Cardiac Myocyte Contractility Research Articles

Abnormal expression of T-type Ca2+ channel modulates contractility in hypertrophied cardiac myocytes

Abnormal expression of T-type Ca2+ channel modulates contractility in hypertrophied cardiac myocytes

Myocardial dysfunction in the patient with sepsis.

The nature of myocardial dysfunction during sepsis and septic shock has been investigated for more than half a century. This review traces the evolution of scientific thought regarding this phenomenon during this period with particular emphasis on the current understanding of both the clinical manifestations and the molecular/cellular basis of septic myocardial dysfunction in critically ill patients. Current data suggest, contrary to older literature, that patients with septic shock develop a hyperdynamic circulatory state after fluid resuscitation and maintain this hyperdynamic circulatory state until death or recovery. Overt myocardial depression, as manifested by decreased cardiac output, is decidedly uncommon, even in the preterminal phase. Nonetheless, myocardial depression, as evidenced by biventricular dilation and depression of the ejection fraction, can be demonstrated in most patients with septic shock by using either radionuclide cineangiography or echocardiography. Depression is reversible over the course of 7 to 10 days in survivors. Available evidence suggests that myocardial hypoperfusion is not responsible for septic myocardial depression, because examination of humans with septic shock demonstrates increased myocardial perfusion, and animal models of septic shock appear to maintain myocardial high-energy phosphates. A circulating factor or factors, including the cytokines tumor necrosis factor alpha and interleukin-1beta, appear to have a significant role in the phenomenon. In addition, septic myocardial depression appears to be mediated in part through combinations of nitric oxide-dependent and -independent alterations of basal and catecholamine-stimulated cardiac myocyte contractility.

P2X4 Receptor Is a Glycosylated Cardiac Receptor Mediating a Positive Inotropic Response to ATP

Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X(4) receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X(4)R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X(4)R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5'-(alpha,beta-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5' region of cP2X(4)R abrogated the P2 agonist-stimulated (45)Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X(4) receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X(4) receptor, antibody against cP2X(4)R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X(4) receptor. The nonglycosylated 44-kDa P2X(4) receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins with N-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X(4)R showed that only the glycosylated 58-kDa P2X(4) receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor's localization on the plasma membrane. These data revealed a novel physiologic function of the P2X(4) receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility.

Increased contractility and calcium sensitivity in cardiac myocytes isolated from endurance trained rats.

Regular exercise enhances cardiac function and modulates myocyte growth in healthy individuals. The purpose of the present study was to assess contractile function and expression of selected genes associated with intracellular Ca2+ regulation after intensity controlled aerobic endurance training in the rat. Female Sprague-Dawley rats were randomly assigned to sedentary control (SED) or treadmill running (TR) 2 h per day, 5 days per week for 2, 4 or 13 weeks. Rats ran 8-min intervals at 85-90% of VO2max separated by 2 min at 50-60%. Myocyte length, intracellular Ca2+ (Fura-2), and intracellular pH (BCECF) were measured in dissociated cells in response to electrical stimulation at a range of stimulation rates. The increase in VO2max plateaued after 6-8 weeks, 60% above SED. After 13 weeks, left and right ventricular weights were 39 and 36% higher than in SED. Left ventricular myocytes were 13% longer, whereas width remained unchanged. After 4 weeks training, myocyte contractility was approximately 20% higher in TR. Peak systolic intracellular Ca2+ and time for the decay from systole were 20-35 and 12-17% lower, respectively. These results suggest that increased myofilament Ca2+ sensitivity is the dominant effect responsible for enhanced myocyte contractility in TR. Intracellular pH progressively decreased as stimulation frequency was increased in the SED group. This decrease was markedly attenuated in TR and the intracellular pH was significantly higher in the TR group at a stimulation rate of 5-10 Hz. This effect may contribute to the increased contractility observed at the higher stimulation frequencies in TR. A higher intrinsic myofilament Ca2+ sensitivity was observed in permeabilised myocytes from the TR group under conditions of constant pH and [Ca2+]. Western blot analysis indicated 21 and 46% higher myocardial SERCA-2 and phospholamban, but unaltered Na+/Ca(2+)-exchanger levels. Competitive RT-PCR revealed that TR significantly increased Na+/H(+)-exchanger mRNA. Intensity controlled interval training increases cardiomyocyte contractility. Higher myofilament Ca(2+)-sensitivity, and enhanced Ca(2+)-handling and pH-regulation are putative mechanisms. Our results suggest that physical exercise induces adaptive hypertrophy in cardiac myocytes with improved contractile function.

