Raymond K. Kudej

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.

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.

H11 kinase is a novel mediator of myocardial hypertrophy in vivo.

Abstract— By subtractive hybridization, we found a significant increase in H11 kinase transcript in large mammalian models of both ischemia/reperfusion (stunning) and chronic pressure overload with hypertrophy. Because this gene has not been characterized in the heart, the goal of the present study was to determine the function of H11 kinase in cardiac tissue, both in vitro and in vivo. In isolated neonatal rat cardiac myocytes, adenoviral-mediated overexpression of H11 kinase resulted in a 37% increase in protein/DNA ratio, reflecting hypertrophy. A cardiac-specific transgene driven by the &agr;MHC-promoter was generated, which resulted in an average 7-fold increase in H11 kinase protein expression. Transgenic hearts were characterized by a 30% increase of the heart weight/body weight ratio, by the reexpression of a fetal gene program, and by concentric hypertrophy with preserved contractile function at echocardiography. This phenotype was accompanied by a dose-dependent activation of Akt/PKB and p70S6 kinase, whereas the MAP kinase pathway was unaffected. Thus, H11 kinase represents a novel mediator of cardiac cell growth and hypertrophy.

Association between outcome and changes in plasma lactate concentration during presurgical treatment in dogs with gastric dilatation-volvulus: 64 cases (2002-2008).

OBJECTIVE To determine whether changes in presurgical plasma lactate concentration (before and after initial fluid resuscitation and gastric decompression) were associated with short-term outcome for dogs with gastric dilatation-volvulus (GDV). DESIGN Retrospective case series. ANIMALS 64 dogs. PROCEDURES Medical records were reviewed, and signalment, history, resuscitative treatments, serial presurgical lactate concentrations, surgical findings, and short-term outcome were obtained for dogs with confirmed GDV. RESULTS 36 of 40 (90%) dogs with an initial lactate concentration <or= 9.0 mmol/L survived, compared with only 13 of 24 (54%) dogs with a high initial lactate (HIL) concentration (> 9.0 mmol/L). Within HIL dogs, there was no difference in mean +/- SD initial lactate concentration between survivors and nonsurvivors (10.6 +/- 2.3 mmol/L vs 11.2 +/- 2.3 mmol/L, respectively); however, there were significant differences in post-treatment lactate concentration, absolute change in lactate concentration, and percentage change in lactate concentration following resuscitative treatment. By use of optimal cutoff values within HIL dogs, survival rates for dogs with final lactate concentration > 6.4 mmol/L (23%), absolute change in lactate concentration <or= 4 mmol/L (10%), or percentage change in lactate concentration <or= 42.5% (15%) were significantly lower than survival rates for dogs with a final lactate concentration <or= 6.4 mmol/L (91%), absolute change in lactate concentration > 4 mmol/L (86%), or percentage change in lactate concentration > 42.5% (100%). CONCLUSIONS AND CLINICAL RELEVANCE Calculating changes in plasma lactate concentration following initial treatment in dogs with GDV may assist in determining prognosis and identifying patients that require more aggressive treatment.

Delayed Enhanced Nitric Oxide–Mediated Coronary Vasodilation Following Brief Ischemia and Prolonged Reperfusion in Conscious Dogs

The goal of this study was to determine both the early and delayed effects of a brief (10-minute) coronary artery occlusion (CAO) and prolonged (5-day) reperfusion (CAR) on coronary endothelial function. Fourteen mongrel dogs were chronically instrumented to measure aortic and left ventricular pressures, wall thickness, and left circumflex coronary blood flow (CBF). Before CAO and during CAR, coronary vascular reactivity was investigated by 15-second CAO and subsequent reactive hyperemia (RH) and by the selective intracoronary infusion of acetylcholine (ACh, 10 micrograms/min) and bradykinin (BK, 2.5 micrograms/min), endothelium-dependent vasodilators, and sodium nitroprusside (SNP, 40 micrograms/min), an endothelium-independent vasodilator. CBF responses to ACh and BK began to increase after 6 hours of CAR, reached a peak after 1 to 2 days of CAR, and then subsided over the subsequent 4 days. After 1 day of CAR, compared with before CAO, enhanced CBF responses (P < .05), associated with increased coronary sinus oxygen content, were observed for-ACh (+66 +/- 20%), BK (+74 +/- 24%), and RH (+24 +/- 5%) but not SNP (-2 +/- 10%). Production of NO metabolites (nitrate and nitrite), measured as their coronary arteriovenous differencexCBF, was significantly increased after 1 to 2 days of CAR, both at baseline (153 +/- 56%) and during BK infusion (220 +/- 76%) (P < .05). Holding CBF at pre-CAO levels during the initial CAR period did not attenuate the delayed enhanced endothelial vasodilation to ACh and BK. However, NO blockade with intracoronary NG-nitro-L-arginine blocked the enhanced coronary vasodilation to ACh and BK. Thus, in contrast to previous studies, these data indicate that brief ischemic episodes induce delayed enhanced coronary endothelial function, which is delayed in onset and prolonged in duration. This can be explained by an upregulation of coronary vascular NO production, potentially involved in the mechanism of the delayed window of preconditioning.

