Down regulation of the L-type Ca2+ channel, GRK2, and phosphorylated phospholamban: protective mechanisms for the denervated failing heart.

We previously found that a canine model of selective surgical ventricular denervation (VD), which does not permit increased sympathetic tone during the pathogenesis of heart failure (HF), tolerated the development of HF better than controls. To investigate the cellular mechanisms, we examined cellular contraction and L-type Ca(2+) channel currents (I(Ca)) and their responses to beta-adrenergic receptor (beta-AR) stimulation in left ventricular myocytes from 1) control, 2) VD, 3) HF induced by rapid pacing, and 4) HF induced in VD (VD-HF) dogs. The magnitude of myocyte contraction and rate of relaxation in VD were similar to control but were depressed in both HF and VD-HF. These changes were associated with reduced protein expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and protein kinase A phosphorylated phospholamban (PLB), which was reduced in HF, but essentially abolished in VD-HF. beta-AR kinase (GRK2) was increased in HF but reduced in VD-HF. Basal I(Ca) density did not differ among control, VD, and HF groups, but VD-HF myocytes showed a markedly reduced I(Ca) density (approximately 40%). Compared to controls, the sensitivity of I(Ca) to isoproterenol (ISO), was significantly higher in VD, but reduced in HF. While I(Ca) responses to ISO in VD-HF were maintained at control levels, the amplitude of the ISO-stimulated I(Ca) was significantly smaller (approximately 50%) compared with HF myocytes. The relative decrease in Ca(2+) influx due to downregulation of I(Ca) density may contribute to the cardioprotective effects in VD-HF hearts by preventing Ca(2+) overload during the development of HF. These findings, in combination with the virtual abolition of phosphorylated PLB in VD-HF and the decrease in GRK2, may explain, in part, why VD dogs tolerate the development of HF better than control dogs.

Gender-specific proteomic alterations in glycolytic and mitochondrial pathways in aging monkey hearts.

We utilized proteomic techniques in a primate model (Macaca fascicularis) of aging to determine potential mechanisms to explain gender differences in protection of the aging heart. The majority of prior work in this field utilized rodent models, and importantly no prior study utilized a proteomic approach in the aging heart. We studied changes in proteins in seven monkeys in each group (young and old males and females (YMs, OMs, YFs, and OFs, respectively)), and used two-dimensional gel electrophoresis in combination with mass spectrometry in five monkeys in each group. We found decreases (P < 0.05) in the expression of key enzymes in glycolysis (e.g. pyruvate kinase, alpha-enolase, triosephosphate isomerase), glucose oxidation (e.g. pyruvate dehydrogenase E1 beta-subunit), and the tricarboxylic acid (TCA) cycle (2-oxoglutarate dehydrogenase) in left ventricular (LV) samples from OM monkeys; these changes in glycolytic, glucose oxidation, and TCA enzymes were not observed either in YMs, YFs or OFs. We found additional gender differences in the reduced expression and function of proteins that are responsible for electron transport and oxidative phosphorylation in mitochondria only in hearts from OM monkeys, with corresponding decreased oxidation rates with NADH and ascorbate-N,N,N',N' ''-tetramethyl-p-phenylenediamine substrates. The changes in glycolytic and mitochondrial metabolic pathways in OM monkey hearts are similar to changes observed in hearts affected by diabetes or LV dysfunction, and could be involved in the mechanism for the cardiomyopathy of aging. The sparing of these changes in OF hearts could be involved in the mechanism mediating delayed cardiovascular risk in OFs.

A novel mechanism for myocardial stunning involving impaired Ca(2+) handling.

The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca(2+) responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca(2+) handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, P<0.01, in stunned myocytes (6.3+/-0.4%) compared with normal myocytes (8.8+/-0.4%). The time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (131+/-8 ms) compared with normal myocytes (105+/-5 ms). The impaired contractile function was associated with decreased Ca(2+) transients (stunned, 0.33+/-0.04 versus normal, 0.49+/-0.05, P<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca(2+)-current density (stunned, -4.8+/-0.4 versus normal, -6.6+/-0.4 pA/pF, P<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca(2+) transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca(2+) handling and dephosphorylation of phospholamban, but not TnI degradation.

