Regulation of Fibrillar Collagen Gene Expression and Protein Accumulation in Volume-Overloaded Cardiac Hypertrophy
Background Interstitial collagen accumulation has been extensively demonstrated to be increased at both mRNA and protein levels in pressure-overloaded cardiac hypertrophy. However, few data are available regarding the effects of volume overload on myocardial collagens.
Methods and Results To determine whether the alterations of collagens may occur in volume-overloaded cardiac hypertrophy, we measured collagen types I and III mRNA levels and protein accumulation in left ventricular (LV) myocardium of rats at 3, 7, and 28 days after the creation of an aortocaval (AC) shunt. Eccentric LV hypertrophy was produced in rats with AC shunting. Northern blot analysis on RNA extracted from LV tissue indicated that the steady state mRNA levels for both type I and III collagen were persistently upregulated in AC shunt rats compared with sham-operated control rats. In contrast, the biochemical collagen protein concentration and morphometric collagen volume fraction were comparable between sham-operated control and AC shunt rats at any study time point. Furthermore, the immunohistochemical staining of types I and III collagen and Sirius red staining on myocardial tissue sections revealed no significant alterations in the distribution or density of fibrillar collagens in AC shunt rats. Tissue collagenase activity was not different between control and AC shunt rats after 28 days.
Conclusions Cardiac volume overload increases LV collagen mRNA as does pressure overload. However, in contrast to pressure-overloaded hypertrophy, the upregulation of collagen transcriptional activity does not result in subsequent myocardial fibrosis in volume-overloaded hypertrophy due to AC shunting. Therefore, the upregulation of collagen gene expression and protein accumulation might be different in pressure-overloaded and volume-overloaded hypertrophy.
It has been well documented that pressure-overloaded cardiac hypertrophy is accompanied by an increased accumulation of fibrillar collagens in the myocardium,1 which might increase myocardial stiffness and ultimately lead to ventricular dysfunction.2 3 The changes in interstitial collagens in response to pressure overload have been extensively documented at both protein and mRNA levels.4 Chapman et al4 demonstrated increased mRNA levels for types I and III collagens in the rat myocardium during the first week after abdominal aortic constriction. Villarreal and Dillmann5 showed a similar transient increase in LV collagen mRNA levels immediately after thoracic aortic banding. In contrast, volume overload caused by AC shunting for 2 months in dogs resulted in no significant changes in the LV collagen volume fraction.6 Similarly, Michel et al7 reported no changes in LV collagen density after 1 or 3 months of cardiac volume overload caused by AC shunting in rats. However, Ruzicka et al8 recently reported decreases in LV collagen after 4 to 10 weeks of volume overload by AC shunting in rats. The previous data concerning the alterations in collagen proteins or fibrosis are conflicting, and the changes in collagen mRNA levels were not evaluated in any studies of volume-overloaded hypertrophy. The above findings suggest that the differential regulation might occur in the biosynthesis of the myocardial collagen in pressure-overloaded and volume-overloaded cardiac hypertrophy.
To investigate the changes of collagen biosynthesis during volume-overloaded hypertrophy, we studied the expression of types I and III collagen genes as well as collagen protein levels in volume-overloaded rat myocardium.
LV volume overload was induced in rats according to the procedures described by Garcia and Diebold.9 Male Wistar rats with a weight of 180 to 250 g and an age of 6 to 7 weeks were used. All operative procedures were carried out under full surgical anesthesia with intraperitoneal administration of sodium pentobarbital (50 mg/kg). Under sterile conditions, a 5-mm segment of the abdominal aorta was exposed through a midline abdominal incision. AC shunting was produced with an 18-gauge disposable needle. The patency of the shunt was verified visually on the basis of the swelling of the vena cava and the mixing of arterial blood with venous blood. These animals were identified as AC shunt rats. We also performed sham operations in control rats using procedures identical to those for AC shunt rats except for hemodynamic interventions (sham-operated rats). The abdomen was closed surgically, and the animals were allowed to recover for 3, 7, and 28 days before the following studies. All procedures and the care of the animals were approved by the guidelines for Institutional Animal Care and Use of Laboratory Animals of Kyushu University School of Medicine.
