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Hypertension. 1997;30:383-391

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(Hypertension. 1997;30:383.)
© 1997 American Heart Association, Inc.

Articles

Contractile Systolic and Diastolic Dysfunction in Renin-Induced Hypertensive Cardiomyopathy

Markus Flesch; Frank Schiffer; Oliver Zolk; Yigal Pinto; Stephan Rosenkranz; Claudia Hirth-Dietrich; Georg Arnold; Martin Paul; Michael Böhm

From the Klinik III für Innere Medizin (M.F., F.S., O.Z., S.R., M.B.) and the Institut für Pathologie (G.A.) der Universität zu Köln, Cologne; the Institut für Klinische Pharmakologie der Freien Universität Berlin, Universitätsklinikum Benjamin Franklin (Y.P., M.P.); and the Institut für Pharmakologische Forschung der Bayer AG, Wuppertal (C.H.-D.), Germany.


*    Abstract
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Abstract The present study investigated whether functional, molecular, and biochemical alterations occurring in chronic heart failure can already be detected in compensated hypertensive cardiac hypertrophy. Force of contraction (isolated papillary muscle strip preparations), sarcoplasmic reticulum (SR) protein and myosin heavy chain isoform expression (Northern and Western blot analysis), myocardial fibrosis (collagen stains, hydroxyproline quantification), myocardial renin mRNA (RT-PCR), and angiotensin II levels and plasma aldosterone concentrations (radioimmunoassay) were studied in hypertrophied myocardium from transgenic rats harboring the mouse Ren-2d gene. Contraction and relaxation velocities of isolated papillary muscle strips were significantly reduced in cardiac hypertrophy. The ß-/{alpha}-myosin heavy chain ratio was significantly increased in the hypertrophied left ventricles, whereas SR Ca2+-ATPase (SERCA 2a) and phospholamban mRNA and protein levels were significantly decreased. The decrease in SERCA 2a was more pronounced than the decrease in phospholamban levels. There was no increased myocardial fibrosis. Left ventricular myocardial renin mRNA and angiotensin II concentrations, as well as plasma aldosterone levels, were higher in transgenic than in control rats. In hypertensive cardiac hypertrophy, myosin heavy chain isoform shift and reduction of SR protein levels are related to systolic and diastolic dysfunction, respectively. These alterations precede the development of myocardial fibrosis. Increased myocardial renin mRNA and angiotensin II concentrations suggest that an activated tissue renin-angiotensin system might contribute to these alterations. Since the alterations in compensated cardiac hypertrophy apparently precede those in chronic heart failure, they might accelerate the transition from hypertrophy to failure and could therefore be targets for pharmacological interventions.


Key Words: hypertrophy • heart failure • hypertension, genetic • renin-angiotensin system • sarcoplasmic reticulum


*    Introduction
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 *Introduction
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Cardiac hypertrophy is an adaptive process of the heart to reduce wall stress when an increased pressure load is imposed on the myocardium.1 The increase in wall thickness is accompanied by a number of biochemical and molecular changes of the myocardial cells, such as a shift to fetal phenotype gene expression like a change of MHC phenotype2 and alterations in the intracellular Ca2+ handling, most likely due to an impaired activity or reduction of the amount of SR proteins.3 In addition, alterations in the nonmyocyte compartment of the myocardium have been observed, leading to excessive myocardial fibrosis.4 The consequence is an altered contractile performance of the heart.5 Indeed, cardiac hypertrophy has been regarded as a major risk factor and precursor of heart failure. However, the precise mechanisms that accelerate the progression from compensated hypertrophy to failure are still unknown.

Activation of the renin-angiotensin system has been postulated to play a major role in the pathophysiology of cardiac hypertrophy and failure.6 In vivo studies have demonstrated that angiotensin II increases left ventricular mass7 and contributes to cardiac phenotype modulation independently from its effect on arterial pressure.8 By in vitro studies, it has been shown that angiotensin II causes cardiac myocyte hypertrophy9 and modulates interstitial fibrosis.9 Since in some animal models of experimental heart failure only an activation of the tissue renin-angiotensin system has been observed, whereas the circulating renin-angiotensin system has been unaltered, a special role has been suggested for the tissue renin-angiotensin system.10

Recently, transgenic rats [TG(mREN2)27] became available that overexpress the mouse renin at particularly high concentrations in multiple tissues.11 These animals develop fulminant arterial hypertension, beginning in their fourth week of life,11 and cardiac hypertrophy.12 Expression of the transgene leads to an activation of the tissue renin-angiotensin system, with an increase in local angiotensin II concentrations, whereas circulating renin and angiotensin concentrations are suppressed.11 Thus, TG(mREN2)27 might be a suitable model for cardiac hypertrophy induced by an activation of the tissue and especially the cardiac renin-angiotensin system.

