James D. Marsh

Impaired chronotropic response to exercise in patients with congestive heart failure. Role of postsynaptic beta-adrenergic desensitization.

The mechanism responsible for the attenuated heart rate (HR) response to exercise in patients with congestive heart failure (CHF) was investigated in 46 normal subjects and 59 patients with CHF stratified by peak exercise oxygen consumption (VO2). The peak exercise HR and the increment in HR from rest to peak exercise were decreased in CHF patients, and both correlated strongly with peak VO2 (r = 0.810, p less than 0.0001; r = 0.863, p less than 0.0001, respectively). Peak exercise norepinephrine level (NE) and the increment in NE from rest to peak exercise were not attenuated in CHF patients. Resting NE was elevated in CHF patients and correlated inversely with peak VO2 (r = -0.595, p less than 0.001). However, no significant correlation occurred between peak VO2 and either peak exercise NE or the exercise increment in NE. The ratio of the exercise increments in HR and NE, and indirect index of sinoatrial node sympathetic responsiveness, was markedly reduced in CHF patients and was inversely related to the severity of exercise impairment. Likewise, the HR response to a graded isoproterenol infusion was markedly reduced in CHF patients. Age-matching of normal subjects and CHF patients did not affect the foregoing observations. Infusion of CHF patients with the phosphodiesterase inhibitor milrinone caused a significant increase in the ratio of the exercise increments in HR and NE. These data strongly suggest that the attenuated HR response to exercise in CHF patients is due, at least in part, to postsynaptic desensitization of the beta-adrenergic receptor pathway.

Androgen Receptors Mediate Hypertrophy in Cardiac Myocytes

BACKGROUND The role of androgens in producing cardiac hypertrophy by direct action on cardiac myocytes is uncertain. Accordingly, we tested the hypothesis that cardiac myocytes in adult men and women express an androgen receptor gene and that myocytes respond to androgens by a hypertrophic response. METHODS AND RESULTS We used reverse transcription-polymerase chain reaction methods to demonstrate androgen receptor transcripts in multiple tissues and [3H]phenylalanine incorporation and atrial natriuretic peptide secretion as markers of hypertrophy in cultured rat myocytes. Messenger RNA encoding androgen receptors was detected in myocytes of male and female adult rats, neonatal rat myocytes, rat heart, dog heart, and infant and adult human heart. Both testosterone and dihydrotestosterone produced a robust receptor-specific hypertrophic response in myocytes, determined by indices of protein synthesis and atrial natriuretic peptide secretion. CONCLUSIONS Androgen receptors are present in cardiac myocytes from multiple species, including normal men and women, in a context that permits androgens to modulate the cardiac phenotype and produce hypertrophy by direct, receptor-specific mechanisms. There are clinical implications for therapeutic or illicit use of androgens in humans.

Myocardial infarction in young patients

Myocardial infarction in persons under the age of 45 years accounts for 6% to 10% of all myocardial infarctions in the United States. In this age group, it is predominantly a disease of men. Important risk factors include a family history of myocardial infarction before age 55 years, hyperlipidemia, smoking, and obesity. Unlike older patients, approximately half of young patients have single-vessel coronary disease, and in up to 20%, the cause is not related to atherosclerosis. Coronary angiography may be warranted in young patients with myocardial infarction to define the anatomy of the disease and to permit optimal management.

Expression of dihydropyridine receptor (Ca2+ channel) and calsequestrin genes in the myocardium of patients with end-stage heart failure.

Cytoplasmic free calcium ions (Ca2+) play a central role in excitation-contraction coupling of cardiac muscle. Abnormal Ca2+ handling has been implicated in systolic and diastolic dysfunction in patients with end-stage heart failure. The current study tests the hypothesis that expression of genes encoding proteins regulating myocardial Ca2+ homeostasis is altered in human heart failure. We analyzed RNA isolated from the left ventricular (LV) myocardium of 30 cardiac transplant recipients with end-stage heart failure (HF) and five organ donors (normal control), using cDNA probes specific for the cardiac dihydropyridine (DHP) receptor (the alpha 1 subunit of the DHP-sensitive Ca2+ channel) and cardiac calsequestrin of sarcoplasmic reticulum (SR). In addition, abundance of DHP binding sites was assessed by ligand binding techniques (n = 6 each for the patients and normal controls). There was no difference in the level of cardiac calsequestrin mRNA between the HF patients and normal controls. In contrast, the level of mRNA encoding the DHP receptor was decreased by 47% (P less than 0.001) in the LV myocardium from the patients with HF compared to the normal controls. The number of DHP binding sites was decreased by 35-48%. As reported previously, expression of the SR Ca(2+)-ATPase mRNA was also diminished by 50% (P less than 0.001) in the HF group. These data suggest that expression of the genes encoding the cardiac DHP receptor and SR Ca(2+)-ATPase is reduced in the LV myocardium from patients with HF. Altered expression of these genes may be related to abnormal Ca2+ handling in the failing myocardium, contributing to LV systolic and diastolic dysfunction in patients with end-stage heart failure.

Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular myocytes.

Transforming growth factor-beta 1 (TGF-beta 1) is a peptide growth factor that may play a role in the myocardial response to hypertrophic stimuli. However, the cellular distribution, mechanism of induction, and source of increased TGF-beta 1 in response to hypertrophic stimuli are not known. We tested the hypothesis that the cardiac myocyte responds to hypertrophic stimuli with the increased expression of TGF-beta 1. In adult rat ventricular myocardium freshly dissociated into myocyte and nonmyocyte cellular fractions, the preponderance of TGF-beta 1 mRNA visualized by Northern hybridization was in the nonmyocyte fraction. Abdominal aortic constriction (7 d) and subcutaneous norepinephrine infusion (36 h) each caused ventricular hypertrophy associated with 3.1-fold and 3.8-fold increases, respectively, in TGF-beta 1 mRNA in the myocyte fraction, but had no effect on the level of TGF-beta 1 mRNA in the nonmyocyte fraction. In ventricular myocytes, norepinephrine likewise caused a 4.1-fold increase in TGF-beta 1 mRNA associated with an increase in TGF-beta bioactivity. This induction of TGF-beta 1 mRNA occurred at norepinephrine concentrations as low as 1 nM and was blocked by prazosin, but not propranolol. NE did not increase the TGF-beta 1 mRNA level in nonmyocytes, primarily fibroblasts, cultured from neonatal rat ventricle. Thus, the cardiac myocyte responds to two hypertrophic stimuli, pressure overload and norepinephrine, with the induction of TGF-beta 1. These data support the view that TGF-beta 1, released by myocytes and acting in an autocrine and/or paracrine manner, is involved in myocardial remodeling by hypertrophic stimuli.

Left Ventricular End-Systolic Pressure-Dimension and Stress-Length Relations in Normal Human Subjects

Abstract To determine whether the left ventricular end systolic pressure-dimension and end-systolic stress-dimension relations in human beings are linear and sensitive to altered contractility, we studied 13 normal subjects during methoxamine infusion and with postextrasystolic potentiation induced by an external mechanical cardiac stimulator. End-systolic diameter was obtained with echocardiography and end-systolic pressure was estimated in six subjects from the dicrotic notch of a simultaneously recorded carotid pulse tracing, standardized by cuff pressure, whereas in seven subjects intraarterial pressure was recorded. For each subject, the end-systolic pressure-dimension relation was linear ( r = 0.83–0.99) over a range of 76 mm Hg (84 to 160) for end-systolic pressure. The mean slope of the end-systolic pressure-dimension line was 62 ± 22 mm Hg/cm. Peak systolic pressure was also linearly related to end-systolic diameter ( r = 0.82–0.99) over a range of 100 mm Hg (104 to 204). End-systolic stress was a linear function of end-systolic dimension as well ( r = 0.93–0.99) over an end-systolic stress range of 181 g/cm 2 . With postextrasystolic potentiation the potentiated beat had a smaller end-systolic dimension for any given end-systolic pressure and thus shifted the end-systolic pressure-dimension relation to the left. Thus, end-systolic pressure-dimension and stress-dimension relations in human subjects appear to be linear and are sensitive to the inotropic state.

Ca2+ Influx–Induced Sarcoplasmic Reticulum Ca2+ Overload Causes Mitochondrial-Dependent Apoptosis in Ventricular Myocytes

Increases in Ca2+ influx through the L-type Ca2+ channel (LTCC, Cav1.2) augment sarcoplasmic reticulum (SR) Ca2+ loading and the amplitude of the cytosolic Ca2+ transient to enhance cardiac myocyte contractility. Our hypothesis is that persistent increases in Ca2+ influx through the LTCC cause apoptosis if the excessive influx results in SR Ca2+ overload. Feline ventricular myocytes (VMs) in primary culture were infected with either an adenovirus (Ad) containing a rat Cav1.2 &bgr;2a subunit-green fluorescent protein (GFP) fusion gene (Ad&bgr;2a) to increase Ca2+ influx or with AdGFP as a control. Significantly fewer &bgr;2a-VMs (21.4±5.6%) than GFP-VMs (99.6±1.7%) were viable at 96 hours. A fraction of &bgr;2a-VMs (20.8±1.8%) contracted spontaneously (SC-&bgr;2a-VMs), and viability was significantly correlated with the percentage of SC-&bgr;2a-VMs. Higher percentages of apoptotic nuclei, DNA laddering, and cytochrome C release were detected in &bgr;2a-VMs. This apoptosis was prevented with pancaspase or caspase-3 or caspase-9 inhibitors. L-type calcium current (ICa-L) density was greater in &bgr;2a-VMs (23.4±2.8 pA/pF) than in GFP-VMs (7.6±1.6 pA/pF). SC-&bgr;2a-VMs had higher diastolic intracellular Ca2+ (Indo-1 ratio: 1.1±0.1 versus 0.7±0.03, P<0.05) and systolic Ca2+ transients (1.89±0.27 versus 0.80±0.08) than GFP-VMs. Inhibitors of Ca2+ influx, SR Ca2+ uptake and release, mitochondrial Ca2+ uptake, mitochondrial permeation transition pore, calpain, and Bcl-2-associated X protein protected &bgr;2a-VMs from apoptosis. These results show that persistent increases in Ca2+ influx through the ICa-L enhance contractility but lead to apoptosis through a mitochondrial death pathway if SR Ca2+ overload is induced.

