Peter M. Buttrick

Hypoxia-associated induction of early growth response-1 gene expression.

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298–8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 ≈ 12 torr) displayed increasedEgr-1 expression because of de novobiosynthesis, with a ≈10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to −424/−65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning −424/−375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation ofEgr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-βII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediatedEgr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1β and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.

Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Localization of types I, III and IV collagen mRNAs in rat heart cells by in situ hybridization

Previous studies investigating the cellular origins of several collagens in young adult rat hearts (Eghbali et al., 1988) demonstrated that the mRNAs for types I and III collagen occurred in non-myocyte cells, mostly fibroblasts, whereas the mRNA for type IV collagen was observed in both myocytes and non-myocyte cells. In the present study, cellular localization of collagen mRNAs has been achieved by in situ hybridization in rat heart tissue and in isolated heart cells. Frozen tissue sections, isolated cardiomyocytes, cultured neonatal cardiomyocytes and fibroblasts were hybridized with DNA probes for type-specific collagens, actin, and myosin heavy chain. Silver grains were visualized by dark field imaging. In heart sections, types I and III mRNAs were observed predominantly adjacent to myocytes and in the interstitium, where fibroblasts are known to be present. In contrast, type IV collagen mRNA was identified both within the myocytes and the interstitium. In freshly isolated adult cardiomyocytes and in cultured neonatal cardiomyocytes, collagen type IV mRNA was observed but type I collagen mRNA was not. In cultured neonatal fibroblasts, both types IV and I collagen mRNAs were abundant.

β-Adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy

To establish whether catecholamines per se in the absence of significant increases in systolic load induce myocardial damage via apoptosis, rats were treated with vehicle or isoproterenol (400 μg ⋅ kg-1 ⋅ h-1). Apoptotic cardiocytes (Apo) were identified in paraffin-embedded sections using terminal deoxynucleotide transferase-mediated dUTP nick end labeling. Results were confirmed using an independent ligase assay. Systolic blood pressures were comparable in isoproterenol-treated and control rats. Twenty-four hours of treatment with isoproterenol resulted in significant numbers of Apo compared with control [7.9 ± 2.5 vs. 0.3 ± 0.3 (SE) cm-2, P < 0.05]. A cohort of animals was subjected to ventricular pacing to induce a tachycardia equivalent to that induced by isoproterenol, and these animals did not show an increase in Apo. The left ventricular hypertrophy induced by 2 wk of abdominal aortic banding also increased Apo (∼7.2-fold); however, 24 h of isoproterenol infusion did not induce additional Apo in these rats. Thus catecholamines, in the absence of altered systolic load, induce Apo which is not mediated solely by tachycardia. Left ventricular hypertrophy secondary to abdominal aortic banding is associated with Apo, but this does not increase sensitivity to isoproterenol-induced Apo.

Adora2b-elicited Per2 stabilization promotes a HIF-dependent metabolic switch crucial for myocardial adaptation to ischemia

Adenosine signaling has been implicated in cardiac adaptation to limited oxygen availability. In a wide search for adenosine receptor A2b (Adora2b)-elicited cardioadaptive responses, we identified the circadian rhythm protein period 2 (Per2) as an Adora2b target. Adora2b signaling led to Per2 stabilization during myocardial ischemia, and in this setting, Per2−/− mice had larger infarct sizes compared to wild-type mice and loss of the cardioprotection conferred by ischemic preconditioning. Metabolic studies uncovered a limited ability of ischemic hearts in Per2−/− mice to use carbohydrates for oxygen-efficient glycolysis. This impairment was caused by a failure to stabilize hypoxia-inducible factor-1α (Hif-1α). Moreover, stabilization of Per2 in the heart by exposing mice to intense light resulted in the transcriptional induction of glycolytic enzymes and Per2-dependent cardioprotection from ischemia. Together, these studies identify adenosine-elicited stabilization of Per2 in the control of HIF-dependent cardiac metabolism and ischemia tolerance and implicate Per2 stabilization as a potential new strategy for treating myocardial ischemia.

Behavior of genes directly injected into the rat heart in vivo.

