oxadiazoles and Cardiomegaly

Investigate if azilsartan protects against myocardial hypertrophy by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated pathways.. Abdominal aortic constriction (AAC)-induced cardiac hypertrophy in rats was applied. Azilsartan or vehicle was administered daily for 6 weeks in sham or AAC rats. Cardiac morphology and ventricular function were determined. Azilsartan effects upon neonatal rat cardiomyocyte (NRCM) hypertrophy and molecular mechanisms were studied in angiotensin (Ang) II-stimulated NRCMs in vitro. Nrf2-small interfering RNA (siRNA) was used to knockdown Nrf2 expression. Messenger RNA (mRNA)/protein expression of Kelch-like erythroid cell-derived protein (Keap)1 and Nrf2 and its downstream antioxidant enzymes was determined by real-time reverse transcription-quantitative polymerase chain reaction and western blotting, respectively.. Azilsartan treatment ameliorated cardiac hypertrophy/fibrosis significantly in AAC rats. Azilsartan increased expression of Nrf2 protein but decreased expression of Keap1 protein. Upregulation of protein expression of Nrf2's downstream antioxidant enzymes by azilsartan treatment was observed. Azilsartan inhibited Ang II-induced NRCM hypertrophy significantly and similar effects on the Keap1-Nrf2 pathway were observed in vivo. Nrf2 knockdown markedly counteracted the beneficial effects of azilsartan on NRCM hypertrophy and the Keap1-Nrf2 pathway.. Azilsartan restrained pressure overload-induced cardiac remodelling by activating the Keap1-Nrf2 pathway and increasing expression of downstream antioxidant enzymes to alleviate oxidative stress.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Animals; Antioxidants; Benzimidazoles; Cardiomegaly; Female; Heart Ventricles; Kelch-Like ECH-Associated Protein 1; Male; Myocardium; Myocytes, Cardiac; NF-E2-Related Factor 2; Oxadiazoles; Oxidative Stress; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Up-Regulation

The possible counteracting effect of angiotensin (Ang)-converting enzyme (ACE)2/Ang-(1-7)/Mas axis against the ACE/Ang II/Ang II type 1 (AT1) receptor axis in blood pressure control has been previously described. We examined the possibility that this pathway might be involved in the anti-hypertensive effect of a newly developed AT1 receptor blocker (ARB), azilsartan, and compared azilsartan's effects with those of another ARB, olmesartan. Transgenic mice carrying the human renin and angiotensinogen genes (hRN/hANG-Tg) were given azilsartan or olmesartan. Systolic and diastolic blood pressure, as determined by radiotelemetry, were significantly higher in hRN/hANG-Tg mice than in wild-type (WT) mice. Treatment with azilsartan or olmesartan (1 or 5 mg kg(-1) per day) significantly decreased systolic and diastolic blood pressure, and the blood pressure-lowering effect of azilsartan was more marked than that of olmesartan. The urinary Na concentration decreased in an age-dependent manner in hRN/hANG-Tg mice. Administration of azilsartan or olmesartan increased urinary Na concentration, and this effect was weaker with olmesartan than with azilsartan. Azilsartan decreased ENaC-α mRNA expression in the kidney and decreased the ratio of heart to body weight. Olmesartan had a similar but less-marked effect. ACE2 mRNA expression was lower in the kidneys and hearts of hRN/hANG-Tg mice than in WT mice. This decrease in ACE2 mRNA expression was attenuated by azilsartan, but not by olmesartan. These results suggest that the hypotensive and anti-hypertrophic effects of azilsartan may involve activation of the ACE2/Ang-(1-7)/Mas axis with AT1 receptor blockade.