The aim of this study was to examine (1) whether disturbances of contractile performance occur already at the stage of compensated cardiac hypertrophy and (2) which molecular and biochemical alterations might possibly underlie alterations in cardiac function. Special emphasis was laid on the question of whether changes of extracellular matrix proteins are necessary for the development of contractile dysfunction. Since understanding of the pathophysiology of cardiac hypertrophy and failure in humans is the final aim, the question of how closely the alterations observed in this model of renin-induced hypertensive cardiomyopathy resemble changes in human cardiac hypertrophy and heart failure was addressed.


*    Methods
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Transgenic Animals
Transgenic animals [TG(mREN2)27] were bred in the animal laboratory of the Max Delbrück Zentrum for Molecular Medicine in Berlin. SP were obtained from the Laboratorium für Versuchstierkunde in Hannover (Germany). From their fifth week of life, animals were housed in the animal laboratory of the University of Cologne according to the guidelines of animal care of the State of Nordrhein-Westfalen (Germany). Experiments were performed at the age of 12 to 14 weeks. At this age, hypertension is fully established, as determined by the tail-cuff method according to Pfeffer et al13 [ie, TG(mREN2)27: 230±8 mm Hg, n=12, versus SP: 115±12 mm Hg, n=12]. Rats were killed by a blow on the head and the hearts were rapidly removed and arrested in ice-cold 0.9% NaCl. In TG(mREN2)27 compared with SP, there was a significant increase of absolute and relative heart weights [TG(mREN2)27: 1.82±0.03 g, 4.92±0.11 mg/g, versus SP: 1.42±0.03 g, 3.78±0.08 mg/g; P<.001]. This increase was entirely due to an increase in left ventricular weight, whereas right ventricular weights did not differ. Hearts of TG(mREN2)27 exhibited concentric hypertrophy but no dilatation. No signs of venous congestion were observed in any other organ, eg, there was no increase in the relative wet weights of the liver [TG(mREN2)27: 3.38±0.13 mg/g versus SP: 3.59±0.07 mg/g]. Moreover, in vivo determination of left ventricular pressure performed in a previous study revealed that TG(mREN2)27 at the age of 12 weeks have an increased systolic but no increased end diastolic pressure (M. Flesch, unpublished data, 1995).

Contraction Studies in Isolated, Electrically Driven Papillary Muscles
Functional experiments were performed on electrically driven (1 Hz) left ventricular papillary muscles. Muscle strips of uniform size with muscle fibers running approximately parallel to the length of the strips (diameter <1.0 mm, length 3 to 6 mm) were dissected under microscopic control in aerated bathing solution (for composition, see discussion following) at room temperature. The preparations were attached to a bipolar platinum stimulating electrode and suspended individually in 75-mL glass tissue chambers for recording isometric contractions. The bathing solution was a modified Tyrode’s solution containing (in mmol/L) NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, and glucose 5.0. It was continuously gassed with 95% O2/5% CO2 and maintained at 35°C; the pH was 7.4. Force of contraction was measured with an inductive force transducer (W. Fleck) attached to a Gould recorder. All preparations were allowed to equilibrate for at least 90 minutes in drug-free bathing solution. The solution was changed once after 45 minutes. During this period, each muscle was gradually stretched to the length at which force of contraction was maximal (Lmax, reached at approximately 5 mN). Once a stable Lmax was reached, the resting force was kept constant throughout the experiment. The preparations were electrically paced at 1 Hz with rectangular pulses of 5 ms duration (Grass stimulator SD 9). The voltage was about 20% greater than threshold. The following parameters were determined: force of contraction, peak rate of tension increase, and peak rate of tension decrease, the latter two parameters being obtained by differentiation of the force signal.

Isolation of Total RNA
Total RNA from frozen left ventricular tissue samples was prepared according to the protocol of Chomczynski and Sacchi.14 Between 50 µg and 100 µg of total RNA was obtained from 150 mg tissue. The mean yield did not significantly differ between myocardium from TG(mREN2)27 and SP.

RT-PCR Determination of Renin mRNA Levels
Left ventricular RNA (5 µg) was reverse transcribed according to standard protocols. For semiquantitative detection of renin mRNA, 10 pg of transcribed cDNA was taken for PCR with specific primers for renin (encoding for both rat and mouse renin) and GAPDH. The sequence of the primers was CCG TGA TCC TCA CCA ACT AC (sense) and AGA CGA CTT TGA AGG TCT GG (antisense) for renin and GAC AAG ATG GTG AAG GTC GG (sense) and GATG GAC TGT GGT CAT GAG C (antisense) for GAPDH. PCR conditions were denaturation over 5 minutes at 95°C, followed by 30 cycles for detection of renin mRNA and 25 cycles for detection of GAPDH mRNA, each cycle consisting of 45 seconds at 55°C, 45 seconds at 72°C, and 45 seconds at 94°C. The number of cycles was within the linear range of PCR. The PCR products were run on a 1% agarose gel stained with ethidium bromide, and the resulting bands were analyzed by using a computer-based imaging system. The resulting density of the renin band was expressed relative to the density of the GAPDH band.