At the time of the study, the rats were anesthetized with intraperitoneal administration of sodium pentobarbital (50 mg/kg). A median sternotomy was performed, and the heart was arrested in diastole with the intravenous administration of 1 mol/L KCl, rapidly excised, and placed into ice-cold saline to allow the heart to remain in diastole and to remove the blood. After removal of the atria and great vessels, the ventricles were blotted dry, the heart was quickly removed, and the atria and great vessels were trimmed away. The right ventricle (free wall) and LV (free wall plus septum) were separated and weighed. The LV was washed again in 0.1 mol/L ice-cold sodium phosphate, pH 6.0, until all visible blood was removed and then blotted dry. The middle slice of the LV was used for assessment of LV wall thickness and internal diameters as previously described by Tsoporis et al.10
Northern Blot Analysis
At the time of the study, myocardial tissue specimens were obtained from the LV free wall of the rats and used for RNA extraction, biochemical hydroxyproline assay, and morphometric analysis of collagens. Frozen LV myocardial tissue (100 to 150 mg) was homogenized with a Polytron homogenizer in a solution containing 4 mol/L guanidinium thiocyanate, and total RNA (100 to 150 μg) was isolated according to the methods of Chomczynski and Sacchi.11 RNA was quantified through absorbance at 260 nm, and the integrity was determined through examination of the 28S and 18S rRNA bands in ethidium bromide–stained agarose gels visualized under UV light. Poly(A)+–containing RNA was enriched with the use of oligo(dT)30-Latex [Oligotex-dt30(Super); Japan Synthetic Rubber Co].12 Poly(A)+–containing mRNA (1 μg) was denatured (60°C, 10 minutes) and size-fractionated through electrophoresis on 1% (wt/vol) agarose gels under denaturing conditions. RNA was transferred to nylon membranes (Hybond N; Amersham International) and immobilized with UV irradiation. Hybridization with cDNA probes was performed overnight at 42°C in buffer containing 50% formamide, 5× SSC buffer (1× SSC contains 0.15 mol/L NaCl, 0.015 mol/L sodium citrate), 5× Denhardt's solution (5× contains 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll), 5% (wt/vol) dextran sulfate, 0.1% SDS, and 100 μg/mL denatured salmon sperm DNA. The following cDNA clones were used: human collagen type I, α1 [α1(I)], clone Hf677,13 human collagen type III, α3 [α1(III)], clone Hf934,14 and human GAPDH, clone pHcGAP.15 All cDNAs were obtained from the American Type Culture Collection. The cDNA clones were radioactively labeled with the use of a random-prime DNA labeling kit (Boehringer-Mannheim Biochemica). [32P]dCTP (DuPont–New England Nuclear) was included in the reaction mixture to obtain a specific activity of 5 to 20×108 cpm/μg of DNA. Blots were washed in 2× SSC/0.1% SDS (55°C, 30 minutes) and 0.2× SSC/0.1% SDS (55°C, 15 minutes). All membranes were exposed at −70°C for varying time periods to X-Omat x-ray film (Eastman Kodak Co) with intensifier screens. Quantification of Northern blots was obtained on the basis of the integrated absorbance increase over background absorbance in a rectangular region of interest. Data were expressed as the densitometric intensity of the hybridization signals for types I and III collagen mRNA levels relative to the consistently expressed GAPDH mRNA.
The dried myocardial tissues (20 to 30 mg) were heated in 6 mol/L hydrochloric acid at 100°C overnight to hydrolyze collagen into its component amino acids. Hydroxyproline concentration was measured spectrophotometrically on the basis of its reaction with Ehrlich's reagent according to the methods described by Stegeman and Stalder.16 The concentration of tissue hydroxyproline was expressed as milligrams of hydroxyproline per gram of myocardial dry weight. Assays were performed in triplicate.
