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(Hypertension. 1996;28:269-275.)
© 1996 American Heart Association, Inc.
Articles |
Advanced Hypertensive Heart Disease in Spontaneously Hypertensive Rats
Lisinopril-Mediated Regression of Myocardial Fibrosis
the Center of Internal Medicine, Division of Cardiology, Philipps University of Marburg (Germany) (C.G.B.), and Department of Internal Medicine, Division of Cardiology, University of Missouri–Columbia.
Correspondence to Christian G. Brilla, MD, PhD, Division of Cardiology, Center of Internal Medicine, Philipps University of Marburg, Baldingerstr., D-35033 Marburg, FRG.
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Abstract |
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Left ventricular hypertrophy (LVH) in spontaneously hypertensive rats (SHR) is accompanied by a structural remodeling of the myocardium that includes myocyte hypertrophy and interstitial and perivascular fibrosis of intramyocardial coronary arteries. The structural abnormalities related to fibrous tissue accumulation lead to increased myocardial diastolic stiffness and ultimately impaired systolic function of the left ventricle. It has been shown in 14-week-old SHR with early hypertensive heart disease that myocardial fibrosis could be reversed and myocardial diastolic stiffness normalized by 12-week treatment with the angiotensin-converting enzyme inhibitor lisinopril. Whether such functional defects of the myocardium, based on adverse structural changes, are also reversible in advanced hypertensive heart disease has been questioned. Therefore, we treated 78-week-old male SHR that had chronic hypertension and advanced LVH with severe myocardial fibrosis and age- and sex-matched normotensive Wistar-Kyoto rats (WKY) with 20 mg/kg per day oral lisinopril for 8 months. Compared with untreated SHR or WKY, we found the following: (1) Systolic arterial pressure was normalized (P<.025) and LVH completely reversed (P<.025) in SHR, with no significant reduction in systolic arterial pressure or left ventricular mass in WKY; (2) morphometrically determined myocardial fibrosis in SHR was significantly reversed (P<.025) and associated with improved diastolic stiffness (P<.05), which was measured in the isolated heart by calculation of the stiffness constant of the myocardium; no significant changes occurred in WKY; (3) reversal of myocardial fibrosis was accompanied by an increase (P<.025) in myocardial matrix metalloproteinase 1 activity determined by degradation of [14C]collagen with myocardial tissue extracts after trypsin activation of myocardial promatrix metalloproteinase 1; matrix metalloproteinase 1 activity remained unchanged in WKY treated with lisinopril; and (4) systolic dysfunction, measured by a significantly (P<.025) diminished slope of the systolic stress-strain relation under isovolumic conditions of the left ventricle, was found in 110-week-old SHR, and it could be prevented by lisinopril treatment. Thus, long-term angiotensin-converting enzyme inhibition with lisinopril normalized arterial pressure and LVH, reversed myocardial fibrosis, and improved abnormal myocardial diastolic stiffness in advanced hypertensive heart disease in SHR. In addition, systolic dysfunction of the left ventricle could be prevented. The fibrolytic response to lisinopril was at least partly due to enhanced collagen degradation by activation of tissue matrix metalloproteinase 1.
Key Words: myocardium • fibrosis • collagen • hypertrophy, left ventricular
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Introduction |
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In various experimental models of renovascular or genetic hypertension, previous studies have demonstrated that in addition to LVH, mediated by an increment in cardiac myocyte size, excessive deposition of fibrillar collagen may also occur within the cardiac interstitium.1 2 3 4 5 This structural remodeling of the myocardial collagen matrix accounts for abnormalities in myocardial stiffness.1 5 6 7 In vitro experiments with adult rat cardiac fibroblasts have shown that the effector hormones of the RAAS, Ang II8 9 and aldosterone,9 stimulate fibroblast-mediated collagen synthesis and that Ang II additionally suppresses collagenase or MMP1 activity,9 which synergistically leads to collagen accumulation. Previously, we found that in 14-week-old SHR with early hypertensive heart disease, which included LVH and myocardial fibrosis, these structural alterations could be reversed along with normalization of blood pressure and myocardial diastolic stiffness by 12-week treatment with the ACE inhibitor lisinopril.10 We subsequently demonstrated that the regression in fibrosis was independent of the antihypertensive effect of lisinopril.7 Whether abnormal myocardial stiffness based on adverse structural changes within the cardiac interstitium is also reversible in advanced hypertensive heart disease has been questioned. This appears to be of particular clinical interest because many patients present with symptoms due to advanced hypertensive heart disease and are commonly free of symptoms during the early course of disease.