Northern Blot Analysis
Total RNA (10 µg) was separated in a 6% formaldehyde/1.2% agarose gel, blotted on nylon membranes by overnight capillary blotting, and fixed by UV irradiation. Blots were prehybridized in 5x SSC, 5x Denhardt’s solution, 11.1 mol/L (50%) formamide, 1% SDS, 50 mmol/L sodium phosphate, pH 6.8, 10% dextran sulfate, and 100 µg/mL salmon sperm DNA at 42°C. Hybridization was performed in 50% formamide solution at 42°C for 16 hours. Membranes were successively hybridized with a 2-kb SERCA 2a cDNA fragment encoding for rabbit heart SERCA 2a (BamHI-BamHI, provided by D.H. MacLennan, Toronto, Canada15 ), a 0.5-kb phospholamban cDNA fragment encoding for rabbit phospholamban (EcoRI-BamHI, provided by D.H. MacLennan, Toronto, Canada16 ), and a 0.7-kb cDNA fragment (Pst I-Pst I) encoding for ANF17 (donated from Suntory Limited, Osaka, Japan). The cDNA for rabbit SERCA 2a showed an 87% homology to the rat heart sequence,18 and the cDNA for rabbit phospholamban showed a 79% homology to rat phospholamban.19 The fragments were excised from the plasmid vector with the appropriate restriction enzymes, separated from the vector DNA on a 1% low-melt agarose gel, and labeled with [{alpha}-32P]dCTP (Amersham Buchler) using the Multiprime DNA labeling kit from Amersham Buchler. The concentration of the respective labeled probe in the hybridization solution was 1x106 cpm/mL. After hybridization, membranes were successively washed twice in 2x SSC/0.1% SDS at room temperature at 42°C for 15 minutes and twice in 0.2x SSC/0.1% SDS at 68°C for 45 minutes. Standardization was performed by hybridization of the same membrane using a 40-base single-stranded synthetic oligonucleotide probe for GAPDH (Dianova). Hybridization conditions were the same as described above. Stringency washes were performed with 2x SSC/0.1% SDS at room temperature, for 30 minutes with 2x SSC/0.1% SDS at 65°C, and twice for 5 minutes with 2x SSC/0.1% SDS at room temperature. Membranes were exposed to Kodak films (Kodak X-OMAT, Kodak Inc). Quantification of the signals was performed by densitometric analysis using the Image Quant Densitometric System (Molecular Dynamics).

Myocardial Protein Preparation
Myocardial tissue (3 to 5 g) was thawed on ice and chilled in 10 mL ice-cold homogenization buffer (in mmol/L: Tris-HCl 20, EDTA 1, and DTT 1, pH 7.4) including the protease inhibitors soybean trypsin inhibitor (2 µg/mL), benzamidine (3 µmol/L), pepstatin (1 µmol/L), leupeptin (1 µmol/L), and PMSF (0.3 µmol/L). Connective tissue was trimmed away and the remaining tissue minced with scissors and homogenized by hand for 1 minute with a glass-glass homogenizer. The suspension was centrifuged at 480g for 15 minutes. The supernatant was diluted with the same volume of 1 mol/L KCl and incubated on ice for 15 minutes. The solution was centrifuged at 100 000g for 30 minutes. The supernatant was discarded and the remaining pellet rehomogenized in 2 mL of homogenization buffer. Finally, the solution was centrifuged again at 100 000g for 30 minutes. The obtained pellet was dissolved in 1 mL homogenization buffer. For determination of {alpha}-MHC and ß-MHC protein levels, the homogenized myocardium was centrifuged at 100 000g for 30 minutes. The supernatant was discarded and the remaining pellet centrifuged again at 100 000g for 30 minutes. The remaining pellet was dissolved in 5 mL of homogenization buffer.

Immunoblotting Techniques
For detection of MHC isoforms, samples of myocardial proteins were denatured by heating to 95°C for 5 minutes in electrophoresis buffer containing 4% SDS, 50 mmol/L Tris-HCl, pH 6.8, 2.7 mol/L (20%) glycerol, 0.1 mmol/L pyronin Y, and 10 mmol/L DTT. For detection of phospholamban, protein samples were denatured for 30 minutes to obtain monomeric proteins. The amount of protein loaded on the gels was 5 µg for MHC detection, 25 µg for SERCA 2a, and 10 µg for phospholamban. Separation of proteins was performed by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane (0.2 µm) by semidry electrophoretic blotting, using an LKB 2117-250 Novablot electrophoretic transfer kit (LKB-Pharmacia; 0.8 mA/cm2, 90 minutes) with a discontinuous buffer system. The sheets were immersed in 100 mL of 5% low-fat dry milk powder in PBS buffer (KH2PO4 100 mmol/L; NaCl 137 mmol/L; KCl 2.68 mmol/L, NaH2PO4xH20 10.44 mmol/L; pH 7.4) and shaken for at least 1 hour at room temperature. After washes in PBS/Tween 20 (0.5% vol/vol), sheets were incubated with the first antibody. Phospholamban was detected by using a phospholamban monoclonal antibody (A1, diluted 1:500) raised against purified phospholamban from canine hearts (Upstate Biotechnology Inc). SERCA 2a was detected by using a monoclonal specific antibody raised in mouse (dilution 1:500, Dianova). For detection of {alpha}-MHC and ß-MHC, monoclonal specific antibodies raised in mouse against bovine MHC isoforms (diluted 1:500, kindly provided by Professor J.J. Leger, Montpellier, France) were used. After repeated washes, immunoreaction was continued by incubation of the nitrocellulose sheets with the respective peroxidase-conjugated goat secondary antibody. Detection was performed by using the enhanced chemiluminescence assay (ECL kit, Amersham). After exposure to X-ray film (Kodak X-OMAT AR, Kodak Inc), signals were quantified by two-dimensional densitometry (Image Quant Densitometric System, Molecular Dynamics).

