Cultured Chick Ventricular Myocytes Research Articles

Regulation of the cAMP-elevating effects of isoproterenol and forskolin in cardiac myocytes by treatments that cause increases in cAMP

Regulation of the cAMP-elevating effects of isoproterenol and forskolin in cardiac myocytes by treatments that cause increases in cAMP. We have found that elevations in cyclic AMP (cAMP) have long-term effects on both the β-adrenergic receptor and adenylyl cyclase in cultured chick ventricular myocytes. Pretreatment with isoproterenol for 15 min markedly reduced the cAMP-elevating effect of a subsequent treatment with isoproterenol 18 h later. Responses to isoproterenol were similarly reduced after overnight treatments with forskolin or phosphodiesterase inhibitors. Furthermore, these same treatments also markedly blunted the cAMP-elevating effect of forskolin, a direct activator of adenylyl cyclase. The blunting of the isoproterenol effect was greater than that of the forskolin effect, at least partially because the pretreatments caused a decrease in the number of β-adrenergic receptors as well as a net decrease in adenylyl cyclase activity. Experiments with a recombinant adenovirus to express luciferase under the control of cAMP responsive elements (CREs) showed that the same treatments elevated cAMP sufficiently to drive the transcription of a gene with CREs in its promoter. The blunting of both the isoproterenol and forskolin responses was blocked by the inhibition of protein synthesis or by infecting cells with a recombinant adenovirus that expresses rabbit muscle cAMP-dependent protein kinase inhibitor (PKI). It is hypothesized that one or more adenylyl cyclase isozymes responsible for the generation of cAMP in the myocytes, along with other proteins previously reported to regulate β-adrenergic receptors and perhaps adenylyl cyclase, are negatively regulated by cAMP, most likely at the level of gene expression, and that this regulation may have therapeutic consequences in the treatment of cardiac diseases.

Ischemic preconditioning in isolated cells.

Ischemic preconditioning describes the increased resistance to myocardial infarction that follows short sublethal periods of ischemia. After the ischemia that triggers preconditioning, there are 2 phases of protection: an early (short-lived) phase termed early or classic preconditioning1 and a late (more prolonged) phase termed late preconditioning or the second window of protection.2 The purpose of the present minireview is to highlight the contributions being made to our current understanding of early preconditioning by models based on isolated cardiac cells. Within the preconditioning literature are disparate findings usually explained by variations in species, maturity, preconditioning trigger, anesthetic, and/or choice of end point. This lack of generality is a particular problem with cell-based models. Variability in the circumstances of the trigger, simulation of ischemia, in vitro maintenance conditions, cell type, and species of origin result in innumerable combinations and permutations (see online data supplement at http://www.circresaha.org), making findings difficult enough to compare between cell models let alone between these models and preconditioning in the whole heart. Given these drawbacks, why are an increasing number of preconditioning investigators adopting a cell-based approach? Little controversy surrounds the surface receptors able to trigger preconditioning. In the whole heart, their successful pharmacological manipulation can be verified by alterations in vascular resistance, rate and strength of contraction, and atrioventricular conduction. However, attention has now shifted to intracellular signaling pathways, and the specificity of pharmacological agents is diminished, their effects on physiology are less certain, and their costs are greatly increased. The cell-based models overcome these disadvantages through a small volume of distribution, through an ability to manipulate signaling proteins by the introduction of cDNAs, antisense RNA, recombinant protein, and interfering peptides, and through the interrogation of altered signaling cascades and their consequences within a homogeneous cell type. However, these advantages are at the expense of …

Additive effects of late preconditioning produced by monophosphoryl lipid A and the early preconditioning mediated by adenosine receptors and KATP channel.

The cardioprotective effect of preconditioning can be exerted within 1 to 2 hours after initial ischemia, termed classical or early preconditioning, or can reappear 24 hours later as second window or late preconditioning. The objective of this study was to study the interaction between late and early preconditioning and to determine the potential underlying mechanism. Adenosine receptor agonists and a KATP channel opener were used to achieve early preconditioning, and Monophosphoryl lipid A (MLA) was used to induce late preconditioning. Cultured chick ventricular myocytes were used as a myocyte model of simulated ischemia and preconditioning. Prior treatment of the myocyte with MLA caused a dose-dependent decrease in the ischemia-induced myocyte injury 24 hours later, consistent with a late preconditioning effect. L-NMMA, glibenclamide, or 5-hydroxydecanoic acid administered during the ischemia blocked the MLA effect. Twenty four hours after MLA treatment, a 5-minute exposure to ischemia, adenosine, adenosine A1 agonist CCPA, or A3 agonist resulted in less myocyte injury during the subsequent prolonged ischemia, as compared with cells pretreated with the vehicle and subsequently exposed to the same early preconditioning stimuli. In addition to its ability to enhance the early preconditioning effect by A1 and A3 agonists, MLA pretreatment also increased the phorbol ester- and pinacidil-mediated early preconditioning effect. This study defined a novel interaction in which the cardioprotective effect of early preconditioning is additive to that of late preconditioning and raised the possibility that both agents can be used as combined therapy in the treatment of ischemic heart disease.