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental Diabetes Is Associated With Functional Activation of Protein Kinase Cε and Phosphorylation of Troponin I in the Heart, Which Are Prevented by Angiotensin II Receptor Blockade

A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)-mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC epsilon isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P < .05). PKC delta, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P < .01). The change in PKC epsilon distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor-mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

Recruitment of Compensatory Pathways to Sustain Oxidative Flux With Reduced Carnitine Palmitoyltransferase I Activity Characterizes Inefficiency in Energy Metabolism in Hypertrophied Hearts

Background— Transport rates of long-chain free fatty acids into mitochondria via carnitine palmitoyltransferase I relative to overall oxidative rates in hypertrophied hearts remain poorly understood. Furthermore, the extent of glucose oxidation, despite increased glycolysis in hypertrophy, remains controversial. The present study explores potential compensatory mechanisms to sustain tricarboxylic acid cycle flux that resolve the apparent discrepancy of reduced fatty acid oxidation without increased glucose oxidation through pyruvate dehydrogenase complex in the energy-poor, hypertrophied heart. Methods and Results— We studied flux through the oxidative metabolism of intact adult rat hearts subjected to 10 weeks of pressure overload (hypertrophied; n=9) or sham operation (sham; n=8) using dynamic 13C–nuclear magnetic resonance. Isolated hearts were perfused with [2,4,6,8,10,12,14,16-13C8] palmitate (0.4 mmol/L) plus glucose (5 mmol/L) in a 14.1-T nuclear magnetic resonance magnet. At similar tricarboxylic acid cycle rates, flux through carnitine palmitoyltransferase I was 23% lower in hypertrophied (P<0.04) compared with sham hearts and corresponded to a shift toward increased expression of the L–carnitine palmitoyltransferase I isoform. Glucose oxidation via pyruvate dehydrogenase complex did not compensate for reduced palmitate oxidation rates. However, hypertrophied rats displayed an 83% increase in anaplerotic flux into the tricarboxylic acid cycle (P<0.03) that was supported by glycolytic pyruvate, coincident with increased mRNA transcript levels for malic enzyme. Conclusions— In cardiac hypertrophy, fatty acid oxidation rates are reduced, whereas compensatory increases in anaplerosis maintain tricarboxylic acid cycle flux and account for a greater portion of glucose oxidation than previously recognized. The shift away from acetyl coenzyme A production toward carbon influx via anaplerosis bypasses energy, yielding reactions contributing to a less energy-efficient heart.

Acute myocardial infarction and chest pain syndromes after cocaine use

Seventy patients hospitalized with chest pain after cocaine use were retrospectively evaluated to define the risk and clinical course of acute myocardial infarction (AMI). AMI developed in 22 patients (31%) and transient myocardial ischemia was seen in an additional 9 patients (13%). Coronary risk factors did not distinguish those who developed AMI from those who did not. The presenting electrocardiogram was abnormal in 20 of 22 patients who evolved AMI and in 19 of 48 of those who did not. Creatine kinase levels were elevated in 75% of the patients, including 65% of those who did not develop AMI, but creatine kinase-MB elevations were only observed in the AMI group. The route of cocaine administration did not predict AMI and there was no predilection for a particular coronary vascular bed. The length of time between drug use and onset of AMI pain was often quite prolonged (median interval, 18 vs 1 hour in the non-AMI group). Eight of the patients with AMI underwent cardiac catheterization and 4 had significant coronary narrowing.

Tetracycline-regulated cardiac gene expression in vivo.

Tight regulation of foreign genes expressed in vivo would facilitate studies of many biologic processes and would be useful for gene transfer-based therapies. To test the ability of a tetracycline-regulated gene expression system to function in vivo, we directly injected chimeric tet repressor-VP16 transactivator expression plasmids and luciferase target genes into the hearts of adult rats. Cardiac luciferase activity increased over two orders of magnitude in response to small changes in input tetracycline-controlled transactivator DNA. Transactivation was repressed to background levels by subtherapeutic concentrations of tetracycline in a dose-dependent manner. Target gene expression could be rapidly and reversibly controlled by manipulating antibiotic administration. This system may be particularly useful for in vivo studies of gene function or gene therapies where the timing or extent of expression are critical variables.