Characterization of G-protein signaling in ventricular myocytes from the adult mouse heart: differences from the rat.

We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3-6x10(6)cells/heart. The cells are rod-shaped, roughly 20 microM x 100 microM and Ca(++)tolerant, with viability of 65-80%. Binding studies with [(125)I]ICYP demonstrate the presence of beta -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the beta(1)-specific antagonist CGP 20712A on [(125)I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are beta(1)and 33% are beta(2), compared to 16-20%beta(2)in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC(50) approximately 110+/-20 n M. A functional G(i)pathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the G(q)-phospholipase C pathway, whereas carbachol, PGF(2 alpha)and alpha(1)-adrenergic receptor agonists show no significant effect. The inability of alpha(1)-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold alpha(1)-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

The quasi-irreversible nature of endothelin binding and G protein-linked signaling in cardiac myocytes.

In experiments on neonatal and adult rat ventricular myocytes, endothelin (ET) binding and the effects of ET on transmembrane signaling are quasi-irreversible. The ET(A) receptor antagonist BQ123 competes for binding and biochemical effects if added simultaneously with ET; when added after ET, the antagonist prevents neither binding nor activation of the Gi and Gq pathways. At 4 degrees C, at which internalization of the ligand should be minimized, the interaction of [125I]ET is still irreversible. After binding of radio-labeled ligand at either 4 degrees C or 37 degrees C, only 50% of ligand is removed by acid washing. Permeabilization of the cells with Triton X-100 fails to release irreversibly bound ligand. Binding experiments in cell membranes mimic this irreversible binding. At 37 degrees C, the addition of mercaptoethanol or dithiothreitol inhibits concurrent ET binding but does not cause the dissociation of previously bound ligand or the reversal of previously activated signaling. We conclude that ET binds irreversibly to myocytes, that this irreversibility is reflected in the biochemical responses of the cells to ET and that the irreversibility is more complex than the formation of S-S bonds between surface receptors and ET or internalization of bound ET. We interpret these findings and others in the literature in light of a testable model of ET(A) receptor/G protein/effector interaction in which quasi-irreversible binding of ET to the ET(A) receptor occurs before the interaction of the ligand/receptor complex with G protein and in which irreversible binding contributes to the prolonged effects of ET and is a prelude to refractoriness and to the slow regeneration of free ET(A) receptor.

Alpha 1-adrenergic stimulation inhibits 3,5,3'-triiodothyronine-induced expression of the rat heart sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene.

The interactions between the beta-adrenergic system and thyroid hormone (T3) on cardiac function have been investigated in detail. In addition to beta-adrenoceptors, alpha 1-adrenergic receptors are present in the mammalian heart. The interactions between T3 and the alpha 1-adrenergic system remain, however, poorly understood. T3 stimulates the expression and transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) gene, a protein vital in the control of cardiac calcium transients and contractility. We show that in rat cardiac myocytes, the stimulatory effect of T3 on SERCA2 messenger RNA expression and gene transcription is inhibited by an alpha 1-adrenergic agonist. We demonstrate that direct activation of the alpha 1-adrenergic signaling pathway, using a mutant constitutively active G protein (Gq) similarly down-regulated the T3 effect on SERCA2 transcription. The combined effect of thyroid hormone receptor and retinoid X receptors on T3-stimulated SERCA2 gene transcription was also markedly attenuated by alpha 1-adrenergic stimulation. These results suggested that activation of the alpha 1-adrenergic signaling pathway has an inhibitor effect on T3-dependent SERCA2 gene transcription. As this inhibitory effect of alpha 1-adrenergic stimulation occurs when only one thyroid hormone response element (TRE) drives reporter expression, it is most likely mediated by an alteration of the nuclear factors binding to the TRE or by influencing the interaction of the TRE complex with the basal transcriptional machinery.

Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways.

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.

Small heat shock proteins and protection against ischemic injury in cardiac myocytes.

BACKGROUND: Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS: Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS: The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake.