Topics: Angiotensin I; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme 2; Animals; Antihypertensive Agents; Benzimidazoles; Blood Pressure; Cardiomegaly; Epithelial Sodium Channels; Imidazoles; Male; Mice; Mice, Inbred C57BL; Oxadiazoles; Peptide Fragments; Peptidyl-Dipeptidase A; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Sodium; Tetrazoles

: In sinoatrial (SA) node cells, nitric oxide (NO) exerts a dual effect on the hyperpolarization-activated current, I(f), i.e. in basal conditions NO enhances I(f) whereas in the presence of beta-adrenergic stimulation it decreases it. Recent studies have shown that I(f) is present in ventricular myocytes from hypertrophied or failing hearts where it may promote abnormal automaticity. Since these pathological conditions are associated with increased sympathetic tone and upregulation of myocardial NO production, we set out to investigate whether I(f) is similarly modulated by NO in hypertrophied ventricular myocytes.. Left ventricular myocytes were isolated from 18-20-month-old spontaneously hypertensive rats (SHRs). Membrane current was measured under whole-cell or amphotericin-perforated patch-clamp conditions, at 35 degrees C.. Application of diethylamine-NO (DEA-NO, 1-100 microM) did not alter the amplitude or voltage dependence of activation of I(f) under basal conditions (half-activation voltage, V(h): control -82.9+/-2.6, DEA-NO -84.0+/-2.6 mV). Similarly, I(f) was not affected by the inhibition of endogenous NO production (L-NMMA, 500 microM) or guanylate cyclase (ODQ, 10 microM). Forskolin (10 microM) or isoprenaline (100 nM) elicited a positive shift in V(h) but subsequent application of DEA-NO did not further affect the properties of I(f).. Our results show that, unlike in SA node cells, in SHR ventricular myocytes basal and adrenergically stimulated I(f) is not modulated by exogenous NO or by constitutive NO or cGMP production.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Amphotericin B; Analysis of Variance; Animals; Arrhythmias, Cardiac; Cardiomegaly; Colforsin; Enzyme Inhibitors; Guanylate Cyclase; Hydrazines; Isoproterenol; Male; Membrane Potentials; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrogen Oxides; omega-N-Methylarginine; Oxadiazoles; Patch-Clamp Techniques; Penicillamine; Pyridines; Rats; Rats, Inbred SHR; Sinoatrial Node

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cardiomegaly; Colforsin; Cyclic AMP; Cyclic GMP; Dogs; Heart; Milrinone; Oxadiazoles; Oxygen Consumption; Purinones; Quinoxalines

Several functional and biochemical characteristics of hypertrophied hearts isolated from rats with renovascular hypertension provide indirect evidence that cellular Ca2+ dynamics during myocardial contraction-relaxation are altered. In this study, intracellular Ca2+ concentration ([Ca2+]i) dynamics were examined in paced left ventricular (LV) myocytes isolated from rats with hypertension (HYP) induced by partial occlusion of the left renal artery and from normotensive rats (Sham). Characteristic myocardial changes produced by renovascular hypertension included a 40% increase in LV weight and a 3.6-fold increase in the fractional expression of the beta-heavy chain of myosin in isolated LV myocytes. In periods of mechanical quiescence between contractions, basal [Ca2+]i values were similar in Sham and HYP LV myocytes. During a contraction-relaxation cycle in HYP myocytes, peak [Ca2+]i, +d[Ca2+]i/dt, and -d[Ca2+]i/dt were reduced, whereas the time required for [Ca2+]i to rise from a basal value to a peak value (time-to-peak [Ca2+]i) was unaffected. In both Sham and HYP myocytes, the fall in [Ca2+]i from peak to basal values could be approximated by a monoexponential rate constant, kf. Values for kf were significantly smaller in HYP than in Sham myocytes. After treatment with 4 microM isoproterenol, peak [Ca2+]i, +[Ca2+]i/dt, -d[Ca2+]i/dt, and kf increased in both Sham and HYP myocytes. In contrast, basal [Ca2+]i and time-to-peak [Ca2+]i did not change. Thus, despite recent reports of inefficiencies of beta-adrenergic receptor coupling, there was no evidence of blunted beta-adrenergic responsiveness in HYP myocytes with respect to [Ca2+]i dynamics during contraction-relaxation. Finally, no Sham vs. HYP differences in the number of specific [3H]-PN200-110 binding sites per cell in quiescent, rod-shaped myocytes were detected, but a significant reduction in [3H]-PN200-110 binding sites in an enriched sarcolemmal membrane fraction isolated from HYP animals was observed. These observations are suggestive of a reduction in slow, Ca2+ channel surface density in HYP myocytes. The results of this study clearly indicate that [Ca2+]i dynamics during contraction-relaxation in single left ventricular myocytes are affected by residence in a chronic setting of renovascular hypertension. In addition, the prolonged [Ca2+]i removal phase observed in HYP myocytes can be restored toward normal by beta-adrenergic agonists.

