Optimal reactive oxygen species concentration and p38 MAP kinase are required for coronary collateral growth

Reactive oxygen species (ROS) are implicated in coronary collateral growth (CCG). We evaluated the requirement for ROS in human coronary artery endothelial cell (HCAEC) tube formation, CCG in vivo, and signaling (p38 MAP kinase) by which ROS may stimulate vascular growth. The flavin-containing oxidase inhibitor diphenyleneiodonium (DPI) or the superoxide dismutase inhibitor diethyldithiocarbamate (DETC) blocked vascular endothelial growth factor-induced HCAEC tube formation in Matrigel. We assessed the effect of DPI and DETC on CCG in a rat model of repetitive ischemia (RI) (40 s left anterior descending coronary artery occlusion every 20 min for 2 h 20 min, 3 times/day, 10 days). DPI or DETC was given intraperitoneally, or the NAD(P)H oxidase inhibitor apocynin was given in drinking water. Collateral-dependent flow (measured by using microspheres) was expressed as a ratio of normal and ischemic zone flows. In sham-operated rats, collateral flow in the ischemic zone was 18 +/- 6% of normal zone; in the RI group, collateral flow in the ischemic zone was 83 +/- 5% of normal zone. DPI prevented the increase in collateral flow after RI (25 +/- 4% of normal zone). Similar results were obtained with apocynin following RI (32 +/- 7% of that in the normal zone). DETC achieved similar results (collateral flow after RI was 21 +/- 2% of normal zone). DPI and DETC blocked RI-induced p38 MAP kinase activation in response to vascular endothelial growth factor and RI. These results demonstrate a requirement for optimal ROS concentration in HCAEC tube formation, CCG, and p38 MAP kinase activation. p38 MAP kinase inhibition prevented HCAEC tube formation and partially blocked RI-induced CCG (42 +/- 7% of normal zone flow), indicating that p38 MAP kinase is a critical signaling mediator of CCG.     

High glucose concentrations induce oxidative damage to mitochondrial DNA in explanted vascular smooth muscle cells

Oxidative stress is considered to be one of the mechanisms leading to atherosclerosis. It occurs in response to injury or to altered metabolic state. Alterations in cell growth (proliferation or apoptosis) can also contribute to the pathogenesis of atherosclerosis and is influenced by oxidative stress. Smooth muscle cells (SMC) from aortic explants of JCR:LA-cp homozygous cp/cp corpulent rats who are genetically predisposed to develop atherosclerosis exhibit increased SMC proliferation, which can be attenuated by exercise and food restriction. This study was conducted to characterize the effects fo oxidative stress and high glucose media on cell growth and its relationship to mitochondrial DNA integrity and gene expression in explanted aortic SMC from corpulent and lean JCR:LA-cp rats. The results show that SMC from the cp/cp rat appear to be resistant to oxidant-induced cell death and that they accumulate mitochondrial DNA mutations, probably as a result of a reduction in apoptosis. These data suggest that susceptibility to age- and glucose-related atherosclerosis may be related to alterations in redox signaling.     

AT2 receptors contribute to acute blood pressure-lowering and vasodilator effects of AT1 receptor antagonism in conscious normotensive but not hypertensive rats

The aims of this study were to determine the contribution of the AT2 receptor to the antihypertensive and regional vasodilatory effects of AT1 receptor blockade in adult spontaneously hypertensive rats (SHR), 2-kidney, 1-clip hypertensive (2K1C) rats, and sham-operated normotensive rats. Several studies have provided evidence to support the notion that the AT2 receptor may have opposing effects to those mediated by the AT1 receptor. We therefore tested the hypothesis that the depressor and vasodilator effects of acute AT1 receptor blockade are dependent on AT2 receptor activation. Heart rate, mean arterial pressure, and regional hemodynamics were measured over a 4-day protocol in rats that received the following treatments in randomized order: saline vehicle, the AT1 receptor antagonist candesartan (0.1 mg/kg iv bolus), the AT2 receptor antagonist PD-123319 (50 microg.kg(-1).min(-1)), or both antagonists. Intravenous candesartan reduced mean arterial pressure in all groups of rats, and this was accompanied by renal and mesenteric vasodilation. Neither saline nor PD-123319 significantly affected these variables. Concomitant PD-123319 administration partially reversed the depressor and mesenteric vasodilator effects of candesartan in sham-operated normotensive rats but not in SHR or 2K1C rats. These data indicate that the AT2 receptor contributes to the blood pressure-lowering and mesenteric vasodilator effects of AT1 receptor blockade in the acute setting in conscious normotensive but not hypertensive rats.     

