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Biomedical subjects

R A Kelly

Publications and source records attributed to R A Kelly.

At least 19 recordsLinked to original sources
PubMed1999

Activation of nitric oxide synthase (NOS3) by mechanical activity alters contractile activity in a Ca2+-independent manner in cardiac myocytes: role of troponin I phosphorylation.

Cardiac myocytes express the calcium-responsive nitric oxide synthase (eNOS or NOS3). Activation of NOS3 by increased intracellular Ca2+ concentration, [Ca2+]i, has been demonstrated to decrease myocyte contractile responsiveness, although this appears to occur in a Ca2+-independent manner. Therefore, the aim of this study was to examine the possibility that contractile activity could be modulated by an NO-mediated alteration in the phosphorylation status of troponin I, which is known to alter myofilament sensitivity to Ca2+. During pacing at 3 Hz, 32P-labeled myocytes exhibited a 59 +/- 9% increase in TnI phosphorylation compared to quiescent cells (p < 0.05), an effect that was significantly attenuated by either methylene blue or l-nitroarginine (l-NA). While exposure to methylene blue significantly increased the contractile amplitude of paced myocytes, this was not accompanied by an alteration in intracellular Ca2+. These data indicate that the NO-mediated effects on myocyte contraction may be elicited through an alteration in myofilament Ca2+ sensitivity that results from an alteration in the phosphorylation status of troponin I.

Actin Cytoskeleton↗
PubMed1999

Digitalis.

Cardiac glycosides have played a prominent role in the therapy of congestive heart failure since William Withering codified their use in his late 18th century monograph on the efficacy of the leaves of the common foxglove plant (Digitalis purpurea). Despite their widespread acceptance into medical practice in the ensuing 200 years, both the efficacy and the safety of this class of drugs continue to be a topic of debate. Moreover, despite the fact that the molecular target for the cardiac glycosides, the alpha-subunit of sarcolemmal Na+K+-ATPase (or sodium pump) found on most eukaryotic cell membranes, has been known for several decades, it remains controversial whether the sympatholytic or positive inotropic effects of these agents is the mechanism most relevant to relief of heart failure symptoms in humans with systolic ventricular dysfunction. Herein, we review the molecular and clinical pharmacology of this venerable class of drugs, as well as the manifestations of digitalis toxicity and their treatment. We also review in some detail recent clinical trials designed to examine the efficacy of these drugs in heart failure, with a focus on the Digoxin Investigation Group data set. Although, in our opinion, the data on balance warrant the continued use of these drugs for the treatment of symptoms of heart failure in patients already receiving contemporary multidrug therapy for this disease, the use of digitalis preparations will inevitably decline with the maturation of newer pharmacotherapies.

Digitalis Glycosides↗
PubMed1999

Tumor necrosis factor-alpha contributes to ischemia- and reperfusion-induced endothelial activation in isolated hearts.

-During myocardial reperfusion, polymorphonuclear neutrophil (PMN) adhesion involving the intercellular adhesion molecule-1 (ICAM-1) may lead to aggravation and prolongation of reperfusion injury. We studied the role of early tumor necrosis factor-alpha (TNF-alpha) cleavage and nuclear factor-kappaB (NF-kappaB) activation on ICAM-1 expression and venular adhesion of PMN in isolated hearts after ischemia (15 minutes) and reperfusion (30 to 480 minutes). NF-kappaB activation (electromobility shift assay) was found after 30 minutes of reperfusion and up to 240 minutes. ICAM-1 mRNA, assessed by Northern blot, increased during the same interval. Functional effect of newly synthesized adhesion molecules was found by quantification (in situ fluorescence microscopy) of PMN, given as bolus after ischemia, which became adherent to small coronary venules (10 to 50 microm in diameter). After 480 minutes of reperfusion, ICAM-1-dependent PMN adhesion increased 2.5-fold compared with PMN adhesion obtained during acute reperfusion. To study the influence of NF-kappaB on PMN adhesion, we inhibited NF-kappaB activation by transfection of NF-kappaB decoy oligonucleotides into isolated hearts using HJV-liposomes. Decoy NF-kappaB but not control oligonucleotides blocked ICAM-1 upregulation and inhibited the subacute increase in PMN adhesion. Similar effects were obtained using BB 1101 (10 microg), an inhibitor of TNF-alpha cleavage enzyme. These data suggest that ischemia and reperfusion in isolated hearts cause liberation of TNF-alpha, activation of NF-kappaB, and upregulation of ICAM-1, an adhesion molecule involved in inflammatory response after ischemia and reperfusion.