Myocardial Collagen Staining
Histological examinations were performed in cryostat sections and sections from paraffin-embedded specimens of left ventricular myocardium of five control animals and seven TG(mREN)27. Besides hemalain eosin, the following connective tissue stains were applied: van Gieson’s and Masson’s trichrome stain for paraffin sections and Engel’s trichrome stain for cryostat sections.

Determination of Myocardial Hydroxyproline Concentrations
Quantification of left ventricular myocardial hydroxyproline concentrations was performed according to the method of Prockop and Udenfriend.20 Left ventricular myocardial specimens of approximately 90 mg weight were dried and hydrolyzed in 6N HCl at 100°C for 22 hours. The hydrolyzed material was dried and reconstituted in 5 mL H2O. Hydrolysate (200 µL) was mixed with 200 µL ethanol and 200 µL chloramine T solution (1.4% in citrate buffer) and allowed to oxidize for 20 minutes at room temperature. Ehrlich’s reagent (400 µL), containing 12 g p-dimethylaminobenzaldehyde in 40 mL ethanol+2.7 mL H2SO4, was added. After 3 hours of incubation at 37°C, extinction at 573 nm was measured.

Determination of Myocardial Angiotensin II Concentrations
Myocardial tissue was thawed on ice and chilled in 10 mL ice-cold homogenization buffer as described earlier. After centrifugation (484g, 15 minutes), the supernatant was diluted with 2 mL of ice-cold 1 mol/L KCl and centrifuged at 100 000g for 30 minutes. The resulting supernatant was purified with Sep-Pak columns. The eluates were lyophilized and the dry residues dissolved in 1.2 mL Tris-HCl reconstitution buffer (pH 7.4). Angiotensin II was determined with a commercially available RIA according to the instructions of the manufacturer (Biermann). The sensitivity of the RIA was 0.7 pg/mL, and the cross-reactivity of the angiotensin II antibody was 0.14% for angiotensin I.

Determination of Plasma Aldosterone Concentrations
Plasma aldosterone concentrations were determined by using a commercially available RIA according to the instructions of the manufacturer (IBL).

Miscellaneous
Protein was determined according to Lowry et al21 and 5'-nucleotidase activity according to the method of Dixon and Purdom.22

Materials
Antibodies, if not indicated otherwise, were from Sigma- Aldrich. Restriction enzymes were purchased from Boehringer. All other chemicals were of analytical grade or the best grade commercially available.

Statistics
Data shown are mean±SEM. Statistical significance was estimated with Student’s unpaired t test. A value of P<.05 was considered significant.


*    Results
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 *Results
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Left Ventricular Myocardial Renin mRNA and Angiotensin II Concentrations
According to semiquantitative mRNA analysis, renin mRNA levels were significantly (P<.001) increased in the left ventricles of TG(mREN2)27. After 30 PCR cycles, renin expression was hardly detectable in the control hearts, whereas a strong PCR signal was obtained from the hypertrophied hearts (Fig 1). The PCR signals for GAPDH obtained after 25 PCR cycles were not significantly different. Also, left ventricular myocardial angiotensin II concentrations were significantly increased in TG(mREN 2)27 compared with control rats (in picograms per gram tissue: 83±13, n=6 versus 49±6, n=6; P<.05).



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  		Figure 1. Semiquantitative detection of renin mRNA concentrations in left ventricular myocardium from TG(mREN2)27 (TG) and SP by RT-PCR. Top, PCR products run on a 1% agarose gel. In TG(mREN2)27, PCR of reverse-transcribed renin mRNA led to a strong amplification signal, whereas there was only a weak signal obtained from SP. GAPDH amplification showed no differences between TG(mREN2)27 and SP. Bottom, Mean densities (±SEM) of the renin bands in relation to the bands obtained for GAPDH, as measured by a computer-based imaging system.

Plasma Aldosterone Concentrations
Plasma aldosterone concentrations were significantly increased in TG(mREN2)27 compared with controls (1126±63 pg/mL, n=8, versus 822±89 pg/mL, n=10; P<.05).