AT(1) and glutamatergic receptors in paraventricular nucleus support blood pressure during water deprivation

Water deprivation activates sympathoexcitatory neurons in the paraventricular nucleus (PVN); however, the neurotransmitters that mediate this activation are unknown. To test the hypothesis that ANG II and glutamate are involved, effects on blood pressure (BP) of bilateral PVN microinjections of ANG II type 1 receptor (AT1R) antagonists, candesartan and valsartan, or the ionotropic glutamate receptor antagonist, kynurenate, were determined in urethane-anesthetized water-deprived and water-replete male rats. Because PVN may activate sympathetic neurons via the rostral ventrolateral medulla (RVLM) and because PVN disinhibition increases sympathetic activity in part via increased drive of AT1R in the RVLM, candesartan was also bilaterally microinjected into the RVLM. Total blockade of the PVN with bilateral microinjections of muscimol, a GABA(A) agonist, decreased BP more (P < 0.05) in water-deprived (-29 +/- 8 mmHg) than in water-replete (-7 +/- 2 mmHg) rats, verifying that the PVN is required for BP maintenance during water deprivation. PVN candesartan slowly lowered BP by 7 +/- 1 mmHg (P < 0.05). In water-replete rats, however, candesartan did not alter BP (1 +/- 1 mmHg). Valsartan also produced a slowly developing decrease in arterial pressure (-6 +/- 1 mmHg; P < 0.05) in water-deprived but not in water-replete (-1 +/- 1 mmHg) rats. In water-deprived rats, PVN kynurenate rapidly decreased BP (-19 +/- 3 mmHg), and the response was greater (P < 0.05) than in water-replete rats (-4 +/- 1 mmHg). Finally, as in PVN, candesartan in RVLM slowly decreased BP in water-deprived (-8 +/- 1 mmHg; P < 0.05) but not in water-replete (-3 +/- 1 mmHg) rats. These data suggest that activation of AT(1) and glutamate receptors in PVN, as well as of AT1R in RVLM, contributes to BP maintenance during water deprivation.     

AT2 receptor and apoptosis during AT1 receptor blockade in reperfused myocardial infarction in the rat

We assessed whether upregulation of the angiotensin II (AngII) type 2 receptor (AT2R) during AngII type 1 receptor (AT1R) blockade might induce apoptosis in the in vivo rat model of reperfused myocardial infarction (RMI) and whether addition of an AT2R blocker abolishes that effect. We measured in vivo hemodynamics and left ventricular (LV) systolic and diastolic function (echocardiograms/Doppler), and ex vivo infarct size (triphenyl tetrazolium chloride), regional AT1R and AT2R proteins (immunoblots), and apoptosis (TUNEL assay and DNA ladder) after regional anterior RMI (60 min ischemia, 90 min reperfusion) in Sprague-Dawley rats randomized to intravenous AT1R blockade with candesartan (1 mg/kg, n = 9) or saline (controls, n = 14) over 30 min before RMI, and sham (n = 8). We also assessed the effect of AT2R blockade (PD123319, 10 mg/kg i.v.) plus candesartan on infarct size and apoptosis. Compared to controls, candesartan significantly (p < 0.001) limited increases in left atrial pressure, improved positive LV dP/dtmax and negative dP/dtmin, normalized LV ejection fraction, improved LV diastolic function, limited infarct expansion, decreased infarct size and apoptosis, and increased AT2R protein (not AT1R) in the reperfused ischemic zone. There were no changes in sham hearts. PD123319 abolished the candesartan-induced decrease in infarct size and LV dysfunction but not the decrease in apoptosis. Thus, during AT1R blockade in the in vivo rat model of RMI, regional AT2R upregulation contributes to the beneficial effect on infarct size and LV dysfunction but not on apoptosis, suggesting that the apoptosis is AT1R not AT2R-mediated.     

