(Hypertension. 1996;28:265-268.)
© 1996 American Heart Association, Inc.
Articles |
Angiotensin II Increases Left Ventricular Mass Without Affecting Myosin Isoform mRNAs
the Laboratory on Hypertension Research, Alton Ochsner Medical Foundation, New Orleans, La.
Correspondence to Edward D. Frohlich, Alton Ochsner Medical Foundation, 1516 Jefferson Hwy, New Orleans, LA 70121.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
We studied the effect of chronic (7 days) angiotensin II (Ang II) infusion in nonpressor and pressor doses on cardiovascular mass and expression of
- and ß-myosin heavy chain genes in the left ventricle in normotensive Wistar rats. An increased left ventricular mass was observed in rats receiving nonpressor and pressor doses of Ang II, but only high doses increased arterial pressure. Normalization of arterial pressure during Ang II infusion by losartan, a specific Ang II receptor antagonist, or hydralazine had different effects on left ventricular mass. Losartan prevented the increased left ventricular mass, and hydralazine did not affect left ventricular mass. Northern blot analysis showed that the switch in left ventricular myosin isoform mRNA from the adult to the fetal pattern occurred only in rats given the pressor Ang II dose. Both losartan and hydralazine, in parallel with the normalization of arterial pressure, prevented this myosin isoform switch. Thus, these data suggest that the Ang II-induced increase in left ventricular mass was not dependent on pressure overload, but the switch in myosin isoform mRNA from the adult to the fetal pattern was dependent on pressure overload.
Key Words: hypertrophy, left ventricular • angiotensin II • myosin • losartan • hydralazine
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The pathogenesis of hypertensive left ventricular hypertrophy (LVH) involves mechanical, hormonal, and neural factors.1 2 A number of earlier reports demonstrated that certain antihypertensive agents, although equipotent in lowering arterial pressure in hypertensive animals, may be more effective in reducing left ventricular (LV) mass than others and that the renin-angiotensin system may be involved independent of its pressor effect.3 4 This notion is further supported by the results of in vivo and in vitro studies showing that angiotensin II (Ang II) increases protein synthesis and myocardial mass and that this stimulatory effect of Ang II on myocardial growth may be blocked by specific Ang II receptor antagonists.2 5 6 7 8 In addition to these quantitative changes, LVH also involves the expression of cardiac genes and their respective proteins from the adult to the fetal pattern.9 Several proteins and their mRNAs are involved, including contractile proteins, creatine kinase, and atrial natriuretic peptide.
The purpose of this study was to define further the cardiovascular effects of Ang II and to distinguish between its hemodynamic actions and its effect on cardiac mass. To this end, we evaluated the effect of prolonged Ang II infusion in pressor and nonpressor doses on systemic hemodynamics, cardiac mass, and myosin isoform mRNA expression in normotensive Wistar rats. To differentiate further the direct cardiac actions of Ang II from its indirect, pressure-mediated effects, we also gave rats receiving Ang II infusion either a specific Ang II receptor antagonist or a direct-acting vasodilator in doses that prevented Ang II pressure elevation and normalized arterial pressure.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Male 16-week-old Wistar rats (Charles River Laboratories, Wilmington, Mass) were given standard rat chow (Ralston Purina) and tap water ad libitum. Rats were kept in an animal facility approved by the American Association for Accreditation of Laboratory Animal Care and were handled in accordance with National Institutes of Health guidelines. The protocol was approved by our institutional Animal Care Committee.
Rats were randomly divided into five groups, with six rats in each. With rats under pentobarbital anesthesia (40 mg/kg IP), an osmotic minipump (model 2001, Alza Corp) was implanted subcutaneously for delivery of either vehicle or Ang II. Ang II acetate salt (Sigma Chemical Co) was dissolved in sterile 0.9 NaCl solution containing 0.01 mol/L acetic acid. The first rat group (control) received vehicle alone. Rats in the second group received a subpressor Ang II dose (96 µg/d). In preliminary experiments, indirect (tail-cuff) systolic pressure was measured and used to determine the highest Ang II dose that did not elevate arterial pressure during a 7-day infusion period. The third rat group received a high Ang II dose (300 µg/d). Rats of the fourth group received, in addition to a high Ang II dose, the type 1 Ang II receptor antagonist losartan (DuPont-Merck, 30 mg/d by gavage). Rats of the fifth group received a high Ang II dose and hydralazine (Sigma, 5 mg/d by gavage).