Characterization of a newly found stretch-activated KCa,ATP channel in cultured chick ventricular myocytes.

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 +/- 8.2 pS (n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl- as estimated from their permeability ratios were PNa/PK = 0.03, PCa/PK = 0.025, and PCl/PK = 0.026. The probability of the channel being open (Po) increased with the Ca2+ concentration in the bath ([Ca2+]b; dissociation constant Kd = 0.51 microM at +30 mV) and membrane potential (voltage at half-maximal Po = 39.4 mV at 0.35 microM [Ca2+]b). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+ (KCa) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface (Kd = 0.74 mM at 0.10 microM [Ca2+]b at +30 mV). From these data taken together, we concluded that the channel is a new type of KCa channel that could be designated as an "SA KCa,ATP channel." To our knowledge, this is the first report of KCa channel in heart cells.

Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes.

Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique. PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa. We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.

Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors.

Preconditioning with brief ischemia before a sustained period of ischemia reduces infarct size in the perfused heart. A cultured chick ventricular myocyte model was developed to investigate the role of adenosine receptor subtypes in cardiac preconditioning. Brief hypoxic exposure, termed preconditioning hypoxia, prior to prolonged hypoxia, protected myocytes against injury induced by the prolonged hypoxia. Activation of the adenosine A1 receptor with CCPA or the A3 receptor with C1-IB-MECA can replace preconditioning hypoxia and simulate preconditioning, with a maximal effect at 100 nM. While activation of the A2a receptor by 1 microM CGS21680 could not mimic preconditioning, its stimulation during preconditioning hypoxia, however, attenuated the protection against hypoxia-induced injury. Blockade of A2a receptors with the selective antagonist CSC (1 microM) during preconditioning hypoxia enhanced the protective effect of preconditioning. Nifedipine, which blocked the A2a receptor-mediated calcium entry, abolished the A2a agonist-induced attenuation of preconditioning. Isoproterenol, forskolin, and BayK 8644, which stimulated calcium entry, also attenuated preconditioning. Nifedipine blocked the increase in calcium uptake by these agents as well as their attenuating effect on preconditioning. The present study provides the first evidence that the adenosine A3 receptor is present on ventricular myocytes and can mediate simulation of preconditioning. The data demonstrate, for the first time, that activation of the A2a receptor antagonizes the preconditioning effect of adenosine, with increased calcium entry during the preconditioning stimuli as a novel mechanism.

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.

Nitric oxide-mediated effects of interleukin-6 on [Ca2+]i and cell contraction in cultured chick ventricular myocytes.

Cytokines have significant roles in some cardiovascular disorders, but direct myocardial effects of cytokines remain to be elucidated. In the present study, we examined both the early and delayed effects of interleukin-6 (IL-6) on cultured chick embryo ventricular myocytes. Exposure of these cells to human recombinant IL-6 significantly decreased peak systolic [Ca2+]i (71.0 +/- 0.6% of the control value) and the amplitude of cell contraction (66.0 +/- 7.4% of the control value) within a few minutes. Pretreatment with NG-monomethyl-L-arginine (L-NMMA) or methylene blue completely inhibited the IL-6-induced early changes. Subsequent addition of L-arginine reversed the effects of L-NMMA. The levels of cGMP were significantly increased after 30 minutes of exposure to IL-6 (134.4 +/- 9.1% of the control value). Pretreatment with L-NMMA or EGTA significantly inhibited the IL-6-induced early elevation of cGMP. These results suggest that IL-6 acutely decreases intracellular Ca2+ transients and depresses cell contraction by nitric oxide (NO)-cGMP-mediated pathway. Therefore, IL-6 may enhance the Ca(2+)-dependent constitutive NO synthase activity in cardiac myocytes. On the other hand, 24-hour exposure to IL-6 also increased the levels of cGMP (159.0 +/- 22.8% of the control value) regardless of pretreatment with EGTA. These delayed increases in cGMP were also shown to be coupled with decreases in intracellular Ca2+ transients and the amplitude of cell contraction. Thus, IL-6 may induce Ca(2+)-independent NO synthase in cardiac myocytes. Together with the previous reports that have suggested the possible roles of IL-6 in myocardial stunning or endotoxic shock, this negative inotropic effect of IL-6 may contribute to these clinical settings.