The decreased expression of the sarcoplasmic reticulum Ca(2+)-ATPase associated with cardiac hypertrophy was investigated in cultured neonatal rat cardiac myocytes. Northern blot analysis indicated a significant 55-60% decrease in Ca(2+)-ATPase mRNA levels and after 12 and 24 h of treatment with the phorbol ester phorbol myristate acetate (PMA). Myocytes treated with the phorbol ester for 80 h showed a significant 34% decrease (relative to vehicle-treated control cells) in the levels of Ca(2+)-ATPase protein, and a significant 38% increase in the levels of alpha-sarcomeric actin, as assessed by Western blot analysis using specific antibodies. Immunocytochemistry of myocytes treated for 72 h with the phorbol ester revealed a hypertrophied cell morphology, and showed a marked decrease in Ca(2+)-ATPase staining intensity. Contractile calcium transients were evaluated through the use of indo-1. It was found that the t1/2 for the decline of calcium transient was significantly prolonged by PMA treatment (0.51 +/- 0.15) when compared to controls (0.38 +/- 0.17, P < 0.001). Treatment of myocytes with endothelin-1 also led to a 35% decrease in sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca(2+)-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca(2+)-ATPase gene expression as observed in vivo in the hypertrophied and failing heart. The observed prolongation in t1/2 for [Ca2+]i decline might be due to the observed depressed levels for sarcoplasmic reticulum Ca(2+)-ATPase in PMA treated cells.

Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

Transmembrane mechanochemical coupling in cardiac myocytes: novel activation of Gi by hyposmotic swelling.

We have studied the effect of hyposmotic swelling on adenosin 3',5'-cyclic monophosphate (cAMP) metabolism in isolated cardiac myocytes. Decreasing extracellular osmolarity by 12.5-50% results in graded inhibition (10-40%) of isoproterenol-stimulated and forskolin-stimulated cAMP accumulation but does not affect basal and hormone-stimulated phosphoinositide hydrolysis or cellular ATP content. Treatment with pertussis toxin does not alter the swelling response but abolishes the inhibitory effect of swelling on cAMP accumulation. The response to swelling seems not to involve the release of effectors known to couple to inhibitory G protein (Gi) in myocytes: BQ-123, atropine, and adenosine deaminase do not alter the inhibitory effect of swelling on isoproterenol-stimulated cAMP accumulation; conditioned medium from swollen cells, with restored osmolarity, has no effect on cAMP accumulation when added to control myocytes. In distinction to these effects on myocytes, swelling enhances hormone-stimulated cAMP accumulation in cultured S49 lymphoma cells. We conclude that swelling of cardiac myocytes inhibits cAMP accumulation through a mechanism that involves activation of a pertussis toxin-sensitive Gi protein. Activation of Gi by this means may contribute to adrenergic hyporesponsiveness in hypoxic and ischemic myocardium.

Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes.

We have immunologically identified the isoforms of protein kinase C (PKC) present in neonatal and adult rat cardiomyocytes and examined their regulation by hormones and phorbol ester. Both cell types express the Ca(2+)-dependent alpha-PKC and the Ca(2+)-independent epsilon- and delta-PKC isoforms. The atypical zeta-PKC isoform is also expressed in neonatal, but only weakly in adult cells. Stimulation of the alpha 1-adrenergic or purinergic receptor with phenylephrine or ATP, respectively, increases membrane-associated immunoreactivity of both epsilon- and delta-PKC in neonatal and adult cells; endothelin and carbachol are also effective in adult cells. In contrast, none of the agonists leads to increases in membrane-associated alpha-PKC in cardiomyocytes. PKC zeta is also unaffected by receptor stimulation. The phorbol ester phorbol 12-myristate 13-acetate causes redistribution and subsequently down-regulation of alpha-, epsilon-, and delta- but not zeta-PKC. The three isoforms are down-regulated at distinctively different rates, with alpha-PKC being the most rapid and epsilon-PKC the slowest. We used selective down-regulation of alpha-, epsilon-, and delta-PKC to investigate the role of these isoforms in PKC phosphorylation-dependent events in neonatal myocytes. Our findings suggest that epsilon-PKC is responsible for the phenylephrine-induced phosphorylation of MARCKS, an endogenous PKC-specific substrate. In contrast, agonist-induced c-fos expression is unlikely to be mediated by epsilon-PKC since the response is rapidly down-regulated and apparently Ca(2+)-dependent. Our finding that the PKC isoforms are differentially responsive to neurohormones suggests that they play distinct and specific roles in cardiac function.

Coupling of the type A endothelin receptor to multiple responses in adult rat cardiac myocytes.

