Elevated collagen content in volume overload induced cardiac hypertrophy

Journal of Molecular and Cellular Cardiology (1980)

Elevated

RITA

12, 929-936

Collagen Content in Volume Induced Cardiac Hypertrophy

A. CAREY, WILLIAM

GANGAIAH P. SANTAMORE

NATARJAN”,

Overload

ALFRED A. BOVE, F. SPANN

AND JAMES

Cardiology Section and Department of Physiology at Temple University, Pennsylvania, U.S.A. (Received 25 January

1980, accepted in revised form 7 April

Philadelphia, 1980)

R. A. CAREY, G.NATARJAN, A. A. BOVE, W. P. SANTAMORE AND J. F. SPANN. Elevated Collagen Content in Volume Overload Induced Cardiac Hypertrophy. Journal of Molecular and Cellular Cardiology (1980) 12, 929-936. This investigation was designed to determine if chronic volume overload is associated with altered collagen content of five regions of the myocardium. Five adult cats were subjected to a 6-week period of chronic volume overload induced by atria1 septotomy and five untreated animals served as controls. Significant (P < 0.05) right ventricular hypertrophy was present as indicated by the right ventricular body weight ratio. For control animals this ratio was 0.68 & O.O4g/kg; for volume overloaded animals it was 0.83 & 0.05 g/kg.) The collagen content was assessed by measuring the hydroxyproline content of the dried cardiac muscle. Right ventricular endocardium hydroxyproline in volume overloaded animals was significantly elevated above that in control animals (in the latter it was 5.30 * 0.36 kg/mg; in the former it was 6.33 & 0.18 pg/mg) while the epicardial collagen content was unchanged. Similarly, the amount of collagen found in the left ventricle was significantly increased in the endocardium and normal in the epicardium. Septal collagen concentration was unaltered in volume overloaded animals. This study demonstrated that alterations in cardiac muscle collagen concentration are associated with volume overload and that these cellular changes are nonuniform.

KEY WORDS: Collagen;

Cardiac

hypertrophy;

Volume

overload.

1. Introduction Cardiac hypertrophy results from increasing the workload on the myocardium. It is, however, becoming increasingly apparent that experimentally induced pressure and volume overload, while producing hypertrophy of equal magnitude, yield hearts with vastly different mechanical and biochemical characteristics. Cooper et al. [8] and Carey et al. [4] have shown that myocardial mechanical function is normal when the ventricle is subjected to chronically elevated preload. We have also demonstrated [4] that cardiac muscle myosin ATPase activity is normal in volume overload induced cardiac hypertrophy. Conversely, myocardial hypertrophy resulting from chronically elevated afterload is associated with * Present address: Department of Medicine, Medical Center, Lexington, Kentucky 40506, 0022-2828/80/090929

+ 08 $02.00/O

Cardiovascular U.S.A. 0

Division,

1980 Academic

University

Press Inc.

of Kentucky

(London)

Limited

930

R.

A.

CAREY

ET AL.

depressed mechanical function [3, 2.51 and depressed myosin ATPase activity [3, 23, 271. Investigators have also shown that chronic pressure overload is associated with an elevated collagen content [2] and the increased collagen may be a factor in determining the diastolic pressure-volume characteristics of the pressure overload myocardium. We have demonstrated [17] that diastolic stiffness is increased in pressure overload. Normal diastolic stiffness is, however, associated with volume overload [ 161. To date, it is not known if volume overload is accompanied by alterations in collagen similar to that observed in pressure overload. Since it is important to define clearly the differences and similarities in biochemistry of cardiac muscle subjected to pressure and volume overload, this study was designed to determine if chronic right ventricular volume overload stress is associated with abnormal myocardial collagen content. Further, theoretical considerations [IS] lead one to postulate that the distribution of stress is nonhomogenous in the myocardium. Since dissimilar stresses may lead to nonuniform accumulation of collagen in the normal and overloaded myocardium, it is important to study connective tissue concentration in different regions of the heart. Thus, the myocardium in this study was dissected into five areas in an effort to evaluate the manner in which different regions of the heart respond to volume overload.