Topics: Analysis of Variance; Animals; Calcium; Calcium Channel Blockers; Cardiomegaly; Cells, Cultured; Fura-2; Heart Ventricles; Hypertension, Renovascular; Isoproterenol; Isradipine; Kinetics; Male; Myocardium; Myosins; Oxadiazoles; Rats; Rats, Inbred Strains; Reference Values; Sarcolemma; Spectrometry, Fluorescence

We explored the effect of age and of hypertrophy on Ca2+ antagonist binding site density (Bmax), affinity (Kd), and selectivity in cardiac membranes harvested from the hearts of young adult (9-week-old) and older (25-week-old) Sprague Dawley (SD) rats, Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Radioligand binding studies with (+)[3H]PN200-110 failed to show a significant difference between the Bmax obtained for the cardiac membranes of 9-week-old SD, WKY, or SHR. Similarly, at 25 weeks, the Bmax values were the same for each group, but in each group the Bmax tended to increase with age. The Kd and selectivity were unchanged. For (-)[3H]D888 binding, the Kd values changed with age, but there was no hypertension or hypertrophy-linked increase in Bmax. In the SD and SHR series, but not in the WKY, there was a tendency for the Bmax to increase with age. We interpreted these results to mean that age may contribute to the different Kd and Bmax values described for cardiac membranes from 25-week-old WKY and SHR.

Topics: Aging; Animals; Blood Pressure; Body Weight; Calcium Channels; Cardiomegaly; Cell Membrane; Dihydropyridines; Indicators and Reagents; Isradipine; Male; Myocardium; Oxadiazoles; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY; Receptors, Nicotinic; Time Factors; Verapamil

Inotropic responsiveness to dihydropyridines (DHP) and characterization of DHP receptors were studied during the onset of hypertrophy in rat hearts. The inotropic responsiveness of isolated hearts to external Ca2+ (0.25-2.50 mM) and nifedipine (10(-9)-10(-7) M), as expressed in percent change in dP/dtmax, was unchanged by the process of hypertrophy. Characterization of DHP receptors by Scatchard plots (Kd = 0.45 and 0.47 nM for nitrendipine), displacement curves (Kd = 0.44 and 0.42 nM for PN 200-110), and dissociation kinetics (k-1 = 4.82 X 10(-2) X min-1 and 4.85 X 10(-2) X min-1) revealed the similarity of the Ca2+ antagonist binding sites in hypertrophied and control hearts, respectively. These results on crude or purified sarcolemmal preparations from left ventricle were consistent with the presence of only one type of binding site of high affinity for DHP. The total number of Ca2+ channels was increased in hypertrophied left ventricle (LV) as compared with left ventricle from sham-operated animals (15,000 fmol/LV and 8900 fmol/LV), respectively. This increased synthesis of Ca2+ channels was observed as early as 5 days after the aortic stenosis and was related to the increase in ventricular mass. Results are in favor of an adaptational process of regulation of the total number of Ca2+ channels as an answer to pressure overload.

Topics: Adaptation, Physiological; Animals; Binding, Competitive; Calcium Channel Blockers; Calcium Channels; Cardiomegaly; Heart; In Vitro Techniques; Isradipine; Kinetics; Male; Myocardium; Nitrendipine; Oxadiazoles; Rats; Rats, Inbred Strains; Receptors, Nicotinic; Sarcolemma