Therefore, we treated 78-week-old male SHR with chronic hypertension, advanced LVH, and severe myocardial fibrosis and age- and sex-matched normotensive WKY for 8 months with lisinopril. Our objectives were (1) to normalize blood pressure in SHR, (2) to reverse LVH, including myocardial fibrosis, (3) to restore diastolic and systolic functions of the left ventricle, and (4) to elucidate the role of myocardial tissue MMP1 in the fibrolytic response that could accompany long-term treatment with an ACE inhibitor.
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Methods |
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Animal Models
Age-matched male SHR and their genetic WKY controls were obtained from Sasco Inc, Omaha, Neb. This strain of SHR developed hypertension early (at 6 weeks of age) and reached a steady-state elevated blood pressure at 12 weeks of age; WKY remained normotensive. Rats were studied in the following manner: (1) 78-week-old SHR (SHR78, n=10) and age-matched WKY (WKY78, n=10) were killed for physiological, morphological, and biochemical studies after appropriate anesthetization (see below); (2) untreated 78-week-old SHR (n=10) and WKY (n=10) were followed for 32 weeks and killed at 110 weeks of age (SHR110 and WKY110); (3) 78-week-old SHR were treated with the ACE inhibitor lisinopril (20 mg/kg per day) in their drinking water for 32 weeks and were killed at 110 weeks of age (SHRL, n=10); and (4) 78-week-old WKY received the same oral dose of lisinopril for 32 weeks (WKYL, n=10) and were killed at 110 weeks of age.
For the initial month, blood pressure was determined weekly (titration of blood pressure) and thereafter monthly in all rats by a standard tail-cuff method to ascertain responses in arterial pressure. All rats were given standard rat chow and water ad libitum and were housed under equal, standardized conditions.
Hemodynamics
After rats were anesthetized (methohexital, 50 mg/kg IP), the right carotid artery was cannulated. Arterial pressure was recorded with rats in a lightly anesthetized state. After additional anesthesia, rats were intubated and mechanically ventilated. The chest was opened by median sternotomy, and the heart and lungs were removed en bloc. Within seconds, the ascending aorta was cannulated, and retrograde perfusion with 37°C modified Krebs-Henseleit solution was established according to the Langendorff technique as previously reported.7 10 A balloon was positioned in the left ventricle via the mitral valve and secured at the apex, which was punctured to permit the egress of any thebesian drainage. The other end of the balloon was fixed to a short catheter whose outer diameter approximated that of the mitral annulus, thereby preventing displacement of the balloon into the left atrium. The catheter was connected by a stopcock to a syringe (to allow volume changes) and to a Statham pressure transducer. Coronary perfusion pressure was increased until maximal LV peak systolic pressure was obtained in the absence of any increase in end-diastolic pressure.2 7 Atrial pacing was used to maintain heart rate at 200 beats per minute in all rats.
Steady-state LV pressure was recorded from isovolumetrically beating hearts during increments (0.02 mL) in balloon volume over the LV end-diastolic pressure range from 0 to 25 mm Hg. For assessment of myocardial stiffness for hearts of different LV weight and size, stress (
, kilopascals), tangent elastic modulus (E, kilopascals), and strain (
) for the midwall at the equator of the left ventricle were calculated assuming spherical geometry and considering the midwall equatorial region as representative of remaining myocardium.11 12 13 Mathematical equations used for calculation of , E, and have been previously reported.7 Myocardial diastolic stiffness was calculated with the stiffness constant (k, dimensionless), ie, the slope of the linear relationship between E and end-diastolic .11 Myocardial contractility was determined with the slope, c, of the systolic - relation of the isovolumetrically beating heart. The total duration of the physiological study did not exceed 30 minutes. After diastolic and systolic pressure-volume data were obtained, atria and great vessels were trimmed away and ventricles (left ventricle plus septum) separated and weighed. LV dry weight was obtained by lyophilization (2 hours) and incubation of myocardial samples in a vacuum oven at 55°C until a constant weight was reached (2 hours).