Mg2+ buffering in cultured chick ventricular myocytes: quantitation and modulation by Ca2+.

To characterize the Mg2+ buffering of cultured chick ventricular myocytes, cytosolic Mg2+ was increased by liberating Mg2+ normally chelated by ATP upon total depletion of ATP content. Because the total Mg content and cell volume remained constant during this time, the difference between the amount of Mg2+ liberated (2.7 mM) and the 0.9 mM increase in cytosolic Mg2+ activity measured fluorometrically with mag-fura-2 indicates a sizable Mg2+ buffering. A new term, the Mg2+ buffer coefficient (BMg), was derived to quantify this buffering. We also determined that cytosolic Mg2+ activity increased by only 0.6 mM in cells acutely exposed to zero external Ca2+ during ATP depletion. In the absence of extracellular Ca2+, the basal cytosolic Ca2+ activity (alpha Ca2+i) was reduced by 72%, whereas the increase in alpha Ca2+i induced by ATP depletion was substantially blunted; no difference in either the time course of adenine nucleotide changes or the Ca and Mg content was observed. The BMg value calculated for these cells indicates that Mg2+ buffering is substantially greater in the absence of extracellular Ca2+ (2.5) than when extracellular Ca2+ is present (1.4), indicating that alpha Ca2+i affects cytosolic Mg2+ activity in ventricular myocytes. Therefore the Mg2+ buffering of ventricular myocytes appears to be comprised of at least two components: 1) a Ca(2+)-insensitive adenine nucleotide pool and 2) a Ca(2+)-sensitive nonadenine nucleotide pool.

ATP depletion causes a reversible decrease in Na+ pump density in cultured ventricular myocytes.

To examine factors contributing to impaired K+ homeostasis induced by prolonged but sublethal ATP depletion, we subjected cultured chick ventricular myocytes to metabolic inhibition with 20 mM 2-deoxy-D-glucose plus 1 mM NaCN for 2 h and then allowed myocytes to recover for 5 days in medium containing 6% fetal calf serum (FCS) or in hormone-supplemented serum-free medium. We measured spontaneous contractions (with a video motion detector), K+ content, K+ uptake, membrane potential, and Na+ pump density ([3H]ouabain binding). Exposure to metabolic inhibition for 2 h caused an acute decrease in Na+ pump site density [8.2 +/- 1.1 to 3.8 +/- 0.8 (SE) pmol/mg protein; n = 9, P < 0.02]. Compared with control cells (no metabolic inhibition, cultured for 5 days in serum-free medium), Na+ pump density remained depressed in cells recovered from metabolic inhibition in serum-free medium (3.0 +/- 0.7 pmol/mg), and this was associated with persistently depressed K+ uptake (54% of control), K+ content (67% of control), and membrane depolarization (-19 +/- 2 mV), a significant decrease in cell number (79% of control), and failure to resume spontaneous contractions. Exposure of cells inhibited for 2 h to culture medium containing 6% FCS resulted in a return of Na+ pump site density toward normal levels by 5 days, associated with recovery of K+ uptake and K+ content, preservation of cell number, and resumption of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Divergent kinetics of 201Tl and 99mTc-SESTAMIBI in cultured chick ventricular myocytes during ATP depletion.