2. MateriaIs Dejnition

and Methods

of exflerimental groufis and production of volume overload hypertrophy

Two groups of normal adult cats were. studied. Five cats served as controls (C), and five were subjected to a 6-week period of chronic volume overload (VO) secondary to atria1 septotomy. Production of VO by atria1 septotomy has been developed in this laboratory [18]. The cats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and allowed to breathe spontaneously. The ascending aorta and right ventricle were catheterized via the carotid artery and jugular vein, respectively. After measurement of cardiac output by a dye dilution technique, a flexible bronchoscopy biopsy forceps was advanced via the femoral vein into the right atrium. When the limits of the right atrium were defined, using small amounts of radio-opaque contrast material, the mouth of the biopsy forceps was placed adjacent to the atria1 septum, opened, and closed on the septum. The catheter then was pulled down, forcibly pinching off the septum. The magnitude of the atria1 septal defect (ASD) thus created was assessed by repeating the dye dilution procedure and calculating the pulmonary blood flow to systemic blood flow ratio (QP/QS) by the method of Carter et al. [5]. The cats were maintained for 41 & 0.7 days (s.E.M.) prior to final study.

COLLAGEN

CONTENT

AND

CARDIAC

HYPERTROPHY

931

Measurement of hemodynamic parameters and assessment of atria1 sepal defect Immediately prior to excision of the heart, the cats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and allowed to breathe spontaneously. A cannula (PE-190) was placed in the ascending aorta, and a No. 4 French catheter was placed in the right ventricle. Right ventricular pressure was recorded, and heart rate was measured from the pressure record. The magnitude of the ASD at the time of final study was determined from the QP/QS ratio determined as described above. i@ocardial

dissection

the right and Ieft ventricle and septum Following excision of the myocardium, were dissected from each other. The two ventricles were then separated into epicardium and endocardium midway between the base and apex of the heart. Approximately 100 mg of tissue from each of the five sections were weighed and placed in an oven for drying. When the dry weights stabilized the tissue samples were assayed for collagen content.

Measurement of collagen content The collagen content of dried cardiac tissue was assessed by measuring the amount of hydroxyproline present. A modification of the spectrophotometric method of Bergman and Loxley [I] was used. Dried tissue (18 to 25 mg) was hydrolyzed in at 110°C in a screw-top Pyrex test tube. The mixture was 6 N HCl overnight evaporated, taken up in 2 ml of water, and 2 ml of isopropanol was added followed by 1 ml of mixture containing 7% chloramine T, acetate-citrate buffer (5.7% sodium acetate+3H,O, 3.75% sodium citrate.2H,O, pH 6.0) and 38.5”/, isopropanol. After 4 min, 13 ml of Ehrlich’s reagent (p-dimethylaminobenzaldehyde) dissolved in 60% perchloric acid and combined with isopropanol were added. The mixture was heated for 25 min at 6O”C, cooled for 2 to 3 min on ice, and the O.D. at 558 nm was recorded within 4 h. The results were expressed as micrograms of hydroxyproline per milligram of dry weight of tissue.

3. Results Magnitude of atria1 septal defects and hemodynamic jndings Body weights 3.02 & 0.12 kg cantly different by the RV/BW

(BW) of the control and VO groups were 2.49 + 0.08 kg and respectively (Table 1). The experimental group was not signififrom control. Right ventricular (RV) hypertrophy was evaluated ratio. Significant hypertrophy developed in the VO group.

5 5

2.49 f 0.08 3.02 f 0.12

LV wt k)

Heart rate (beats/min) QI’IQS

7.64 f 7.64 f

5

vo

6.33 f

0.52

0.18”

5.30 * 0.36

0.52

Endocardium

Right ventricle kg/mg dry wt) Epicardium

5

No. of cats

content

Control

Group

2. Hydroxyproline

Values are expressed as S.E.M. VO, volume overload. * VO significantly greater than control (P < 0.05).

TABLE

Systole

Diastole

Arterial pressure (mm Hd Systole

5.57 + 0.20

5.55 & 0.38*

0.31

4.47 f 5.18 f

0.46

Endocardium

Epicardium

Left ventricle b-&w dry wt)

to systemic blood flow.

dry wt)

Septum

3.85 f

0.39

3.31 * 0.44

k/w

2.74 & 1.16 1.03 + 1.18

Diastole

RV pressure (mm J&d

157.40 zt 12.07 129.20 i 10.78 25.37 5 1.16 0.68 f 0.04 6.28 5 0.35 121.20 i 22.72 0.83 f 0.05* 6.90 & 0.40 123.50 5 26.68 1.90 & 0.21 175.00 & 8.89 143.25 f 9.86 23.00 + 2.70

RVjBW k/W

data

Values are expressed as S.E.M. VO, volume overload. QP/QS, ratio of pulmonary * Symbols show VO to be significantly greater than control; * P < 0.05.

