Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat

The transgenic (mRen2)27 (Ren2) rat overexpresses mouse renin in extrarenal tissues, causing increased local synthesis of ANG II, oxidative stress, and hypertension. However, little is known about the role of oxidative stress induced by the tissue renin-angiotensin system (RAS) as a contributing factor in pulmonary hypertension (PH). Using male Ren2 rats, we test the hypothesis that lung tissue RAS overexpression and resultant oxidative stress contribute to PH and pulmonary vascular remodeling. Mean arterial pressure (MAP), right ventricular systolic pressure (RVSP), and wall thickness of small pulmonary arteries (PA), as well as intrapulmonary NADPH oxidase activity and subunit protein expression and reactive oxygen species (ROS), were compared in age-matched Ren2 and Sprague-Dawley (SD) rats pretreated with the SOD/catalase mimetic tempol for 21 days. In placebo-treated Ren2 rats, MAP and RVSP, as well as intrapulmonary NADPH oxidase activity and subunits (Nox2, p22phox, and Rac-1) and ROS, were elevated compared with placebo-treated SD rats (P < 0.05). Tempol decreased RVSP (P < 0.05), but not MAP, in Ren2 rats. Tempol also reduced intrapulmonary NADPH oxidase activity, Nox2, p22phox, and Rac-1 protein expression, and ROS in Ren2 rats (P < 0.05). Compared with SD rats, the cross-sectional surface area of small PA was 38% greater (P < 0.001) and luminal surface area was 54% less (P < 0.001) in Ren2 rats. Wall surface area was reduced and luminal area was increased in tempol-treated SD and Ren2 rats compared with untreated controls (P < 0.05). Collectively, the results of this investigation support a seminal role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling.     

Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat

Angiotensin II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type 1 receptor (AT(1)R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT(1)R blockade (AT(1)B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6-7 wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac1, and p22(phox), heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT(1)B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT(1)B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT(1)R-mediated increases in NADPH oxidase activity.     

Nebivolol improves diastolic dysfunction and myocardial remodeling through reductions in oxidative stress in the transgenic (mRen2) rat

Angiotensin II contributes to myocardial tissue remodeling and interstitial fibrosis through NADPH oxidase-mediated generation of oxidative stress in the progression of heart failure. Recent data have suggested that nebivolol, a third-generation β-blocker, improves diastolic dysfunction by targeting nitric oxide (NO) and metabolic pathways that decrease interstitial fibrosis. We sought to determine if targeting NO would improve diastolic function in a model of tissue renin-angiotensin system overactivation. We used the transgenic (TG) (mRen2)27 rat, which overexpresses the murine renin transgene and manifests insulin resistance and left ventricular dysfunction. We treated 6- to 7-wk-old TG (mRen2)27 rats and age-matched Sprague-Dawley control rats with nebivolol (10 mg·kg(-1)·day(-1)) or placebo via osmotic minipumps for a period of 21 days. Compared with Sprague-Dawley control rats, TG (mRen2)27 rats displayed a prolonged diastolic relaxation time and reduced initial filling rate associated with increased interstitial fibrosis and left ventricular hypertrophy. These findings were temporally related to increased NADPH oxidase activity and subunits p47(phox) and Rac1 and increased total ROS and peroxynitrite formation in parallel with reductions in the antioxidant heme oxygenase as well as the phosphorylation/activation of endothelial NO synthase and PKB/Akt. Treatment with nebivolol restored diastolic function and interstitial fibrosis through increases in the phosphorylation of 5'-AMP-activated protein kinase, Akt, and endothelial NO synthase and reductions in oxidant stress. These results support that targeting NO with nebivolol treatment improves diastolic dysfunction through reducing myocardial oxidative stress by enhancing 5'-AMP-activated protein kinase and Akt activation of NO biosynthesis.     

Estrogen, nitric oxide, and hypertension differentially modulate agonist-induced contractile responses in female transgenic (mRen2)27 hypertensive rats

Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17beta-estradiol (E(2)) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg E(2) or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. E(2) treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with N(omega)-nitro-L-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in E(2)-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen.     