Pair feeding-mediated changes in metabolism: stress response and pathophysiology in insulin-resistant, atherosclerosis-prone JCR:LA-cp rats

Rats of the JCR:LA-cp strain, which are homozygous for the cp gene (cp/cp), are obese, insulin-resistant, and hyperinsulinemic. They exhibit associated micro- and macrovascular disease and end-stage ischemic myocardial lesions and are highly stress sensitive. We subjected male cp/cp rats to pair feeding (providing the rats each day with the amount of food eaten by matched freely fed animals), a procedure that alters the diurnal feeding pattern, leading to a state of intermittent caloric restriction. Effects on insulin, glucose, and lipid metabolism, response to restraint stress, aortic contractile/relaxant response, and myocardial lesion frequency were investigated. Pair-fed young (12-wk-old) cp/cp rats had lower insulin and glucose levels (basal and following restraint), consistent with increased insulin sensitivity, but a greater increase in plasma nonesterified fatty acids in response to restraint. These effects were unrelated to lipolytic rates in adipose tissue but may be related to reduced fatty acid oxidation in skeletal muscle. Older (24-wk-old) pair-fed cp/cp rats had significantly reduced plasma triglyceride levels, improved micro- and macrovascular function, and reduced severity of ischemic myocardial lesions. These changes indicate a significant amelioration of end-stage disease processes in this animal model and the complexity of metabolic/physiological responses in studies involving alterations in food intake. The effects illustrate the sensitivity of the JCR:LA-cp rat, an animal model for the metabolic syndrome and associated cardiovascular disease, to the environmental and experimental milieu. Similar stress-related mechanisms may play a role in metabolically induced cardiovascular disease in susceptible human beings.     

Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat.

The corpulent JCR:LA-cp rat (cp/cp) is a useful model for study of the metabolic consequences of obesity and hyperinsulinemia. To assess the effect of hyperinsulinemia on VLDL secretion in this model, we measured rates of secretion of VLDL in perfused livers derived from cp/cp rats and their lean littermates. Livers of cp/cp rats secreted significantly greater amounts of VLDL triglyceride and apolipoprotein, compared with lean littermates. The content of apoB, apoE, and apoCs in both perfusate and plasma VLDL was greater in the cp/cp rat, as was the apolipoprotein (apo)C, apoA-I, and apoA-IV content of plasma HDL. Triglyceride content was also greater in cp/cp livers, as was hepatic lipogenesis and expression of lipogenic enzymes and sterol regulatory element binding protein-1 (SREBP-1). Hepatic mRNAs for apoE, and apoA-I were higher in livers of cp/cp rats. In contrast, the steady state levels of apoC-II, apoC-III, and apoB mRNAs were unchanged. Thus, livers of obese hyperinsulinemic cp/cp JCR:LA-cp rats secrete a greater number of VLDL particles that are enriched in triglyceride, apoE, and apoC. Greater secretion of VLDL in the cp/cp rat in part results from higher endogenous fatty acid synthesis, which in turn may occur in response to increased expression of the lipogenic enzyme regulator SREBP-1c.     