After 7 days of the respective treatments, rats were anesthetized with pentobarbital (40 mg/kg IP), and PE-50 tubing was inserted into the femoral artery and right jugular vein for arterial pressure measurement and saline injection, respectively. The catheter in the jugular vein was advanced into the right atrium, guided by pressure waveforms. A thermistor microprobe (type IT-18, Physitemp Instruments Inc) was introduced into the ascending aorta through the right carotid artery. The arterial catheter was connected to a multichannel recorder (SensorMedics Corp) through a P23Db Statham pressure transducer, and arterial pressure (systolic, diastolic, and mean), heart rate, and thermodilution curves were recorded. A high-precision syringe (CR-700-200, Hamilton Co) was connected to the venous catheter for saline injections at room temperature. The thermistor microprobe was connected to a thermodilution device (Cardiotherm 500, Columbus Instruments) for cardiac output measurement. Cardiac output was obtained from a digital screen, normalized for body weight, and expressed in milliliters per minute per 100 g.10 Total peripheral resistance was calculated from mean arterial pressure and cardiac output, with the assumption that right atrial pressure was zero. After hemodynamic studies were completed, rats were killed with an overdose of pentobarbital and the hearts were removed. The atria were excised, and the right ventricular lateral wall was separated from the left ventricle and septum and their wet weights determined. A small piece of left ventricle (about 200 mg) was cut for determination of the ratio of wet to dry weight. The remaining ventricular tissue was frozen immediately in liquid nitrogen and stored at -70°C until analysis. For determination of the ratio of wet to dry weight, a piece of left ventricle was weighed and then dried to a constant weight in a vacuum oven.
Total LV RNA was extracted with the acid guanidinium thiocyanate/phenol/chloroform method.11 The ratio of the absorbance at 260/280 nm was 1.8 or more in all samples. RNA integrity was ascertained by the appearance of the 18S and 28S rRNA bands after agarose gel electrophoresis and ethidium bromide staining. For Northern blot analysis, 20 µg total RNA was denatured with formaldehyde and formamide and size fractionated on a 1% agarose gel.12 Transfer of RNA to nylon membranes (GeneScreen Plus, DuPont) was done by capillary blotting. Hybridization to oligonucleotide probes for rat - and ß-myosin heavy chains13 was then performed. Probes were purchased from Oncogene Science and were 5' end labeled with [
-32P]dCTP and the T4 polynucleotide kinase kit (GIBCO BRL). All hybridizations were carried out at 42°C for 18 hours in a buffer containing 25 mmol/L KH2PO4, 50% formamide, 5x SSC, 5x Denhardt's solution, 10% dextran, 1% sodium dodecyl sulfate, and 0.5 mg/mL salmon sperm DNA. After washing (2x SSC, 0.1% sodium dodecyl sulfate), membranes were autoradiographed with the use of Kodak X-Omat AR film and intensifying screens. To check for evenness of loading and transfer, membranes were stripped and rehybridized to GAPDH probe. Oligonucleotide GAPDH probe was purchased from Oncogene Science and 5' end labeled, and hybridization was carried out as already described. Autoradiographic films were analyzed densitometrically, and after normalization for GAPDH, relative amounts of - and ß-myosin mRNAs were expressed in relation to controls.
Values are expressed as mean±SE. One-way ANOVA and Bonferroni's modification of the t test for multigroup comparisons were used to test the differences among groups.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of the hemodynamic studies and of cardiovascular mass measurements are presented in Figs 1 through 3
. A significant reduction in body weight was observed in rats receiving the high Ang II dose, and losartan prevented this effect (Fig 1). Systolic (Fig 2) and mean (Fig 3) arterial pressures were significantly increased in rats receiving the high Ang II dose. Both losartan and hydralazine prevented this Ang II-induced rise in arterial pressure. No significant differences in heart rate and cardiac index were observed among the groups, although rats receiving the high Ang II dose had somewhat higher heart rates and lower cardiac outputs (Fig 2). An increased total peripheral resistance was observed in rats given the high Ang II dose; this was also prevented by both losartan and hydralazine (Fig 2). Both LV mass and LV mass index were significantly increased in rats receiving either the subpressor or pressor Ang II dose (Figs 1 and 3). Right ventricular mass index was also increased in rats receiving Ang II (Fig 1). Losartan prevented this effect but hydralazine did not. There was no difference in the ratio of wet to dry LV weight among the groups (Fig 1).