Sex differences in baroreflex sensitivity, heart rate variability, and end organ damage in the TGR(mRen2)27 rat

The aim of this investigation was to evaluate sex differences in baroreflex and heart rate variability (HRV) dysfunction and indexes of end-organ damage in the TG(mRen2)27 (Ren2) rat, a model of renin overexpression and tissue renin-angiotensin-aldosterone system overactivation. Blood pressure (via telemetric monitoring), blood pressure variability [BPV; SD of systolic blood pressure (SBP)], spontaneous baroreflex sensitivity, HRV [HRV Triangular Index (HRV-TI), standard deviation of the average NN interval (SDNN), low and high frequency power (LF and HF, respectively), and Poincaré plot analysis (SD1, SD2)], and cardiovascular function (pressure-volume loop analysis and proteinuria) were evaluated in male and female 10-wk-old Ren2 and Sprague Dawley rats. The severity of hypertension was greater in Ren2 males (R2-M) than in Ren2 females (R2-F). Increased BPV, suppression of baroreflex gain, decreased HRV, and associated end-organ damage manifested as cardiac dysfunction, myocardial remodeling, elevated proteinuria, and tissue oxidative stress were more pronounced in R2-M compared with R2-F. During the dark cycle, HRV-TI and SDNN were negatively correlated with SBP within R2-M and positively correlated within R2-F; within R2-M, these indexes were also negatively correlated with end-organ damage [left ventricular hypertrophy (LVH)]. Furthermore, within R2-M only, LVH was strongly correlated with indexes of HRV representing predominantly vagal (HF, SD1), but not sympathetic (LF, SD2), variability. These data demonstrated relative protection in females from autonomic dysfunction and end-organ damage associated with elevated blood pressure in the Ren2 model of hypertension.     

Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6-8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P < 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P < 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue.     

Estrogen and salt sensitivity in the female mRen(2). Lewis rat

The present study determined whether early loss of estrogen influences salt-sensitive changes in blood pressure, renal injury, and cardiac hypertrophy as well as the effects on the circulating renin-angiotensin-aldosterone system (RAAS) in the hypertensive female mRen(2). Lewis strain. Ovariectomy (OVX) of heterozygous mRen(2). Lewis rats on a normal salt (NS) diet (0.5% sodium) increased systolic blood pressure from 137+/-3 to 177+/-5 mmHg (P<0.01) by 15 wk but did not show any changes in cardiac-to-body weight index (CI), proteinuria, or creatinine clearance. Maintenance with a high-sodium (HS) diet (4%) increased blood pressure (203+/-4 mmHg, P<0.01), proteinuria (3.5+/-0.3 vs. 6.4+/-0.7 mg/day, P<0.05), and CI (4.0+/-0.1 vs. 5.2+/-0.1 mg/kg, P<0.01) but decreased creatinine clearance (0.89+/-0.15 vs. 0.54+/-0.06 ml/min, P<0.05). OVX exacerbated the effects of salt on the degree of hypertension (230+/-5 mmHg), CI (5.6+/-0.2 mg/kg), and proteinuria (13+/-3.0 mg/day). OVX increased the urinary excretion of aldosterone approximately twofold in animals on the NS diet (3.8+/-0.5 vs. 6.6+/-0.5 ng.mg creatinine-1.day-1, P<0.05) and HS diet (1.4+/-0.2 vs. 4.5+/-1.0 ng.mg creatinine-1.day-1, P<0.05). Circulating renin, angiotensin-converting enzyme, and angiotensin II were also significantly increased in the OVX group fed a HS diet. These results reveal that the protective effects of estrogen apart from the increase in blood pressure were only manifested in the setting of a chronic HS diet and suggest that the underlying sodium status may have an important influence on the overall effect of reduced estrogen.     

Vasopressin V1a, V1b and V2 receptors mRNA in the kidney and heart of the renin transgenic TGR(mRen2)27 and Sprague Dawley rats.

