Left Ventricular Hypertrophy, Cardiac Myocyte Adaptation, and Collagen/Parenchymal Distribution in Response to Subpressor and Pressor Doses of Angiotensin II in Sprague-Dawley Rats
P Reaves, O Kirksey, E Britt, M Holder
angiotensin ii, cardiac myocyte, left ventricular hypertrophy
P Reaves, O Kirksey, E Britt, M Holder. Left Ventricular Hypertrophy, Cardiac Myocyte Adaptation, and Collagen/Parenchymal Distribution in Response to Subpressor and Pressor Doses of Angiotensin II in Sprague-Dawley Rats. The Internet Journal of Laboratory Medicine. 2008 Volume 3 Number 2.
The alteration of cardiovascular myocytes is an important compensatory response to hypertension; however the pressor effect of angiotensin-II (A-II) on the correlation of myocyte morphological adaptation and collagen/parenchyma distribution in the ventricles has not been determined. Previous observations that the AII peptide is involved in the etiology of hypertension, suggested that ventricular collagen remodeling and associated pathophysiological alterations may be induced by a dysfunctional renin-angiotensin system. The objectives of this study were to 1) determine the contribution and distribution of collagen and parenchyma remodeling in the left ventricles after chronic exposure to subpressor (Sd) or pressor doses (Pd) of angiotensin II (A-Il); and 2) correlate the morphological adaptations of cardiomyocytes to weight changes of the entire heart after A-Il. The increase was 0.03 to 0.11 in the Pd-treated rats which was accompanied by a higher level of hypertrophic response than with the Sd treatment. Left-ventricular cell lengths (CL) of Pd-treated rats increased by 13%, while the heart weight to body weight ratio (HW/BW) increased by 22%. The myocardial interstitium response to the hypertrophic stimulation by A-Il included disproportionate collagen/parenchyma distribution to myocyte enlargement that is more pronounced with larger doses of A-Il and level of hypertrophy. This suggests that the A-II peptide is involved in the etiology of hypertension and a local increase is a primary factor of sustained myocardial remodeling.
Cardiovascular function integrally involves the renin angiotensin system (RAS). Classically, endogenous angiotensin II (A-Il), the central product of the RAS, is well known to produce potent vasoconstrictive responses resulting in increases in arterial blood pressure. A growing body of evidence on A-lI’s actions, from several labs, supports the conclusion that the peptide is involved in the etiology of hypertension, as well as the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, atherosclerosis and glomerulosclerosis (Yamamoto
The aim of this study was to determine whether cardiomegaly (due to ventricular hypertrophy) induced by chronic subpressor (Sd) and pressor (Pd) doses of A-Il can be characterized by significant alterations in morphometry of myocytes and/or quantitative and qualitative changes in collagen underlying a remodeling process.
Materials and Methods
Three sets of experiments were performed in this study. The first experimental designed established pressor and subpressor doses of A-Il and their systemic effects; and the second was investigated to establish changes in collagen/parenchyma makeup of myocardial tissue in response to A-Il and accompanying hypertrophic response. In each set, adult male Sprague-Dawley (SD) rats (Harlan Sprague Dawley, Inc., Indianapolis, Indiana, USA) (n = 144) weighing 290-385 grams were used. Rats were grouped and housed in plastic cages and maintained in an animal facility. The facility was maintained at 23 degrees Celsius, 35-45 % relative humidity and a fixed l2-hour light 12-hour dark cycle. Animals were fed a standard lab chow (Purina # 5001, Ralston Purina Co., St. Louis, MO) and tap water
Dose-Dependent Effects of Angiotensin II
In order to study the dose effect of A-Il (Sigma Chemical Comp., St. Louis, MO) on blood pressure and heart weight, seven doses (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 5 microgram per kilogram per day (5.0 µg/ kg/day) of the hormone were prepared and put into mini-osmotic pumps before the pumps were subcutaneously implanted into animals. Pumps remained in place throughout the experimental period but had a possible injection span of 14 days. Sham controls received 0.9% physiological saline. At the end of the treatment period, and at the end of the first five days blood pressure was recorded. At the end of 10 days animals and excised hearts were weighed. Hearts were divided into ventricular and atrial components and the sections were weighed. Total heart weight to body weight ratios (HW/BW) were calculated and compared to control animals that received normal saline injections during the same time period. From this data a clear-cut pressor dose (Pd) (5.0 µg/kg/day) was chosen while the subpressor dose (Sd) (1.5 µg /kg/day) was selected as the lowest dose at which blood pressure remained relatively close to the control values but HW/BW was elevated. After the drug treatment period, rats were weighed, anaesthetized with Equithesin, at 0.15 milliliters per 100 grams (ml/100 gm) by intraperitoneal injections (i.p.), and prepared for hemodynamic recording as described below.
