Blockade of the renin-angiotensin system (RAS) by the use of angiotensin-converting enzyme (ACE) inhibitors has proven to be an effective means of controlling hypertension and managing congestive heart failure (1-3). However, because of the diverse activity of ACE inhibitors, affecting bradykinin and prostaglandin metabolism, development of more specific inhibitors of the RAS has occurred. In particular, specific angiotensin II subtype 1 receptor (AT1R) antagonists hold promise in being as effective as or more effective than ACE inhibitors with fewer side effects (4,5). Whether these drugs prove as effective or more effective in the long-term control of hypertension and the cardiac hypertrophy that precedes heart failure remains controversial (2). Recent studies in rat models suggest that ACE inhibition may influence cardiac hypertrophy by altering gene expression (6). Although use of either an ACE inhibitor or an AT1R antagonist will limit myocyte hypertrophy (7), it is unknown whether these drugs have a similar influence on gene activation during cardiac hypertrophy.
The SHHF/Mcc-facp rat (hereafter abbreviated as SHHF) is a model of spontaneous hypertension and congestive heart failure (8). As in humans, development of congestive heart failure is associated with activation of the RAS as well as other neurohumoral systems (9). As hypertension and hypertrophy progress in this model, there is an increase in plasma renin activity (PRA), aldosterone, and atrial natriuretic peptide (ANP) concentrations (8,9). Hypertrophy of cardiac myocytes is associated with activation of known markers of compensatory hypertrophy, including increased expression of left ventricular ANP messenger RNA (mRNA) and conversion from predominantly V1 to V3 myosin isozymes (8). Treatment of SHHF rats with an ACE inhibitor has been shown to reduce blood pressure effectively, with lesser effects on cardiac mass (10-12). It is unknown whether treatment with an AT1R antagonist would significantly improve outcome.
This study examined the effects of long-term treatment with a specific AT1R antagonist, irbesartan (SR 47436; BMS-186295), on blood pressure control, expression of early markers of adaptive cardiac hypertrophy, and metabolism in lean male SHHF rats. Irbesartan is a nonpeptide, orally active, selective AT1R antagonist (13). Like losartan, irbesartan lacks agonist activity. It has proven effective in reducing blood pressure in hypertensive patients (14), but little is known about specific effects on cardiac hypertrophy or gene expression. In this study, effects of treatment with the specific AT1R antagonist, irbesartan, are compared with those of an ACE inhibitor, captopril, and placebo-treated controls.
Thirty 4-month-old, lean male SHHF rats were obtained from Dr. Sylvia McCune's breeding colony at The Ohio State University. These rats were housed two to a cage in shoebox-style polycarbonate cages on Beta-chip hardwood bedding in a temperature-controlled room with a 12-h light-dark cycle. Animals were fed ad libitum Prolab Rat/Mouse/Hamster 3000 diet (Agway, Syracuse, NY, U.S.A.). Water was provided free choice in water bottles. The animal facility is AAALAC-accredited, and the animal protocol defining experimental procedures was approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee.
At the beginning of the study, all rats had blood pressures taken and baseline serum, plasma, and urine samples collected after 24-h fasting in metabolic cages. Systolic blood pressure was measured in awake animals by the standard tail-cuff method by using a Gilson Duograph (model ICT-2H; Gilson Medical Electronics, Middleton, WI, U.S.A.). The rats were ranked by blood pressure and then randomized into either of two drug-treatment groups or a control group (n = 10/group). The first group received 100 mg/kg/day of the ACE inhibitor, captopril, in drinking water. The second group received 50 mg/kg/day of the AT1R antagonist, irbesartan (Bristol-Myers Squibb, Princeton, NJ, U.S.A.), in drinking water. Controls received buffered water. Based on preliminary studies (data not shown), these doses were chosen to provide comparable blood pressure control. The medication for both groups was mixed and provided fresh each day. Irbesartan was dissolved in 0.035 M Na3PO4, followed by neutralization with 0.035 M NaH2PO4, to form a stock solution of 7 mg of drug/ml. Water intake was monitored daily and drug concentration adjusted by dilution with distilled water to maintain appropriate doses. To eliminate differences in sodium intake, captopril and control groups had similar amounts of sodium phosphate buffer added to their drinking water.