AKAP-mediated targeting of protein kinase a regulates contractility in cardiac myocytes.

Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca(2+) cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca(2+) but no significant difference between Ht31-expressing and control cells in the rate of Ca(2+) cycling or amplitude of the Ca(2+) transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs.

Age-dependent variation in contractility of adult cardiac myocytes

This study was designed to examine the influence of the age of adults on the contractile characteristics of the myocardium and to ascertain whether the age dependent variation is related to variation in sarcolemmal calcium channels. Cardiomyocytes were isolated from 2, 6 and 12-month-old, male Sprague–Dawley rats and the extent and velocity of contraction were recorded as a function of change in cell length. Age dependent increase in cell length and sarcomere length was significant ( P<0.05). Extent of contraction increased with age and the velocities of contraction and relaxation normalized to total contraction decreased with age ( P<0.05). Sensitivity to the L-type channel antagonist (verapamil, 1 μM) and the T-type channel antagonist (nickel chloride, 40 μM) was significant in 6 and 12-month-old animals. This differential response to calcium channel antagonists suggests that the age-dependent variation in contractility may be mediated by the variation in the distribution/function of sarcolemmal calcium channels.

Pathogenesis of diverse clinical and pathological phenotypes in hypertrophic cardiomyopathy

Myocardial contractility is generally believed to be increased in hypertrophic cardiomyopathy. I propose the opposite--that cardiac myocyte contractility is decreased in this disorder. Accordingly, the contractile deficit provides the primary stimulus for increased expression of trophic factors in the heart, which leads to hypertrophy, interstitial fibrosis, and other phenotypes. Variation among individuals in expression of trophic factors would account for the variability of phenotypes. Gene transfer studies in cardiac myocytes showing impaired contractility and increased expression of trophic factors in the myocardium of patients with hypertrophic cardiomyopathy support this hypothesis. Testing of the hypothesis would require measurement of contractility of cardiac myocytes isolated from patients with hypertrophic cardiomyopathy, identification of the main trophic factors in the hearts of these patients, and investigation of whether their inhibition can prevent or lead to regression of the cardiac phenotypes.

Effects of mutant and antisense RNA of phospholamban on SR Ca(2+)-ATPase activity and cardiac myocyte contractility.

The delayed cardiac relaxation in failing hearts has been attributed to a reduced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure. Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied. One mPLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and resulted in a 45% increase in the affinity of SERCA2 for Ca(2+) and 27% faster diastolic Ca(2+) decline as determined by SR (45)Ca uptake assays and by indo 1-facilitated Ca(2+) transient measurement, respectively. Edge-detection analysis of adult myocyte contractility showed a 74% increase in fractional shortening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of neonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca(2+) affinity of SERCA2 and 21% faster diastolic Ca(2+) decline. However, in adult cardiac myocytes, Adv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters. K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.

Activated macrophages decrease rat cardiac myocyte contractility: importance of ICAM-1-dependent adhesion.