Gene program for cardiac cell survival induced by transient ischemia in conscious pigs.

Therapy for ischemic heart disease has been directed traditionally at limiting cell necrosis. We determined by genome profiling whether ischemic myocardium can trigger a genetic program promoting cardiac cell survival, which would be a novel and potentially equally important mechanism of salvage. Although cardiac genomics is usually performed in rodents, we used a swine model of ischemia/reperfusion followed by ventricular dysfunction (stunning), which more closely resembles clinical conditions. Gene expression profiles were compared by subtractive hybridization between ischemic and normal tissue of the same hearts. About one-third (23/74) of the nuclear-encoded genes that were up-regulated in ischemic myocardium participate in survival mechanisms (inhibition of apoptosis, cytoprotection, cell growth, and stimulation of translation). The specificity of this response was confirmed by Northern blot and quantitative PCR. Unexpectedly, this program also included genes not previously described in cardiomyocytes. Up-regulation of survival genes was more profound in subendocardium over subepicardium, reflecting that this response in stunned myocardium was proportional to the severity of the ischemic insult. Thus, in a swine model that recapitulates human heart disease, nonlethal ischemia activates a genomic program of cell survival that relates to the time course of myocardial stunning and differs transmurally in relation to ischemic stress, which induced the stunning. Understanding the genes up-regulated during myocardial stunning, including those not previously described in the heart, and developing strategies that activate this program may open new avenues for therapy in ischemic heart disease.

Cyclosporine reduces left ventricular mass with chronic aortic banding in mice, which could be due to apoptosis and fibrosis.

A tacit assumption in studies of left ventricular (LV) hypertrophy is that left ventricular/body weight (LV/BW) reflects the extent of myocyte hypertrophy. The goal of the current investigation was to determine if there was another explanation for the reduced LV/BW observed after inhibiting calcineurin with cyclosporine during the development of pressure overload LV hypertrophy as compared with animals that did not receive cyclosporine. Accordingly, we examined the prevalence of fibrosis and apoptosis and measured cell size in the hearts from mice at 1 and 3 weeks after transverse aortic banding with and without chronic cyclosporine. Although LV/BW, compared to aortic banded vehicle treated mice, was reduced by 30% in aortic banded cyclosporine treated mice, myocyte cross sectional area was similar in both banded groups (346+/-9 microm2 v 336+/-13 microm2). The volume percent interstitial fibrosis was greater in aortic banded cyclosporine treated animals (1.4+/-0.2%) compared with aortic banded vehicle treated animals (0.9+/-0.2%, P<0.05) or in sham animals (0.6+/-0.1%). Surprisingly, lesions including myocytes containing iron were observed and were most prominent in aortic banded cyclosporine treated animals. Apoptosis, quantitated with TUNEL staining as percent of myocytes, was increased in aortic banded cyclosporine treated animals at 7 days (1.6+/-0.4%) compared with aortic banded vehicle treated animals (0.4+/-0.1%, P<0.01) and was still increased at 21 days. Immunoblotting demonstrated a decrease in the phosphorylation of Akt and Bad, and also Bcl-2 levels were reduced in aortic banded cyclosporine treated animals at 7 days compared with aortic banded vehicle treated animals. These proteins protect against apoptosis, and support the concept that cyclosporine inhibited the calcineurin pathway, resulting in enhanced apoptosis. Thus, the decrease in LV/BW in the aortic banded cyclosporine treated animals actually may be due, at least in part, to cell loss and death, as reflected by the enhanced fibrosis and apoptosis and the focal iron deposits in myocytes.

Paradoxically enhanced endothelin-B receptor-mediated vasoconstriction in conscious old monkeys.