Blood pressure measurements were taken at baseline and at 1, 2, 4, 8, 12, and 16 weeks after starting medication. At baseline and at 4, 8, 12, and 16 weeks after starting treatment, all rats were placed in metabolic cages for 24-h urine collection to determine daily urinary protein excretion. Water containing the drug as appropriate was provided ad libitum, but food was withheld during this 24-h period. After urine collection, blood was collected from the tail vein. Approximately 0.5 ml of blood for plasma was collected into a microtube containing 5 μl of 1,000 U heparin/ml and 250 KIU of aprotinin. An additional 0.5 ml of blood was collected for serum. Both samples were immediately placed on ice until they were centrifuged to collect plasma and serum. Plasma samples were immediately frozen and stored at −70°C until assayed for ANP. Serum was used in the analysis for cholesterol, glucose, and triglycerides.
At week 15 of treatment, half of the rats from each group were randomly selected for two-dimensional (2D) echocardiography, M-mode echocardiography, and color and spectral Doppler studies. The studies were performed by using a phased array, color-flow, Doppler-echocardiographic system with a 7.5-mHz pediatric transducer (Sonos 1000; Hewlett-Packard, Inc., Waltham, MA, U.S.A.). For these studies, rats were anesthetized with xylazine (10 mg/kg) and ketamine (50 mg/kg), given intraperitoneally.
At the end of week 16 of treatment, half of the rats from each group were randomly selected for cannulation and measurement of blood pressure dose-response curves to angiotensin I (AngI) and angiotensin II (AngII). The remaining rats were killed and tissues harvested for determination of renal renin and left ventricular ANP gene expression.
Urine protein was determined by the dye-binding method of Bradford (15) and was expressed as total milligrams of protein excreted per day. Urinary sodium and potassium concentrations were determined by flame photometry, and 24-h excretion calculated. Serum triglycerides were measured with an enzymatic triglyceride Test Set GPO-PAP Method (Stanbio Laboratory, San Antonio, TX, U.S.A.), and cholesterol was determined by enzymatic method with Direct Cholesterol Test Set (Stanbio Laboratory). Glucose was measured by using the hexokinase enzymatic analysis (16).
Plasma ANP concentrations were determined on samples taken at the beginning of the study and at the end of 4 and 16 weeks of treatment. The radioimmunoassay was performed as previously described (11) by using a modification of a commercially available rat ANP radioimmunoassay kit (Peninsula Labs, Belmont, CA, U.S.A.).
AngI and AngII dose-response curves
At the end of the 16-week treatment period, half of the rats from each group were randomly selected for a study to obtain dose-response curves to AngI and AngII. Each rat was anesthetized with 45 mg/kg of pentobarbital sodium given intraperitoneally. During the procedure, rats were given 5-10 mg/kg/h of pentobarbital sodium, intravenously, to maintain a constant state of anesthesia. Once anesthetized, polyethylene PE-50 catheters were placed in the carotid artery and advanced into the aorta for measurement of blood pressure and in the jugular vein for administration of drugs. The arterial catheter was then attached to a Statham P23ID transducer on a Grass model 7D polygraph (Grass Instruments, West Warwick, RI, U.S.A.) for measurement of mean arterial blood pressure. Once the rats were instrumented and steady blood pressure readings obtained, each rat was given a 0.1-ml bolus injection of 0.001 ng of AngI via the jugular catheter, and the line was immediately flushed with 0.1 ml of saline. Once the blood pressure had returned to a steady baseline, the rat was given a 0.1-ml bolus injection of 0.001 ng of AngII. Alternating drug doses were increased by a factor of 10 until the maximal dose of 0.01 mg was given for each drug. The maximal change in mean arterial blood pressure was recorded in response to the individual doses of the angiotensin peptides. Curves were analyzed, and half-maximal drug concentrations (EC50) were calculated by using a curve-fitting program (Graph Pad). On completion of the dose-response curves, rats were killed with an intravenous overdose (100 mg/kg) of pentobarbital sodium. Heart, kidneys, brain, and liver were removed and weighed.