Macrophages are found in the heart as part of the inflammatory response. To determine whether macrophages could contribute to myocardial dysfunction, rat ventricular myocytes were isolated and cocultured with elicited peritoneal macrophages in media containing tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, or endotoxin for 4 h. Cardiac myocytes were electrically stimulated, and fractional shortening was determined using videomicroscopy. When myocytes alone or myocytes in coculture with macrophages separated by a membrane were challenged with TNF-alpha, lipopolysaccharide, or IL-1, fractional shortening did not decrease. When macrophages were allowed to contact myocytes, fractional shortening decreased from 20. 1 +/- 0.9% in unchallenged macrophage-myocyte cocultures to 15.5 +/- 0.9, 16.3 +/- 0.8, and 15.8 +/- 0.6% when challenged for 4 h with TNF-alpha, endotoxin, or IL-1beta, respectively (P < 0.05). Myocytes had a mean adherence of 4.2 +/- 0.2 macrophages after TNF-alpha challenge compared with 2.6 +/- 0.3 for controls (P < 0.05). The number of adherent macrophages was associated with the decrease in fractional shortening. Anti-intercellular adhesion molecule-1 (ICAM-1) reduced macrophage adherence and prevented the decrease in fractional shortening. This decrease was also prevented by desferoxamine, superoxide dismutase, and nitro-L-arginine methyl ester. This suggests that activated macrophages adhere to myocytes via ICAM-1, and adherent macrophages decrease their contractile function via TNF-alpha, oxygen free radicals, and nitric oxide.

Effects of epinephrine and amrinone on contractility and cyclic adenosine monophosphate generation of tumor necrosis factor alpha-exposed cardiac myocytes.

This study utilized an in vitro neonatal rat cardiac myocyte assay to evaluate potential differences in the response of TNF-alpha-exposed myocytes to stimulation with the adrenergic agent, epinephrine, and the phosphodiesterase III inhibitor, amrinone. Contractility was assessed by measuring the maximum extent of the contraction of electrically paced neonatal rat cardiac myocytes in tissue culture using a closed-loop video tracking system. Myocytes were incubated in control or media containing TNF-alpha (50 ng/mL) for 20 mins and were then stimulated with increasing concentrations of either epinephrine (0.1 to 100 ng/mL) for 15 mins or amrinone lactate (0.25 to 10 microg/mL) for 20 mins. Compared with control myocytes, TNF-alpha-exposed myocytes stimulated with increasing concentrations of epinephrine demonstrated a decreased peak augmentation of contractility (p<.0001 analysis of variance). This decrease was paralleled by a decrease in epinephrine-stimulated generation of cyclic AMP, as measured by enzyme-linked immunoassay (p = .05 polynomial regression). In contrast, increasing concentrations of amrinone produced increased peak augmentation of contractility (p = .003 analysis of variance) in TNF-alpha-exposed cardiac myocytes (relative to controls). However, this increase was not reflected by increased amrinone-stimulated generation of cyclic AMP relative to control myocytes not exposed to TNF-alpha (p = NS polynomial regression). Our data suggest that TNF-alpha induces a defect in beta-adrenergic signal transduction and catecholamine-stimulated contractility in neonatal rat cardiac myocytes. In addition, TNF-alpha augments the inotropic response of myocardial tissue to phosphodiesterase inhibitors through a mechanism independent of cyclic AMP generation. Phosphodiesterase inhibitors such as amrinone may be found to exert significant inotropic effects in catecholamine-refractory septic shock with myocardial depression and other conditions of inflammatory myocardial dysfunction.

A soluble neuronal factor alters contractile function of ventricular myocytes without effect on troponin T isoform expression.

