The effect of growth hormone on rat myocardial collagen

Growth Hormone & IGF Research 1999, 9, 123–130 Article No. ghir.1999.0097, available online at http://www.idealibrary.com on

The effect of growth hormone on rat myocardial collagen Annemarie Brüel and Hans Oxlund Dept of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, Denmark

Summary Growth hormone (GH) can increase cardiac performance, but conditions with GH excess, such as acromegaly, are associated with hypertrophy and fibrosis of the heart. The aim of this study was to examine the effect of GH administration on rat myocardial collagen. Female rats were injected with GH (5 mg/kg/day) for 80 days. The weight of the right ventricle (RV) and the left ventricle (LV) was increased in the GH-treated group compared with the control group (P < 0.001). No differences in the ratio of heart weight/body weight or ventricle weight/body weight were found. The total amount of RV and LV collagen was increased in the GH-treated group (P < 0.001), but the collagen concentration was decreased (P < 0.001). Histomorphometry showed that the area fraction of collagen relative to myocytes remained unchanged. The composition of ventricular collagen in the GH- injected group did not differ from that of the control group concerning the relative amounts of collagen types I and III and pyridinoline, a mature collagen cross-link. We conclude that GH induced a substantial, but proportionate growth of the myocardium without formation of fibrosis. GH actually decreased the collagen concentration, and did not change the composition of myocardial collagen. © 1999 Churchill Livingstone

Key words: rat, myocardium, growth hormone, collagen, collagen types, cross-links.

INTRODUCTION Several studies suggest that administration of growth hormone (GH) can improve the cardiac performance in vivo. In normal humans, GH administration can increase cardiac contractility and cardiac output1. One unrandomized, unblinded study suggested that GH can improve cardiac function in patients with dilated cardiomyopathy2. However, this has not been confirmed3. In rats, administration of GH has been shown to increase cardiac output and reduce peripheral resistance4,5. Studies on isolated rat hearts, papillary muscles and

Received 1/9/98 Revised 7/4/99 Accepted 14/4/99 Correspondence to: Annemarie Brüel, Dept of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, DK-8000 C, Denmark. Tel: + 45 8942 3012; Fax: +45 8613 7539; E-mail: [email protected]

1096–6374/99/020123+08 $18.00/0

skinned muscle fibres have shown that GH treatment can increase the contractility of myocardial tissue6–8. Furthermore, GH may increase cardiac performance in rats with experimental cardiac failure9,10. These experimental results may provide indications for therapeutic use of GH in cardiac disease in the future. GH administration induces growth of the rat heart, and histological examinations have shown that no significant fibrosis developed when GH was given for 4 weeks5,6. In a previous study, we found that short-term GH administration (7 days) increased the collagen deposition rate in aorta and cardiac musculature of young rats11, suggesting that GH can increase the turnover of collagen. We have also shown that administration of GH for 80 days significantly increased collagen concentration and the relative amount of collagen type I of rat aorta accompanied by increased mechanical stiffness of the aorta12. These findings suggest that GH influences the collagen metabolism of the cardiovascular system. However, the © 1999 Churchill Livingstone

124 A. Brüel and H. Oxlund

effect of GH administration for a longer period on the concentration and composition of myocardial collagen has not yet been studied. The aim of the present study was to examine the effect of GH administration (80 days) on the concentration of collagen and the relative amounts of type I and type III collagen. We also measured the content of pyridinoline cross-links, a mature cross-link in collagen. The myocardial content of collagen fibres relative to myocytes was analysed by histomorphometry.

removed, as were any visible part of the cusps. The investigation was approved by the Danish Animal Experiment Inspectorate and conforms with “Guide for the Care and Use of Laboratory Animals” published by the US National Institute of Health (NIH publication No. 85–23, revised 1985). Determination of IGF-I Serum IGF-I levels were determined by radioimmunoassay13.