Left Ventricular Myocardial mRNA Levels for ANF
Determination of left ventricular myocardial ANF mRNA levels by Northern blot analysis revealed a hybridization band at a position corresponding to an mRNA size of 900 bp. ANF mRNA was detectable in the hypertrophied myocardium of TG(mREN2)27, but hardly at all in undiseased myocardium (Fig 2).



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  		Figure 2. Northern blot analysis of left ventricular myocardial SERCA 2a, phospholamban (PLB), and ANF mRNA. Top, Representative hybridization signals obtained from TG(mREN2)27 (TG) and SP at the expected position, corresponding to an mRNA size of 4 kb, 3.3 kb, and 0.6 kb, respectively. The hybridization signal for GAPDH is shown as a control. Bottom, Mean values (±SEM) for SERCA 2a, PLB, and ANF mRNA levels in relation to GAPDH mRNA levels, as obtained by densitometric analysis.

Cardiac Force of Contraction
Isometric force of contraction was determined in isolated, electrically driven left ventricular papillary muscle strip preparations under baseline conditions. Mean maximum force of contraction was similar in both TG(mREN2)27 and controls [TG(mREN2)27: 4.11±0.7 mN, n=32, versus SP: 4.25±0.2 mN, n=37, NS]. The maximum rate of tension increase (+dF/dT) was significantly smaller in TG(mREN2)27 than in controls [TG(mREN2)27: 8.66±0.4 mN/s versus SP: 9.52±0.4 mN/s, P<.05], as was the maximum rate of tension decrease [-dF/dT, TG(mREN2)27: 7.49±0.3 mN/s versus SP: 9.11±0.4, P<.001], indicating an impairment of both systolic and diastolic contractile function.

{alpha}-MHC and ß-MHC Protein Levels
Western blot analysis of left ventricular myocardial {alpha}-MHC and ß-MHC protein revealed specific immunoreactive signals at the expected position corresponding to a molecular weight of 200 kD (Fig 3, top). Specificity of the antibody used for detection of {alpha}-MHC was controlled by immunostaining of purified ß-MHC (kindly donated by Prof J.J. Leger, Montpellier, France). Only the ß-MHC antibody, but not the {alpha}-MHC antibody, reacted with purified ß-MHC (not shown). Specificity of the ß-MHC antibody had been checked previously (Prof J.J. Leger, Montpellier, France, personal communication, 1995). According to densitometric analysis, there was a significant decrease in left ventricular myocardial {alpha}-MHC protein content in TG(mREN2)27 compared with controls (P<.01), whereas ß-MHC protein levels were not significantly increased in TG(mREN2)27 (Table). The ratio between ß-MHC and {alpha}-MHC was significantly higher in TG(mREN2)27 than in controls (P<.01; Fig 3, bottom).



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  		Figure 3. Immunochemical detection (Western blot analysis) of {alpha}-MHC and ß-MHC isoforms in left ventricular myocardium from renin transgenic rats (TG) and SP. Top, Representative signals occurring at a position corresponding to a molecular weight of {approx}200 kD. Bottom, Mean values (±SEM) for {alpha}-MHC and ß-MHC protein levels, as obtained from densitometric analysis and mean ratio between the isoforms (ß-MHC/{alpha}-MHC).


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  		Table 1. SR Protein Levels in Left Ventricular Myocardium of 12-Week-Old Renin Transgenic Rats and Age-Matched Control Rats

SERCA 2a and Phospholamban mRNA Levels
Northern blot hybridization of left ventricular myocardial total RNA from transgenic and control animals with the 2-kb cDNA fragment encoding for SERCA 2a and a 0.5-kb cDNA fragment encoding for rabbit phospholamban revealed single hybridization signals at the expected position, corresponding to an mRNA size of approximately 4 kb and 3.3 kb, respectively (Fig 2, top). According to densitometric analysis, mean left ventricular levels of SERCA 2a mRNA and phospholamban mRNA related to GAPDH mRNA levels were significantly decreased in TG(mREN2)27 compared with SP (Fig 2, bottom). The intensity of the GAPDH hybridization signals as determined by densitometry did not differ between transgenic and control hearts, indicating that there was no difference in GAPDH mRNA levels between the groups.

SERCA 2a and Phospholamban Protein Levels
Immunochemical detection of SERCA 2a and phospholamban protein levels by Western blot analysis using specific monoclonal antibodies revealed a single immunoreactive band at the expected position, corresponding to a molecular weight of {approx}115 kD and {approx}6 kD, respectively (Fig 4, top). According to densitometric analysis, mean protein levels of SERCA 2a and phospholamban were significantly decreased in TG(mREN2)27 compared with control (P<.001 and P<.001, respectively; Fig 4, bottom). The reduction of phospholamban was less pronounced than the reduction of SERCA 2a protein, resulting in an inverse SERCA 2a/phospholamban ratio (Table). However, this inversion was not significant. The results were similar when the oligomeric form ({approx}29 kD) or the monomeric form ({approx}6 kD) of phospholamban was analyzed. The reductions in SR protein levels were also significant when densitometric values were related to 5'-nucleotidase activity as a marker for the amount of membrane protein or to ß-MHC levels as a marker for the amount of contractile proteins (Table).