Vasopressin plays significant role in regulation of blood pressure by means of V1 and V2 receptors, however regulation of synthesis of these receptors in hypertension is only poorly recognized. The purpose of the present study was to compare expression of V1a, V1b and V2 vasopressin (R) mRNA in the renal cortex, renal medulla and the heart of hypertensive renin transgenic TGR(mRen2)27 rats (TGR) and of their parent normotensive Sprague Dawley (SD) strain. The study was performed on 12 weeks old TGR and SD rats. Competitive PCR method was used for quantitative analysis of V1a, V1b and V2 receptors mRNA in fragments of renal cortex, renal medulla and apex of the left ventricle of the heart. In both strains expression of V1aR and V2R mRNA was significantly greater in the renal medulla than in the renal cortex. In the renal medulla but not in the cortex expression of V1aR mRNA was significantly greater in TGR than in SD rats. V2R mRNA expression was similar in the renal cortex and renal medulla of both strains. V1aR mRNA was well expressed in the heart of SD and TGR rats, however there was no significant difference between these two strains. V2R mRNA was not present in the heart. V1bR mRNa could not be detected either in the kidney or in the heart. The results provide evidence for specific increase of expression of V1a receptors mRNA in the renal medulla of TGR rats.     

Increased expression of IL-6 and LIF in the hypertrophied left ventricle of TGR(mRen2)27 and SHR rats

Cytokines from the interleukin-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin II-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)27 (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)27 and SHR. (Mol Cell Biochem 269: 95–101, 2005)     

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague-Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats

The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague-Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17beta-estradiol (E2). We evaluated estrogen's influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1-7) [Ang-(1-7)] and on aminopeptidase (AMP), which degrades Ang-(1-7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31+/-0.03 nmol/min/mg versus 0.18+/-0.04 nmol/min/mg of protein, p<0.05) and Tg(+) (0.26+/-0.04 nmol/min/mg versus 0.13+/-0.02 nmol/min/mg of protein, p<0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p<0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1-7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.     

Circadian pacemaker function and entrainment during maturation of transgenic hypertensive TGR(mREN2)27 and Sprague-Dawley rats

TGR(mREN2)27 (TGR) transgenic rats develop hypertension due to the mouse mRen-2 gene inserted in their genome. At 5 weeks of age, the blood pressure of TGR rats starts rising, until a maximum is reached at 10 weeks of age. Adult TGR rats show peak values of blood pressure (BP) during the light phase, while heart rate (HR) and motor activity (MA) peak at night. In the present experiment, we evaluated the evolution of circadian rhythms in motor activity, heart rate, and blood pressure of TGR and Sprague-Dawley (SD) rats under 12h light-dark cycles (LD 12:12). Results confirmed that the blood pressure of TGR rats starts to increase at 5 weeks of age, reaching a plateau by the 11th week. Parallel to the increase in blood pressure levels, there was a decrease in the period length of the blood pressure rhythm, a delay in the onset of the alpha phase of the blood pressure rhythm with respect to that of motor activity and heart rate, and a decrease in heart rate levels. In all of the variables studied, the alpha phase of SD rats always started before darkness, whereas that of TGR rats started after lights off. In general, heart rate and motor activity levels of TGR rats were higher than those of SD rats. The amplitude of the circadian rhythms studied was greater in TGR rats than in SD rats. The present results suggest that the different evolution of circadian rhythms in TGR and SD rats might be due to differences in the functioning of the entrainment pathway or the circadian clock itself, which can be detected in young rats and that are probably caused by the expression of the mouse transgene. (Chronobiology International, 18(4), 627-640, 2001)     

Regulation of angiotensin II receptors in the prostate of the transgenic (mRen-2)27 rat: effect of angiotensin-converting enzyme inhibition