Coronary arteriolar vasoconstriction to angiotensin II is augmented in prediabetic metabolic syndrome via activation of AT1 receptors

The metabolic syndrome is associated with activation of the renin-angiotensin system. However, whether the coronary vascular response to ANG II is altered under this condition is unknown. Experiments were conducted in control and chronically high-fat-fed dogs with the prediabetic metabolic syndrome both in vitro (isolated coronary arterioles, 60-110 microm) and in vivo (anesthetized and conscious). We found that plasma renin activity and ANG II levels are elevated in high-fat-fed dogs and that this increase in ANG II is associated with a significant increase in ANG II-mediated coronary vasoconstriction in isolated coronary arterioles and in anesthetized open-chest dogs. The vasoconstriction to ANG II is abolished by ANG II type 1 (AT1) receptor blockade. In conscious chronically instrumented dogs, AT1 receptor blockade with telmisartan improved the balance between coronary blood flow and myocardial oxygen consumption in the high-fat-fed dogs but not in normal control dogs, i.e., the relationship between coronary venous Po2 and myocardial oxygen consumption was shifted upward, toward normal control values. Quantitative assessment of coronary arteriolar AT1 and ANG II type 2 (AT2) receptor mRNA levels by real-time PCR revealed no significant difference between normal control and high-fat-fed dogs; however, Western blot analysis showed a significant increase in AT1 receptor protein level with no change in AT2 receptor protein density. These findings indicate that AT1 receptor-mediated coronary constriction is augmented in the prediabetic metabolic syndrome and contributes to impaired control of coronary blood flow via increases in circulating ANG II and/or coronary arteriolar AT1 receptor density.     

Cardiovascular disease in the JCR:LA-cp rat

The JCR:LA-cp rat is one of a number of strains that carry the mutant autosomal recessive cp gene. Animals, of all strains, that are homozygous, for the gene (cp/cp) become obese, insulin resistant, and hypertriglyceridemic. Heterozygotes or homozygous normal rats (+/+)are lean and metabolically normal. The JCR:LA-cp rat is unique in the development of a frank vasculopathy with atherosclerotic lesions and associated ischemic myocardial lesions. The cardiovascular disease is strongly correlated with the hyperinsulinemia, which develops as the animals mature from 4 to 8 weeks of age. The hyperinsulinemia can be decreased by marked food restriction, ethanol consumption, or reduction of the postprandial glucose and insulin responses through the use of a-glucosidase inhibitors. Any treatment that reduces plasma insulin levels is associated with a reduction in cardiovascular disease. In contrast, a reduction in plasma triglyceride concentrations, alone, has no effect on end-stage lesions. JCR:LA-cp rats, particularly those that are cp/cp, are, however, sensitive to cholesterol in the diet, unlike other strains that are highly resistant. Further, the rats have abnormal vascular smooth muscle cells that, especially in the cp/cp animals, are hyperplastic and activated and migrate into the intimal space. Our findings suggest that susceptibility to cardiovascular disease requires hypermsulinemic stress coupled with excessive dietary intake and the presence of one or more other necessary, but not sufficient, genetic factors. One of these may be a genetic abnormality of vascular smooth muscle cells. A similar situation may occur in humans.     

Adaptive mechanisms to compensate for overnutrition-induced cardiovascular abnormalities

In conditions of overnutrition, cardiac cells must cope with a multitude of extracellular signals generated by changes in nutrient load (glucose, amino acids, and lipids) and the hormonal milieu [increased insulin (INS), ANG II, and adverse cytokine/adipokine profile]. Herein, we review the diverse compensatory/adaptive mechanisms that counter the deleterious effects of excess nutrients and growth factors. We largely focus the discussion on evidence obtained from Zucker obese (ZO) and Zucker diabetic fatty (ZDF) rats, which are useful models to evaluate adaptive and maladaptive metabolic, structural, and functional cardiac remodeling. One adaptive mechanism present in the INS-resistant ZO, but absent in the diabetic ZDF heart, involves an interaction between the nutrient sensor kinase mammalian target of rapamycin complex 1 (mTORC1) and ANG II-type 2 receptor (AT2R). Recent evidence supports a cardioprotective role for the AT2R; for example, suppression of AT2R activation interferes with antihypertrophic/antifibrotic effects of AT1R blockade, and AT2R agonism improves cardiac structure and function. We propose a scenario, whereby mTORC1-signaling-mediated increase in AT2R expression in the INS-resistant ZO heart is a cardioprotective adaptation to overnutrition. In contrast to the ZO rat, heart tissues of ZDF rats do not show activation of mTORC1. We posit that such a lack of activation of the mTOR?AT2R integrative pathway in cardiac tissue under conditions of obesity-induced diabetes may be a metabolic switch associated with INS deficiency and clinical diabetes.     