BACKGROUND: We investigated the effects of aging on the responses to endothelin (ET) in conscious old (19.8+/-0.6 years) and young adult (6.8+/-0.3 years) monkeys and compared these results with those of other vasoconstrictors, eg, phenylephrine (PE) and angiotensin II (Ang II). METHODS AND RESULTS: The monkeys (Macaca fascicularis) were chronically instrumented. Baseline total peripheral resistance (TPR) was not different between the 2 groups. As expected, TPR rose less (P<0.05) with PE (5 microgram/kg) in old monkeys (34+/-3%) than in young monkeys (57+/-6%); TPR also rose less with Ang II. Surprisingly, TPR rose more (P<0.05) with endothelin-1 (ET-1, 25 ng. kg(-1). min(-1)) in old monkeys (36+/-6%) than in young monkeys (10+/-2%). An ET(B) receptor agonist, sarafotoxin (S6c, 30 ng. kg(-1). min(-1)) was administered in the presence of an ET(A) receptor antagonist, BQ-123 (1 mg/kg). Under these conditions, TPR increased more (P<0.05) in old monkeys (59+/-10%) than in young monkeys (31+/-4%). In the presence of nitric oxide synthase (NOS) inhibition with N(W)-nitro-L-arginine methyl ester (60 mg/kg), vasoconstriction induced by S6c no longer differed with age, because it was enhanced in young monkeys (P<0.05) (68+/-9% versus 31+/-4%) but not in old monkeys (58+/-6% versus 59+/-10%). Thus, after NOS inhibition, vasoconstrictor responses to ET were no longer enhanced in old monkeys. CONCLUSIONS: Peripheral vasoconstriction (PE and Ang II) is reduced in old monkeys, as expected. Paradoxically, vasoconstriction induced by ET-1 was actually enhanced in old monkeys, which appears to be a result of impaired endothelium-dependent vasodilation, which with ET-1 should involve the ET(B) receptor.

Beta-adrenergic cardiac hypertrophy is mediated primarily by the beta(1)-subtype in the rat heart.

Myocardial beta-adrenergic receptors (beta -ARs) consist of beta(1)- and beta(2)-subtypes, which mediate distinct signaling mechanisms. We examined which beta-AR subtype mediates cardiac hypertrophy. The beta(2)-subtype is predominant in neonatal rat cardiac myocytes (beta(1), 36%vbeta(2), 64%), while the beta(1)-subtype predominates in the adult rat heart (59%v 41%). Stimulation of cultured cardiac myocytes in vitro with isoproterenol (ISO), an agonist for beta(1)- and beta(2)-ARs, caused hypertrophy of myocytes along with increases in transcription of atrial natriuretic factor (ANF) and actin reorganization. All of these ISO-mediated myocyte responses in vitro were inhibited by a beta(1)-AR antagonist, betaxolol, but not by a beta(2)-AR antagonist, ICI 118551. Pertussis toxin failed to affect ISO-induced increases in total protein/DNA content and ANF transcription in vitro. ISO increased LV weight/body weight and ANF transcription in the adult rat in vivo, which were also inhibited by betaxolol but not by ICI 118551. These results suggest that beta -AR stimulated hypertrophy is mediated by the beta(1)-subtype and by a pertussis toxin-insensitive mechanism

Enhanced cAMP-induced nitric oxide-dependent coronary dilation during myocardial stunning in conscious pigs.

The goal of the current study was to determine the effects of cAMP-mediated coronary reactivity in conscious pigs with stunned myocardium induced by 1.5 h coronary stenosis (CS) and 12 h coronary artery reperfusion (CAR). Domestic swine (n = 5) were chronically instrumented with a coronary artery blood flow (CBF) probe, hydraulic occluder, left ventricular pressure gauge, wall-thickening crystals in the ischemic and nonischemic zones, and a coronary sinus catheter. The hydraulic occluder was inflated to induce a CS with a stable 38 +/- 1% reduction in CBF for 1.5 h. Before flow reduction and during CAR, cAMP-induced coronary vasodilation was investigated by forskolin (20 nmol. kg(-1). min(-1)). Enhanced CBF responses [+62 +/- 9%, P < 0.05, compared with pre-CS (+37 +/- 3%)] were observed for forskolin at 12 h after CAR as well as for bradykinin and reactive hyperemia. With the use of a similar protocol during systemic nitric oxide (NO) synthase inhibition with N(omega)-nitro-L-arginine (30 mg. kg(-1). day(-1) for 3 days), the enhanced CBF responses to forskolin, bradykinin, and reactive hyperemia were not observed after CS. Isolated microvessel preparations from pigs (n = 8) also demonstrated enhanced NO production to direct stimulation of adenylyl cyclase with forskolin (+71 +/- 12%) or NKH-477 (+60 +/- 10%) and administration of 8-bromo-cAMP (+74 +/- 13%), which were abolished by protein kinase A or NO synthase inhibition. These data indicate that cAMP stimulation elicits direct coronary vasodilation and that this action is amplified in the presence of sustained myocardial stunning after recovery from CS. This enhanced cAMP coronary vasodilation is mediated by an NO mechanism that may be involved in myocardial protection from ischemic injury.