The purpose of this investigation was to establish a model system to facilitate identification of the sympathetic neuronal factor(s) that promotes improved contractility in neonatal cardiac myocytes. Conditioned medium from PC12 cells with sympathetic phenotype served as the source of the neuronal factor. Contraction frequency, amplitude and velocity of cultured neonatal rat cardiac myocytes were measured by online video analysis. Interventions included in vitro sympathetic innervation, exposure to PC12 conditioned medium, neurotransmitters and antagonists. Metabolic activity was assayed by 2-deoxyglucose uptake. Troponin T isoform expression was analyzed by SDS-polyacrylamide gel electrophoresis. Medium conditioned by neuronal PC12 cells induced contractility changes similar to those induced by in vitro sympathetic innervation. These effects of PC12 conditioned medium and innervation were not suppressed by adrenergic or muscarinic antagonists nor reproduced by neuropeptide Y or somatostatin. Neuronal PC12 conditioned medium but not chromaffin PC12 conditioned medium, increased metabolic activity of the myocytes as detected by [3H]-2-deoxyglucose, indicating that the effect was specific to the neuronal PC12 cells. The in vitro switch of troponin T isoform expression was not altered by exposure to PC12 conditioned medium. Increased contractile function induced by sympathetic innervation is reproduced by PC12 conditioned medium, but neither is mediated by sympathetic or muscarinic neurotransmitters. Troponin T isoform expression is not related to the contractility changes. This model system will allow identification of the factor(s).

Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility.

Previous studies have demonstrated the existence of a circulating myocardial depressant substance during human septic shock. We have recently identified this substance as a synergistic combination of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). This study utilized an in vitro cardiac myocyte assay to evaluate the potential mechanistic role of nitric oxide (NO) and cGMP in depression of myocyte contractility induced by TNF-alpha, IL-1beta, TNF-alpha + IL-1beta (at low concentrations), and human septic shock serum (HSS). TNF-alpha, IL-1beta, TNF-alpha + IL-1beta, and each of 5 sera from patients with acute septic shock caused depression of both maximum extent and peak velocity of cardiac myocyte shortening and an increase in intracellular cGMP concentration during 30 min of exposure (minimum P < 0.01). NO synthetase (NOS) and guanylate cyclase inhibitors such as N-methyl-L-arginine (L-NMA) and methylene blue prevented these effects; an excess of L-arginine with L-NMA restored them (minimum P < 0.01). In contrast, D-arginine failed to reestablish cytokine-induced myocyte depression and cGMP accumulation prevented by L-NMA. Exposure of myocytes to TNF-alpha, IL-1beta, or TNF-alpha + IL-1beta produced a concentration-dependent increase in intracellular cGMP that paralleled the depression of cardiac myocyte contractility (minimum P < 0.001). In addition, TNF-alpha, IL-1beta, TNF-alpha + IL-1beta, or HSS application to cardiac myocytes resulted in increased NO gas generation, which was inhibited by L-NMA (minimum P < 0.01). Furthermore, unstimulated cardiac myocytes were shown to harbor constitutive but not inducible NOS activity. These data suggest that the sequential generation of NO by a constitutive NOS and cGMP by guanylate cyclase represents an important mechanism of cardiac myocyte depression by TNF-alpha, IL-1beta, TNF-alpha + IL-1beta, and the myocardial depressant substance(s) of septic shock.

T- and L-type Ca2+-channel antagonists reduce contractility in guinea pig cardiac myocytes.

The aim of this study was to investigate the influence of L- and T-type Ca2+-channel blockade on myocardial contractility in guinea pig cardiomyocytes. Left ventricular myocardium from guinea pig contains both L- and T-type Ca2+ channels. The T-type Ca2+ influx was inhibited with mibefradil (1-100 microM), a novel compound with a threefold higher affinity for T- compared with L-type Ca2+ channels. In comparison, L-type Ca2+ influx was reduced by the benzodiazepine diltiazem (1-100 microM). The effect of mibefradil and diltiazem on electrically driven (0.5 Hz) isolated cardiomyocytes (n = 12) was studied in a concentration-dependent manner. The change of the contraction amplitude (percentage of cell shortening) was continuously recorded with an one-dimensional high-speed camera. Both mibefradil and diltiazem concentration-dependently reduced (p < 0.05 vs. control) the contraction amplitude in isolated myocytes from guinea pig. The concentration at which the contraction amplitude of guinea pig cardiomyocytes was reduced by 50% (EC50) was 31.6 microM for diltiazem and 6.3 microM for mibefradil, indicating that the T-type Ca2+-channel blocker mibefradil is more potent in reducing contractility in guinea pig cardiac myocytes in comparison with the L-type Ca2+-channel antagonist diltiazem. Mean values for cell shortening in percentage +/- SEM for mibefradil (0, 1, 10, 100 microM) were 100%, 78 +/- 9.2%, 36 +/- 5.4%, and 24 +/- 3.6%. The corresponding values for diltiazem were 100%, 92 +/- 12.5%, 79 +/- 8.9%, and 35 +/- 2.6%. In contrast, the increase of the extracellular Ca2+ concentration (2-7.5 mM) resulted in a significant increase of the contraction amplitude (+213 +/- 14%). Therefore, blockade of the Ca2+ influx through voltage-dependent T- or L-type Ca2+ channels decreases contraction in isolated cardiac myocytes from guinea pigs containing L- and T-type Ca2+ channels.

