Sanford P. Bishop

Adverse Effects of Chronic Endogenous Sympathetic Drive Induced by Cardiac Gsα Overexpression

To study the physiological effect of the overexpression of myocardial Gsalpha (protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3 +/- 0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF) was not different from that of control mice at baseline (LVFS, 40 +/- 3% versus 36 +/- 2%; LVEF, 78 +/- 3% versus 74 +/- 3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 micrograms/kg per minute) infusion were higher than the values in control mice (LVFS, 68 +/- 4% versus 48 +/- 3%; LVEF, 96 +/- 1% versus 86 +/- 3%; P < .05). Norepinephrine (NE, 0.2 micrograms/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59 +/- 4% versus 47 +/- 3%; LVEF, 93 +/- 2% versus 85 +/- 3%; P < .05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33 +/- 2% with ISO (0.02 micrograms/kg per minute) and by only 13 +/- 2% in three wild-type control mice (P < .01). NE (0.1 micrograms/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65 +/ 8% versus 28 +/- 4%, P < .05). Pathological and histological analyses of older transgenic mouse hearts (16.0 +/- 0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2 +/- 1.4% versus 1.2 +/- 0.3%, P < .05) and in the cross-sectional area of myocytes (298 +/- 29 versus 187 +/- 12 micron2, P < .05) compared with control mouse hearts. These results suggest that Gsalpha overexpression enhances the efficacy of the beta-adrenergic receptor-Gs-adenylyl cyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study.

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.

Apoptosis of Cardiac Myocytes in Gsα 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.

β-Adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic G sα 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.

An α-cardiac myosin heavy chain gene mutation impairs contraction and relaxation function of cardiac myocytes

Left Ventricular (LV) myocytes were isolated from 15-wk-old male mice bearing the Arg403 → Gln α-cardiac myosin heavy chain missense mutation (α-MHC403/+), a model of familial hypertrophic cardiomyopathy. LV myocytes were classified morphologically: type I, rod shaped with parallel myofibrils; type II, irregularly shaped, shorter and wider than wild-type (WT) control cells, with parallel myofibrils; and type III, irregularly shaped with disoriented myofibrils. Compared with WT myocytes, α-MHC403/+ myocytes had fewer type I cells (WT = 74 ± 3%, α-MHC403/+ = 41 ± 4%, P < 0.01) and more type III cells (WT= 12 ± 3%, α-MHC403/+ = 49 ± 7%, P < 0.01). In situ histology also demonstrated marked myofibrillar disarray in the α-MHC403/+ hearts. With the use of video edge detection, myocytes were paced at 1 Hz (37°C) to determine the effects of the mutation on myocyte function. End-diastolic length was reduced in mutant myocytes, but fractional shortening (% contraction) and sarcomere length were not. Velocity of contraction (-d L/d t max) was depressed in mutant cells, but more in type II and III cells (-31%) than in type I cells (-18%). Velocity of relaxation (+d L/d t) was also depressed more in type II and III cells (-38%) than in type I cells (-16%). Using fura 2 dye with intracellular Ca2+ transients, we demonstrated that in α-MHC403/+ myocytes, the amplitude of the Ca2+ signal during contraction was unchanged but that the time required for decay of the signal to decrease 70% from its maximum was delayed significantly (WT = 159 ± 8 ms; α-MHC403/+ = 217 ± 14 ms, P < 0.01). Sarco(endo)plasmic reticulum Ca2+-ATPase mRNA levels in α-MHC403/+ and WT mice were similar. These data indicate that the altered cardiac dysfunction of α-MHC403/+ myocytes is directly due to defective myocyte function rather than to secondary changes in global cardiac function and/or loading conditions.