Peripheral vascular endothelial dysfunction and apoptosis in old monkeys.

To determine the effects of aging on vasoactivity in a primate model (Macaca fascicularis), 13 young male monkeys (aged 7.1+/-0.4 years) and 9 old male monkeys (aged 19.8+/-0.6 years) were chronically instrumented for measurement of left ventricular and aortic pressures and cardiac output. Total cholesterol, triglyceride, and fasting blood sugar levels were not different between the 2 groups. There were no significant differences in baseline mean aortic pressure and total peripheral resistance (TPR) in the young monkeys versus the old monkeys. TPR fell less (P<0.05) with acetylcholine (1 microg/kg) in old monkeys (-25+/-1%) than in young monkeys (-34+/-2%), whereas decreases in TPR with sodium nitroprusside were similar in old and young monkeys. There was no evidence of atherosclerosis, but apoptosis of endothelial cells was enhanced (P<0.05) in the aortas and femoral arteries, but not in the media, of the old monkeys. There was a relationship (r=0.62, P=0.013) between the incidence of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive endothelial cells and endothelial cell density in the femoral artery. The reduced endothelial cell density was also correlated (r=0.82, P<0.01) with depressed TPR responses to acetylcholine. Thus, vascular endothelial dysfunction was present in old monkeys without evidence of atherosclerosis, which may be due to endothelial apoptosis and reduced endothelial cell density.

Determinants of the cardiomyopathic phenotype in chimeric mice overexpressing cardiac Gsalpha.

Mice with overexpressed cardiac Gsalpha develop cardiomyopathy, characterized by myocyte hypertrophy and extensive myocardial fibrosis. The cardiomyopathy likely involves chronically enhanced beta-adrenergic signaling, because it can be blocked with long-term propranolol treatment. It remains unknown whether the genotype of the myocyte is solely responsible for the progressive pathological changes. A chimeric population in the heart should answer this question. Accordingly, we developed a chimeric animal, which combined cells from a transgenic overexpressed Gsalpha parent and a Rosa mouse containing the LacZ reporter gene, facilitating identification of the non-Gsalpha cells, which express a blue color with exposure to beta-galactosidase. We studied these animals at 14 to 17 months of age (when cardiomyopathy should have been present), with the proportion of Gsalpha cells in the myocardium ranging from 5% to 88%. beta-Galactosidase staining of the hearts demonstrated Gsalpha and Rosa cells, exhibiting a mosaic pattern. The fibrosis and hypertrophy, characteristic of the cardiomyopathy, were not distributed randomly. There was a direct correlation (r=0.85) between the extent of myocyte hypertrophy (determined by computer imaging) and the quantity of Gsalpha cells. The fibrosis, determined by picric acid Sirius red, was also more prominent in areas with the greatest Gsalpha cell density, with a correlation of r=0.88. Thus, the overexpressed Gsalpha can exert its action over the life of the animal, resulting in a local picture of cardiomyopathic damage in discrete regions of the heart, where clusters of the overexpressed Gsalpha cells reside, sparing the clusters of normal cells derived from the normal Rosa parent.

Beta-adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic G(salpha) mouse.