|
|
|
The Northern hybridization analysis (Fig 4) showed that chronic Ang II infusion in the pressor but not subpressor dose decreased expression of -myosin mRNA and increased expression of ß-myosin mRNA (Fig 5). Normalization of blood pressure with either losartan or hydralazine prevented this switch in myosin isoform mRNA expression (Fig 5).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of this study clearly indicate that the Ang II-induced increase in LV mass was not pressure dependent. Thus, the increased LV mass was induced by prolonged Ang II infusion in doses that either did or did not increase arterial pressure. Furthermore, the specific Ang II receptor antagonist losartan normalized arterial pressure and prevented the increased mass; but whereas hydralazine also normalized arterial pressure, it did not prevent the Ang II-induced LVH. Since the ratio of wet to dry LV weight was not different between the groups, it is obvious that the observed increased LV mass was not the result of pressure or Ang II-induced changes in cardiac fluid content. These results are in agreement with previously reported findings showing that Ang II promotes LVH in vivo independently of its effect on arterial pressure2 5 7 8 14 ; Ang II increases myocardial cell growth and protein synthesis in cultures of cardiac myocytes5 6 ; Ang II is involved in the development of LVH induced by pressure overload15 16 ; and Ang II also attenuates cardiac atrophy after transplantation.17
It should be noted that Ang II-induced LVH observed in the present study may be due to the direct effect of Ang II on cardiac tissue but that it may also result from indirect actions such as stimulation of sympathetic activity or increased aldosterone synthesis and release.2 18 19 Furthermore, these data do not indicate which of the components of myocardial tissue are involved in the hypertrophic response. The available evidence suggests that Ang II may exert a trophic influence on cardiac myocytes, fibroblasts, vascular smooth muscle cells, and myocardial collagen content.5 6 7 20
The development of hypertensive LVH, in addition to an increase in mass, also involves changes in the expression of a number of cardiac genes and their respective proteins, usually referred to as a switch from the adult to the fetal pattern.9 This response includes changes in the expression of - and ß-myosin heavy chains, and hypertensive LVH in rats has been shown to increase the relative proportion of ß-myosin heavy chain and to downregulate that of -myosin heavy chain.21 22 Myosin heavy chain gene expression has also been shown to be influenced by developmental and hormonal factors.23 24 Our data showing an increase in ß-myosin heavy chain mRNA and a decrease in -myosin heavy chain mRNA in rats receiving a prolonged infusion of high-dose Ang II are also in agreement with these results. Finally, these data demonstrate that the switch in the expression of myosin isoform mRNA occurred only when arterial pressure was increased and indicate that Ang II was not involved in the regulation of myosin heavy chain gene expression; mechanical factors (pressure/stretch) most likely are involved in this biological response. In an apparent inconsistency with our results, Kim et al25 have recently reported that in rats given chronic Ang II infusion and hydralazine, there is a transient (days 2 and 3) increase in ß-myosin heavy chain mRNA. However, on day 7, ß-myosin heavy chain mRNA expression was at control levels in rats given Ang II and hydralazine, whereas in rats given only Ang II, an increased expression of the same gene was noted.25 Therefore, our findings are in agreement with the described results. It is possible that hydralazine-induced changes in heart rate and cardiac output may be responsible for the transient changes in myosin heavy chain gene expression in this experimental model.26
In summary, the presented results suggest that the Ang II-induced increase in LV mass is not dependent on pressure overload and that the switch in myosin isoform mRNA from the adult to the fetal pattern is dependent on pressure.
Received September 20, 1995; first decision December 4, 1995; accepted April 2, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Frohlich ED, Tarazi RC. Is arterial pressure the sole factor responsible for hypertensive heart hypertrophy? Am J Cardiol. 1979;44:959-963.[Medline] [Order article via Infotrieve]
2.
Morgan HE, Baker KM. Cardiac hypertrophy: mechanical, neural, and endocrine dependence. Circulation. 1991;83:13-25.
3. Frohlich ED. Overview of hemodynamic and nonhemodynamic factors associated with left ventricular hypertrophy. J Mol Cell Cardiol. 1989;21(suppl V):3-10.