Control VO

Body wt (kg)

1. Hemodynamic

Group No. of cats

TABLE

COLLAGEN

CONTENT

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HYPERTROPHY

933

As shown in Table 1, the ratio of pulmonary blood flow to systemic blood flow averaged 1.90 + 0.2 1, indicating that a left to right shunt was present. At the time of final study, the arterial pressures and right ventricular pressure of each group were within the normal range and not significantly different (P > 0.05) from each other. The heart rate of both groups was within the normal range, and no significant difference (P > 0.05) between the groups was observed.

Collagen

content

The hydroxyproline contents (Table 2) of the right ventricular epicardium in the control of VO groups were 7.64 & 0.52 and 7.64 + 0.52 mg/mg dry wt respectively and not significantly (P < 0.05) different from each other. In the right ventricular endocardium the hydroxyproline content of 6.33 i 0.18 mg/mg dry wet in the VO group was significantly (P < 0.05) greater than the value of 5.30 & 0.36 mg/mg dry wt found in the controls. In the left ventricle a similar pattern was noted. The epicardial collagen content was unchanged in VO animals while endocardial hydroxyproline was significantly (P < 0.05) elevated. There was no significant (P > 0.05) difference in the hydroxyproline content in the septum of the control and VO groups. 4. Discussion The data presented in this study show that chronic volume overload secondary to an atria1 septal defect leading to 22O1; right ventricular hypertrophy is associated with significantly elevated right ventricular endocardial collagen content and left ventricular endocardial collagen normal epicardial collagen. Similarly, content was elevated while the epicardial connective tissue content was normal. Septal collagen concentration was unaltered by the volume overload in this study. Perhaps dissecting the septum into right and left portions would have yielded significant changes in septal collagen content. It is well established that the collagen content of the myocardium is elevated during chronic pressure overload [2, 11, 241. The mechanism for increased collagen in pressure overload has been found to involve an elevated protocollagen protein hydroxylase [13]. Further, it has been suggested [IO] that the elevated hydroxyproline content of the heart observed in pressure overload does not return to normal following relief of the hemodynamic stress. It is reasonable to speculate that the increased collagen plays a role in the depressed contractile function [Z, 31 and increased diastolic stiffness [17] characteristic of pressure overload induced hypertrophy and failure. Aortic insufficiency of 1 to 3 months [26] d uration in the rabbit was associated with normal hydroxyproline content. These investigators did not separate right from left ventricle nor epicardium from endocardium. The results of the current

934

R. A. CAREY E’TAL.

investigation differ from those observed in aortic insufficiency. The dissimilar results may be attributed to the procedure of separating the regions of the heart employed in this study. Measuring the collagen content of the intact myocardium could mask the difference observed in the endocardium of both the right and left ventricles. Since collagen content may be related to mechanical performance it is of interest that a volume overload identical to that employed in the present study was not a stress of sufficient magnitude to depress mechanical function [4] or to alter diastolic stiffness of the papillary muscle [26]. The possibility remains that an elevated collagen content may influence mechanical function in the presence of a greater volume overload associated with more hypertrophy and myocardial failure. However, in the presence of the relatively mild volume overload stress and hypertrophy in the present study, the increased collagen was tolerated with no change in cardiac muscle performance. It has become increasingly important to understand that overload stresses affect the various regions of the myocardium differently [15]. In this study only endocardial hydroxyproline content is changed in the presence of a 47%. left-toright side shunt. Thus, this study demonstrates yet another way in which the adaptation of the myocardium to stress is nonuniform. It is of interest that overload stresses on either the right or left ventricle are often associated with biochemical changes, not only on the stressed ventricle, but also in the contralateral ventricle. Pressure overload on the right ventricle results in increased collagen [Z], depressed myofibrillar ATPase [S], depressed creatine and creatine phosphate [19], and depleted norepinephrine stores [7, 91 of both the right and left ventricles. Similarly, volume overload on the right ventricle has been shown to reduce both right and left ventricular norepinephrine [12]. Mechanical performance changes and increases in myocardial mtiss associated with pressure and volume overload on one ventricle are known to result in altered function and increased mass of the contralateral ventricle [12, 14, 19-221. Thus, stress placed on one ventricle often results in qualitatively similar adaptations in both ventricles. This study provides yet another example of this commonly observed phenomenon. Acknowledgements

This investigation was supported in part by N.I.H. grants HL 17631, HL 19425 and HL 23979. R.A.C. and W.P.S. are Special Investigators for the South-East Pennsylvania Heart Association; A.A.B. is an Established Investigator for the American Heart Association. We wish to acknowledge MS Gracella Wilson for her expert technical assistance in this investigation. REFERENCES 1.

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and simplified methods for the spectroAnalytic Chemistry 35, 1961-1965 (1963).

COLLAGEN 2.

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IO.

11. 12.

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15.

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18.

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