Quantitative Morphometry
For morphological studies, hearts were perfusion fixed at 100 mm Hg with buffered glutaraldehyde (2.5%, pH 7.40) for 15 minutes. Entire coronal sections of the left ventricle, obtained from its equator, were prepared for light microscopy as previously reported.2 Paraffin-embedded sections (5-µm-thick) were stained with the collagen-specific dye sirius red F3BA.
Interstitial collagen volume fraction was determined by quantitative morphometry of sirius-stained sections with an automated image analyzer (Quantimet 520, Cambridge Laboratories, Inc). Each section was placed in a projection microscope (x40). On the basis of their gray levels, and where collagen fibers appeared black in direct light, images were digitized. Collagen volume fraction was calculated as the sum of all connective tissue areas of the entire coronal section divided by the sum of all connective tissue and muscle areas in all fields of the section.7 This morphometric approach for measurement of fibrillar collagen within the cardiac interstitium has been validated previously.2 3 7 10 We and others14 15 have shown that total collagen volume fraction is closely related to hydroxyproline concentration of the left ventricle.
Collagenase Assay
LV myocardial tissue samples of the different experimental groups were immediately frozen at -80°C until MMP1 activity was measured with [14C]collagen (10 mCi/mmol per liter) according to Moore and Spilburg.16 In 1.5-mL test tubes, 50 µL of [14C]collagen (1 mg/mL) and 50 µL of buffer solution (1 mol/L glucose, 0.1 mol/L Tris, 0.4 mol/L NaCl, 0.02 mol/L CaCl2; pH 7.5) were incubated at 35°C for 10 minutes. Frozen myocardial tissue (approximately 25 mg) was washed three times with cold saline and incubated in 2 mL of extraction buffer (10 mmol/L cacodylic acid at pH 5.0, 0.15 mol/L NaCl, 1 µmol/L ZnCl2, 20 mmol/L CaCl2, 1.5 mmol/L NaN3, 0.01% Triton X-100) at 4°C with continuous agitation for 24 hours. This step was repeated with fresh buffer. The extraction buffer (4 mL) was collected and pH raised to 7.5 by the addition of 0.1 mol/L Tris buffer (pH 7.6). Various volumes of tissue extract (1100, 900, 700, or 500 µL) were concentrated with a Minicon B15 concentrator (Amicon Inc) and were incubated with 1 µL of 1 mg/mL trypsin (dissolved in 1 mmol/L HCl, Sigma Chemical Co) for 20 minutes to activate proMMP1 as previously reported.9 Activation was terminated by addition of 1 µL of 10 mg/mL soybean trypsin inhibitor (Sigma) (dissolved in 0.05 mol/L Tris, 0.01 mol/L CaCl2 at pH 7.5). Thereafter, 100 µL of sample containing activated MMP1 was incubated with 100 µL [14C]collagen-Tris buffer solution at 35°C for 30 minutes. The reaction was terminated by addition of 20 µL of 0.08 mol/L 1,10-phenanthroline in 50% dioxane. After 1 hour of incubation at 35°C, samples were cooled to room temperature for 15 minutes. Thereafter, 200 µL of 100% dioxane was added and the sample mixed to precipitate uncleaved collagen. After final centrifugation at 11 000 rpm for 10 minutes, the supernatant containing degraded 14C-labeled collagen was harvested, and 350-µL aliquots were counted in 5 mL PicoFluor 40 scintillation fluid (Packard) for 5 minutes with a scintillation counter.