RNA isolation and Northern blots
RNA was isolated from freshly frozen left ventricle and kidney samples harvested at the end of the study. These rats were not part of the group cannulated for the dose-response curves. After an overdose of pentobarbital, the heart was quickly removed, blotted of excess blood, and weighed. The left ventricle was separated and quickly frozen and stored at −70°C until isolation of myosin isozymes and RNA. Kidneys were removed, weighed, frozen, and stored at −70°C until RNA isolation. Remaining organs were collected and weighed.
RNA was extracted from samples by homogenizing frozen tissue in a guanidine isothiocyanate solution as described by Sambrook et al. (17). The RNA was separated from the solution by ultracentrifuging over a cesium chloride gradient. After washing, the extracted RNA was dissolved in sterile water, and the concentration was measured spectrophotometrically. The samples were stored at −70°C until used.
Separation of mRNA was done by agarose gel electrophoresis (17). The RNA was transferred to nylon membranes by Northern blotting. The resulting blots were examined under ultraviolet light to determine location of 28S and 18S ribosomal RNA bands, uniformity of loading and migration, as well as degree of degradation. RNA was fixed to the blot by ultraviolet transilluminator (254 nm, 500 μW/cm2) for 1.5 min and by baking at 80°C for 2 h. Kidney blots were probed with a full-length cDNA probe corresponding to rat renin mRNA (obtained from Dr. Kevin Lynch, University of Virginia). The probe was labeled by random priming with [α-32P]deoxycytidine triphosphate (dCTP) by using Prime-a-Gene reagents (Promega Corp., Madison, WI, U.S.A.). The blots were hybridized and washed by using the method of Church and Gilbert (18). Left ventricle was probed with a cDNA probe corresponding to rat ANP mRNA (obtained from Dr. Christopher Glembotski, San Diego State University). Labeling was the same as with renin, but prehybridization and hybridization were done at 42°C, by using 50% formamide. The blots were dried and placed on phosphorimaging screens for 24 h and then scanned by using a Molecular Dynamics Phosphor Imager with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA, U.S.A.). As a permanent record, blots were placed on Kodak X-Omat AR film at −70°C in the presence of an intensifying screen for 5-7 days and then developed.
Myosin isozyme determination
Isolation and separation of myosin were accomplished by using procedures described by Hoh et al. (19). In brief, myosin was extracted from freshly frozen left ventricle by homogenizing frozen tissue in ice-cold buffer [600 mM NaCl, 20 mM magnesium sulfate, 0.1 mM dithiothreitol, 0.1 mM EGTA, 10 mM ATP-Na, 5 mM sodium phosphate (NaH2)]. The homogenate was centrifuged and the supernatant removed and combined with an equal volume of glycerol. The protein concentration of samples was determined by using the Bradford method (15), and then samples were stored at −70°C until used. Myosin was separated into individual isozymes by electrophoresis using a 3.5% polyacrylamide gel in sodium pyrophosphate buffer. Gels were stained with 0.025% Coomassie blue in 25% isopropanol and 10% acetic acid. After destaining with 1% acetic acid, isozymes were analyzed by scanning with a LKB densitometer (LKB Gelscan XL; LKB Probkter AB, Bromma, Sweden). The relative percentage of each isozyme was calculated from the area under the peak height for each isozyme.