Potential mechanisms and consequences of cardiac triacylglycerol accumulation in insulin-resistant rats

The accumulation of intracellular triacylglycerol (TG) is highly correlated with muscle insulin resistance. However, it is controversial whether the accumulation of TG is the result of increased fatty acid supply, decreased fatty acid oxidation, or both. Because abnormal fatty acid metabolism is a key contributor to the pathogenesis of diabetes-related cardiovascular dysfunction, we examined fatty acid and glucose metabolism in hearts of insulin-resistant JCR:LA-cp rats. Isolated working hearts from insulin-resistant rats had glycolytic rates that were reduced to 50% of lean control levels (P < 0.05). Cardiac TG content was increased by 50% (P < 0.05) in the insulin-resistant rats, but palmitate oxidation rates remained similar between the insulin-resistant and lean control rats. However, plasma fatty acids and TG levels, as well as cardiac fatty acid-binding protein (FABP) expression, were significantly increased in the insulin-resistant rats. AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid and glucose metabolism. When activated, AMPK increases fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels, and it decreases TG content by inhibiting glycerol-3-phosphate acyltransferase (GPAT), the rate-limiting step in TG synthesis. The activation of AMPK also stimulates cardiac glucose uptake and glycolysis. We thus investigated whether a decrease in AMPK activity was responsible for the reduced cardiac glycolysis and increased TG content in the insulin-resistant rats. However, we found no significant difference in AMPK activity. We also found no significant difference in various established downstream targets of AMPK: ACC activity, malonyl-CoA levels, carnitine palmitoyltransferase I activity, or GPAT activity. We conclude that hearts from insulin-resistant JCR:LA-cp rats accumulate substantial TG as a result of increased fatty acid supply rather than from reduced fatty acid oxidation. Furthermore, the accumulation of cardiac TG is associated with a reduction in insulin-stimulated glucose metabolism.     

Vascular wall dysfunction in JCR:LA-cp rats: effects of age and insulin resistance

We tested thehypothesis that aging and insulin resistance interact to increasevascular dysfunction by comparing the function of isolated mesentericresistance arteries in obese, insulin-resistant JCR:LA-cp rats andlean, insulin-sensitive rats of the same strain at 3, 6, 9, and 12 moof age. The peak constrictor responses to norepinephrine,phenylephrine, and high potassium were elevated in arteries from obeserats. Responses to these agents increased with age in both obese andlean rats. An eicosanoid constrictor contributed substantially tovasoconstriction in the arteries from both lean and obese animals.Inhibition of nitric oxide synthase increased the vasoconstrictorresponse to norepinephrine in both obese and lean rats. This effectincreased with age in lean rats only. Vascular relaxation in responseto acetylcholine and sodium nitroprusside was impaired in the obeserats and did not alter with age. The results suggest that obeseJCR:LA-cp rats have enhanced maximal constriction, which originates inthe arterial smooth muscle and increases with age. There is evidencethat the ability of the arteries to compensate for the enhancedcontractility is impaired in obese rats, particularly with advanced age.

     

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.     

Oxidative stress alters renal D1 and AT1 receptor functions and increases blood pressure in old rats