A transmural specimen of LV myocardium obtained from the free wall at the level of papillary muscles was fixed in 6% formaldehyde and embedded in paraffin. Morphometric analysis of collagen volume fraction was performed with 5-μm-thick tissue sections stained with Masson's trichrome.17 Briefly, each section was photographed under a microscope and magnified (final magnification, ×200). Three or four fields were randomly selected from one or two coronal sections in each animal. Thus, collagen volume fraction was measured at approximately five to seven fields for each animal. Within each field, segments representing connective tissue and muscle were identified and manually traced with a digitizing pad and a computer to calculate the traced area. Collagen volume fraction was then calculated for the heart as the sum of all connective tissue areas divided by the sum of all connective tissue and muscle areas in all fields. Collagen surrounding intramyocardial coronary arteries was excluded from the calculation. The operator was blinded to the experimental group during the analysis.
For visualization of the appearance of types I and III collagen, tissue staining was performed with avidin-biotin assay. Specificity of polyclonal antibodies directed against rat collagen types I and III (1:200 dilution; Chemicon International, Inc) had been evaluated with both a solid-phase radioimmunoassay18 19 and immunoblot analysis20 by Vialle-Presles et al.21 We further confirmed the specificity of the antibodies used in this study through immunoblotting analysis according to the procedures described by Yoshioka et al.22 Briefly, the samples of type I and III collagens were solubilized with the pepsin digestion from the newborn rat skin and purified through differential salt precipitation.23 A mixture of type I and type III collagens (15 μg) was applied onto the gel, and collagen α-chains were separated through interrupted gel electrophoresis with 7% polyacrylamide gel.24 The samples were then transferred to polyvinylidene difluoride membranes (Immobilon; Millipore). After blocking with 5% nonfat milk in PBS for 30 minutes, the membrane was incubated with a 1:500 dilution of antiserum directed against rat type I or type III collagen for 2 hours at room temperature. After washing with PBS, the membranes were further incubated with peroxidase-labeled anti-rabbit IgG (1:500 dilution; Jackson ImmunoResearch Laboratories, Inc) for 1 hour. After washing, an immunoreaction was detected with Konica Immunostaining HRP-100 (Konica, Inc). The polyclonal antibodies to collagen type I or type III recognized mainly each isoform of collagens (Fig 1⇓). However, the antibody to collagen type III slightly interacted with collagen type I (Fig 1⇓, lane 3). In a control experiment with normal rabbit serum instead of antisera, no bands were detected.
The frozen sections (4 μm thick) were cut at −20°C in a cryostat and mounted onto glass slides coated with poly-l-lysine. After blocking with 2% nonfat milk in PBS for 30 minutes, the sections were incubated with antibodies directed against rat collagen types I and III for 1 hour at room temperature. After extensive washing in PBS, the sections were incubated with a biotinylated secondary antibody for 10 minutes at room temperature and further incubated with peroxidase-conjugated streptavidin. After extensive washing with PBS, sections were incubated with 0.1% 3,3′-diaminobenzidine tetrahydrochloride (Merck) for color development. After further washing in buffer, the slides were lightly counterstained with hematoxylin. As a negative control, parallel sections were incubated with IgG prepared from rabbit preimmune serum. Positive staining with 3,3′-diaminobenzidine tetrahydrochloride appeared as brownish-black.
Sirius Red Staining
To confirm our findings of immunohistochemistry, we stained collagens in paraffin-embedded tissue sections (5 μm thick) with Sirius red. Briefly, after being washed in tap water (10 minutes) and in distilled water (2 minutes twice), the sections were treated with 0.2% phosphomolybdic acid for 5 minutes and then with 0.1% Sirius red solution for 90 minutes. The sections were further treated with 0.01N HCl for 2 minutes and dehydrated. Types I and III collagen fibers could be identified as yellow-red and green fibers, respectively, through polarization microscopy with appropriate band-pass filters.25 This method allowed us to observe both type I and type III collagen fibers on the same section and thus compare the spatial distribution of these two collagens.