The blank containing buffer/substrate ([14C]collagen) solution, trypsin, and soybean trypsin inhibitor was handled in the same way as the sample. Control experiments were carried out as reported previously.9 We observed no liberation of soluble 14C counts, indicating that neither trypsin nor trypsin–soybean trypsin inhibitor complex was able to degrade [14C]collagen. MMP1 activity was measured as released counts per minute and calculated as micrograms of degraded [14C]collagen per milliliter per 30 minutes per milligram of protein; the protein content of each dish was determined according to the Lowry method.17
Statistics
All data are expressed as mean±SEM and were compared by one-way ANOVA. If the omnibus hypothesis was rejected, post hoc pairwise comparisons were performed in a conservative manner with t statistics. We elected to address the following five comparisons: SHR78/WKY78, SHR110/WKY110, SHRL/WKY110, WKYL/WKY110, and SHRL/SHR78. These multiple comparisons were corrected according to Bonferroni: P=P'xk, where k is the number of comparisons and P' is the probability of error before Bonferroni correction. Thus, the reported P values are corrected according to Bonferroni. Comparisons of the diastolic E- or systolic - relations among the different experimental groups were performed by multiple regression analysis. The following sequential procedure was applied: (1) ANOVA, and (2) method of dummy variables to test for intercept and slope differences.18 Either regression lines with 95% confidence limits or bars with SEM are shown for each group. Significance was assumed at a value of P<.05.
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Results |
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Blood Pressure and LVH
Severe arterial hypertension was present in SHR78 and SHR110. Systolic arterial pressure averaged 220±6 and 225±7 mm Hg, respectively (P<.025 versus WKY78, 128±7 mm Hg, or versus WKY110, 135±7 mm Hg). In untreated SHR, systolic arterial pressure was stable throughout the 32-week observation period. LVH was determined by LV weight normalized to body weight, which was markedly elevated in SHR78 and SHR110 compared with age- and sex-matched WKY (Table
). Of note, SHR110 showed severe LVH, with a mean LV weight of 1798 mg compared with WKY110 (LV weight=1108 mg).
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In SHRL, systolic arterial pressure was normalized within the first treatment week and was 134±8 mm Hg at the end of the study when LVH (ratio of LV weight to body weight [LV/BW], 2.5±0.1 mg/g, P<.025, versus 3.6±0.2 mg/g in SHR78) was completely reversed; ie, LV/BW did not differ significantly between SHRL and WKY110. No treatment effect of lisinopril on blood pressure or LV mass was found in WKYL at 110 weeks of age after 32 weeks of lisinopril administration (systolic arterial pressure=132±4 mm Hg; LV/BW=2.3±0.2 mg/g).
Ratios of LV wet weight to dry weight did not differ significantly among the groups (Table
), indicating that myocardial edema was not present.
Myocardial Fibrosis and Collagenase Activity
Diffuse myocardial fibrosis was present in SHR78 and SHR110 (Fig 1). There was a severalfold increase of interstitial collagen volume fraction of the left ventricle in untreated SHR compared with age-matched WKY (Fig 2) (8.2±1.0% and 9.0±1.2% in SHR78 and SHR110; 2.5±0.4% and 1.8±0.2% in WKY78 and WKY110, respectively). Interstitial fibrosis was significantly (P<.05) reversed with long-term lisinopril treatment down to 5.3±0.7% in 110-week-old SHRL compared with SHR78. However, this regression was not complete because a significant difference between SHRL compared with age-matched WKY was found (P<.05). In keeping with increased collagen turnover in LVH, MMP1 activity was elevated in untreated SHR78 and SHR110 compared with age- and sex-matched WKY, in which no LVH was present. Parallel to the regression of interstitial fibrosis with long-term ACE inhibition in SHR, the activity of the key enzyme of collagen degradation, MMP1, was significantly (P<.025) further increased in SHRL compared with untreated SHR (MMP1 activity=3.7±0.5 and 2.9±0.2 µg degraded collagen/mL/30 min/mg protein, respectively) and compared with age-matched WKY (Fig 3). In contrast, 32 weeks of lisinopril treatment in WKY did not alter collagen volume fraction (2.0±0.4%) or MMP1 activity (1.7±0.2 µg degraded collagen/mL/30 min/mg protein).