Aging is associated with an increase in oxidative stress and blood pressure (BP). Renal dopamine D1 (D1R) and angiotensin II AT1 (AT1R) receptors maintain sodium homeostasis and BP. We hypothesized that age-associated increase in oxidative stress causes altered D1R and AT1R functions and high BP in aging. To test this, adult (3 mo) and old (21 mo) Fischer 344 × Brown Norway F1 rats were supplemented without/with antioxidant tempol followed by determining oxidative stress markers (urinary antioxidant capacity, proximal tubular NADPH-gp91phox, and plasma 8-isoprostane), D1R and AT1R functions, and BP. The D1R and AT1R functions were determined by measuring diuretic and natriuretic responses to D1R agonist (SKF-38393; 1 μg·kg(-1)·min(-1) iv) and AT1R antagonist (candesartan; 10 μg/kg iv), respectively. We found that the total urinary antioxidant capacity was lower in old rats, which increased with tempol treatment. In addition, tempol decreased the elevated NADPH-gp91phox and 8-isoprostane levels in old rats. Systolic, diastolic, and mean arterial BPs were higher in old rats and were reduced by tempol. Although SKF-38393 produced diuresis in both adult and old rats, urinary sodium excretion (UNaV) increased only in adult rats. While candesartan increased diuresis and UNaV in adult and old rats, the magnitude of response was greater in old rats. Tempol treatment in old rats reduced candesartan-induced increase in diuresis and UNaV. Our results demonstrate that diminished renal D1R and exaggerated AT1R functions are associated with high BP in old rats. Furthermore, oxidative stress may cause altered renal D1R and AT1R functions and high BP in old rats.     

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.     

Restoration of diabetes-induced abnormal local Ca2+ release in cardiomyocytes by angiotensin II receptor blockade

Stimulation of local renin-angiotensin system and increased levels of oxidants characterize the diabetic heart. Downregulation of ANG II type 1 receptors (AT(1)) and enhancement in PKC activity in the heart point out the role of AT(1) blockers in diabetes. The purpose of this study was to evaluate a potential role of an AT(1) blocker, candesartan, on abnormal Ca(2+) release mechanisms and its relationship with PKC in the cardiomyocytes from streptozotocin-induced diabetic rats. Cardiomyocytes were isolated enzymatically and then incubated with either candesartan or a nonspecific PKC inhibitor bisindolylmaleimide I (BIM) for 6-8 h at 37 degrees C. Both candesartan and BIM applied on diabetic cardiomyocytes significantly restored the altered kinetic parameters of Ca(2+) transients, as well as depressed Ca(2+) loading of sarcoplasmic reticulum, basal Ca(2+) level, and spatiotemporal properties of the Ca(2+) sparks. In addition, candesartan and BIM significantly antagonized the hyperphosphorylation of cardiac ryanodine receptor (RyR2) and restored the depleted protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6). Furthermore, candesartan and BIM also reduced the increased PKC levels and oxidized protein thiol level in membrane fraction of diabetic rat cardiomyocytes. Taken together, these data demonstrate that AT(1) receptor blockade protects cardiomyocytes from development of cellular alterations typically associated with Ca(2+) release mechanisms in diabetes mellitus. Prevention of these alterations by candesartan may present a useful pharmacological strategy for the treatment of diabetic cardiomyopathy.     

Cardiomyocyte Antihypertrophic Effect of Adipose Tissue Conditioned Medium from Rats and Its Abrogation by Obesity is Mediated by the Leptin to Adiponectin Ratio

White adipocytes are known to function as endocrine organs by secreting a plethora of bioactive adipokines which can regulate cardiac function including the development of hypertrophy. We determined whether adipose tissue conditioned medium (ATCM) generated from the epididymal regions of normal rats can affect the hypertrophic response of cultured rat ventricular myocytes to endothelin-1 (ET-1) administration. Myocytes were treated with ET-1 (10 nM) for 24 hours in the absence or presence of increasing ATCM concentrations. ATCM supressed the hypertrophic response to ET-1 in a concentration-dependent manner, an effect enhanced by the leptin receptor antagonist and attenuated by an antibody against the adiponectin AdipoR1 receptor. Antihypertrophic effects were also observed with ATCM generated from perirenal-derived adipose tissue. However, this effect was absent in ATCM from adipose tissue harvested from corpulent JCR:LA-cp rats. Detailed analyses of adipokine content in ATCM from normal and corpulent rats revealed no differences in the majority of products assayed, although a significant increase in leptin concentrations concomitant with decreased adiponectin levels was observed, resulting in a 11 fold increase in the leptin to adiponectin ratio in ATCM from JCR:LA-cp. The antihypertrophic effect of ATCM was associated with increased phosphorylation of AMP-activated protein kinase (AMPK), an effect abrogated by the AdipoR1 antibody. Moreover, the antihypertrophic effect of ATCM was mimicked by an AMPK activator. There was no effect of ET-1 on mitogen-activated protein kinase (MAPK) activities 24 hour after its addition either in the presence or absence of ATCM. Our study suggests that adipose tissue from healthy subjects exerts antihypertrophic effects via an adiponectin–dependent pathway which is impaired in obesity, most likely due to adipocyte remodelling resulting in enhanced leptin and reduced adiponectin levels.     