Ineffective Perfusion-Contraction Matching in Conscious, Chronically Instrumented Pigs With an Extended Period of Coronary Stenosis

Several models purported to represent hibernating myocardium involve a coronary stenosis (CS) to reduce blood flow (BF) and function without eliciting necrosis in anesthetized pigs. The goal of the present study was to determine whether sustained moderate reduction in coronary BF in conscious pigs induced hibernating myocardium, ie, perfusion-contraction matching with no necrosis. These experiments were conducted in conscious pigs chronically instrumented with a coronary artery BF probe and hydraulic occluder, left ventricular (LV) pressure gauge, and wall thickening (WT) crystals in the potentially ischemic and nonischemic zones. The hydraulic occluder was inflated to induce a stable 41+/-4% reduction in BF for 24 hours. Ischemic zone systolic WT fell initially with CS and then continued to decline during the period of CS even though blood flow did not change further, suggesting the induction of myocardial stunning. At 2 days after release of CS, WT was still depressed by 48+/-15%. Assessment of necrosis by histology or triphenyltetrazolium chloride showed 40+/-5% multifocal patchy necrosis interspersed with normal tissue involving the inner one third to one half of the ventricular wall. Regional myocardial BF (radioactive microsphere technique) was assessed by dividing the entire LV into an average of 488+/-59 pieces and examining the spatial distribution of BF within the area at risk (AAR). BF in the samples in the area of patchy necrosis was reduced (-66+/-4% from a baseline of 1.55+/-0.27 mL x min(-1) x g(-1)), whereas BF was maintained in samples in the AAR without necrosis (-2+/-7% from a baseline of 1.25+/-0.22 mL x min(-1) x g(-1)). These findings indicate that when hypoperfusion induced by CS in conscious pigs is sustained, the result is necrosis rather than hibernating myocardium. The remainder of the AAR, which lacked necrosis, might have been mistaken for hibernating myocardium had only histology been evaluated and BF not been measured and found to be at normal levels.

Regional myocyte size in normotensive and spontaneously hypertensive rats.

SUMMARY Regional differences in cell size in the hearts of rats with and without cardiac hypertrophy were studied using isolated muscle cells. Isolated cardiac myocytes were prepared from left ventricular free wall inner and outer halves and the right ventricle of six male 12-week-old spontaneously hypertensive (SHR), Wistar-Kyoto (WKY) and Fischer-344 rats. In SHR, blood pressure was increased to 188 ± 4 (SEM) mm Hg versus 143 ± 2 and 133 ± 10 for WKY and Fischer rats, respectively (p < 0.001). Total heart weight was increased to 1103 ± 29 mg in SHR compared to 824 ± 21 in WKY and 951 ± 23 in Fischer rats (p < 0.001). Isolated cardiac myocytes were prepared by perfusion of isolated hearts with Ca++ free Hanks solution containing EGTA followed by collagenase-containing media. Mean length, width and volume of 150 cells stained with hematoxylin and eosin from each site were measured with a sonic digitizer. Two nuclei were present in 85 to 87% of isolated cells from all strains and regions. There was no difference among strains in right ventricular cell length, width, or volume, nor between left ventricular inner and outer halves within each strain. Left ventricular cells were larger than right ventricular cells (p < 0.05) in all strains. Left ventricular cells of SHR were larger than left ventricular cells of WKY or Fischer rats in proportion to the increase in total heart weight, indicating that cardiac enlargement in SHR is due to increased cell size rather than increased cell number.

Nitric Oxide Mediates Benefits of Angiotensin II Type 2 Receptor Overexpression During Post-Infarct Remodeling