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Diastolic Stiffness and Myocardial Contractility
A leftward shift and steepening of diastolic - relations was found in untreated SHR compared with age-matched WKY, revealing an increase in diastolic stiffness in SHR; lisinopril treatment reversed this displacement of the diastolic function curves (data not shown). No change of the diastolic - relation was seen with lisinopril treatment in WKY. The stiffness constant of LV myocardium, ie, the slope of the linear relationship between E and end-diastolic that characterizes myocardial tissue stiffness, was significantly (P<.05) increased in SHR78 and SHR110 compared with age-matched WKY and was significantly (P<.05) reduced from 19.9±1.2 to 15.1±1.4 (dimensionless) with lisinopril treatment (Fig 4). No significant difference of myocardial diastolic stiffness remained after 32 weeks of long-term ACE inhibition compared with age-matched WKY (k=12.3±1.1) at the end of the study. Lisinopril treatment in WKY had no effect on diastolic stiffness of the left ventricle.
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Myocardial contractility, as determined by the slope of the isovolumic systolic - relation, was normal in SHR78 compared with WKY78. However, at 110 weeks of age, depressed systolic function of the left ventricle was seen. The slope, c, of the systolic - relation was reduced to 110.1±9.8 kPa from 142.5±11.1 kPa in SHR78 and compared with WKY110 (c=139.6±10.9 kPa, P<.025). In keeping with the overall reduced LV function in these rats with advanced hypertensive heart disease, body weight was significantly (P<.05) higher in SHR110 compared with WKY110 (Table), most likely because of RAAS-mediated salt and water retention. Pleural effusions were present in four of nine SHR110 and in none of the lisinopril-treated rats. One untreated SHR died suddenly during the course of the study; all SHRL survived until the end of the study. The decline in myocardial contractility and rise in body weight were prevented by lisinopril (Fig 5, Table). No change in myocardial contractility occurred with lisinopril treatment in WKY.
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Discussion |
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In recent years, remodeling of the myocardial collagen matrix in hypertensive heart disease3 4 5 6 7 14 19 20 21 has interested many investigators. It is now well accepted that in addition to the structural changes of the myocyte compartment involving cardiac myocyte hypertrophy22 and phenotype changes within these parenchymal cells, eg, isomyosin pattern23 or sarcoplasmic ATPase function,24 the structural alterations within the nonmyocyte compartment or cardiac interstitium are an integral part of the attempt of the cardiac muscle to cope with chronic pressure or volume overload.
We have focused on the role of the myocardial collagen matrix2 3 7 9 10 14 20 and have demonstrated that the development of myocardial fibrosis and not muscle mass per se is the major determinant of myocardial diastolic stiffness in hypertensive heart disease.7 Conversely, no sign of diastolic dysfunction of the left ventricle has been reported in forms of LVH in which myocardial fibrosis is absent, eg, hyperthyroidism,25 chronic volume overload,26 or the athletic heart.27 In addition, we have previously shown that the development of myocardial fibrosis in LVH is related to a stimulated circulating RAAS.3 20 We have shown this to be true in in vivo studies in renovascular hypertension or with long-term administration of aldosterone.3 Likewise, a local cardiac Ang II–generating system may be operative in the SHR model of genetic hypertension.7 28 At the cellular level, we have shown that Ang II significantly stimulates collagen synthesis in cultured adult rat cardiac fibroblasts in a dose-dependent manner.9 This has been confirmed in several laboratories.8 29 Furthermore, we have found that Ang II significantly inhibits MMP1 activity in adult rat cardiac fibroblast culture, where MMP1 is a key enzyme involved in fibrillar collagen degradation.9
Therefore, it appears to be a logical treatment goal in hypertensive heart disease to counteract the circulating RAAS or local generation of Ang II by long-term ACE inhibition and to promote collagenolytic activity in the heart. We proved this to be feasible in the SHR model of genetic hypertension,10 which is an analogous model for human primary hypertension, the major cause of high blood pressure and hypertensive heart disease. However, in these earlier studies we used young rats in which a modest degree of myocardial fibrosis resulted in diastolic LV dysfunction with preserved systolic function.7 10 In early hypertensive heart disease, relaxation abnormalities caused by reduced sarcoplasmic Ca2+-ATPase activity contribute also to the development of diastolic LV dysfunction,24 whereas in later stages, impaired ventricular filling due to fibrosis-mediated increases in myocardial stiffness plays the dominant role in the progression of LV dysfunction.20 23 25 Therefore, it is of major clinical interest whether such regression of interstitial and perivascular fibrosis can also be achieved in advanced hypertensive heart disease.