Decompensation of pressure-overload hypertrophy in G alpha q-overexpressing mice.

Receptor-mediated activation of myocardial Gq signaling is postulated as a biochemical mechanism transducing pressure-overload hypertrophy. The specific effects of Gq activation on the functional and morphological adaptations to pressure overload are not known. To determine the effects of intrinsic myocyte G alpha q signaling on the left ventricular hypertrophic response to experimental pressure overload, transgenic mice overexpressing G alpha q specifically in the heart (G alpha q-25) and nontransgenic siblings underwent microsurgical creation of transverse aortic coarctation and the morphometric, functional, and molecular characteristics of these pressure-overloaded hearts were compared at increasing times after surgery. Before aortic banding, isolated G alpha q-25 ventricular myocytes exhibited contractile depression (depressed +dl/dt and -dl/dt) and G alpha q-25 hearts showed a pattern of fetal gene expression similar to the known characteristics of nontransgenic pressure-overloaded mice. Three weeks after transverse aortic banding, G alpha q-25 left ventricles hypertrophied to a similar extent (approximately 30% increase) as nontransgenic mice. However, whereas nontransgenic mice exhibited concentric left ventricular remodeling with maintained ejection performance (compensated hypertrophy), G alpha q-25 left ventricles developed eccentric hypertrophy and ejection performance deteriorated, ultimately resulting in left heart failure (decompensated hypertrophy). The signature hypertrophy-associated progress of fetal cardiac gene expression observed at baseline in G alpha q-25 developed after aortic banding of nontransgenic mice but did not significantly change in aortic-banded G alpha q-25 mice. Intrinsic cardiac myocyte G alpha q activation stimulates fetal gene expression and depresses cardiac myocyte contractility. Superimposition of the hemodynamic stress of pressure overload on G alpha q overexpression stimulates a maladaptive form of eccentric hypertrophy that leads to rapid functional decompensation. Therefore G alpha q-stimulated cardiac hypertrophy is functionally deleterious and compromises the ability of the heart to adapt to increased mechanical load. This finding supports a reevaluation of accepted concepts regarding the mechanisms for compensation and decompensation in pressure-overload hypertrophy.

Vascular endothelial cell-cardiac myocyte crosstalk in achieving a balance between energy supply and energy use.

In isolated perfused hearts, endothelial cells in the coronary arterial vascular system release substances that can alter the contractility of the cardiac myocytes. There are at least two different substances, one that increases and another that decreases the contractility of cardiac myocytes. The rate of release of these endothelial-derived cardioactive substances depends on the oxygen tension in the immediate vicinity of the cardiac myocytes. As the local oxygen tension increases the contractility changes in the same direction. The oxygen sensor in this regulatory system is the cardiac myocyte, which then releases substances that regulate the secretion of endothelin and a relaxant by endothelial cells. The result is a loop involving cross talk between coronary endothelial cells and cardiac myocytes to modulate cardiac contractility in accordance with the oxygen supply to the cardiac myocytes. Preliminary data suggest that the change in contractility is related to a change in structure and position of the cross bridge due to phosphorylation of a protein in the thick filament.