Abstract—We hypothesized that nitric oxide (NO) mediates the benefits of cardiac angiotensin II type 2 (AT2-R) overexpression during postmyocardial infarction (post-MI) remodeling. Eleven wild-type (WT) C57BL/6 mice and 28 transgenic (TG) mice with AT2-R overexpression were studied by cardiac magnetic resonance imaging (CMR) at baseline and days 1 and 28 post-MI induced by left anterior descending artery occlusion and reperfusion. Sixteen TG mice were treated from day 1 through 28 post-MI with the NO synthase inhibitor NG-nitro-l-arginine methyl ester in drinking water at 1 mg/mL (TG-Rx). Left ventricular mass index (LVMI), end-diastolic volume index (EDVI) and end-systolic volume index (ESVI), wall thickness, percent thickening, and ejection fraction (EF) were measured. Infarct size on day 1 was assessed by post-contrast CMR. Interstitial collagen was quantified in noninfarcted regions. At baseline, heart rate (HR), blood pressure (BP), LVMI, EDVI, and ESVI were similar between groups, as were infarct size and weekly post-MI HR and systolic BP. By day 28 post-MI, EDVI and ESVI were similar in WT and TG-Rx, but significantly lower in TG (ESVI: 1.41±0.18 &mgr;L/g versus 2.53±0.14 &mgr;L/g in WT; 2.17±0.14 &mgr;L/g in TG-Rx; P <0.008 for both). At day 28, EF was higher in TG (46.3%±2.9%) compared with WT and TG-Rx (32.7±2.3% and 33.7±2.3, respectively; P <0.003 for both). Wall thickening at day 28 post-MI was greater in the base and mid-LV in TG than WT and TG-Rx. Noninfarcted region interstitial collagen was similar between groups. Thus, the NO pathway may mediate much of the benefits of cardiac AT2-R overexpression during post-MI remodeling.

Regional myocyte size in compensated right ventricular hypertrophy in the ferret

The effect of pressure-induced right ventricular hypertrophy on regional myocyte size and shape and regional myocardial wet weight was studied by comparing 20 ferrets which underwent pulmonary artery banding at a weanling age to 19 non-operated siblings. Using isolated myocyte preparations from six myocardial regions in 10 banded and seven non-banded ferrets, a 60% increase in cell volume in the right ventricular outflow tract and the right ventricular free wall was shown to be due primarily to an increased cross-sectional area of individual myocytes. The right side of the interventricular septum exhibited an intermediate increase in cell volume, while the left side of the interventricular septum did not respond to the pulmonary artery banding procedure. These findings confirm that localized hemodynamic changes produce hypertrophy of individual myocytes in selected regions of the heart and that a pressure-induced model of hypertrophy involves an increased cross-sectional area of myocytes, with minimal change in cell length.

The angiotensin II type 2 receptor and improved adjacent region function post-MI.

Angiotensin II type 2 receptor (AT2-R) overexpression in the mouse heart preserves left ventricular (LV) size and global LV function during post-MI remodeling. We hypothesized that CMR tagging would localize regional improvements in myocardial function during post-MI remodeling in AT2-R cardiac overexpressed transgenic mice (TG), which could explain the preservation of global LV function post-MI. Six male wild-type (WT) C57BL/6 mice and 10 TG mice were studied by CMR at baseline (day 0) and days 1, 7, and 28 post-MI. MI was induced by 1 hour occlusion of the LAD followed by reperfusion. On day 1 post-MI, gadolinium-DTPA was injected to assess infarct size. LV size and function was assessed by cine CMR. Mean % circumferential shortening (%CS) was calculated within infarcted, adjacent, and remote regions at each time point in WT and TG mice. Quantitative interstitial collagen and mean myocyte cross-sectional area was measured postmortem at day 28 post-MI. LV end-systolic volume was lower and ejection fraction higher at baseline in the TG group and these differences were maintained post-MI. Within infarcted and remote zones, although %CS was higher in TG mice at day 0, there was no difference by day 28 between groups. Within adjacent regions, while there was no difference at day 0 or 1 in TG vs. WT, %CS was significantly higher in TG mice by day 7, and these changes persisted out to day 28 post-MI. Regional interstitial collagen and myocyte size were similar between groups. Thus, myocardial tagging can detect regional differences in contractile function post-MI in TG mice, and AT2-R overexpression is associated with improved contractile function in adjacent noninfarcted myocardium.