Transgenic (TG) mice with cardiac G(salpha) overexpression exhibit enhanced inotropic and chronotropic responses to sympathetic stimulation, but develop cardiomyopathy with age. We tested the hypothesis that cardiomyopathy in TG mice with G(salpha) overexpression could be averted with chronic beta-adrenergic receptor (beta-AR) blockade. TG mice and age-matched wild-type littermates were treated with the beta-AR blocker propranolol for 6-7 months, starting at a time when the cardiomyopathy was developing but was not yet severe enough to induce significant cardiac depression (9.5 months of age), and ending at a time when cardiac depression and cardiomyopathy would have been clearly manifest (16 months of age). Propranolol treatment, which can induce cardiac depression in the normal heart, actually prevented cardiac dilation and the depressed left ventricular function characteristic of older TG mice, and abolished premature mortality. Propranolol also prevented the increase in myocyte cross-sectional area and myocardial fibrosis. Myocyte apoptosis, already apparent in 9-month-old TG mice, was actually eliminated by chronic propranolol. This study indicates that chronic sympathetic stimulation over an extended period is deleterious and results in cardiomyopathy. Conversely, beta-AR blockade is salutary in this situation and can prevent the development of cardiomyopathy.

Reduced subendocardial ryanodine receptors and consequent effects on cardiac function in conscious dogs with left ventricular hypertrophy.

The goal of this study was to examine the transmural distribution of ryanodine receptors in left ventricular (LV) hypertrophy (LVH) and its in vivo consequences. Dogs were chronically instrumented with an LV pressure gauge, ultrasonic crystals for measurement of LV internal diameter and wall thickness, and a left circumflex coronary blood flow velocity transducer. Severe LVH was induced by chronic banding of the aorta (12+/-1 months), which resulted in a 78% increase in LV/body weight. When ryanodine was infused directly into the circumflex coronary artery, it did not affect LV global function or systemic hemodynamics; however, it reduced LV wall thickening and delayed relaxation in the posterior wall in control dogs but was relatively ineffective in dogs with LVH. In LV sarcolemmal preparations, [3H]ryanodine ligand binding revealed a subendocardial/subepicardial gradient in normal dogs. In LVH there was a 45% decrease in ryanodine receptor binding and a loss in the natural subendocardial/subepicardial gradient, which roughly correlated inversely with the extent of LVH and directly with regional wall motion. Both mRNA and Western analyses revealed similar findings, with a reduction of the transmural mRNA levels and a loss in the natural gradient between subendocardial and subepicardial layers in LVH. Thus, ryanodine receptor message and binding in LVH is reduced preferentially in the subendocardium with consequent attenuation of the action of ryanodine in vivo. The selectively altered ryanodine regulation subendocardially in LVH could reconcile some of the controversy in this field and may play a role in mediating decompensation from stable LVH.

Mechanisms of desensitization to a PDE inhibitor (milrinone) in conscious dogs with heart failure.

The goal of this study was to determine the extent to which the effects of milrinone were desensitized in heart failure (HF) and to determine the mechanisms, i.e., whether these effects could be ascribed to changes in cAMP or phosphodiesterase (PDE) activity in HF. Accordingly, we examined the effects of milrinone in seven conscious dogs before and after HF was induced by rapid ventricular pacing at 240 beats/min. The dogs were chronically instrumented for measurements of left ventricular (LV) pressure and first derivative of LV pressure (dP/dt), arterial pressure, LV internal diameter, and wall thickness. Milrinone (10 micrograms . kg-1. min-1 iv) increased LV dP/dt by 1,854 +/- 157 from 2,701 +/- 105 mmHg/s (P < 0.05) before HF. After HF the increase in LV dP/dt in response to milrinone was attenuated significantly (P < 0.05); it increased by 615 +/- 67 from 1,550 +/- 107 mmHg/s, indicating marked desensitization. In the presence of ganglionic blockade the increases in LV dP/dt (+445 +/- 65 mmHg/s) in response to milrinone were markedly less (P < 0.01), and milrinone increased LV dP/dt even less in HF (+240 +/- 65 mmHg/s). cAMP and PDE activity were measured in endocardial and epicardial layers in normal and failing myocardium. cAMP was decreased significantly (P < 0.05) in LV endocardium (-26%) but not significantly in LV epicardium (-14%). PDE activity was also decreased significantly (P < 0.05) in LV endocardium (-18%) but not in LV epicardium (-4%). Thus significant desensitization to milrinone was observed in conscious dogs with HF. The major effect was autonomically mediated. The biochemical mechanism appears to be due in part to the modest reductions in PDE activity in failing myocardium, which, in turn, may be a compensatory mechanism to maintain cAMP levels in HF. Reductions in cAMP and PDE levels were restricted to the subendocardium, suggesting that the increased wall stress and reduced coronary reserve play a role in mediating these changes.