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  		Figure 4. Immunochemical detection (Western blot analysis) of SERCA 2a ({approx}105 kD) and the monomeric form of phospholamban (PLB, {approx}6 kD) in left ventricular myocardium from TG(mREN2)27 (TG) and SP. Top, Representative immunochemical signals. Bottom, Mean protein values (±SEM), as determined by densitometric analysis.

Collagen Staining of Left Ventricular Myocardial Tissue
Collagen staining of left ventricular myocardial sections revealed no major differences between hypertrophied and control myocardium. All control animals exhibited an inconspicuous myocardium with regularly distributed and sparse perivascular and interstitial collagenous connective tissue (Fig 5a). In TG(mREN2)27, the myocyte diameter was slightly increased, but there was no increase in the amount of interstitial tissue (Fig 5b). Only in two of five hypertrophied left ventricles were there some subendocardial microfoci of diffuse interstitial fibrosis (Fig 5c). The other three hypertrophied ventricles did not show any alterations in their collagen pattern compared with control ventricles.



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  		Figure 5. Collagen stains of left ventricular tissue sections of TG(mREN2)27 and SP. a, Left ventricular myocardium of a 12-week-old control animal. The sparse perivascular and interstitial connective tissue is barely perceptible even in a special connective tissue stain (van Gieson’s, x575). b, Left ventricular myocardium of a 12-week-old TG(mREN2)27 exhibiting the same sparse interstitial and perivascular connective tissue as the control myocardium (van Gieson’s, x575). Notice slightly hypertrophied cardiac myocytes. c, Small subendocardial area of diffuse interstitial fibrosis between individual myocytes, as found in two of five hypertrophied hearts (Masson’s, x575).

Quantification of Left Ventricular Myocardial Hydroxyproline Content
There was no significant difference between mean hydroxyproline concentrations in the hypertrophied and nonhypertrophied left ventricles (heart weight: TG(mREN2)27: 2.30±0.1 mg/g, n=5, versus SP: 2.02±0.1 mg/g, n=7, NS). Assuming that collagen contains 14% hydroxyproline and thus that the hydroxyproline content parallels the total collagen content, there is no evidence for an increase in myocardial collagen concentrations.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Cardiac hypertrophy due to long-standing arterial hypertension is associated with a high incidence of heart failure.23 Indeed, one might assume that cardiac hypertrophy and chronic heart failure form one disease at different stages. In this context, it is of interest which alterations regarded as typical for heart failure occur already at the stage of compensated cardiac hypertrophy. These alterations themselves might contribute to the progression of the disease. Experiments using human hypertrophied myocardium are sparse owing to the limited access to human tissue. Thus, there is need for suitable animal models of cardiac hypertrophy, which allow the study of functional, biochemical, and molecular alterations at this early stage of the disease.

The Ren-2 transgenic rat [TG(mREN2)27] is an animal model of fulminant arterial hypertension and cardiac hypertrophy11 12 characterized by an activation of the tissue renin-angiotensin system.11 Recently, a reduced inotropic responsiveness to isoproterenol has been demonstrated in hypertrophied hearts of TG(mREN2)27, which might be explained by a heterologous desensitization of the adenylyl cyclase.24 Interestingly, changes in the ß-adrenoceptor adenylyl cyclase system observed in this model were closer to those in human heart failure than changes in any other studied model of cardiac hypertrophy.24 In this study, functional alterations of myocardial performance in TG(mREN2)27 and possible biochemical and molecular causes were characterized at a stage when there is a significant hypertrophy of the left ventricle but no signs of overt heart failure. At this stage of compensated cardiac hypertrophy, there is already an impairment of systolic and diastolic contractile function in TG(mREN2)27, which can at least in part be explained by a shift in myosin isoform expression and a decrease in SERCA 2a and phospholamban mRNA and protein levels. Interestingly, there were no signs of increased myocardial fibrosis at this stage of the disease.

Parameters of contractile function were determined in isolated papillary muscles in vitro. This technique allows the muscles to be stretched to the length at which force of contraction is maximal. This is important, because in hypertensive cardiomyopathy the heart is used to an increased workload. Even under optimized conditions, maximum rates of tension increase and decrease were significantly decreased in papillary muscle preparations from TG(mREN2)27. A similar prolongation of the time course of contraction has been described for isolated left ventricular papillary muscle strips from failing human hearts.25 Thus, in the stage of compensated hypertensive cardiac hypertrophy, there is already an impairment of systolic as well as diastolic function, which precedes the onset of heart failure.