Physical, contractile and calcium handling properties of neonatal cardiac myocytes cultured on different matrices.

Extracellular matrix components play a vital role in the determination of heart cell growth, development of spontaneous contractile activity and morphologic differentiation. In this work we studied the physical and contractile changes in neonatal rat cardiac myocytes over the first four days of growth on three different extracellular matrices. We compared commercial laminin and fibronectin, plus a fibroblast-derived extracellular matrix, which we have termed cardiogel. Myocytes cultured on cardiogel were characterized by greater cellular area and volume when compared to cells cultured on the other single-component matrices. Spontaneous contractile activity appeared first in the cells grown on cardiogel, sometimes as early as the first day post-plating, in contrast to day three in the cells cultured on laminin. Measurements of cardiac myocyte contractility i.e. percent shortening and time to peak contraction, were made on each of the first four days in each culture. Myocytes cultured on cardiogel developed maximum shortening more rapidly than the other cultures, and an earlier response to electrical pacing. Histochemical staining for myocyte mitochondrial content, revealed that the cardiogel-supported cells exhibited the earliest development of this organelle and, after four days, the greatest abundance. This reflects both a greater cell size, as well as response to increasing energy demands. Due to the increase in volume and contractile activity exhibited by the cardiogel grown myocytes, we employed calcium binding and uptake experiments to determine the comparative cellular capacities for calcium and as an indicator of sarcoplasmic reticulum development. Also whole cell phosphorylation in the presence of low detergent was assayed, to correlate calcium uptake with phosphorylation, in an attempt to examine possible increases in calcium pump number and other phosphorylatable proteins. In agreement with our physical and contractile data, we found that the cells grown on cardiogel showed a greater calcium uptake over the first four days of culture, and increased phosphorylation. However, calcium binding was not dramatically different comparing the three culture matrices. Based on our data, the fibroblast-derived cardiogel is the matrix of choice supporting earliest maturation of neonatal cardiomyocytes, in terms of spontaneous contractions, calcium handling efficiency, cell size and development of a subcellular organelle, the mitochondrion.

Lipopolysaccharide Depresses Cardiac Contractility and β-Adrenergic Contractile Response by Decreasing Myofilament Response to Ca 2+ in Cardiac Myocytes

Lipopolysaccharide (LPS) plays a key role in the pathogenesis of sepsis. Cardiac function and the inotropic response to beta-adrenergic stimulation are impaired in sepsis. We hypothesized that LPS, in clinically relevant levels (1 ng/mL), directly depresses contractility and beta-adrenergic responses in cardiac myocytes. Cardiac myocytes were isolated from the left ventricle of adult rabbits using digestive enzymes (collagenase and protease). We depyrogenated the enzymes (LPS contamination lowered from 100 to 300 ng/mL to < 0.7 ng/mL) to minimize development of LPS tolerance during cell isolation. After 6 hours of incubation with 1 ng/mL LPS, there was a decrease in the extent of active cell shortening with no change in Ca2+ transients (measured with indo 1 fluorescence), indicating decreased myofilament responsiveness to Ca2+. This was related to NO pathways, since cGMP (a second messenger of NO) increased in cardiac myocytes and LPS effects were completely reversed with a 1 mmol/L NG-monomethyl-L-arginine (L-NMMA, a NO synthase inhibitor). LPS did not alter the intracellular Ca2+ response to beta-adrenergic stimulation with isoproterenol but attenuated the contractile response (maximal cell shortening, 15.5 +/- 1.0% versus 23.3 +/- 1.1% in control myocytes; P < .001). LPS attenuation of the contractile response to isoproterenol was restored completely by L-NMMA and almost completely restored (to 86% of the control response) by an inhibitor of cGMP-dependent protein kinase. We conclude that LPS depresses cardiac contractility and the contractile response to beta-adrenergic stimulation by a NO-cGMP-mediated decrease in myofilament responsiveness to Ca2+. The direct effects of low levels of LPS on cardiac myocytes may contribute to cardiac depression and hemodynamic decompensation during sepsis.