Differential regulation of inotropy and lusitropy in overexpressed Gsalpha myocytes through cAMP and Ca2+ channel pathways.

We investigated the mechanisms responsible for altered contractile and relaxation function in overexpressed Gsalpha myocytes. Although baseline contractile function (percent contraction) in Gsalpha mice was similar to that of wild-type (WT) mice, left ventricular myocyte contraction, fura-2 Ca2+transients, and Ca2+ channel currents (ICa) were greater in Gsalpha mice in response to 10(-8) M isoproterenol (ISO) compared with WT mice. The late phase of relaxation of the isolated myocytes and fura-2 Ca2+ transients was accelerated at baseline in Gsalpha but did not increase further with ISO. In vivo measurements using echocardiography also demonstrated enhanced relaxation at baseline in Gsalpha mice. Forskolin and CaCl2 increased contraction similarly in WT and Gsalpha mice. Rp-cAMP, an inhibitor of protein kinase, blocked the increases in contractile response and Ca2+ currents to ISO in WT and to forskolin in both WT and Gsalpha. It also blocked the accelerated relaxation in Gsalpha at baseline but not the contractile response to ISO in Gsalpha myocytes. Baseline measurements of cAMP and phospholambation phosphorylation were enhanced in Gsalpha compared with WT. These data indicate that overexpression of Gsalpha accelerates relaxation at end diastolic but does not affect baseline systolic function in isolated myocytes. However, the enhanced responses to sympathetic stimulation partly reflect increased Ca2+ channel activity; i.e the cellular mechanisms mediating these effects appear to involve a cAMP-independent as well as a cAMP-dependent pathway.

Apoptosis of cardiac myocytes in Gsalpha transgenic mice.

-The stimulatory GTP-binding protein Gsalpha transmits signals from catecholamine receptors to activate adenylyl cyclase and thereby initiate a cascade leading to cardiac chronotropy and inotropy. Transgenic mice overexpressing the Gs alpha subunit (Gsalpha) selectively in their hearts exhibit increased cardiac contractility in response to beta-adrenergic receptor stimulation. However, with aging, these mice develop a cardiomyopathy. This study sought morphological and biochemical evidence that overexpression of Gsalpha is associated with increased myocyte apoptosis in the older animals and to determine whether such overexpression can promote apoptosis of isolated neonatal cardiac myocytes exposed to beta-adrenergic receptor agonists. In the hearts of 15- to 18-month-old Gsalpha transgenic mice, histochemistry and electron microscopy illustrated the existence of numerous myocytes with abnormal nuclei embedded in collagen-rich connective tissue. Terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL, for in situ labeling of DNA breaks) demonstrated that approximately 0.6% of myocyte nuclei contained fragmented DNA. Agarose gel electrophoresis provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation. Cultured cardiac myocytes from newborn Gsalpha transgenic mice showed increased TUNEL staining and internucleosomal DNA fragmentation compared with wild-type controls when treated with the beta-agonist isoproterenol. Thus, enhanced activation of beta-adrenergic signaling by overexpression of Gsalpha in the hearts of transgenic mice induces apoptosis of cardiac myocytes. This represents a potential mechanism that may contribute to the development of cardiomyopathy in this model.

Beta-adrenergic receptor-G protein-adenylyl cyclase signal transduction in the failing heart.