Animals↗
PubMed1999

Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane.

-The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations </=1 micromol/L, induced myocyte programmed cell death within 24 hours, as defined by several complementary techniques. In contrast, daunorubicin concentrations >/=10 micromol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 micromol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of alpha-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 micromol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (>/=10 micromol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 micromol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs.

Animals↗
PubMed1999

Muscarinic cholinergic signaling in cardiac myocytes: dynamic targeting of M2AChR to sarcolemmal caveolae and eNOS activation.

The isoform of nitric oxide synthase (eNOS or NOS3) originally described in endothelial cells is also expressed in a number of other cell types, including cardiac myocytes. eNOS is activated in both atrial and ventricular myocytes, including specialized pacemaker cells, by M2AChR agonists, among other stimuli. In cardiac myocytes, as in endothelial cells, eNOS is targeted to sarcolemmal caveolae, due to both co-translational myristoylation and later palmitoylation, and by the presence of a caveolin binding domain in eNOS which interacts with the caveolin scaffolding domain. In the absence of ligand, the M2AChR is not associated with caveolar microdomains, but translates into caveolae upon agonist (but not antagonist) binding. Finally, the role of M2AChR-induced eNOS activation in regulating I(Ca-L) via activation of guanylyl cyclase has been confirmed in ventricular myocytes of mice that lack functional eNOS (i.e., eNOS(null)).

Animals↗
PubMed1998

Modulation of the endothelial nitric-oxide synthase-caveolin interaction in cardiac myocytes. Implications for the autonomic regulation of heart rate.

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr-) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr- eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.

Acylation↗
PubMed1998

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase.

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

Animals↗
PubMed1998

Mechanical strain suppresses inducible nitric-oxide synthase in cardiac myocytes.

We investigated the effects of precisely controlled mechanical strain on nitric-oxide synthase activity in cultured neonatal rat cardiac myocytes. Incubation of cardiac myocytes for 24 h with 4 ng/ml interleukin-1beta and 100 units/ml interferon-gamma stimulated an increase in nitric oxide production, inducible nitric-oxide synthase (iNOS) mRNA, and iNOS protein. Mechanical strain suppressed nitric oxide production, iNOS mRNA, and iNOS protein stimulated by cytokines in an amplitude-dependent manner. Losartan (1 microM), an angiotensin II type 1 receptor antagonist, weakly inhibited the effect of strain, suggesting that paracrine angiotensin II is not the mediator of the strain effect. In addition, cycloheximide (10 microM), a protein synthesis inhibitor, inhibited the effect of strain by 46%. Transforming growth factor-beta (1 ng/ml) suppressed iNOS mRNA expression, but anti-transforming growth factor-beta antibody (30 microg/ml) did not block the effect of strain. In contrast, staurosporine (100 nM; a nonselective protein kinase inhibitor), calphostin C (1 microM; a selective protein kinase C inhibitor), and pretreatment with phorbol 12-myristate 13-acetate abolished the effect of strain. Genistein (100 microM), a tyrosine kinase inhibitor, partially inhibited the effect of strain. Thus, cyclic mechanical deformation suppresses cytokine-induced iNOS expression in cardiac myocytes, and this effect is mediated at least partially via activation of protein kinase C.

Animals↗
PubMed1998

Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes.

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.

Aging↗
PubMed1998

Regulation of cardiac myocyte contractile function by inducible nitric oxide synthase (iNOS): mechanisms of contractile depression by nitric oxide.