4. Frohlich ED. The heart in hypertension: a 1991 overview. Hypertension. 1991;18(suppl III):III-62-III-68.
5. Khairallah PA, Robertson AL, Davila D. Effect of angiotensin II on DNA, RNA and protein synthesis. In: Genest J, Koiw E, eds. Hypertension. New York, NY: Springer-Verlag; 1972:212-220.
6.
Baker KM, Aceto JF. Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells. Am J Physiol. 1990;259:H610-H618.
7.
Crawford DC, Chobanian AV, Brecher P. Angiotensin II induces fibronectin expression associated with cardiac fibrosis in the rat. Circ Res. 1994;74:727-739.
8. Black MJ, Bertram JF, Campbell JH, Campbell GR. Angiotensin II induces cardiovascular hypertrophy in perindopril-treated rats. J Hypertens. 1995;13:683-692.[Medline] [Order article via Infotrieve]
9. Parker TG, Schneider MD. Growth factors, proto-oncogenes and plasticity of the cardiac phenotype. Annu Rev Physiol. 1991;53:179-200.[Medline] [Order article via Infotrieve]
10. Soria F, Frohlich E, Aristizabal D, Kaneko K, Kardon M, Hunter J, Pegram B. Preserved cardiac performance with reduced left ventricular mass in conscious exercising spontaneously hypertensive rats. J Hypertens. 1994;12:485-589.
11. Chomczynski P, Sacchi J. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]
12. Grierson D. Electrophoresis of RNA in horizontal slab gels after denaturation with glyoxal or formamide. In: Rickwood D, Hames BD, eds. Gel Electrophoresis of Nucleic Acids. A Practical Approach. New York, NY: Oxford University Press; 1990:225-247.
13.
Mahdavi V, Chambers AP, Nadal-Ginard B. Cardiac - and ß-myosin heavy chain genes are organized in tandem. Proc Natl Acad Sci U S A. 1984;81:2626-2630.
14.
Golomb E, Abassi ZA, Cuda G, Stylianou M, Panchal VR, Trachewsky D, Keiser HR. Angiotensin II maintains, but does not mediate, isoproterenol-induced cardiac hypertrophy in rats. Am J Physiol. 1994;267:H1496-H1506.
15.
Suzuki J, Matsubara H, Urakami M, Inada M. Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy. Circ Res. 1993;73:439-447.
16.
Kojima M, Shiojima I, Yamazaki T, Komuro I, Yunzeng Z, Ying W, Mizuno T, Ueki K, Tobe K, Kadowaki T, Nagai R, Yazaki Y. Angiotensin II receptor antagonist TCV-116 induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intracellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro. Circulation. 1994;89:2204-2211.
17.
Geenen D, Malhotra A, Scheuer J. Angiotensin II increases cardiac protein synthesis in adult rat heart. Am J Physiol. 1993;265:H238-H243.
18.
Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT. Remodeling of the rat right and left ventricles in experimental hypertension. Circ Res. 1990;67:1355-1364.
19.
Robert V, Thiem NV, Cheav SL, Mouas C, Swyngheduaw B, Delcayre C. Increased cardiac types I and III collagen mRNAs in aldosterone-salt hypertension. Hypertension. 1994;24:30-36.
20. Schelling P, Fisher H, Ganten D. Angiotensin II and cell growth: a link to cardiovascular hypertrophy? J Hypertens. 1991;9:3-15.[Medline] [Order article via Infotrieve]
21.
Imamura SI, Matsuoka R, Hiratsuka M. Adaptational changes of MHC gene expression and isoenzyme transition in cardiac overloading. Am J Physiol. 1991;260:H73-H79.
22.
Haddad F, Bodell PW, Baldwin KM. Pressure-induced regulation of myosin expression in rodent heart. J Appl Physiol. 1995;78:1489-1495.
23.
Lompre AM, Nadal-Ginard B, Mahdavi V. Expression of the cardiac ventricular alpha- and beta-myosin heavy chain genes is developmentally and hormonally regulated. J Biol Chem. 1984;259:6437-6446.
24.
Rupp H, Wahl R. Influence of thyroid hormones and catecholamines on myosin of swim-exercised rats. J Appl Physiol. 1990;68:973-978.
25.
Kim S, Ohta K, Hamaguchi A, Yukimura T, Miura K, Iwao H. Angiotensin II induces phenotypic modulation and remodeling in vivo in rats. Hypertension. 1995;25:1252-1259.