Tissue Collagenase Measurements
For tissue collagenase measurement, we created sham-operated control (n=5) and AC shunt (n=5) rats as described above. The preparation of samples and the collagenase assay were performed according to the methods previously described by Charney et al26 with some modifications. The heart was perfused with potassium phosphate buffer (pH 7.4, 4°C) to remove plasma protease inhibitors. Approximately 100 to 200 mg myocardial tissue was suspended in 2 mL of 50 mmol/L Tris-HCl buffer containing 0.1% Triton-X, 0.2 mol/L NaCl, and 5 mmol/L CaCl2. The tissue was minced and homogenized for 15 seconds at 4°C and then sonicated for 15 seconds. The homogenate was centrifuged at 2000g for 10 minutes at 4°C, and the resultant supernatant was used for collagenase assay. Collagenase assay was performed in duplicate with appropriate controls. The reconstituted collagen fibril assay contained fluorescein isothiocyanate–labeled guinea pig type I collagen as substrate (50 μL) in 50 mmol/L Tris-HCl buffer containing 0.2 mol/L NaCl and 5 mmol/L CaCl2 at 37°C (Yagai Co). Tissue collagenase activity was normalized and expressed in units per hour per 100 mg of tissue, in which 1 unit of collagenase degrades 1 μg of native collagen at 37°C.
All values are presented as mean±SEM. An unpaired Student's t test was used to compare values between sham-operated control and AC shunt rats. Differences were considered statistically significant at a level of P<.05.
Characteristics of Experimental Models
LV weight–to–body weight ratio was comparable between sham-operated control (n=6) and AC shunt (n=7) rats after 3 days (Fig 2⇓). LV weight–to–body weight ratio in rats with AC shunt at 7 (n=7) and 28 (n=4) days after surgery was significantly increased compared with sham-operated control rats (n=5 for 7 days, n=5 for 28 days). Fig 3⇓ shows LV internal diameter (Fig 3⇓A) and LV wall thickness (Fig 3⇓B) in sham-operated control and AC shunt rats at 3 (n=5 for sham; n=5 for AC shunt), 7 (n=4 for sham; n=7 for AC shunt), and 28 (n=4 for sham; n=5 for AC shunt) days after surgery. An AC shunt increased the LV internal diameter by 28 days (Fig 3⇓A), with only little effect on LV wall thickness at the same time points (Fig 3⇓B). These data indicate that the eccentric cardiac hypertrophy is produced in rats by an AC shunt.
Collagen Gene Expression
Fig 4⇓ shows the representative Northern blot analysis of the steady state mRNA levels for collagen types I and III and GAPDH at 3, 7, and 28 days after AC shunting. As is evident, mRNA levels for both types I and III collagens (relative to the consistently expressed mRNA encoding GAPDH) were substantially increased as early as 3 days after AC shunting (Fig 5⇓). Importantly, the upregulation of these transcript levels was persistent until 28 days after AC shunting.
Biochemical and Morphological Collagen Concentration
Total tissue collagen concentration, as derived from the tissue hydroxyproline concentration, was comparable between sham-operated control (n=6, n=5, and n=5) and AC shunt (n=7, n=7, and n=4) rats at 3, 7, and 28 days, respectively, after surgery (Fig 6⇓A). In addition, the collagen volume fraction in AC shunt rats did not differ significantly compared with the control values (Fig 6⇓B). To exclude any lag-time between the upregulation of mRNA expression and the accumulation of the protein, we measured hydroxyproline concentration in rats 8 weeks after the creation of an AC shunt. Even at a later time, there were no significant differences in hydroxyproline concentration between control (3.1±0.1 mg/g dry tissue; n=5) and AC shunt (3.2±0.1 mg/g dry tissue; n=6) rats. Thus, in contrast to the persistent upregulation in mRNA levels, no significant increases occurred in tissue collagen accumulation on the basis of biochemical or morphometric determinations at any study time points.