All results are presented as the mean ± one standard error of the mean (SEM) unless specifically stated otherwise. Group effects were analyzed by analysis of variance. If significance (p < 0.05) was noted, mean separation was performed by using Tukey's multiple comparison test. Plasma ANP concentrations were analyzed by using the Kruskal-Wallis analysis of variance for nonparametric data and Dunn's multiple comparison test.
Systolic blood pressure
There was a statistically significant decrease in systolic blood pressure in both treatment groups compared with the controls by the end of the first week (Fig. 1). Systolic blood pressure for captopril and irbesartan groups continued to decline over 7 weeks, at which point they reached a plateau. Blood pressure for the control group demonstrated a slow increase over the length of the study. By the end of the fourth week, there was also a difference between captopril and irbesartan treatment groups. These differences continued throughout the study. Both drugs gave continuous control of blood pressure, and there was no difference in the change in blood pressure from baseline over 16 weeks between irbesartan and captopril (Table 1).
Doppler flow studies revealed that both drug-treatment groups had significantly lower maximum aortic velocity compared with controls (Table 2). No other functional parameters were affected by either drug treatment.
Physical and metabolic characteristics
There were no significant differences in body weights or organ weights among the three groups (Table 3). However, there was a trend for the heart weight (p = 0.08), heart/brain weight ratio (p = 0.06), and heart/body weight ratio (p = 0.08) to be lower in the treated groups compared with controls. Serum cholesterol, triglyceride, and glucose concentrations also were unaffected by treatment. Despite a significant decrease in plasma ANP concentration (Fig. 2), urinary sodium excretion was comparable between the groups receiving irbesartan and captopril and the controls. No differences were observed in urinary potassium or protein excretion between the groups.
Plasma atrial natriuretic peptide
At the end of 4 and 16 weeks, treatment with captopril and irbesartan resulted in suppression of plasma ANP (Fig. 2). Those samples that were below the detectable limits of this assay were assigned the value of 20 pg/ml (the detection limit of this assay). There was a significant decrease in plasma ANP concentration for both drug-treatment groups, whereas ANP concentration increased in the controls (Fig. 2).
There was a significant right shift in the AngI dose-response curve with both captopril (EC50, 95 ng; range, 9-275 ng) and irbesartan (EC50, 313 ng; range, 204-457 ng) compared with the control group (EC50, 14 ng; range, 4-35 ng), indicating that the effects of AngI were being blocked (Fig. 3). There was no significant difference between the EC50 values for captopril and irbesartan. When AngII was administered, only the irbesartan group (EC50, 110 ng; range, 48-178 ng) had a significant right shift in the dose-response curve (Fig. 4), as compared with both captopril (EC50, 18 ng; range, 11-55 ng) and control groups (EC50, 14 ng; range, 2-38 ng), indicating that irbesartan was effectively blocking the AT1 receptor. The similar blood pressure response for captopril, as compared with the control group after AngII administration, demonstrated as expected that captopril was only inhibiting conversion of AngI to AngII. Neither captopril nor irbesartan affected maximal pressor response to either AngI or AngII.
A change in myosin from predominantly V1 to V3 isozyme is a marker of adaptive hypertrophy to hypertension in the SHHF rat and becomes pronounced as the rats develop congestive heart failure. Both irbesartan and captopril treatments slowed the conversion from V1 to V3 myosin isozymes in the left ventricle compared with controls (Fig. 5).
mRNA analysis of renin and ANP
Renal renin mRNA expression (Fig. 6) was significantly higher in captopril group versus the control group (p < 0.01). Expression was also higher in the irbesartan group versus the control group, although the increase was not statistically significant (p = 0.08).
The relative level of left ventricular expression of ANP mRNA for the control group was >10 times higher than the captopril and irbesartan groups (p < 0.01; Fig. 7). There was no difference between captopril and irbesartan groups. Left ventricular ANP gene expression was significantly correlated with plasma ANP concentrations (R = 0.83, p = 0.0015) and systolic pressure (R = 0.51, p = 0.0056) at 16 weeks. ANP mRNA expression was inversely correlated to the amount of left ventricular V1 isozyme (R = −0.83, p = 0.0016).