Activation of renal angiotensin type 1 receptor contributes to the pathogenesis of progressive renal injury in a rat model of chronic cardiorenal syndrome

Although chronic cardiac dysfunction is known to progressively exacerbate renal injury, a condition known as type 2 cardiorenal syndrome (CRS), the mechanism responsible is largely unknown. The present study was undertaken to clarify the mechanism of renal injury in rats with both unilateral nephrectomy (NX) and surgically induced myocardial infarction (MI), corresponding to a model of type 2 CRS. Compared with a control group, rats with both MI and NX (MI+NX) exhibited progressive proteinuria during the experimental period (34 wk after MI surgery), whereas proteinuria was not observed in rats with MI alone and was moderate in rats with NX alone. The proteinuria in rats with MI+NX was associated with renal lesions such as glomerulosclerosis and infiltration of mononuclear cells and upregulation of the renal proinflammatory and -fibrotic cytokine and angiotensin II type 1a receptor (AT1aR) genes. In contrast, plasma renin activity was lowered in rats with MI+NX. Immunohistochemistry revealed that the increased AT1R protein was present mainly in renal interstitial mononuclear cells. Olmesartan medoxomil, an AT1R blocker, markedly reduced the proteinuria and infiltration of mononuclear cells, whereas spironolactone, a mineralocorticoid receptor blocker, did not. The present findings demonstrate the pathogenetic role of renal interstitial AT1R signaling in a model of type 2 CRS, providing evidence that AT1R blockade can be a useful therapeutic option for this syndrome.     

Long-term AT1 receptor blockade improves metabolic function and provides renoprotection in Fischer-344 rats

Fischer-344 (F344) rats exhibit proteinuria and insulin resistance in the absence of hypertension as they age. We determined the effects of long-term (1 yr) treatment with the angiotensin (ANG) II type 1 (AT(1)) receptor blocker L-158,809 on plasma and urinary ANG peptide levels, systolic blood pressure (SBP), and indexes of glucose metabolism in 15-mo-old male F344 rats. Young rats at 3 mo of age (n = 8) were compared with two separate groups of older rats: one control group (n = 7) and one group treated with L-158,809 (n = 6) orally (20 mg/l) for 1 yr. SBP was not different between control and treated rats but was higher in young rats. Serum leptin, insulin, and glucose levels were comparable between treated and young rats, whereas controls had higher glucose and leptin with a similar trend for insulin. Plasma ANG I and ANG II were higher in treated than untreated young or older rats, as evidence of effective AT(1) receptor blockade. Urinary ANG II and ANG-(1-7) were higher in controls compared with young animals, and treated rats failed to show age-related increases. Protein excretion was markedly lower in treated and young rats compared with control rats (young: 8 +/- 2 mg/day vs. control: 129 +/- 51 mg/day vs. treated: 9 +/- 3 mg/day, P < 0.05). Long-term AT(1) receptor blockade improves metabolic parameters and provides renoprotection. Differential regulation of systemic and intrarenal (urinary) ANG systems occurs during blockade, and suppression of the intrarenal system may contribute to reduced proteinuria. Thus, insulin resistance, renal injury, and activation of the intrarenal ANG system during early aging in normotensive animals can be averted by renin-ANG system blockade.