Qualitative assessment of the immunostained myocardial sections revealed anti-collagen types I and III antibody staining corresponded with fibers within the interstitial space. Representative immunohistochemical micrographs of sham-operated control and AC shunt rats for 28 days are given in Fig 7⇓. There were no detectable differences in the staining of fibrillar types I and III collagen in the LV myocardium between control and AC shunt rats. We also examined the distribution of collagen by using a monoclonal antibody to human collagen type III, which had been shown to cross-react specifically to rat collagen type III (kindly provided by Dr Akira Ooshima, First Department of Pathology, Wakayama Prefectural Medical College). The distributions of collagen stained by the two different antibodies against collagen type III were similar (data not shown).
In Sirius red–stained myocardial tissue sections, interstitial fibrosis was identified with polarized light (Fig 8⇓). With band-pass filters, yellow-red fibers (type I collagen) and green fibers (type III collagen) could be observed on the same tissue sections. Type I and type III collagen fibers were colocalized in all interstitial areas of collagen deposition. The densities of interstitial yellow-red and green collagen fibers were not apparently different between sham-operated control and AC shunt rats. Even though there is an apparent discrepancy in the density and distribution between the Sirius red–positive collagen fibers and the collagen matrix labeled by antibodies, we consider our methods of Sirius red staining to still be valid because a similar pattern of birefringent collagen fiber distribution has been reported with the use of the same techniques6 and the Sirius red–stained fibers appeared less intense than those stained with antibodies in parallel tissue sections.27
Tissue Collagenase Activity
Tissue collagenase activity in AC shunt rats for 28 days was 32.6±3.6 units·h−1·100 mg of tissue−1 (n=5), which was not significantly (P>.05) different from that in sham-operated control rats (27.7±1.2 units·h−1·100 mg of tissue−1; n=5).
The present study demonstrated that mRNA levels encoding types I and III fibrillar collagen were increased as early as 3 days after AC shunting, which was maintained until 28 days. However, myocardial collagen accumulation, determined on the basis of biochemical hydroxyproline concentration and morphological collagen volume fraction, was not altered in volume-overloaded hypertrophy.
Collagen Protein and Gene Expression in Volume-Overloaded Hypertrophy
The present study demonstrated that interstitial collagen protein accumulation did not change in response to volume overload induced by AC shunting. This is consistent with previous reports using a similar model in the dog or rat. Michel et al7 reported that the hydroxyproline concentration of the hypertrophied heart was not increased with the volume overload due to an AC shunt. Weber and colleagues6 have also shown that the fibrillar collagen matrix remains normal in the dilated LV secondary to an AC shunt. However, Ruzicka et al8 recently reported a decrease in hydroxyproline concentration in the same rat model of volume-overloaded hypertrophy. The collagen concentration was increased in a cat model of right ventricular volume-overloaded hypertrophy due to atrial septal defect.28 Although the reasons for the discrepancies among these studies are unclear, it may be in part due to the differences between the animal models and the degree of volume overload.
Despite the lack of increase in collagen protein (hydroxyproline and fibrosis) in LV myocardium from rats with an AC shunt, the steady state levels of their corresponding mRNAs were demonstrated to be increased in the present study. It appears reasonable, based on the earlier observations in pressure-overloaded hypertrophy, to predict that collagen protein levels would also increase in parallel with their mRNA levels in volume-overloaded hypertrophy. However, this did not occur in our rat model of AC shunting. A similar discrepancy between collagen protein synthetic rates and the protein accumulation has been demonstrated in thyroxine-induced cardiac hypertrophy, in which a threefold increase in collagen synthesis after 8 weeks of daily thyroxine administration to juvenile rats resulted in the decrease (rather than increase) in tissue collagen concentration.29
The mechanisms responsible for the absence of the increase in collagen accumulation and interstitial fibrosis despite the persistent upregulation in collagen gene expression remain undetermined in the present study. One possible mechanism is the concomitant increase in the degradation rates of collagen proteins. Alternatively, the increase in collagen mRNA levels, which could result from either an increase in transcription rates or a decrease in mRNA degradation rates, might reflect compensatory responses by cardiac fibroblasts to the increased rates of degradation of collagen proteins. We also could not exclude the possibility that the increase in intracellular degradation of collagen30 might account for the discrepancy between collagen biosynthesis and tissue accumulation in AC shunt rats. To determine the role of collagenase in the degradation of collagen protein in this model, we measured tissue collagenase activity in LV myocardium obtained from sham-operated control and AC shunt rats. However, the tissue collagenase activities in AC shunt rats were not increased compared with those in control rats, which indicates that local sources of collagenase are unlikely to play a significant role in the discrepancy between the collagen synthesis and protein accumulation in volume-overloaded cardiac muscle. Further clarification will ultimately require the quantification of collagen synthesis and degradation rates in volume-overloaded hypertrophy.