The beta-adrenergic receptor signal transduction pathway is critical for rapid adjustments to increased cardiovascular demand (e.g., during exercise). In the face of chronic stimulation of this pathway, as occurs in the pathogenesis of heart failure, beta-adrenergic receptor stimulation may become maladaptive. Under these conditions, elevation of circulating catecholamines and depletion of cardiac tissue stores of norepinephrine occur in the failing heart, resulting in desensitization. Whether or not stimulation or inhibition of the beta-adrenergic receptor signaling pathway is beneficial in heart failure is controversial. One approach to address this question is to specifically overexpress a component of the beta-adrenergic receptor signaling pathway in a transgenic mouse heart. We have characterized young and old adult mice with overexpressed cardiac G(s alpha) which couples the beta-adrenergic receptor to adenylyl cyclase. In younger animals, beta-adrenergic receptor stimulation results in an augmented heart rate and cardiac contractility. Over the life of the animal, however, a picture of cardiomyopathy develops. The result is a dilated heart with a large amount of fibrosis and myocyte hypertrophy, degeneration atrophy, and apoptosis. Conversely, chronic beta-adrenergic receptor blockade prevents the development of cardiomyopathy. These experiments support the point of view that chronic beta-adrenergic stimulation during the development of heart failure is deleterious and that protecting the heart with chronic beta-adrenergic receptor blockade is salutary, conceptually consistent with results of recent clinical trials examining the effects of beta-adrenergic receptor blockers in patients with heart failure.

Physiological and biochemical adrenergic regulation of the stunned myocardium.

A dual approach was employed to study beta-adrenergic receptor signal transduction in post ischemic (stunned) myocardium, examining physiological interventions in awake, chronically instrumented pigs and biochemical, cellular mechanisms in sarcolemmal preparations from the stunned hearts using the contralateral non-ischemic zone as a control. Ten min of coronary artery occlusion (CAO) and 30 min coronary artery reperfusion (CAR) resulted in depressed posterior wall-thickening (myocardial stunning). Isoproterenol increased transmural wall thickening more in stunned myocardium than in non-ischemic myocardium. In contrast, the responses of wall thickening to forskolin, actually decreased during stunning compared with control. NKH 477, a water soluble forskolin derivative, that does not activate cardiac nerves, increased wall thickening in non-ischemic tissue similarly to the effects on stunned myocardium. Increasing cardiac neural tone reflexly with inferior venal caval occlusion (IVCO) elicited similar results to forskolin, i.e., stunned myocardium responded with less of an increase in wall thickening as compared with non-ischemic myocardium. Beta-adrenergic receptor density, as determined with 125I-cyanopindolol binding, was significantly increased in stunned subendocardium and subepicardium compared with respective values in non-ischemic myocardium. There were no differences in the response of adenylyl cyclase to isoproterenol in stunned and non-ischemic myocardium. The enhanced responsiveness of the beta-adrenergic receptor to isoproterenol stimulation in stunned myocardium corresponded to the increase in beta-adrenergic receptor density. The combination of enhanced responses to isoproterenol, and decreased responses to forskolin and to IVCO and preserved responsiveness to NKH 477, suggest that stunned myocardium is characterized by transient sympathetic neural stunning. The enhanced sensitivity to beta-adrenergic receptor stimulation has important clinical implications, both in terms of therapy of stunned myocardium and detection of stunned and/or hibernating myocardium, i.e., low dose dobutamine echocardiography.

Cardiac Gsalpha overexpression enhances L-type calcium channels through an adenylyl cyclase independent pathway.

The alpha subunit of the stimulatory heterotrimeric G protein (Gsalpha) is critical for the beta-adrenergic receptor activation of the cAMP messenger system. The role of Gsalpha in regulating cardiac Ca2+ channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac Gsalpha were used to assess the role of Gsalpha on the whole-cell Ca2+ currents (ICa). Cardiac myocytes from transgenic mice had a 490% higher peak ICa compared with those of either wild-type controls or Gsalpha-nonexpressing littermates. The effect of Gsalpha overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPgammaS-activated Gsalpha. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated Gsalpha. The data indicate that Gsalpha overexpression is associated with a constitutive enhancement of ICa which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.