Marc Heidbreder

Hypoxia rapidly activates HIF-3α mRNA expression

The role of the hypoxia‐inducible factor (HIF) subunits 1α and 1β in cellular response to hypoxia is well established, whereas little is known about HIF‐2α and HIF‐3α with respect to organ distribution and transcriptional regulation by hypoxia. We investigated mRNA levels of all HIF subunits and of their target genes erythropoietin (EPO) and glucose‐transporter 1 (GLUT1) in rats undergoing systemic hypoxia for 30 or 120 min by quantitative real‐time RT‐PCR. In normoxia, persistently high mRNA levels of all HIF subunits were detected in cerebral cortex, hippocampus, and lung; the heart contained the lowest amounts. Hypoxia did not affect mRNA levels of HIF‐1α, ‐1β, and ‐2α. HIF‐3α mRNA levels increased in all organs examined after 2 h of hypoxia. A significant rise of EPO and GLUT1 mRNA levels occurred in cortex, heart, liver, and kidney after 2 h of hypoxia, indicating activation of the HIF system. Protein levels of all HIF subunits, determined in brain and lung by immunoblotting, showed a marked increase corresponding to the duration of hypoxia. Our results suggest that induction at the transcriptional level is a unique feature of HIF‐3α, which therefore may represent a rapidly reacting component of the HIF system in protection against hypoxic damage.

Remote preconditioning protects the heart by activating myocardial PKCϵ-isoform

Objective: Myocardial protection can be achieved by brief ischemia-reperfusion of remote organs, a phenomenon described as remote preconditioning (RPC). Since the intracellular mechanisms of RPC are not known, we tested the hypothesis that RPC might activate myocardial PKCϵ, an essential mediator of classical ischemic preconditioning. Furthermore, we tried to delineate the mechanisms by which RPC is transduced to the heart with respect to the possible contribution of kinins and neuronal reflexes. Methods: Anesthetized rats were randomised to undergo either 30 min of waiting (controls) or RPC (brief mesenteric artery occlusion followed by reperfusion) in the absence or presence of chelerythrine (5 mg kg−1), a specific PKC inhibitor. Myocardial infarct size was measured by TTC staining after 30 min of coronary artery occlusion followed by 150 min of reperfusion. In separate sets of experiments RPC was performed with or without pretreatment with HOE140, a selective B2-antagonist or hexamethonium was used to explore the influence of ganglion blockade on RPC. Translocation of PKCϵ from cytosol to the particulate fraction was measured by quantitative immunoblotting. Results: RPC significantly reduced infarct size which was completely blocked by the PKC inhibitor. RPC shifted the ratio between cytosolic and particulate PKCϵ, an indicator for PKC-activation, from 0.95±0.06 in controls to 0.41±0.09 ( P <0.05), and this effect was abolished by HOE140. Activation of PKCϵ could not be achieved after pretreatment with HEX (0.69±0.06 in HEX vs. 0.78±0.06 in HEX+RPC). Conclusions: RPC activates myocardial PKCϵ through a neuronal and bradykinin-dependent pathway. We assume that activation of PKCϵ is an important step in cardioprotection induced by remote preconditioning.

Acute reduction of myocardial infarct size by a hydroxymethyl glutaryl coenzyme A reductase inhibitor is mediated by endothelial nitric oxide synthase.

In addition to their lipid-lowering properties, statins improve endothelial function by increasing the activity of endothelial nitric oxide synthase (eNOS). It was hypothesized that, by this mechanism, statins protect the myocardium from ischemia/reperfusion injury in normocholesterolemic animals. Rats were pretreated for 1 week with either cerivastatin (0.3 mg/kg/d) or placebo. Anesthetized animals underwent 30 minutes of coronary artery occlusion (CAO) followed by 180 minutes of reperfusion. In a separate set of experiments, the NOS inhibitor l-NAME (15 mg/kg; Nω-nitro-l-arginine methyl ester) was administered 15 minutes before CAO. Cerivastatin decreased infarct size by 49% (P < 0.05) without reducing plasma cholesterol levels. Cerivastatin increased myocardial eNOS mRNA and NOS activity and by 52% and 58% (P < 0.05), respectively. Cardioprotection and upregulation of eNOS activity evoked by cerivastatin were not observed in rats cotreated with l-NAME. These results show that statins reduce the extent of myocardial necrosis in normocholesterolemic rats after acute ischemia/reperfusion injury by increasing myocardial eNOS activity. Therefore, statins may protect the heart not only by reducing the incidence of ischemic events, but also by limiting cell damage during acute myocardial infarction.

Calcitonin gene related peptide mediates cardioprotection by remote preconditioning.