26.
Geenen DL, Malhotra A, Buttrick PM, Scheuer J. Increased heart rate prevents the isomyosin shift after cardiac transplantation in the rat. Circ Res. 1992;70:554-558.
This article has been cited by other articles:
 |
|
 |
|
  J. Xu, O. A. Carretero, C.-X. Lin, M. A. Cavasin, E. G. Shesely, J. J. Yang, T. L. Reudelhuber, and X.-P. Yang Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1900 - H1907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. McBride AT1 receptors are necessary for eccentric training-induced hypertrophy and strength gains in rat skeletal muscle Exp Physiol, March 1, 2006; 91(2): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R Porrello, C. E Huggins, C. L Curl, A. A Domenighetti, T. Pedrazzini, L. M. Delbridge, and T. O Morgan Elevated dietary sodium intake exacerbates myocardial hypertrophy associated with cardiac-specific overproduction of angiotensin II Journal of Renin-Angiotensin-Aldosterone System, December 1, 2004; 5(4): 169 - 175. [Abstract] [PDF]
|
|
|
|
 |
|
 A. H. Maass, K. Ikeda, S. Oberdorf-Maass, S. K.G. Maier, and L. A. Leinwand Hypertrophy, Fibrosis, and Sudden Cardiac Death in Response to Pathological Stimuli in Mice With Mutations in Cardiac Troponin T Circulation, October 12, 2004; 110(15): 2102 - 2109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N C Sundgren, G D Giraud, P J S Stork, J G Maylie, and K L Thornburg Angiotensin II stimulates hyperplasia but not hypertrophy in immature ovine cardiomyocytes J. Physiol., May 1, 2003; 548(3): 881 - 891. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Rugale, S. Delbosc, J.-P. Cristol, A. Mimran, and B. Jover Sodium restriction prevents cardiac hypertrophy and oxidative stress in angiotensin II hypertension Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1744 - H1750. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Delbosc, J.-P. Cristol, B. Descomps, A. Mimran, and B. Jover Simvastatin Prevents Angiotensin II-Induced Cardiac Alteration and Oxidative Stress Hypertension, August 1, 2002; 40(2): 142 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Segar, G. B. Dalshaug, K. A. Bedell, O. M. Smith, and T. D. Scholz Angiotensin II in cardiac pressure-overload hypertrophy in fetal sheep Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R2037 - R2047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. de Simone, F. Pasanisi, and F. Contaldo Link of Nonhemodynamic Factors to Hemodynamic Determinants of Left Ventricular Hypertrophy Hypertension, July 1, 2001; 38(1): 13 - 18. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Gordon, B. S. Davis, C. J. Carlson, and F. W. Booth ANG II is required for optimal overload-induced skeletal muscle hypertrophy Am J Physiol Endocrinol Metab, January 1, 2001; 280(1): E150 - E159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Katz, J. A. Opsahl, S. E. Wernsing, L. M. Forbis, J. Smith, and L. J. Heller Myocardial renin is neither necessary nor sufficient to initiate or maintain ventricular hypertrophy Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2000; 278(3): R578 - R586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Mandinov, F. R. Eberli, C. Seiler, and O. M. Hess Diastolic heart failure Cardiovasc Res, March 1, 2000; 45(4): 813 - 825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Paradis, N. Dali-Youcef, F. W. Paradis, G. Thibault, and M. Nemer Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling PNAS, January 18, 2000; 97(2): 931 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Lamb, H. P. Beyerbacht, A. van der Laarse, B. C. Stoel, J. Doornbos, E. E. van der Wall, and A. de Roos Diastolic Dysfunction in Hypertensive Heart Disease Is Associated With Altered Myocardial Metabolism Circulation, May 4, 1999; 99(17): 2261 - 2267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ehmke, J. Faulhaber, K. Munter, M. Kirchengast, and R. J. Wiesner Chronic ETA Receptor Blockade Attenuates Cardiac Hypertrophy Independently of Blood Pressure Effects in Renovascular Hypertensive Rats Hypertension, April 1, 1999; 33(4): 954 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bohlender, A. Fukamizu, A. Lippoldt, T. Nomura, R. Dietz, J. Menard, K. Murakami, F. C. Luft, and D. Ganten High Human Renin Hypertension in Transgenic Rats Hypertension, January 1, 1997; 29(1): 428 - 434. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||