One explanation for systolic contractile dysfunction in TG(mREN2)27 is a change in the ß-MHC/{alpha}-MHC ratio, with an increase in the relative amount of ß-MHC.2 It is known that the predominant expression of the ß-MHC phenotype is associated with not only a decreased myosin ATPase activity and shortening velocity26 but also an energetically more efficient contraction.27 Interestingly, the altered ß-MHC/{alpha}-MHC ratio in TG(mREN2)27 was due to a decrease in {alpha}-MHC, not an increase in ß-MHC protein. This observation resembled findings in hearts of spontaneously hypertensive rats.28 However, the change in MHC isoform expression in the latter study was present only in the stage of chronic heart failure and not of cardiac hypertrophy.28

Disturbances, especially of diastolic contractile performance, have been related to an impaired uptake of Ca2+ into the SR, as has been demonstrated in different animal models29 and in human dilated cardiomyopathy.30 Despite partly contradictory results in humans,31 it has been suggested that a reduction in the density of SERCA 2a molecules and a reduced ATPase activity could be responsible for this alteration in diastolic Ca2+ handling.32 33 In the present study, a significant reduction in SERCA 2a and phospholamban mRNA and protein levels was observed in the hypertrophied left ventricular myocardium of TG(mREN2)27, which goes along with the decrease in papillary muscle shortening velocity. The decrease in SERCA 2a protein levels by 77% was more pronounced than the decrease in phospholamban protein levels by 67%. This relative increase of phospholamban protein levels might be important, since phospholamban in its unphosphorylated state is known to inhibit SERCA 2a activity.34 Decreased phospholamban gene expression leads to an attenuation of this inhibitory effect,35 whereas enhanced phospholamban gene expression increases it.36

Interestingly, the decrease in SERCA 2a mRNA levels was even more pronounced in myocardium from 16-week-old TG(mREN2)27 than in the 12-week-old animals in the present study.37 One might hypothesize that there is a progressive decrease in SERCA 2a gene expression, as has been demonstrated recently in a model of pressure-overload cardiac hypertrophy.38 A progressive decrease of SERCA 2a activity would explain why maximum force of contraction was not altered in the hypertrophied hearts of 12-week-old TG(mREN2)27. Given that maximum force of contraction correlates with the amount of Ca2+ taken up into the SR during diastole and released during systole, Ca2+ uptake into the SR might be delayed, but not sufficiently reduced to produce diminished Ca2+-triggered Ca2+ release during the next beat.

Despite alterations within cardiac myocytes, changes in the nonmyocyte compartment of the myocardium, especially increased cardiac fibrosis and ventricular stiffening, have been demonstrated to be important causes of cardiac contractile dysfunction.4 Interestingly, there was virtually no sign of increased myocardial fibrosis in the hypertrophied hearts of TG(mREN2)27, as determined by collagen staining of myocardial slices and quantification of hydroxyproline content. These findings are in accordance with recent observations by Villareal et al,37 who observed even a decrease in left ventricular hydroxyproline levels and in collagen area fraction in TG(mREN)27. Only perivascular fibrosis seemed to be increased at this age.37 Bachmann et al12 observed some foci of perivascular fibrosis predominantly in the hypertrophied hearts of male TG(mREN2)27, suggesting a special role of androgen steroids in this condition.

The absence of myocardial fibrosis distinguishes renin transgenic rats from other models of cardiac hypertrophy or heart failure. In spontaneously hypertensive rats, Boluyt et al28 found that the transition from cardiac hypertrophy to heart failure was characterized by an increase in pro-{alpha}1(I) and pro-{alpha}1(III) collagen and in fibronectin mRNA levels, as markers for an increasing interstitial fibrosis, whereas SERCA 2a mRNA levels remained unaltered. Similarly, Conrad et al39 described an increase in collagen concentrations and increased interstitial fibrosis in failing left ventricles of spontaneously hypertensive rats, which was accompanied by increased papillary muscle stiffness and impaired tension development. These recent observations led to the suggestion that myocardial fibrosis might be the distinctive marker between cardiac hypertrophy and heart failure.39 40 The results of this study demonstrate that alterations in the cardiac contractile cycle can occur independent from extracellular matrix changes. Indeed, Luo et al41 demonstrated that simply by altering phospholamban gene expression, contractile parameters of isolated myocytes and beating hearts can be influenced. Thus, cardiac fibrosis and myocardial stiffening are not the prerequisites necessary for the development of contractile dysfunction. In contrast, alterations in the level of SR and contractile proteins may precede cardiac fibrosis, and these alterations on the myocyte level can be sufficient to alter cardiac contractility.