Over the course of this 4-month study, irbesartan and captopril had similar effects on most of the parameters examined. At the doses used, irbesartan and captopril treatment produced equivalent shifts in the dose-response curve to AngI. Irbesartan blocked the response to AngII, resulting in right shift of the dose-response curve, but no alteration in the maximal contraction. Both captopril and irbesartan were effective in reducing systolic blood pressure, producing a similar change from initial systolic blood pressure. As expected with the SHHF model, the systolic blood pressure of the control group steadily increased throughout the course of the study. Inhibition of ACE by captopril resulted in a marked, 17.7-fold increase in renin gene expression in the kidney, as a result of activation of feedback loops. Irbesartan produced an intermediate, 8.6-fold increase in expression of renin mRNA, suggesting that feedback within the RAS may have been less stimulated compared with ACE inhibition.
In other studies, ACE inhibitors have been shown to have a beneficial or neutral effect on glucose and lipid metabolism (20). Although the mechanisms for metabolic improvement have not been clearly identified, it may be related to peripheral vasodilation, resulting in increasing blood flow to skeletal muscles, improving insulin availability and glucose uptake. Another possible mechanism is related to ACE inhibitors' effect on kinins, which are vasodilatory and local modulators of insulin sensitivity in muscle tissue (21-23). Because AT1R antagonists are a relatively new group of drugs, there is limited information regarding their effects on glucose or lipid metabolism, but a few studies indicate a similar or lesser effect on metabolism, as described for ACE inhibitors (23,24). In our study, both irbesartan and captopril had a neutral effect, with glucose, triglyceride, and cholesterol concentrations remaining the same for both treatment groups as compared with the controls. Further study is needed before conclusions can be made regarding AT1R antagonists' effects on glucose and lipid metabolism.
Based on echocardiograms, the only statistically significant difference in cardiac function was lower aortic peak velocities in both captopril and irbesartan groups compared with controls. Aortic peak velocity is an index of left ventricular contractility and is correlated to maximal dP/dt, peak flow, and flow rate (25). Compared with normotensive rat strains, peak aortic velocity is increased in the SHHF rats during the development of hypertrophy (26), but the mechanism underlying this increase in the SHHF rat is unknown. Aortic peak velocity will increase in normotensive animals after administration of isoproterenol (27), and the increases in untreated SHHF rats may be secondary to activation of the sympathetic nervous system. Alternatively, increased aortic peak velocity may reflect an alteration in excitation/contraction coupling intrinsic to the myocyte. Such increases occur when calcium uptake or release by the sarcoplasmic reticulum is altered and is one of the echocardiographic changes observed in transgenic mice with phospholamban deficiency (28) or with hyperthyroidism (29). Alterations in calcium handling have been demonstrated in the SHHF strain (30), but its relation to increased aortic peak velocities has not been defined. The mechanism by which irbesartan and captopril decrease aortic peak velocity in SHHF rats is unknown, and decreasing afterload may not entirely explain this effect. It is possible that blockade of the effects of AngII diminished sympathetic activation. Both drugs alleviated some of the early compensatory changes associated with cardiac hypertrophy, such as expression of ANP mRNA and the myosin isozyme conversion. Amelioration of the increase in aortic peak velocity may be another indicator that myocyte hypertrophy is attenuated by these drugs.
