ventricular muscles of guinea-pig.

Electron probe microanalysis of rapidly-frozen small ventricular trabeculae of guinea-pig demonstrates that the distribution of total intracellular calcium varies under positive inotropy depending on the type of inotropic intervention. The sarcoplasmic reticulum (SR) (or part of it) localized at the level of the z-lines reveals high calcium accumulation at the end of diastole whenever a stimulus is followed by a contraction with a short time to peak of force. After paired pulse stimulation, only this cell compartment accumulates calcium at the end of diastole. Since this cell compartment is "Ca-empty" in muscles frozen during contraction, SR is considered to be the source of activator Ca. In several cases of inotropy (after application of ARL, caffeine or after lowering the extracellular Na+ concentration), calcium is also detectable on the mitochondria, suggesting that these organelles participate in slow regulation of cytosolic calcium. In some cases, total calcium located on the sarcomeres is increased. The interpretation of this finding is intriguing and requires the assumption of supplementary cytosolic Ca-sinks as yet unknown.

Myocellular calcium regulation by the sarcolemmal membrane in the adult and immature rabbit heart

Previous studies have suggested ontogenic differences in Ca-mediated excitation-contraction coupling in mammalian heart. Sarcolemmal (SL) Ca regulation may predominate prior to the development of the specialized Ca-regulatory properties of the sarcoplasmic reticulum (SR). The effect of development on selected Ca-regulatory properties of cardiac SL was evaluated utilizing membrane vesicles obtained from immature (14 to 21-day-old) and adult rabbit heart. Methods were adapted to comparably enrich SL membrane vesicles from immature and adult rabbit heart. The global fluidity characteristics were determined by the polarized fluorescence of diphenylhexatriene passively incorporated into enriched SL membrane vesicles. No age-related differences in either the membrane microviscosity of the lipid-order parameter between 10 degrees C and 37 degrees C were observed. The membrane characteristics of the voltage-gated Ca channel were determined by the membrane binding characteristics of 3[H]-nitrendipine. Scatchard analysis of high affinity specific nitrendipine binding demonstrated comparable binding affinity (KD; 511 +/- 40 vs 484 +/- 40 pM) and theoretical maximal binding site density (Bmax; 218 +/- 19 vs 240 +/- 40 fmoles/mg prot.) in immature and adult respectively. ATP-independent Ca binding to SL membrane vesicles was determined between 1.5 and 10 mM [Ca]. Ca binding was greater in the immature at 10 mM [Ca] as compared to the adult (840 +/- 120 vs 350 +/- 30 nmoles/mg). Ca bound to SL over this Ca concentration range is indicative of a "pool" of Ca for cellular influx across the SL by the Na-Ca exchange mechanism and the voltage-gated Ca channel. In view of electrophysiologic evidence also suggesting that Ca-channel-mediated Ca conductance is greater in the immature than the adult, it is proposed that the number of voltage-activatable Ca channels localized to the SL is greater in the immature than the adult. The larger transsarcolemmal Ca fluxes plays a larger role in the beat-to-beat- regulation of cardiac contraction in the developing mammalian heart prior to full expression of the specialized Ca regulatory properties of the SR.