Inflammatory cytokines have been implicated in the reversible depression of cardiac contractile function accompanying local or systemic immune stimulation. Incubation of cardiac myocytes with soluble components in the supernatant from cultured rat lung macrophages activated with endotoxin decreases their contractile response to beta-adrenergic stimulation through the induction of iNOS and the subsequent production of nitric oxide by these cells. In the present study, we characterize the mechanisms underlying NO's attenuation of adrenergic responsiveness in cardiac myocytes. iNOS was induced in cultured ventricular myocytes from adult rats by incubation for 20 h with conditioned medium from lipopolysaccharide (LPS)-activated macrophages. iNOS induction did not induce any alteration in beta-adrenergic receptor density or affinity, Galphai protein abundance, or adenylyl cyclase activity in cultured myocytes. Myocyte exposure to activated macrophage-conditioned medium markedly attenuated the elevation of cAMP in response to isoproterenol (Iso, 2 nM). Induction of iNOS with the macrophage-conditioned medium also potentiated the Iso-induced increase in myocyte cGMP. This cGMP increase was totally abolished by NOS inhibitors. NOS inhibition also returned the attenuated cAMP response to 2 nM Iso to levels observed in control cells. Pre-incubation of the cells in isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, also partly reversed the attenuation of cAMP increase with 2 nM Iso in cells expressing iNOS. Brief (15 min) exposure of myocytes to the NO donor, S-nitrosoacetylcysteine (SNAC, 100 micro M) which produced a three-fold increase in intracellular cGMP, also decreased by half the contractile response of cardiac myocytes to Iso (2 nM). We conclude that NO endogenously produced by iNOS decreases the intracellular levels of cAMP in response to beta-adrenergic stimulation in isolated cardiac myocytes, in part through a cGMP-mediated mechanism. This effect may participate in the NO-dependent depression of cardiac function following cytokine exposure.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Reversible S-nitrosation of creatine kinase by nitric oxide in adult rat ventricular myocytes.

We have previously demonstrated that the nitric oxide (NO) donor S-nitroso-N-acetylcysteine (SNAC) reversibly decreases the activity of creatine kinase (CK) in an isolated rat heart preparation, markedly suppressing myocardial contractile responsiveness to an inotropic challenge. We wished to further examine the role of exogenous and endogenous sources of NO species on S-nitrosation of CK and subsequent enzyme activity in adult rat ventricular myocytes (ARVM). Two S-nitrosothiol groups were formed in the CK dimer after nitrosation of rabbit skeletal muscle CK in solution. CK inactivation due to S-nitrosation was time- and concentration-dependent in solution and in ARVM lysate for both NO donors S-nitroso-N-acetylpenicillamine (SNAP) and SNAC, and was rapidly reversible with the sulfhydryl dithiothreitol (DTT). Similarly, SNAC or SNAP dose-dependently decreased CK activity in intact ARVM, which was further attenuated by increasing the metabolic activity of the cells with electrical pacing for 1 h. Co-cultures of ARVM with interleukin 1 beta (IL-1 beta)- and interferon gamma (IFN gamma)-pretreated cardiac microvascular endothelial cells (CMEC) caused no detectable decline in myocyte CK activity. Increasing GSH levels attenuated the decline in myocyte CK activity with SNAC, while decreases in myocyte GSH levels enhanced the inhibitory effect of SNAC on intact myocyte CK activity. These data indicate that the degree of inhibition of cardiac myocyte CK by NO is dependent on the extent of myocyte metabolic activity and the intracellular GSH content.

Acetylcysteine↗
PubMed1998

Human ventricular myocytes in vitro exhibit both early and delayed preconditioning responses to simulated ischemia.

Myocardial tissue has been demonstrated to exhibit, in response to brief periods of ischemia, both an immediate period of cytoprotection [i.e. early or "first window" preconditioning response (EPR)], and a later period of cytoprotection [i.e. delayed or "second window" preconditioning response (DPR)], when exposed to a subsequent prolonged hypoxic insult. EPR has been documented in vitro in isolated cardiac myocytes, as well as in situ in intact hearts or trabeculae, for a number of vertebrate species, including humans. However, there are no reports to date of DPR in human cardiac myocytes. To address this question, human ventricular myocytes (HVM) primary isolates were prepared from fetal ventricular muscle, grown to confluency, and studied in primary culture in serum-free medium (> 90%) ventricular myocytes as determined by immunohistochemical analysis with an anti-myosin chain antibody). Using cell viability as determined by trypan blue exclusion, an EPR response could readily be detected following 15, 30, or 60 min of simulated ischemia (SI) in a hypoxic (< 1 tau pO2) buffer containing 11 mmol/l 2-deoxyglucose, followed by a prolonged (c. 17 h) SI challenge. In addition, HVM exposed to 60 min of SI, followed after 24 h by a period of SI, also exhibited a "second window" DPR (80 +/- 10% compared to 71 +/- 11% survival, in preconditioned and non-preconditioned cultures; P < 0.05; n = 18 independent experiments). Thus, in response to short periods of SI, human ventricular myocytes in vitro exhibit both "first window" and "second window" cytoprotective responses to subsequent, prolonged ischemic stress.