Comparative Effects of Subpressor and Pressor Doses of Angiotensin II on Myocyte Morphometry and Collagen
In this experiment the animals received either Sd or Pd doses of A-II for fourteen days. The control animals received saline for the aforementioned 14 days. Following the onset of drug regimen, animals were weighed and blood pressures taken daily by the tail-cuff method utilizing standard instrumentation (Iyer and Katovich, 1996). Body weights were determined and recorded. Hemodynamic recordings were done on the eleventh day after onset of A-II infusion. The method for hemodynamic recording is given below. At the end of the hemodynamic recording session animals were removed from the apparatus while under light anesthesia, hearts were excised, rinsed and weighed. Some hearts were utilized for morphometry while others were used for collagen determination.
At the end of the drug treatment period, a standard instrumentation procedure (Gerdes
Animals were sacrificed and hearts were rapidly removed, rinsed in cold saline and weighed. For hearts selected for morphometry analysis the aorta was cannulated and attached by a cannula to a modified Langendorf retrograde perfusion apparatus. Isolated myocytes were obtained according to the techniques of Gerdes
The isolated cells were stored in 2% glutaraldehyde (Sigma Chemical Comp., St. Louis, MO) until morphological examination. Determination of cell length (the longest end-to-end distance parallel to the longitudinal axis of the cell) was measured with the use of a phase microscope. A minimum of 40 cells from each region of each heart was sized in this manner. Cell volume was determined with a Coulter Channelyzer Model C256 (Coulter Corp. Inc., Hialeah, FL). The Coulter system determines cell volume by measuring the change in electrical resistance accompanying the displacement of electrolyte as cells move through a sized aperture. A correction factor of 1.05 was used to normalize the shape values. Cross-sectional area of the myocyte was calculated by dividing cell volume by cell length.
Tissue Processing and Collagen Volume Fraction
For hearts undergoing collagen studies following hemodynamic recordings, hearts were rapidly excised, rinsed and weighed. Atria, great vessels, and valvular structures were trimmed away and discarded while ventricles were spared. Ventricles were separated from each other and transmural sections of right ventricle and left ventricle plus septum were saved for specific acidic dye (sirius red F3BA staining) (Orlandi
Collagen and Parenchymal Distribution
The collagen matrix of cardiac or skeletal muscle as being broken down into constituent elements, including an epimysium that surrounds muscle, a perimysium that is an extension of the epimysium serving to separate muscle fiber bundles, and a endomysium or final arborization of the perimysium. The endomysium includes a collagenous weave that surrounds muscle cells (Macchiarelli
The picrosirius red F3BA technique polarization microscopy was used to enhance collagen fiber birefringence. This methodology indicates several different patterns of myocardial fibrosis as well the collagen types. According to Orlandi
Values presented for all variables are the means ± standard deviation. Student’s t-test and analysis of variance (ANOVA) were used to establish any significant difference between groups of data.