We examined the regulation/expression of angiotensin II (Ang II) receptors in the transgenic (TG) (mRen-2)27 rat compared to the normal Sprague-Dawley (SD) rat. Ang II receptor binding and mRNA expression were determined by quantitative autoradiography and real-time PCR, respectively. Ang II receptors in the rat prostate rat were of the AT(1) receptor subtype and were significantly reduced in the prostate of the TG rat compared to the normal SD rat. However, AT(1) receptor binding was significantly higher in the prostate of the TG rat treated with the ACE inhibitor lisinopril compared to the untreated TG rat and comparable to the control SD rat. In contrast to the protein, AT(1) receptor mRNA expression was not reduced in the prostate of the TG rat compared to the SD rat. However, AT(1) receptor mRNA was markedly reduced in the prostate of the lisinopril-treated TG rat compared to the untreated TG rat or control SD rat. In conclusion, the findings suggest that AT(1) receptors are present in the rat prostate at a protein level and are subject to down-regulation in the TG rat which may be due to receptor internalisation as a consequence of receptor hyper-stimulation by increased local tissue levels of Ang II. Moreover, AT(1) receptor protein and mRNA expression in the prostate may be inversely modulated.     

Generation and characterization of a Tet-On (rtTA-M2) transgenic rat

Background  

The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter.     

alpha-Tropomyosin mutations Asp(175)Asn and Glu(180)Gly affect cardiac function in transgenic rats in different ways

To study the mechanisms by which missense mutations in alpha-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing alpha-tropomyosin with one of two disease-causing mutations, Asp(175)Asn or Glu(180)Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-alpha-tropomyosin antibody, ARG1. In transgenic rats with either alpha-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca(2+) sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp(175)Asn, but not Glu(180)Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca(2+) waves were detected only in cardiomyocytes from animals with mutation Asp(175)Asn, suggesting an increase in intracellular Ca(2+) concentration compensating for the reduced Ca(2+) sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp(175)Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by alpha-tropomyosin mutation Asp(175)Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca(2+) handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.     

Ovariectomy is protective against renal injury in the high-salt-fed older mRen2. Lewis rat

Studies in experimental animals and younger women suggest a protective role for estrogen; however, clinical trials may not substantiate this effect in older females. Therefore, the present study assessed the outcome of ovariectomy in older mRen2. Lewis rats subjected to a high-salt diet for 4 wk. Intact or ovariectomized (OVX, 15 wk of age) mRen2. Lewis rats were aged to 60 wk and then placed on a high-salt (HS, 8% sodium chloride) diet for 4 wk. Systolic blood pressures were similar between groups [OVX 169 +/- 6 vs. Intact 182 +/- 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 +/- 0.2 vs. Intact 11.5 +/- 2.6 mg/mg creatinine; P < 0.002, n = 6], renal interstitial fibrosis, glomerular sclerosis, and tubular casts were lower in OVX vs. Intact rats. Kidney injury molecule-1 mRNA, a marker of tubular damage, was 53% lower in the OVX HS group. Independent from blood pressure, OVX HS rats exhibited significantly lower cardiac (24%) and renal (32%) hypertrophy as well as lower C-reactive protein (28%). Circulating insulin-like growth factor-I (IGF-I) levels were not different between the Intact and OVX groups; however, renal cortical IGF-I mRNA and protein were attenuated in OVX rats [P < 0.05, n = 6]. We conclude that ovariectomy in the older female mRen2. Lewis rat conveys protection against salt-dependent increase in renal injury.     

H2S generated by heart in rat and its effects on cardiac function

Hydrogen sulfide (H2S), which was considered as a novel gasotransmitter, is produced endogenously from L-cysteine in mammalian brain and vessels, and might be a physiological function regulator to these organs. Here, we showed that mRNA for H2S producing enzyme, cystathionine gamma-lyase, was expressed in myocardial tissues and H2S could endogenously be produced in myocardial tissues. Negative inotropic effect of H2S was proved in present study in vitro and in vivo experiments, and the effect could partly be blocked by glibenclamide, a KATP channel blocker. An intravenous bolus injection of NaHS provoked a decrease in central venous pressure. The present findings suggested that H2S could be endogenously produced by heart tissues, as a physiological cardiac function regulator, mediated by KATP channel pathway.     