Intracoronary infusion of dobutamine to patients with and without severe congestive heart failure. Dose-response relationships, correlation with circulating catecholamines, and effect of phosphodiesterase inhibition.

We infused dobutamine into the left main coronary artery of 24 patients with severe congestive heart failure (CHF) and 8 normal subjects without hemodynamic dysfunction. The maximal +dP/dt response to intracoronary (IC) dobutamine in CHF patients was only 37% of that in normals. This decrease in maximal response was not associated with a rightshift in the EC50 for dobutamines effect on +dP/dt, or a decrease in the affinity of myocardial beta adrenergic receptors for dobutamine determined in vitro. In nine of the CHF patients, IC dobutamine infusion was followed by IC infusion of the phosphodiesterase inhibitor milrinone, and subsequently, by a second IC infusion of dobutamine. After IC milrinone, the increase in +dP/dt caused by IC dobutamine (74 +/- 10%) was significantly greater than that caused by the first infusion of dobutamine (52 +/- 11%; P less than 0.003) or milrinone (42 +/- 6%; P less than 0.001). Resting plasma norepinephrine was markedly elevated in CHF patients (837 +/- 208 ng/liter), but not in normal subjects (142 +/- 32 ng/liter); and the increase in +dP/dt caused by IC dobutamine was inversely related to resting plasma norepinephrine levels (r = -0.653; P less than 0.001). IC dobutamine caused a dose-related decrease in plasma norepinephrine (maximal effect, -160 +/- 31 ng/liter; P less than 0.001). Thus, (a) the maximal inotropic response to dobutamine is markedly depressed in patients with severe CHF, and is significantly greater after pretreatment with the phosphodiesterase inhibitor milrinone; (b) the impairment in inotropic response to dobutamine is inversely related to circulating norepinephrine levels; and (c) myocardial stimulation by dobutamine results in withdrawal of sympathetic tone.

Angiotensin II-induced hypertrophy of adult rat cardiomyocytes is blocked by nitric oxide

The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 ± 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 ± 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 ± 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 +/- 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 +/- 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 +/- 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.

Beta-adrenergic receptor number and adenylate cyclase function in denervated transplanted and cardiomyopathic human hearts.

To test the hypothesis that there is up-regulation of beta-adrenergic receptor density or supersensitivity of beta-adrenergic receptor-stimulated adenylate cyclase in the denervated transplanted human heart, we studied myocardium from transplanted, normal, and failing hearts. Myocardium was obtained from 10 patients 9 +/- 3 months after cardiac transplantation, from 10 patients without cardiac disease, and from eight patients with symptomatic congestive heart failure due to idiopathic cardiomyopathy. beta-Adrenergic receptor density in transplanted myocardium (15 +/- 3 fmol/mg protein, 1.20 +/- 0.14 fmol/mg DNA) was not different from that in normal myocardium (22 +/- 3 fmol/mg protein, 1.46 +/- 0.13 fmol/mg DNA; p = NS for both). In myocardium from cardiomyopathic hearts, beta-adrenergic receptor density was markedly reduced (8 +/- 2 fmol/mg protein, 0.84 +/- 0.13 fmol/mg DNA; p less than 0.05 and p less than 0.01 vs. normal myocardium, respectively). Likewise, the response of adenylate cyclase to isoproterenol in transplanted myocardium was not significantly different from that in normal myocardium, but the response was markedly depressed in cardiomyopathic myocardium. Although forskolin-stimulated adenylate cyclase activity was similar in all three groups, guanine nucleotide-stimulated adenylate cyclase activity was markedly reduced in transplanted myocardium (20 +/- 17 vs. 78 +/- 13 pmol/mg/min for normal myocardium, p less than 0.01) and to a lesser degree in cardiomyopathic myocardium (39 +/- 14 pmol/mg/min, p less than 0.03 vs. normal myocardium). Thus, there is no evidence of beta-adrenergic receptor up-regulation or supersensitivity in denervated transplanted human myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)