Table 1 summarizes the results obtained from animals treated with A-Il. Six doses of A-Il and saline are shown in the table. Shown in the figure are the differences in hearts normalized to body weights (HW/BW) between the treated and the control rats. Average results ± SEM from 7 rats are given for each group in the table. Significant differences in absolute heart weight (HW) and HW/BW occurred with doses above l.0 µg/kg/day (
In Figure 1 results of the Sd and Pd Angiotensin II (A-Il) treatment on blood pressure levels are shown. Only mean blood pressure levels and HW/BW values are shown after the treatment period. The values represent percent differences between the tail-cuff blood pressure records for the animals receiving pressor (Pd) or subpressor (Sd) doses, and those receiving saline for 7 days. The mean blood pressure (BP) of the Pd group was significantly elevated (135 millimeters of mercury (mmHg) by 26 % ± 2 (
Left Ventricular Myocyte Morphometry
Table 2 summarizes the morphometric data for left ventricular myocytes. Animals treated with the Pd showed significant increases in cell length (CL) (144 ± 13.0) (
Collagen and Parenchymal Distribution
The fibrillar collagen and parenchymal distribution in the left ventricles are shown in Table 3. The levels were significantly elevated in both the Sd (0.049 ± 0.01) (
It has been demonstrated that the identification of patients at high risk for cardiovascular events is imperative in the reduction of cardiovascular mortality and morbidity (Javed
In the United States, hypertension is a blood pressure condition that reflects increased tone of the arteries and arterioles. The patient without recognizable symptoms is at extreme risk of pathophysiological alterations such as stroke, blindness, diabetic nephropathy, heart and renal disease (Daull
Several investigations over the last several years have established that A-Il is a potent stimulus to cardiac enlargement (Schnee
Pressor and subpressor doses of A-Il were used in this study to ascertain differences in cardiac tissue responses when animals were chronically exposed to different circulating levels above normal. The use of Alzet mini-osmotic pumps provided a continuous source of A-Il infusions. Understandably, it may be hypothesized that extrinsic A-Il application could inhibit the conversion pathway for intrinsic A-Il production. However, there were no differences in plasma renin activity (PRA) between Pd, Sd and control rats (unpublished data). Also, there were significant alterations in hemodynamics expressed after the completion of A-Il Infusions. Such results suggest that there may be catecholamine contribution to hemodynamic changes accompanying the A-Il treatment.
Other reports have indicated an A-Il adrenergic interdependence in both myocardial (Ahmed
Individual myocardial cells have been shown to be non-mitotic beyond the neonatal stage of development; also, there are marked changes in length, width and volume of myocytes that are obtained from the growing heart. However, during non-mitotic cardiac enlargement there is also a clear indication of changes in myocyte morphometry (Gerdes
Angiotensin has been reported to enhance endothelial permeability in small coronary arteries (Sano
The present results imply that there is a direct relationship between collagen remodeling in the interstitium of the myocardium and the treatment with subpressor or pressor doses of angiotensin II. The significant hypertrophic response that accompanies Pd treatment along with the changes in collagen synthesis and matrix of the interstitium are active participants in the enlargement process of the heart. This is expressed as an accumulation of connective tissue, and fibrillar collagen in particular. These are well-recognized responses to myocardial infarction and myocyte necrosis (Murray
In conclusion, the proliferation of fibrillar collagen in the structural remodeling of the cardiac interstitium may be a definable mechanism in A-Il induced cardiac enlargement. The progressive and possible extensive nature of this collagen remodeling may lead to more severe pathologic hypertrophy with muscle fiber entrapment. This is apparent with the increase in collagen volume fraction associated with the remodeling of myocardial matrix in established pressure overload. On the other hand, there is limited information on the association between collagen volume fraction and collagen remodeling occurring during the evolutionary period of A-Il induced cardiac hypertrophy. These results suggests that in time of cardiac enlargement (hypertrophy) circulating A-Il could be a stimulus to cardiac fibroblast proliferation in both the early and late phases of that process the etiology of hypertension, and a that a local increase is a primary factor of sustained myocardial remodeling. In light of this, the myocardial matrix should be regarded as a complex system of collagen within the cardiac interstitium representing a major determinant of pathological hypertrophy. Further studies and specific strategies that are targeted at modifying activity along this matrix journey will likely alter the course of myocardial remodeling and compensatory heart failure.
Reaves P. School of Allied Health Sciences, Florida Agricultural & Mechanical University, Tallahassee, Florida. E-mail: email@example.com