The RAS appears to play a role in mediating some of the adaptive changes observed during myocyte hypertrophy in response to pressure overload, including activation of fetal genetic pathways (3). Characteristic markers of adaptive hypertrophy of cardiac myocytes to overload in the rat include increased ventricular expression of ANP and a shift in myosin isozyme pattern to a decreased relative proportion of V1 with increased amounts of V3(31). This shift in myosin isozymes occurs in pathologic hypertrophy but not in physiologic hypertrophy induced by exercise. The exact mechanism by which this shift occurs is not known, but appears to be mediated at the level of gene transcription and translation, with the decrease in V1 expression being related to changes in the adenylate cyclase system (32). In SHHF rats, this shift begins early during development of hypertension and becomes marked by the time overt congestive heart failure is manifested (8). Treatment of other rat models of hypertension, such as the spontaneously hypertensive rat (SHR) with an ACE inhibitor, attenuated both cardiac hypertrophy and the shift from V1 to V3 isozymes (33). Although less is known about AT1R antagonists, drugs such as losartan have been shown to have effects similar to ACE inhibitors on cardiac mass and myosin isozyme pattern in renovascular hypertensive rats (34). With the SHHF rats, captopril and irbesartan both tended to reduce heart weight, although this did not reach significance. Other studies with this model have shown that, whereas ACE inhibitors provide adequate blood pressure control, effects on cardiac mass may be less pronounced (10-12) and may relate to the age at which treatment is initiated or duration of therapy. Irbesartan and captopril had similar effects on %V1 myosin isozyme, partially preventing the decline in %V1. The proportion of V1 isozyme inversely correlated with the amount of ANP mRNA expression in the ventricle, consistent with the observation that both of these markers parallel the development of hypertrophy in the SHHF strain of rat.
As is typical for the SHHF rat strain, plasma ANP concentrations nearly doubled over the course of study in the control group, and there was prominent expression of ANP mRNA detected in the left ventricle at termination. Under normotensive conditions, ANP expression level in the ventricles is very low, ∼1% of that in the atria. In response to ventricular overload and, to a lesser degree, aging, ANP gene expression in the ventricles increases (35). In relative tissue levels, the quantity of ANP in atrial tissue remains several hundred times higher than in the ventricles, but studies suggest that a high proportion of the ANP synthesized in the atria is stored, whereas most of the ventricular ANP is continuously released. As a result, the ventricular contribution to plasma ANP concentrations may be relatively higher (36). One study reported ventricular contribution to plasma ANP to account for 9% of the total in normotensive rats and 15% in hypertensive animals (37). Another report showed levels as high as 74% of the total in cardiomyopathic hamsters with heart failure (38). Therefore increased ANP secretion by the ventricles may be an important adaptive mechanism during hypertrophy to maintain normal blood pressure and prevent sodium and water retention.
Both irbesartan and captopril significantly reduced plasma ANP concentrations and decreased ANP gene expression in the left ventricle. This is consistent with findings in SHRs, in which captopril treatment initiated before failure prevents the increase in ANP gene expression associated with the transition to failure (6). ANP mRNA levels in both irbesartan and captopril groups were similarly decreased to <10% that of the control group. The concentration of ANP in the plasma correlated with the expression of ANP mRNA in the ventricle, suggesting that the ventricle significantly contributed to circulating ANP in the SHHF rats. Activation of the RAS in some conditions may contribute to blunting of the renal response to ANP, which can be corrected by administration of an AT1R antagonist (39). SHHF rats showed a progressive increase in PRA with age (9). It is possible that plasma ANP may increase secondary to a blunting of the renal natriuretic response, thus contributing to fluid retention and hypertension. Despite the marked decrease in plasma ANP concentrations in the rats receiving either the AT1R antagonist or the ACE inhibitor, rats in both treatment groups maintained sodium and potassium secretion, possibly reflecting enhanced renal sensitivity to ANP.
In summary, the AT1R antagonist, irbesartan, had effects similar to those of the ACE inhibitor, captopril, in the SHHF rat model of hypertension and cardiac hypertrophy. Both drugs effectively reduced and maintained reductions in systolic blood pressure, while having neutral effects on metabolic parameters. From this study, it appears that direct antagonism of the AT1R and ACE inhibition have similar effects on ventricular gene expression of early markers of pathologic cardiac hypertrophy, including V1 myosin isozyme and ANP. Irbesartan and captopril treatment resulted in downregulation of left ventricular ANP gene expression, accompanied by lower plasma ANP concentrations. Despite the decrease in plasma ANP levels, urinary sodium excretion was maintained in the treatment groups. The %V1 myosin isozyme was also similar in both treatment groups. In general, at the single doses used in this study, neither drug appeared to be superior to the other during this prefailure, hypertensive stage of cardiac hypertrophy in the SHHF rat model. However, complete comparison of the effects of these drugs on cardiac hypertrophy would require assessment of multiple dose levels.