In adult rat cardiac myocytes, endothelin (ET) receptors couple to multiple signaling pathways, including stimulation of phosphoinositide hydrolysis (pertussis toxin insensitive) and inhibition of adenylyl cyclase via Gi. We have used ET-1 and congeners to characterize the subtypes of ET receptors on isolated rat myocytes. The rank orders of potency for stimulating phosphoinositide hydrolysis, inhibiting hormone-sensitive adenylyl cyclase, and competing with 125I-ET-1 for binding to myocytes are the same and show the pattern characteristic of an ETA receptor interaction, i.e., ET-1 approximately ET-2 > sarafotoxin 6b > ET-3; the corresponding EC50 values for the effects of ET on signal transduction are approximately 0.5 nM (ET-1), 0.7 nM (ET-2), 7 nM (sarafotoxin 6b), and 60 nM (ET-3). The ETA receptor antagonist BQ-123 abolishes the cellular responses to ET-1 and competes fully for 125I-ET-1 binding in a concentration-dependent manner. Sarafotoxin 6c, an ETB-specific agonist, does not diminish the responses to ET-1 or compete for 125I-ET-1 binding; no specific binding of the ETB-specific ligand 125I-IRL-1620 is detectable on myocytes. Myocytes express approximately 4 x 10(5) ET-1 binding sites/cell. The association of 125I-ET-1 with myocytes is largely irreversible, as are the biochemical responses to ET-1; thus, constants derived from analyses that assume reversible equilibria are in error. We conclude that the effects of ET on transmembrane signaling in rat ventricular myocytes result from occupation of ETA receptors and that the responses are likely to be long lived, compared with those of the readily dissociable neurotransmitters released by the autonomic nervous system.

Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes.

We have assessed the effects of endothelin-1 (ET-1) on transmembrane signaling in adult rat ventricular myocytes. ET-1 stimulates phosphoinositide hydrolysis with an EC50 of 0.3-0.8 nM. This stimulation is linear for up to 30 min in the presence of a protease inhibitor, is additive with the effects of other stimulators of phosphoinositide hydrolysis, is not inhibited by the Ca2+ entry blocker, nifedipine, and is insensitive to pertussis toxin. ET-1 also reduces cyclic AMP production in myocytes in response to isoproterenol and forskolin (EC50, 1 nM). This cyclic AMP-lowering effect of ET-1 is sensitive to pertussis toxin, can be demonstrated directly in assays of adenylate cyclase activity of myocyte membranes, and seems to be mediated by Gi. These data indicate that the effects of endothelin on adult cardiac myocytes involve multiple signaling pathways, including enhanced activity of the inositol phosphate pathway and a decrease in cyclic AMP-mediated responses, neither of which seems likely to account for the positive contractile effects of endothelin.

Cyclic AMP in myocytes isolated from hypertrophied rat hearts.

Impaired inotropic responsiveness to isoproterenol stimulation has been reported in the hypertrophied hearts of spontaneously hypertensive rats and renal hypertensive rats. This study was carried out in order to investigate the possibility that a defect in cyclic AMP production by cardiac myocytes is responsible for the impaired inotropic responsiveness of these hearts. Basal and isoproterenol stimulated cyclic AMP levels were measured in ventricular myocytes isolated from hypertrophied rat hearts. Cyclic AMP accumulation was also measured in the presence of isobutyl-methyl-xanthine, a phosphodiesterase inhibitor, and the results were compared to the appropriate controls. In the spontaneously hypertensive rat, no changes were detected in the basal or isoproterenol stimulated cyclic AMP formation. This suggests that the biochemical alterations leading to a diminished inotropic response in this model of cardiac hypertrophy involve abnormalities in mechanisms other than cyclic AMP production. In the renal hypertensive rat, basal and isoproterenol stimulated cyclic AMP levels were significantly depressed as compared to controls. This suggests that abnormalities in the signal transduction mechanism and formation of cyclic AMP are, at least in part, responsible for the impaired inotropic responsiveness seen in this model. These results confirm that cardiac hypertrophy is a heterogeneous process. Reduced inotropic responsiveness to isoproterenol stimulation in the hypertrophied hearts of the SHR and the RHR, both models of pressure overload hypertrophy, involve different biochemical alterations. Results of this study suggest that the physiologic response of cardiac hypertrophy may not be as important as the underlying cause of hypertrophic stimuli in determining the pathophysiological consequences.