MATERIALS AND METHODS Materials

Determination of collagen

Twenty-six female SPF Wistar rats, 105 days old with an average body weight of 240 g, were randomized by weight into two groups. One group received biosynthetic human GH, 5 mg/kg/day, given as two daily injections subcutaneously in the nape of the neck. The other group, serving as a control group, received a corresponding volume of vehicle, i.e. physiological saline. The GH preparation was Norditropin® (Novo Nordisk, Gentofte, Denmark), with a specific activity of 2.93 IU/mg. The animals were weighed once a week and had free access to tap water and pellet food (Altromin Diet 1314, Chr. Pedersen Ltd, Ringsted, Denmark). The experiment lasted for 80 days, and no rats died during the experiment. At the end of the experiment, the mean blood pressure was measured with a tailcuff and a pneumatic pulse transducer (Narco BioSystems Inc., Houston, TX, USA) coupled to an amplifier. The coefficient of variation was 4.2%. During the experiment the rats were trained for measurement of the blood pressure. Fasting blood glucose was measured using stix (BM-Test/BG®, Boehringer Mannheim, Germany) and employing a reflectance meter (Reflolux S®, Boehringer Mannheim, Germany). The animals were anaesthetized with pentobarbital intraperitoneally and blood samples were obtained from aorta abdominalis and placed on ice in tubes containing EDTA as anticoagulant. After 15 min, the blood was centrifuged and plasma isolated and stored at –20°C. The heart was carefully removed, rinsed in Ringer’s solution and the wet weight (WW) of the hearts was determined after removal of the atria. The hearts were frozen in Ringer’s solution and stored at –80°C. The ventricles were thawed in Ringer’s solution, 21°C, and the free wall of the right (RV) and left ventricle (LV) was isolated. The thickness of the ventricle walls was measured between two slides using an electronic caliper. The ventricles were then blotted dry and weighed, giving the WW. Standardized samples were prepared for determination of collagen, collagen types I and III, content of pyridinoline cross-links and for histomorphometric studies. The papillary muscles and chordae tendineae were

The dry weight (DW) and dry defatted weight (DDW) of the tissue samples were determined after thawing. Hydroxyproline content of the tissue was determined according to Stegeman and Stalder14 and multiplied by 7.46, giving the collagen content15. The collagen content was expressed as the total collagen content of the ventricles and the collagen concentration as a percentage of the DDW. Determination of collagen types I and III Determination of the proportion of collagen types I and III was based on SDS-polyacrylamide-gel-electrophoresis (SDS-PAGE) of CNBr-digested collagen of the tissue samples12,16. Each sample was minced, defatted with acetone, washed and centrifuged three times with 2% SDS, four times in phosphate buffered saline, and three times in distilled water. The samples were then suspended in 1 ml 70% formic acid and flushed with N2. An amount of CNBr equal to the weight of the samples was added under N2. The samples were then placed in a water bath for 4 h at 30°C and centrifuged at 15 750 × g for 15 min. The supernatant was evaporated until dryness, resuspended in distilled water and freeze-dried for 48 h. The peptides were dissolved in 50–100 µl sample buffer (30 mmol/1 TRIS, 1 mmol/l EDTA, 2.5% SDS, pH 8.0), placed in boiling water for 10 min, and separated by SDS-PAGE using an automated system with preformed gels, sample applicator and buffer strips (PhastSystem®, Pharmacia, Uppsala, Sweden). The gels were PhastGels™, Homogeneous Media 12.5%®, with a thickness of 0.45 mm and a length of 45 mm, including a 13 mm stacking gel zone. The buffer system in the strips was 200 mmol/l tricine, 200 mmol/l TRIS, and 0.55% SDS in 2% agarose, pH 7.5. Application of samples, running of the gels and staining the peptide bands with PhastGel Blue® was done as prescribed by the manufacturer. After drying, the gels were scanned at 500 and 540 nm using Dual-Wavelength Thin Layer Chromate Scanner Model CS-930® (Shimadzu, Koyto, Japan), and the relevant scanned peaks were cut

The effect of GH on rat myocardial collagen 125

out and weighed. The bands used for calculation of the relative amounts of type I and type III collagen were the α1(I) CB7 and the α1(III) CB5. The value of the α1(I) CB7 peak was multiplied by 3/2 because only two of the three αchains of type I collagen are α1-chains. Furthermore, the values of the α1(I) CB7 peak was multiplied by 0.848, since the α1(I)CB7 has a larger molecular weight than the α1(III)CB5 peptide. In a previous study12, we found that the amount of collagen solubilized by the CNBr-treatment was more than 90%, that the collagen peptides did stem from collagen (i.e. were decomposed by bacterial collagenase) and were pure (i.e. no changes in relative density of the electrophoretic bands after treatment with pepsin). Finally, the relative amounts of hydroxyproline in the electrophoretic bands determined by a reversed-phase HPLC amino acid analysis were consistent with the relative amounts of types I and III collagen determined by SDSPAGE. Less than 1% of the total hydroxyproline of the ventricles was extracted by the SDS treatment.