Identification of a cis-acting regulatory element conferring inducibility of the atrial natriuretic factor gene in acute pressure overload.

To identify the cis-acting regulatory element(s) which control the induction of the atrial natriuretic factor (ANF) gene in acute pressure overload, DNA constructs consisting of promoter elements linked to a reporter gene were injected into the myocardium of dogs, which underwent aortic banding or were sham-operated. Expression of a reporter gene construct harboring the ANF promoter (-3400ANF) was induced 6-12-fold after 7 d of pressure overload. An internal deletion of 556 bp (nucleotide sequence -693 to -137) completely abrogated the inducibility of the ANF reporter gene construct. An activator protein-1 (AP1)-like site (-496 to -489) and a cAMP regulatory element (CRE) (-602 to -596) are located within the deleted sequence. Site-directed mutagenesis of the AP1-like site but not the CRE completely prevented the induction of this construct to acute pressure overload. Further, the AP1-like site was able to confer inducibility of a heterologous promoter (beta-myosin heavy chain) to higher values than controls. Gel mobility shift assay (GMSA) supershift analysis was performed using a radiolabeled probe of the ANF promoter (-506/-483) that included the AP1-like site (ATGAATCA) sequence, as well as a probe converted to contain an AP1 consensus sequence (ATGACTCA). GMSA analysis demonstrated that the ANF AP1-like element could bind both a constitutively expressed factor and the AP1 proteins, and conversion to a true AP1 site increased its affinity for AP1. However, 7 d after the onset of pressure overload, the AP1 proteins were present only at low levels, and the major complex formed by the ANF AP1-like probe was not supershifted by a jun antibody. Using a large animal model of pressure overload, we have demonstrated that a unique cis-acting element was primarily responsible for the overload induction of the ANF gene.

Paradoxical Cellular Ca2+ Signaling in Severe but Compensated Canine Left Ventricular Hypertrophy

In conscious dogs with severe left ventricular (LV) hypertrophy (H) (doubling of LV/body weight), which developed gradually over 1 to 2 years after aortic banding, baseline LV function was well compensated. The LV was able to generate twice the LV systolic pressure without an increase in LV end-diastolic pressure, or decrease in LV dP/dt or LV wall thickening. However, LV myocytes isolated from LVH dogs exhibited impaired contraction at baseline and in response to Ca2+. There was no change in L-type Ca2+ channel current (ICa) density but the ability of ICa to trigger Ca2+ release from the sarcoplasmic reticulum (SR) was reduced. Immunoblot analysis revealed a 68% decrease in SERCA2a, and a 35% decrease in the number of ryanodine receptors (RyR2), with no changes in protein level of calsequestrin, Na+/Ca2+ exchanger or phospholamban (PLB), but with both RyR2 and PLB hyperphosphorylated. Spontaneous Ca2+ sparks in LVH cells were found to have prolonged duration but similar intensities despite the reduced SR Ca2+ load. A higher Ca2+ spark rate was observed in LVH cells, but this is inconsistent with the reduced SR Ca2+ content. However, Ca2+ waves were found to be less frequent, slower and were more likely to be aborted in Ca2+-challenged LVH cells. These paradoxical observations could be accounted for by a nonuniform SR Ca2+ distribution, RyR2 hyperphosphorylation in the presence of decreased global SR Ca2+ load. We conclude that severe LVH with compensation masks cellular and subcellular Ca2+ defects that remain likely contributors to the limited contractile reserve of LVH.

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.

Downregulation of caveolin by chronic β-adrenergic receptor stimulation in mice

Caveolae, flask-shaped invaginations of cell membranes, are believed to play pivotal roles in transmembrane transportation of molecules and cellular signaling. Caveolin, a structural component of caveolae, interacts directly with G proteins and regulates their function. We investigated the effect of chronic β-adrenergic receptor stimulation on the expression of caveolin subtypes in mouse hearts by immunoblotting and Northern blotting. Caveolin-1 and -3 were abundantly expressed in the heart and skeletal muscles, but not in the brain. Continuous (-)-isoproterenol, but not (+)-isoproterenol, infusion via osmotic minipump (30 μg ⋅ g-1 ⋅ day-1) for 13 days significantly downregulated both caveolin subtypes in the heart. The expression of caveolin-1 was reduced by 48 ± 6.1% and that of caveolin-3 by 28 ± 4.0% ( P < 0.01, n = 8 for each). The subcellular distribution of caveolin subtypes in ventricular myocardium was not altered as determined by sucrose gradient fractionation. In contrast, the expression of both caveolin subtypes in skeletal muscles was not significantly changed. Our data suggest that the expression of caveolin subtypes is regulated by β-adrenergic receptor stimulation in the heart.