To explore this possibility, we treated 78-week-old SHR with the same ACE inhibitor used in our previous regression studies.7 10 We chose a treatment period long enough to allow removal of a significant amount of collagen from the myocardium, in keeping with the long half-life of fibrillar collagen.30 Our findings indicate that the accumulation of collagen within the interstitium of the hypertrophied left ventricle in 78-week-old rats with genetic hypertension is accompanied by abnormal diastolic myocardial stiffness but preserved systolic function, as determined by the slope of the systolic - relation of the isovolumetrically beating isolated heart. This implies that at this stage of hypertensive heart disease, myocardial fibrosis associated with diastolic dysfunction has occurred in the absence of systolic dysfunction. In more advanced hypertensive heart disease in 110-week-old SHR, myocardial contractility was depressed in addition to the presence of diastolic dysfunction; ie, combined diastolic-systolic dysfunction of the left ventricle has appeared compared with age- and sex-matched normotensive WKY. We have chosen a well-established method of calculating and .11 12 13 There are potential errors in the calculation of myocardial stiffness in that we assumed homogeneity in myocardial structure, and isotropic behavior of the myocardium. Since interstitial collagen fibers of the myocardial collagen network are distributed diffusely within the myocardium, a gross inhomogeneity in collagen accumulation is not likely.
In this study, LVH, ie, myocyte hypertrophy, was completely reversed, even in this late stage of hypertensive heart disease. It is now well established that ACE inhibitors have a clear effect on LVH regression that is more powerful than other antihypertensive agents.31 With oral lisinopril treatment, dosed to normalize blood pressure, it was possible to reverse myocardial fibrosis. However, under these conditions of advanced hypertensive heart disease with severe LVH, collagen volume fraction decreased to values that were still higher than those in genetic normotensive controls, presumably because of the fact that reparative fibrosis32 due to myocyte necrosis (scarring) is pronounced in this late stage of hypertensive heart disease and depends on regulatory systems other than the RAAS, eg, transforming growth factor-ß1.33
In SHR, it is known that aging or persistent elevations in afterload due to an activated vascular renin-angiotensin system are associated with a progressive rise of myocardial collagen concentration, leading to the occurrence of systolic dysfunction, measured by a drop in cardiac output.34 Using papillary muscles from SHR between 20 and 23 months of age, Conrad et al5 found increased passive stiffness and impaired tension development associated with myocardial fibrosis in SHR with clinical signs of heart failure (labored respiration, left atrial thrombi, pleural and pericardial effusions, right ventricular hypertrophy) compared with either age-matched WKY or SHR without heart failure. In addition, the same group observed a marked increase in the expression of genes encoding extracellular matrix components, including types I and III collagens, in these SHR with advanced hypertensive heart disease and combined diastolic-systolic dysfunction relative to age-matched WKY and SHR with nonfailing hearts.4 In the present study, we assessed myocardial contractility in the isolated Langendorff heart under isovolumic conditions using the systolic - relation, which is a linear function. The slope of the systolic - relation is an established measure of myocardial contractility under isovolumic conditions because of its independence of afterload when heart rate is held constant by atrial pacing.35 Although systolic function was still normal in 78-week-old SHR, it deteriorated at 110 weeks of age. These 110-week-old SHR showed an increased body weight compared with age-matched WKY most likely because of an activated RAAS and subsequent sodium and water retention following impaired systolic LV function since both the decline in myocardial contractility and the rise in body weight could be prevented by long-term ACE inhibition with lisinopril. Lisinopril treatment did not influence body weight in WKY, excluding any adverse effect on normal growth. Prevention of the progression of heart failure in patients by long-term ACE inhibition has been confirmed in several large-scale studies.36 37 However, the underlying mechanism for these beneficial effects of ACE inhibition in heart failure are not well understood. The influence of the RAAS on the regulation of the myocardial extracellular matrix3 9 19 20 21 28 and its counteraction by long-term ACE inhibition could be important determinants.