Area fraction (%) = Pc × 100/(Pc + Pm)

The grid was placed randomly over the histological slide and counting was performed on two sections per animal at 12 different sites of the LV and eight different sites of the RV (magnification × 400, 25 µm between gridpoints). Figure 1 shows a section of LV from a GHinjected rat in normal light (a) and in polarized light (b). Statistical analysis Mean values with SEM are given. Analysis of differences between the groups was done by the Student’s t-test, and differences were considered significant when P was less than 0.05 (two-tailed).

Determination of pyridinoline cross-links The samples were washed in distilled water, defatted with acetone and hydrolysed in 6 M HCl, 110°C for 16 h. An aliquot was taken for determination of hydroxyproline as described above. The pyridinoline cross-links were concentrated on a CF-1 column as described by Black et al.17, the fraction containing the cross-links was evaporated until dryness and dissolved in 0.5 ml 1% (v/v) n-hepta-fluorobutyric acid (HFBA) and centrifuged. The concentrations of pyridinoline and deoxy-pyridinoline were determined as described by Eyre et al.18 using HPLC. The standards used for calibration of the column were pyridinoline and deoxy-pyridinoline (Metra Biosystems Inc., Palo Alto, CA, USA). Results are given as mol pyridinoline per mol hydroxyproline. The intra-assay coefficient of variation was 7.76% and the inter-assay coefficient of variation was 8.59%. The amount of deoxy-pyridinoline in the samples was too small for reliable measurements.

(a)

Histomorphometric studies The samples were immersion-fixed in formalin, embedded in paraffin, cut into 4-µm-thick sections and stained with Picro-Sirius Red19. An eyepiece grid with a square side length of 10 mm and 100 intersection points was used to determine the area fraction of collagen and myocytes by point counting. Using polarization microscopy the number of grid points on the collagen (Pc) and the number of grid points on the myocytes (Pm) were calculated, and the area fraction of collagen relative to myocytes was obtained by the following equation:

(b) Fig. 1 Example of a 4-µm section (stained with Picro-Sirius Red) of the left ventricle from a GH- injected rat showing the appearance of a collagen fibre in normal light (a) and polarized light (b). Magnification × 108 (reduced to 90% for reproduction). Arrows pointing at collagen fibres. Bar = 100 µm.

126 A. Brüel and H. Oxlund

Weight (g)

500

GH

400

300 Controls

200

0 16

20

24

28

Age (weeks) Fig. 2 The effect of GH (5 mg/kg/day) on the body weight of the rats during the experiment. Mean values ± SEM.

RESULTS Figure 2 shows the changes in the body weight of the animals. The body weight of the GH-injected group increased substantially during the experimental period. The final body weight of the GH-injected group was increased by 75% compared with the control group (278 ± 5 vs 487 ± 7 g, P < 0.001). Number of animals and values of blood pressure, blood glucose and serum IGF-I are given in Table 1. There were no differences in blood glucose and mean blood pressure, between the groups. A two-fold increase in serum IGF-I was found in the GHinjected group when compared with the control group (1270 ± 56 vs 605 ± 28 ng/ml, P < 0.001). In the GHinjected group, the WW of the ventricles + septum was significantly increased by 67% (1100±28 vs 659±17 mg, P < 0.001). The WW of the ventricles are shown in Fig. 3(A). Comparing the GH-injected group with the control group, the WW of the RV was significantly increased by 79% (215±5 vs 120±5 mg, P < 0.001) and the WW of the LV was significantly increased by 68% (637±24 vs 379±11