Differences in Collagen Biosynthesis Between Pressure Overload and Volume Overload
Collagen gene expression can be potentiated not only in pressure overload but also in volume overload. This indicates that the increases in diastolic wall stress can also upregulate collagen transcription. The increases in collagen type I and III mRNAs caused by systolic wall stress result in the increased accumulation of collagen protein and fibrosis, whereas those in diastolic wall stress have no such effects. It has been postulated that wall stress is a major mechanical stimulus to the two types of hypertrophy, with increased systolic wall stress in pressure overload leading to the concentric hypertrophy and increased diastolic wall stress in volume overload leading to the eccentric hypertrophy. Therefore, it is possible that the systolic wall stress might be a more effective stimulus for collagen accumulation than diastolic wall stress.31 The differences in collagen biosynthesis between pressure and volume overload are similar to the disparity in the magnitude of hypertrophic response to the mechanical stress (ie, volume overload induced by mitral regurgitation produced significantly less ventricular hypertrophy than did a stroke work–matched pressure overload).32
Type I collagen represents a stiff fibrillar protein that provides substantial tensile strength to biological structures such as tendons, and type III collagen forms a reticular supportive network. Both collagens are recognized as structures that are essential in the maintenance of myocardial structural integrity. The lack of collagen accumulation despite the increased diastolic wall stress can lead to the failure of the connective tissue network to maintain the normal ventricular shape. This might contribute, at least in part, to the progressive LV dilatation during volume overload because ventricular aneurysm and rupture can occur in copper-deficient hearts in which the formation of type I collagen fibril is inhibited.33
In the present study, we did not examine the relative biochemical alterations in the tissue composition of types I and III collagen after AC shunting. Rather, the tissue collagen concentration was derived from hydroxyproline concentration, which might have failed to detect small differences in the amounts of types I and III collagen proteins. Furthermore, morphometric analysis of collagen volume fraction may not accurately reflect changes occurring in the tissue, especially immediately after AC shunting. However, Weber and colleagues1 34 confirmed a good correlation between two indices of tissue collagen content (ie, morphometric collagen volume fraction measurement and biochemical hydroxyproline concentration). The consistency of our data with the use of these two analyses suggests the absence of increase in collagen content in volume-overloaded hypertrophy.
Volume overload produced the persistent upregulation of transcriptional signals for types I and III collagen. However, these alterations in mRNAs did not result in the biochemical and histological increases in collagen matrix concentration. Therefore, the regulation of collagen accumulation and gene expression might be different in pressure-overloaded and volume-overloaded hypertrophy. The precise mechanisms of collagen biosynthesis in the diseased myocardium, including transcriptional, post-transcriptional, and translational control of gene expression and collagen protein degradation, should be clarified.
Selected Abbreviations and Acronyms
|LV||=||left ventricular; left ventricle|
|SDS||=||sodium dodecyl sulfate|
|SSC||=||standard saline citrate|
This work was supported in part by grants 06274223, 07266220, and 07670789 from the Ministry of Education, Science, and Culture. We thank Fumiko Amano and Erina Tazima for their technical assistance and Dr Akira Ooshima (First Department of Pathology, Wakayama Prefectural Medical College) for assistance in performing immunoblot analysis.
Presented in part at the 68th Scientific Session of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-528).
- Received August 28, 1996.
- Revision received November 21, 1996.
- Accepted December 13, 1996.