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus.

The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for beta-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of beta-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were beta-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 d postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.

Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes.

Cardiac myocytes coexpress multiple protein kinase C (PKC) isoforms which likely play distinct roles in signaling pathways leading to changes in contractility, hypertrophy, and ischemic preconditioning. Although PKC has been reported to be activated during myocardial ischemia, the effect of ischemia/hypoxia on individual PKC isoforms has not been determined. This study examines the effect of hypoxia on the subcellular distribution of individual PKC isoforms in cultured neonatal rat ventricular myocytes. Hypoxia induces the redistribution of PKC alpha and PKC epsilon from the soluble to the particulate compartment. This effect (which is presumed to represent activation of PKC alpha and PKC epsilon) is detectable by 1 h, sustained for up to 24 h, and reversible within 1 h of reoxygenation. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) prevents the hypoxia-induced redistribution of PKC alpha and PKC epsilon, whereas chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) blocks the redistribution of PKC alpha, but not PKC epsilon; D609 and BAPTA do not influence the partitioning of PKC alpha and PKC epsilon in normoxic myocytes. Hypoxia, in contrast, decreases the membrane association of PKC delta via a mechanism that is distinct from the hypoxia-induced translocation/activation of PKC alpha/PKC epsilon, since the response is slower in onset, slowly reversible upon reoxygenation, and not blocked by D609 or BAPTA. The hypoxia-induced shift of PKC delta to the soluble compartment does not prevent subsequent 4-beta phorbol 12-myristate-13-acetate-dependent translocation/activation of PKC delta. Hypoxia does not alter the abundance of any PKC isoform nor does it alter the subcellular distribution of PKC lambda. The selective hypoxia-induced activation of PKC isoforms through a pathway involving phospholipase C (PKC alpha/PKC epsilon) and intracellular calcium (PKC alpha) may critically influence cardiac myocyte contractility, gene expression, and/or tolerance to ischemia.

Interactions between endothelin-1 and atrial natriuretic peptide influence cultured chick cardiac myocyte contractility

We have previously shown that rat atrial natriuretic peptide (ANP) reduces the contractility of cultured, spontaneously beating chick embryo ventricular cells, an effect opposite to that of endothelin-1. Endothelin-1 has been described as a secretagogue for natriuretic peptides in vitro and in vivo. Natriuretic peptides can inhibit endothelin-1 secretion from cultured endothelial cells, suggesting a negative feedback mechanism between endothelial cells and cardiomyocytes. The aim of this study was to determine whether ANP attenuated the endothelin-1-induced increase in myocyte contractility. Using a video-microscopy system we studied the contractility of isolated cultured chick ventricular myocytes in response to endothelin-1, chicken natriuretic peptide (ChNP), and both. We also used Northern blot analysis to study the time course of ChNP expression in response to endothelin-1. Endothelin-1 (10 −8 M) increased chick cardiomyocyte contractility by 20–25% between 5 and 15 min ( P < 0.05). Although ChNP (3 × 10 −7 M) did not significantly change the amplitude of contraction in basal conditions, it prevented the endothelin-1-induced increase in contractility ( P < 0.05) when perfused prior to endothelin-1, and reversed it when perfused 5 min after endothelin-1 exposure ( P < 0.05). Endothelin-1 significantly increased the accumulation of ChNP mRNA in chick ventricular myocytes as early as the 30 min after exposure ( P < 0.05), with a maximal effect after 2 h of stimulation ( P < 0.01); no effect was observed after 4 h. These data support an interaction between endothelin-1 and natriuretic peptides as autocrine/paracrine factors regulating the contractile function of chick cardiac myocytes, as well as their antagonistic effects on cardiac cell contractility. The early and transient expression of ChNP mRNA in response to endothelin-1 may be involved in this interaction.