Thallous chloride (201Tl) and hexakis(2-methoxyisobutyl isonitrile) technetium (I) (99mTc-SESTAMIBI) are myocardial perfusion imaging agents with biological properties that also reflect tissue viability. Initial myocellular uptake rates of 201Tl reflect activity of Na,K-ATPase, whereas those of 99mTc-SESTAMIBI reflect mean plasma membrane potential. To better understand the mechanistic responses of these tracers to myocellular injury, cultured chick embryo cardiac myocytes were metabolically inhibited in iodoacetate (1 mM) and rotenone (10 microM) for up to 2 hours, and initial uptake rates of each agent were determined at successive intervals along with correlative cellular contents of ATP, sodium, and potassium and lactate dehydrogenase release. ATP content fell from 30.5 +/- 1.4 to 2.7 +/- 0.9 nmol.(mg protein)-1 within 2 minutes, whereas sodium and potassium contents ran down their thermodynamic gradients more slowly (t 1/2 approximately 60 minutes). Modestly severe cell injury was produced at 2 hours as estimated by lactate dehydrogenase release (18% of total). Initial uptake rates of 201Tl declined from 6.9 +/- 0.8 to 4.0 +/- 0.4 fmol.(mg protein)-1.(nMo)-1.(min)-1 by 20 minutes and remained depressed and ouabain (100 microM)-insensitive at 30 +/- 13% of control. Conversely, initial uptake rates of 99mTc-SESTAMIBI increased from 10.6 +/- 0.8 to 15.0 +/- 0.6 fmol.(mg protein)-1.(nMo)-1.(min)-1 within 10 minutes, remained elevated for 40-60 minutes, and later declined to low values. Injury-induced enhancement of initial uptake rates of 99mTc-SESTAMIBI were insensitive to ouabain (100 microM), carbonyl cyanide-m-chlorophenyl hydrazone (5 microM), and valinomycin (1 microgram/ml) but were significantly inhibited by 130 mM Ko buffer, Ba2+ (1 mM), glybenclamide (100 microM), and quinacrine (10 microM). Uptake rates of 201Tl monotonically decline, correlating with Na-K pump inhibition from ATP depletion. Conversely, uptake rates of 99mTc-SESTAMIBI at first increase above control for 40-60 minutes, indicating a mean plasma membrane hyperpolarization possibly resulting from opening of ATP-sensitive and arachidonic acid-activated potassium channels, before declining to low values with more severe cell injury. Correlative non-flow-dependent relations between 201Tl and 99mTc-SESTAMIBI contain information regarding the degree of myocellular injury.

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change K d for Ca 2+

Quantitative measurement of [Ca 2+] i with the fluorescent Ca 2+-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (K d) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) ( 400 500 nm ) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca 2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca 2+ (EGTA-Ca 2+ buffer). Subsequently cells were permeabilized to Ca 2+ by a 1 min exposure to 25 μM digitonin in the presence of ‘zero’ Ca 2+ (10 mM EGTA) and saturating 1 mM Ca 2+ to obtain R min, R max and β. We then calculated [Ca 2+] i from the formula ( [Ca 2+] i = K d [(R − R min) (R max − R)]β ). With K d = 250 nM, calculated systolic [Ca 2+] i was 750 ± 44 nM and diastolic 269 ± 19 nM (means ± SEM, n = 16). The R value calculated for an assumed [Ca 2+] i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 ± 0.03: Br A-23187, 0.66 ± 0.03, ns). As the Br A-23187 method is independent of the value chosen for K d, we conclude that the K d of 250 nM for Indo-1 measured in free solutions closely approximates the K d for intracellular Indo-1 in these cells, and that therefore the K d of Indo-1 for Ca 2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.

Mechanisms of reoxygenation injury in cultured ventricular myocytes.

To investigate factors contributing to reperfusion and reoxygenation myocardial injury, we exposed layers of cultured chick ventricular myocytes to severe hypoxia for up to 3 hours in the presence of 20 mM 2-deoxyglucose, zero glucose, and 5 mM pyruvate, and then exposed the myocytes to reoxygenation. Lactate dehydrogenase (LDH) release was moderately increased during 3 hours of hypoxia but was increased markedly during reoxygenation. Coincident changes in intracellular calcium concentration ([Ca2+]i) and cell motion were also measured during hypoxia and reoxygenation. During hypoxia, [Ca2+]i increased to more than 1 microM, and with reoxygenation, [Ca2+]i abruptly decreased slightly but remained elevated more than 1 microM. Cells developed a stable rigor after 30 minutes of hypoxia. Reoxygenation caused a marked hypercontracture within 5 minutes. Pretreatment of myocytes with either 2,3-butanedione monoxime, which inhibits Ca2(+)-dependent force development, or cyanide inhibited reoxygenation hypercontracture. LDH release after reoxygenation was also significantly reduced in the presence of 2,3-butanedione monoxime. Treatment of myocytes with superoxide dismutase and catalase during hypoxia also resulted in a decrease in LDH release during reoxygenation. We conclude that an abrupt increase in [Ca2+]i during reoxygenation does not account for reoxygenation injury. However, in the presence of elevated [Ca2+]i, reoxygenation and the resulting probable resynthesis of ATP causes [Ca2+]i-dependent myofilament crossbridge cycling, and the resulting hypercontracture contributes to myocyte damage. The generation of oxygen free radicals after reoxygenation also appears to contribute to cell injury in this system.