Determination by RIA confirmed that in TG(mREN2)27, plasma aldosterone concentrations are significantly increased compared with control. Also, myocardial angiotensin II concentrations have been demonstrated to be increased in TG(mREN2)27, and previously catecholamine release from cardiac nerves has been demonstrated to be increased.24 All three hormones are important triggers for the development of cardiac fibrosis.28 42 43 In consequence, one might expect that collagen synthesis and myocardial fibrosis should be enhanced in TG(mREN2)27. One explanation for the obvious discrepancy between this theoretical consideration and the findings in this study might be the age of the transgenic animals, with myocardial fibrosis occurring at a later stage of disease. Villareal et al37 observed an increase in left ventricular transforming growth factor ß1 and collagen type I mRNA levels in 16-week-old TG(mREN2)27, which has been demonstrated to precede collagen deposition.44 Thus, the beginning of perivascular fibrosis observed by Villareal et al37 or the single foci of diffuse interstitial fibrosis in some of the hypertrophied hearts in this study might indicate initialized collagen deposition. On the other hand, not only have mineralocorticoid plasma levels been demonstrated to be increased in TG(mREN2)27 but there is also evidence for enhanced urinary excretion of deoxycorticosterone, 18-OH-corticosterone, and corticosterone.45 Deoxycorticosterone has been demonstrated to increase interstitial and perivascular fibrosis, whereas corticosterone B deposition has little effect on collagen deposition.46 Since mineralocorticoid receptors have a similar affinity for aldosterone, corticosterone, and cortisol,47 one might speculate whether enhanced corticosterone concentrations antagonize the proliferative effects of aldosterone and thus prevent or delay the onset of myocardial fibrosis in TG(mREN2)27.

Despite the well-defined monogenetic basis of hypertension in TG(mREN2)27, the factors finally contributing to the development of cardiac hypertrophy remain to be resolved. Those include increased workload on the myocardium and activation of the sympathetic nervous system or the renin-angiotensin system. Semiquantitative detection of renin mRNA levels in left ventricular myocardium of SP and TG(mREN2)27 revealed that there is a significant increase in renin mRNA levels in the latter as a consequence of transgene expression. Obviously, as further consequence, tissue angiotensin II concentrations have been found to be increased in the left ventricular myocardium of TG(mREN2)27. Thus, there is evidence for an activation of the tissue renin-angiotensin system in the myocardium, and one might speculate that some cardiac alterations found in this study might be caused by increased tissue angiotensin II concentrations. Indeed, many of the alterations found in the myocardium of TG(mREN2)27 have been shown to be inducible by angiotensin II treatment. Examples are the increase in left ventricular ANF mRNA levels or fibronectin levels, the increase in the ratio between ß-MHC and {alpha}-MHC gene expression,9 and the increase in transforming growth factor ß1 and type I and III collagen mRNA levels.8 Activation of the cardiac renin-angiotensin system might also contribute to the downregulation of SERCA 2a mRNA and protein levels in the hypertrophying heart of TG(mREN2)27. Normalization of angiotensin-converting enzyme has been demonstrated to lead to a normalization of SERCA 2a gene expression in failing rat hearts.48 In contrast, increased workload on the myocardium alone does not lead to a decrease in SERCA 2a mRNA levels, as has been demonstrated in spontaneously hypertensive rats.39 Since in TG(mREN2)27 the activation of the cardiac tissue renin-angiotensin systems occurs independently from the increase in arterial blood pressure, future studies using this animal model offer a unique chance to distinguish between the effects of pressure overload, pressure overload leading to activation of the renin-angiotensin system,49 and direct effects of increased tissue angiotensin II concentrations in the progression of cardiac hypertrophy.

In summary, in the stage of compensated cardiac hypertrophy due to renin-induced hypertension, an impairment of systolic and diastolic myocardial function occurs, which is accompanied by an MHC isoform shift and alterations of SR proteins. These alterations are independent of quantitative changes in extracellular matrix proteins. Thus, pathological changes within cardiac myocytes can be sufficient to cause cardiac contractile dysfunction. Since functional disturbances and alterations of SR proteins precede the onset of chronic heart failure, one might speculate whether these initial alterations might accelerate the progression from cardiac hypertrophy to chronic heart failure.


*    Selected Abbreviations and Acronyms
 

ANF = atrial natriuretic factor
MHC = myosin heavy chain
PCR = polymerase chain reaction
RIA = radioimmunoassay
RT-PCR = reverse-transcribed PCR
SERCA 2a = SR Ca2+-ATPase
SP = Sprague-Dawley control rats
SR = sarcoplasmic reticulum
TG(mREN2)27 = renin transgenic rats


*    Acknowledgments
 
This study was supported by the Deutsche Forschungsgemeinschaft through the Gerhard Hess Program (Dr Böhm). The work contains parts of the doctoral thesis of F. Schiffer (University of Cologne). The SERCA 2a cDNA fragment encoding for rabbit heart SERCA 2a and the phospholamban cDNA fragment encoding for rabbit phospholamban were provided by D.H. MacLennan, Toronto, Canada. The cDNA fragment encoding for ANF was donated by Suntory Limited, Osaka, Japan. Purified ß-MHC was kindly donated by Prof J.J. Leger, Montpellier, France.


*    Footnotes
 
Reprint requests to M. Flesch, MD, Klinik III für Innere Medizin der Universität zu Köln, Joseph Stelzmann-Straße 9, 50924 Cologne, Germany.

Received June 24, 1996; first decision August 5, 1996; accepted February 5, 1997.


*    References
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