The mechanism by which long-term ACE inhibition might decrease myocardial collagen is largely unexplored but has been partly addressed in the present study. Any removal of collagen from the cardiac interstitium can be achieved either by inhibition of collagen synthesis or by enhanced collagen degradation. We followed the hypothesis that an active removal of collagen occurs when regression of myocardial fibrosis occurs. Therefore, we focused on the key enzyme involved in collagen degradation—interstitial collagenase or MMP1. This enzyme is known to be present in the rat38 39 and human40 myocardium. This enzyme initially cleaves collagen and promotes an unfolding of the triple helix of the collagen peptide chains so that other collagenolytic enzymes, such as gelatinase A and B or MMP2 and MMP9, respectively, and nonspecific proteolytic enzymes, such as trypsin, can further digest collagen fibrils.41 In the hypertrophied heart, collagen metabolism is increased,30 in keeping with an upregulated MMP1 activity as measured in this study by the degradation of [14C]collagen. In SHR but not in WKY, MMP1 activity was further increased with oral lisinopril treatment, because myocardial Ang II concentrations are elevated in SHR compared with WKY28 and Ang II suppresses MMP1 activity.9 Since morphometrically determined collagen volume fraction of the LV myocardium was decreased by lisinopril treatment, we can conclude that activation of MMP1 is at least partly responsible for the regression of myocardial fibrosis in the SHR model of hypertensive heart disease.
Besides MMP1, only MMP8 is able to cleave native types I and III collagen fibrils.42 Only in states of marked infiltration of the myocardium by polymorphonuclear leukocytes that express MMP8, eg, in myocarditis or postmyocardial infarction,43 does MMP8 play an important role in interstitial collagen degradation. This degradation leads to myocyte slippage and acute dilatation, thereby promoting what has been termed "remodeling" after myocardial infarction. We routinely checked our tissue samples for signs of myocarditis and did not find any evidence of significant infiltration by leukocytes. We did not choose zymography to measure collagenolytic activity of the myocardium because this method would focus on the less specific MMP2 and MMP9, which further degrade unfolded collagen proteins. Using quantitative polymerase chain reaction (data not shown), we were able to detect MMP1, MMP2, and MMP9 in the myocardium of WKY and in the hypertrophied myocardium of SHR. We were not able to detect MMP8. This may be due to the fact that we perfused our isolated rat hearts with modified Krebs-Henseleit perfusate, making these hearts nicely "washed." Therefore, we were sure to measure MMP1 activity using our 30-minute assay with [14C]collagen. We did not, however, examine collagen synthesis in the myocardium, which also could be changed because of ACE inhibition, since Ang II is known to stimulate collagen synthesis in cultured cardiac fibroblasts.9 42 This will require further study.
Thus, in genetic hypertension with advanced hypertensive heart disease, abnormal diastolic myocardial stiffness can be improved along with regression of myocardial fibrosis by long-term ACE inhibition with lisinopril. In addition, the occurrence of systolic dysfunction or impairment of myocardial contractility can be prevented. These beneficial effects of the ACE inhibitor lisinopril appear to be partly mediated by upregulation of the activity of MMP1, the key enzyme for interstitial collagen degradation, and are able to balance the excessive growth of the myocardial collagen matrix in advanced hypertensive heart disease.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grant R05-HL-31701 and Deutsche Forschungsgemeinschaft grant Br-1029-1/2 and was performed at the Division of Cardiology, University of Missouri–Columbia. The authors wish to thank the staff of the Hemodynamic Lab of the Division of Cardiology of the University of Missouri–Columbia (Dalton Research Center, Columbia, Mo) for excellent technical assistance.
Received October 16, 1995; first decision November 14, 1995; accepted March 13, 1996.
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Weber KT, Brilla CG. Pathologic hypertrophy and the cardiac interstitium: fibrosis and the renin-angiotensin-aldosterone system. Circulation. 1991;83:1849-1865.
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