Analysis of Variance↗
PubMed1998

Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes.

ANG II has been implicated in the hypertrophic response in ventricular myocytes by acting at the angiotensin type 1 (AT1) receptor. However, the role of the angiotensin type 2 (AT2) receptor in the adult heart is not as clearly understood. In adult rat ventricular myocytes (ARVM) and cardiac microvascular endothelial cells (CMEC), we examined the role of ANG II signaling, via AT1 and AT2 receptors, on the activation of the extracellular signal-regulated protein kinases (ERKs) and on the expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1. ANG II caused no detectable increase in ERK activity or in c-fos mRNA abundance in ARVM but increased ERK activity within 5 min in CMEC and increased c-fos mRNA levels. However, in the presence of the selective phosphoprotein phosphatase (PP-2A/PP-1) inhibitor okadaic acid (OA), a sustained increase in ERK activity, as well as in c-jun NH2-terminal protein kinase activity, in ARVM was observed. ANG II increased MKP-1 mRNA levels within 15 min in ARVM and CMEC. In contrast to the response in endothelial cells, however, ANG II activation of MKP-1 in ARVM was mediated by AT2-receptor activation. Thus there is constitutive as well as inducible suppression of ERKs and c-jun NH2-terminal protein kinases by MKP and PP-2A/PP-1 in the adult cardiac myocyte phenotype.

Angiotensin II↗
PubMed1997

Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes.

In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand [3H]quinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that [3H]QNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this [3H]QNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the [3H]QNB binding detected in the low density fractions. Moreover, the [3H]QNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells.

Animals↗
PubMed1997

Regulation by cAMP of post-translational processing and subcellular targeting of endothelial nitric-oxide synthase (type 3) in cardiac myocytes.

Cardiac myocytes express the nitric-oxide synthase isoform originally identified in endothelial cells, termed eNOS or NOS3, where it plays a role in regulating myocyte responsiveness to both adrenergic and muscarinic cholinergic autonomic nervous system agonists. eNOS in endothelial cells has been shown to undergo extensive post-translational processing, and in cardiac myocytes as well as endothelial cells, eNOS has been shown to be targeted to plasmalemmal caveolae, a process that is dependent on myristoylation and palmitoylation. Other post-translational modifications essential for the correct subcellular targeting of eNOS have not been described previously. We demonstrate, using [35S]methionine pulse-chase experiments, that native eNOS in adult rat ventricular myocytes is initially translated as a nonpalmitoylated 150-kDa isoform, which is associated with cytosolic and intracellular membrane-enriched fractions. This is subsequently processed to a palmitoylated 135-kDa isoform, which is found only in a sarcolemma-enriched membrane fraction. Forskolin, an agent that elevates intracellular cAMP, rapidly inhibited processing of the 150-kDa isoform to the 135-kDa isoform and transport of eNOS to the sarcolemma, effects paralleled by protein kinase A-dependent phosphorylation of the larger eNOS isoform. Forskolin also decreased palmitoylation of the 135-kDa isoform, although it did not accelerate depalmitoylation of sarcolemmal eNOS, as determined by pulse-chase experiments with [3H]palmitate. Thus, post-translational processing of a 150-kDa isoform of myocyte eNOS appears to be necessary for intracellular trafficking of the enzyme to sarcolemmal caveolae. Both the post-translational processing and subcellular targeting of eNOS appear to be modified by changes in intracellular cAMP, an effect that may have important implications for cardiac myocyte responsiveness to autonomic agonists in vivo.

Acylation↗