Excitation of sensory nerves and activation of myocardial protein kinase C (PKC) epsilon contribute to the transduction of remote preconditioning (RPC) to the heart. Since calcitonin gene related peptide (CGRP) is an important mediator of sensory neurons we tried to delineate whether CGRP a) protects the heart from ischemic injury, b) is involved in cardioprotection after RPC, and c) leads to an activation of myocardial PKCepsilon. RPC was achieved by brief mesenteric artery occlusion followed by reperfusion. Myocardial infarct size (IS) was measured by TTC staining after temporary coronary artery occlusion (CAO) in rats. CGRP plasma levels were determined by radioimmunoassay and PKCepsilon was measured by quantitative immunoblotting. CGRP infusion reduced infarct size by 57%, an action that was abolished after co-treatment with the PKC inhibitor chelerythrine. RPC significantly increased CGRP plasma levels, reduced infarct size, and activated myocardial PKCepsilon. Infarct size reduction was abolished and PKCepsilon activation was significantly attenuated by CGRP(8-37), a specific CGRP receptor antagonist. Ganglion blockade with hexamethonium did not influence CGRP release by RPC but abolished CGRP mediated myocardial PKCepsilon activation. In conclusion, CGRP protects the heart from ischemic injury and is involved in RPC, presumably by activating myocardial PKCepsilon.

Overfeeding-Induced Obesity in Spontaneously Hypertensive Rats: An Animal Model of the Human Metabolic Syndrome

Background/Aims: The metabolic syndrome (MS) has become an epidemiological problem in Western countries. We developed a diet-induced obese rat model that mimics all the symptoms of MS in humans, but whose insulin resistance, hyperphagia and hyperleptinemia are caused by nutrition rather than genetic modifications. Methods: Spontaneously hypertensive rats (SHR) were allowed for 12 weeks to choose between a cafeteria diet (CD, 20.3 kJ/g) and standard rat chow (11.7 kJ/g). Controls received rat chow. Results: Body weight (BW) exceeded control levels when SHR were fed with CD. The increase in BW was attributed to enhanced energy intake. The abundance of abdominal fat as well as the plasma levels of leptin and triglycerides increased concomitant with glucose, insulin and C-peptide. This prediabetic condition was further confirmed by a markedly increased insulin response following glucose challenge and by impaired glucose utilization after insulin tolerance tests. Conclusion: Increases in food intake and BW despite hyperleptinemia indicate leptin resistance following CD feeding. CD-fed SHR feature leptin and insulin resistance, hypertension and obesity, thus mimicking the situation of MS patients. As such, our model is more suitable than the genetically modified rat models used to study human MS.

Suppression of cardiac phosphatidate phosphohydrolase 1 activity and lipin mRNA expression in Zucker diabetic fatty rats and humans with type 2 diabetes mellitus.

Lipin functions in mammalian phospholipid biosynthesis through its phosphatidate phosphohydrolase 1 (PAP(1)) activity. Here, we studied cardiac PAP(1) activity and lipin expression ex vivo in 8-month-old Zucker diabetic fatty (ZDF) rats and humans with type 2 diabetes mellitus undergoing open heart surgery for coronary bypass grafting. Compared to non-diabetic littermates (ZDF-fa/+), left ventricular PAP(1) activity was 29% lower in diabetic ZDF-fa/fa rats. Left ventricular PAP(1) activities were 2.1-fold (ZDF-fa/fa) and 3.6-fold (ZDF-fa/+) higher than the respective atrial activities, indicating marked differences in cardiac distribution of PAP(1). PAP(1) activity was highly related with cardiac lipin-1 and lipin-3 mRNA expression in ZDF rats (r=0.99 and 0.96). Consistent with the findings in experimental animals, human atrial tissue displayed PAP(1) activity that was 33% lower in those having diabetes than in non-diabetic controls. Accordingly, atrial lipin-1 and lipin-3 mRNA expression in diabetic patients was 50% and 59% lower as in non-diabetic patients, respectively. Insulin therapy increased both PAP(1) activity and lipin mRNA expression in diabetic patients. We conclude that suppression of cardiac PAP(1) activity/lipin expression may contribute to metabolic dysfunction of the diabetic heart.

Improved Insulin Sensitivity after Long-Term Treatment with AT1 Blockers Is Not Associated with PPARγ Target Gene Regulation

In both cell culture experiments and in vivo studies, a number of angiotensin II type 1 (AT(1)) receptor antagonists activated the peroxisome proliferator-activated receptor-γ (PPARγ). This mechanism has been discussed to be, at least in part, responsible for the improvement in glucose metabolism observed in animal studies and clinical trials. To investigate whether the PPARγ-dependent mechanism may represent a valid target for chronic therapy, spontaneously hypertensive rats (SHR) were fed either with a cafeteria diet (CD) or standard chow. CD-fed SHR were simultaneously treated with either telmisartan (TEL; 8 mg/kg(body weight)· d) or candesartan (CAND; 10 mg/kg(body weight)· d) for 3 months because TEL, but not CAND, has been demonstrated to be a strong activator of PPARγ. After 3 months, chow- and CD-fed controls were hypertensive, whereas TEL and CAND treatment resulted in normalized blood pressures in SHR. Body weight and the amount of abdominal fat (determined by magnetic resonance imaging) were higher in CD- than in chow-fed SHR. After TEL or CAND, body weight, abdominal fat quantity, and adipocyte size returned to normal. In glucose tolerance tests, the glucose responses were comparable in the TEL- and CAND-treated SHR and obese controls, whereas the insulin response was almost halved by AT(1) blockade. Expression of PPARγ target genes aP2, FAT CD36, FASn, and PEPCK remained unaltered at the protein level in visceral fat after TEL and CAND compared with the CD-fed controls. Because the expression of examined PPARγ target genes was not affected, we concluded that improved insulin sensitivity after long-term treatment with AT(1) blockers was not related to a PPARγ-dependent mechanism.