Acknowledgment: This study was supported by a grant from Bristol-Myers Squibb/Sanofi. This work also was supported in part by US Public Health Service Grant HL48835. Salaries and research support were provided in part by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University.
1. Mancia G. Angiotensin-converting enzyme inhibitors in the treatment of hypertension. J Cardiovasc Pharmacol
2. Cleland JGF, Morgan K. Inhibition of the renin-angiotensin-aldosterone system in heart failure: new insights from basic clinical research. Curr Opin Cardiol
3. Awan NA, Mason DT. Direct selective blockade of the vascular angiotensin II receptors in therapy for hypertension and severe congestive heart failure. Am Heart J
4. Rush JE, Rajfer SI. Theoretical basis for the use of angiotensin II antagonist in the treatment of heart failure. J Hypertens
5. Burnier M, Waeber B, Brunner HR. The advantages of angiotensin II antagonism. J Hypertens
6. Brooks WW, Bing OH, Conrad CH, et al. Captopril modifies gene expression in hypertrophied and failing hearts of aged spontaneous hypertensive rats. Hypertension
7. Taylor K, Patten RD, Smith JJ, et al. Divergent effects of angiotensin converting enzyme inhibition and angiotensin II-receptor antagonism on myocardial cellular proliferation and collagen deposition after myocardial infarction in rats. J Cardiovasc Pharmacol
8. McCune SA, Radin MJ, Jenkins JE, Shu Y, Park S, Peterson RG. SHHF/Mcc-facp
rat model: effects of gender and genotype on age of expression of metabolic complications and congestive heart failure and on response to drug therapy. In: Shafrir E, ed. Lessons from animal diabetes V.
London: Smith-Gordon, 1996:255-70.
9. Holycross BJ, Summers BM, Dunn RB, McCune SA. Plasma renin activity in heart failure prone (SHHF/Mcc-facp
) rats. Am J Physiol
10. Haas GJ, McCune S, Brown DM, Cody RJ. Reversal of myocardial hypertrophy in a rodent model of heart failure. Circulation
11. Radin MJ, Jenkins JE, McCune SA, Hamlin R. Effects of enalapril and clonidine on glomerular structure, function, and atrial natriuretic peptide receptors in SHHF/Mcc-cp rats. J Cardiovasc Pharmacol
12. Radin MJ, Chu YY, Hoepf TM, McCune SA. Treatment of obese female and male SHHF/Mcc-facp
rats with antihypertensive drugs, nifedipine and enalapril: effects on body weight, fat distribution, insulin resistance, and systolic pressure. Obesity Res
13. Cazaubon C, Gougat J, Bousquet F, et al. Pharmacological characterization of SR 47436: a new nonpeptide AT1
subtype angiotensin II receptor antagonist. J Pharmacol Exp Ther
14. van den Meiracker AH, Admiraal PJJ, Janssen JA, et al. Hemodynamic and biochemical effects of the AT1
receptor antagonist in hypertension. Hypertension
15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
16. Bergmeyer HU, Bernt E, Schmidt F, Stock H. D-Glucose determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis,
2nd ed. Vol 3. Deerfield Beach, FL: Verlag Chemic International, 1974:1196-201.
17. Sambrook J, Fristsch EF, Maniatis T. Molecular cloning: a laboratory manual.
2nd ed. Vol 1. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989:4-5-8 and 2-24-30.
18. Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A
19. Hoh JF, McGrath PA, Hale PT. Electrophoretic analysis of multiple forms of rat cardiac myosin: effects of hypophysectomy and thyroxine replacement. J Mol Cell Cardiol
20. Poulter NR. Beyond blood pressure. J Hum Hypertens
21. Gambardella S, Frontoni S, Pellegrinotti M, Testa G, Spallone V, Menzinger G. Carbohydrate metabolism in hypertension: influence of treatment. J Cardiovasc Pharmacol
22. Donnelly R. Angiotensin-converting enzyme inhibitors and insulin sensitivity: metabolic effects in hypertension, diabetes, and heart failure. J Cardiovasc Pharmacol
23. Chow L, De Gasparo M, Levens N. Improved glucose metabolism following blockage of angiotensin converting enzyme but not angiotensin AT1
receptors. Eur J Pharmacol
24. Moan A, Hoieggen A, Seljeflot I, Risanger T, Arnesen H, Kjeldsen SE. The effect of angiotensin II receptor antagonism with losartan on glucose metabolism and insulin sensitivity. J Hypertens
25. Wallmyer K, Wann S, Sagar KB, Kalbfleisch J, Klopfenstein HS. The influence of preload and heart rate on Doppler echocardiographic indexes of left ventricular performance: comparison with invasive indexes in an experimental preparation. Circulation
26. Park SC, Leszcynski J, McCune SA, Bonagura JD. Echocardiographic studies of progression to congestive heart failure in lean male SHHF/Mcc-facp
rats (1994 Ab). FASEB J
27. Hartley CJ, Michael LH, Entman ML. Noninvasive measurement of ascending aortic blood velocity in mice. Am J Physiol
28. Hoit BD, Khoury SF, Kranias EG, Ball N, Walsh RA. In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency. Circ Res
29. Taffet GE, Hartley CJ, Wen X, Pham T, Michael LH, Entman ML. Noninvasive indexes of cardiac systolic and diastolic function in the hyperthyroid and senescent mouse. Am J Physiol
30. Gomez AM, Valdivia H, Cheng H, et al. Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science
31. Buttrick PM, Kaplan M, Leinwand LA, Scheuer J. Alterations in gene expression in the rat heart after chronic pathological and physiological loads. J Mol Cell Cardiol
32. Morano I, Adler K, Weismann K, Knorr A, Erdmann E, Bohm M. Correlation of myosin heavy chain expression in the rat with cAMP in different models of hypertension-induced cardiac hypertrophy. J Mol Cell Cardiol
33. Sen S, Young D. Effect of antihypertensive therapy upon myosin isozyme distribution in spontaneously hypertensive rats. J Hypertens
34. Ling Q, Guo ZG, Su Z, Guo X. Regression of cardiac hypertrophy and myosin isoenzyme patterns by losartan and captopril in renovascular hypertensive rats. Acta Pharmacol Sinica
35. Younes A, Boluyt MO, O'Neill L, Meredith AL, Crow MR, Lakatta EG. Age-associated increase in rat ventricular ANP gene expression correlates with cardiac hypertrophy. Am J Physiol
36. McKenzie JC, Kelly KB, Merisko-Liversidge ME, Kennedy J, Klein RM. Developmental pattern of ventricular atrial natriuretic peptide (ANP) expression in chronically hypoxic rats as an indicator of the hypertrophic process. J Mol Cell Cardiol
37. Kinnunem P, Vuolteenaho P, Usima P, Ruskoaho H. Passive mechanical stretch releases atrial natriuretic peptide from rat ventricular myocardium. Circ Res
38. Thibault G, Nemer M, Drouin J, et al. Ventricles as a major site of atrial natriuretic factor synthesis in cardiomyopathic hamsters with heart failure. Circ Res
39. Abassi ZA, Kelly G, Golomb E, Klein H, Keiser HR. Losartan improves the natriuretic response to ANF in rats with high-output heart failure. J Pharmacol Exp Ther