Metoprolol improves cardiac function and modulates cardiac metabolism in the streptozotocin-diabetic rat

The effects of diabetes on heart function may be initiated or compounded by the exaggerated reliance of the diabetic heart on fatty acids and ketones as metabolic fuels. beta-Blocking agents such as metoprolol have been proposed to inhibit fatty acid oxidation. We hypothesized that metoprolol would improve cardiac function by inhibiting fatty acid oxidation and promoting a compensatory increase in glucose utilization. We measured ex vivo cardiac function and substrate utilization after chronic metoprolol treatment and acute metoprolol perfusion. Chronic metoprolol treatment attenuated the development of cardiac dysfunction in streptozotocin (STZ)-diabetic rats. After chronic treatment with metoprolol, palmitate oxidation was increased in control hearts but decreased in diabetic hearts without affecting myocardial energetics. Acute treatment with metoprolol during heart perfusions led to reduced rates of palmitate oxidation, stimulation of glucose oxidation, and increased tissue ATP levels. Metoprolol lowered malonyl-CoA levels in control hearts only, but no changes in acetyl-CoA carboxylase phosphorylation or AMP-activated protein kinase activity were observed. Both acute metoprolol perfusion and chronic in vivo metoprolol treatment led to decreased maximum activity and decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA. Metoprolol also increased sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and prevented the reexpression of atrial natriuretic peptide in diabetic hearts. These data demonstrate that metoprolol ameliorates diabetic cardiomyopathy and inhibits fatty acid oxidation in streptozotocin-induced diabetes. Since malonyl-CoA levels are not increased, the reduction in total carnitine palmitoyltransferase I activity is the most likely factor to explain the decrease in fatty acid oxidation. The metabolism changes occur in parallel with changes in gene expression.     

Defective insulin signaling in skeletal muscle of the hypertensive TG(mREN2)27 rat

Essential hypertension is frequently associated with insulin resistance of skeletal muscle glucose transport, with a potential role of angiotensin II in the pathogenesis of both conditions. The male heterozygous TG(mREN2)27 rat harbors the mouse transgene for renin, exhibits local elevations in angiotensin II, and is an excellent model of both hypertension and insulin resistance. The present study was designed to investigate the potential cellular mechanisms for insulin resistance in this hypertensive animal model, including an assessment of elements of the insulin-signaling pathway. Compared with nontransgenic, normotensive Sprague-Dawley control rats, male heterozygous TG(mREN2)27 rats displayed elevated (P < 0.05) fasting plasma insulin (74%), an exaggerated insulin response (108%) during an oral glucose tolerance test, and reduced whole body insulin sensitivity. TG(mREN2)27 rats also exhibited decreased insulin-mediated glucose transport and glycogen synthase activation in both the type IIb epitrochlearis (30 and 46%) and type I soleus (22 and 64%) muscles. Importantly, there were significant reductions (approximately 30-50%) in insulin stimulation of tyrosine phosphorylation of the insulin receptor beta-subunit and insulin receptor substrate-1 (IRS-1), IRS-1 associated with the p85 subunit of phosphatidylinositol 3-kinase, Akt Ser473 phosphorylation, and Ser9 phosphorylation of glycogen synthase kinase-3beta in epitrochlearis and soleus muscles of TG(mREN2)27 rats. Soleus muscle triglyceride concentration was 25% greater in the transgenic group compared with nontransgenic animals. Collectively, these data provide the first evidence that the insulin resistance of the hypertensive male heterozygous TG(mREN2)27 rat can be attributed to specific defects in the insulin-signaling pathway in skeletal muscle.     

Reduction of cardiac hypertrophy in TGR(mREN2)27 by angiotensin II receptor blockade

TGR(mREN2)27 is a transgenic rat harboring the murine Ren-2 gene and exhibit fulminant hypertension and marked heart hypertrophy. In order to study the role of angiotensin II in the increase of cardiac mass, these animals were treated with anti-hypertensive and non-antihypertensive doses of the angiotensin II receptor AT₁ antagonist Telmisartan for 9 weeks. All doses led to significant reductions of heart hypertrophy detected by the evaluation of the diameter of cardiac muscle bundles. We conclude from this study that cardiac hypertrophy in TGR(mREN2)27 is characterized by an increased volume of cardiomyocytes and an unchanged amount of fibrous tissue and that angiotensin II plays an important role in the mechanisms leading to this phenotype.