- Copyright © 1997 by American Heart Association
Weber KT, Janicki JS, Shroff SG, Pick R, Chen RM, Bashey RI. Collagen remodeling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res. 1988;62:757-765.
Jalil JE, Doering CW, Janicki JS, Pick R, Shroff SG, Weber KT. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle. Circ Res. 1989;64:1041-1050.
Brilla CG, Janicki JS, Weber KT. Impaired diastolic function and coronary reserve in genetic hypertension: role of interstitial fibrosis and medial thickening of intramyocardial coronary arteries. Circ Res. 1991;69:107-115.
Chapman D, Weber KT, Eghbali M. Regulation of fibrillar collagen types I and III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium. Circ Res. 1990;67:787-794.
Villarreal FJ, Dillmann WH. Cardiac hypertrophy-induced changes in mRNA levels for TGF-β1, fibronectin, and collagen. Am J Physiol. 1992;262:H1861-H1866.
Weber KT, Pick R, Silver MA, Moe GW, Janicki JS, Zucker IH, Armstrong PW. Fibrillar collagen and remodeling of dilated canine left ventricle. Circulation. 1990;82:1387-1401.
Ruzicka M, Keeley FW, Leenen FHH. The renin-angiotensin system and volume overload-induced changes in cardiac collagen and elastin. Circulation. 1994;90:1989-1996.
Garcia R, Diebold S. Simple, rapid, and effective method of producing aortocaval shunts in the rat. Cardiovasc Res. 1990;24:430-432.
Tsoporis J, Fields N, Lee RMKW, Leenen FHH. Arterial vasodilation and cardiovascular structural changes in normotensive rats. Am J Physiol. 1991;260:H1944-H1952.
Kuribayashi K, Hikata M, Hiraoka O, Miyamoto C, Furuichi Y. A rapid and efficient purification of poly(A)-mRNA by oligo(dT)20-Latex. Nucleic Acids Res. 1988;19:61-64.
Chu ML, Myers JC, Bernard MP, Ding JF, Ramirez F. Cloning and characterization of five overlapping cDNAs specific for the human proα1(I) collagen chain. Nucleic Acids Res. 1982;10:5925-5934.
Chu ML, Weil D, de Wet W, Bernard M, Sippola M, Ramirez F. Isolation of cDNA and genomic clones encoding human pro-α1(III) collagen. J Biol Chem. 1985;260:4357-4363.
Tso JY, Sun XH, Kao T, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985;13:2485-2502.
Charriere G, Hartmann DJ, Ville G. Dosage des anticorps anti-collagenes par technique radioimmunologique en phase solide. CR Soc Biol. 1984;178:160-170.
Demarchez M, Hartmann DJ, Herbage D, Ville G, Prunieras M. Wound healing of human skin transplanted onto the nude mouse, II: an immunohistological and ultrastructural study of the epidermal basement membrane zone reconstruction and connective tissue reorganization. Dev Biol. 1987;121:119-129.
Hartmann DJ, Magloire H, Ricard-Blum S, Joffre A, Couble M-L, Ville G, Herbage D. Light and electron immunoperoxidase localization of minor disulfide-bonded collagens in fetal calf epiphyseal cartilage. Collagen Rel Res. 1983;3:349-357.
Charney RH, Takahashi S, Zhao M, Sonnenblick EH, Eng C. Collagen loss in the stunned myocardium. Circulation. 1992;85:1483-1490.
Karim MA, Ferguson AG, Wakim BT, Samarel AM. In vivo collagen turnover during development of thyroxine-induced left ventricular hypertrophy. Am J Physiol. 1991;260:C316-C326.
Mays PK, McAnulty RJ, Campa JS, Laurent GJ. Age-related changes in collagen synthesis and degradation in rat tissues. Biochem J. 1991;276:307-313.
Grossman W, Jones D, McLaurin LP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975;56:56-64.
Carabello BA, Zile MR, Tanaka R, Cooper G IV. Left ventricular hypertrophy due to volume overload versus pressure overload. Am J Physiol. 1992;263:H1137-H1144.
Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium. Circulation. 1991;83:1849-1865.