mg, P < 0.001). Figure 3(B) shows that the wall thickness of both ventricles was significantly increased by 43%, when comparing the GH-injected group with the control group (RV: 0.96±0.03 vs 0.67±0.03 mm, P < 0.001; LV: 2.80±0.08 vs 1.96±0.05 mm, P < 0.001). No differences in the ratios of ventricles+septum weight/body weight or ventricle weight/body weight were found between the groups. The total collagen content of RV was significantly increased by 63% (1.42±0.05 vs 0.87±0.04 mg, P < 0.001) in the GH-injected group compared with the control group, and the collagen content of the left ventricle was increased by 52% (2.97±0.13 vs 1.95±0.04 mg, P < 0.001) [Fig. 4(A)]. However, the collagen concentration, expressed as a percentage of the DDW, was significantly reduced by 17% in both RV and LV of the GH-injected group compared with the control group (RV: 4.06±0.10 vs 4.73±0.11%, P < 0.001; LV: 2.62±0.05 vs 3.06±0.07%, P < 0.001) [Fig. 4(B)] Table 2 gives the area fraction of collagen, collagen types I and III and pyridinoline content. The GH-treatment did not influence the area fraction of collagen relative to myocytes, or the relative amounts of type I and type III collagen in either RV or LV. No differences in the content of pyridinoline, expressed as pmol/nmol hydroxyproline, were found between the groups. DISCUSSION The classic indication for GH therapy is GH deficiency, where GH is administered in substitutional doses. There is, however, a growing interest in using pharmacological doses of GH for catabolic patients, e.g. patients with burns, chronic renal failure, chronic obstructive pulmonary disease20–22, patients that may benefit from the anabolic effects of GH therapy. This, and recent experimental reports of improved cardiac function induced by GH5,8,10, underline the need for thorough elucidation of the effects of GH on the cardiovascular system. As shown in previous studies5,6, GH administration induced a significant growth of the heart. In contrast to these studies, no selective cardiac hypertrophy was found in the present study, as the ratios ventricles + septum weight/body weight and ventricle weight/body weight were unchanged. However, in the previous experiments GH was administered for 4 weeks only, and

Table 1 The effect of GH (5 mg/kg/day) on blood pressure, blood glucose and serum IGF-I. Number of rats

Final mean blood pressure (mmHg)

Final blood glucose (mmol/l)

Serum IGF-I (ng/ml)

Controls

14

115 ± 2

3.8 ± 0.2

605 ± 28

GH

12

119 ± 3

3.8 ± 0.3

1270 ± 56a

Mean values ± SEM. a : P < 0.001 compared with controls

The effect of GH on rat myocardial collagen 127

(A) 800

(B) 3

a

a

2

Thickness (mm)

Weight (mg)

Controls GH

Controls GH

600

400

a

1

a 200

0

RV

0

LV

RV

LV

Fig. 3 The effect of GH (5 mg/kg/day) on (A) the wet weight and (B) the thickness of the ventricles at the end of the experiment. Mean values ± SEM. RV: right ventricle; LV: left ventricle.a: P < 0.001. (A)

(B) 5 a a Collagen concentration (% of DDW)

Total amount of collagen (mg)

3 Controls GH

2 a

1

0

Controls GH

4

3

a

2

1

0 RV

LV

RV

LV

Fig. 4 The effect of GH (5 mg/kg/day) on (A) the total amount of collagen and (B) the collagen concentration of the ventricles at the end of the experiment. Mean values ± SEM. RV: right ventricle; LV: left ventricle; DDW: dry defatted weight. a: P < 0.001.

furthermore a lower dose of GH was used. A different animal model using rats with a transplantable GH-secreting tumour has also been used to study the effect of GH excess on the cardiovascular system. Using this model, rats were exposed to GH excess for 18 weeks and no differences in the ratios RV or LV weight to body weight

were found23. In similar studies of shorter duration, the results have been conflicting4,8,24–26. The myocardium consists of cardiac myocytes, capillaries, fibroblasts and an extracellular matrix. This extracellular matrix is composed of collagen, elastin and a ground substance. Collagen fibres types I and III are the

128 A. Brüel and H. Oxlund

Table 2 The effect of GH (5 mg/kg/day) on the composition of the myocardial collagen. Area fraction of collagen (% of collagen+myocytes)

Type I collagen (% of I+III)

Pyridinoline content (pmol pyr/nmol hydroxyproline)

RV

LV

RV

LV

RV

LV

Controls

5.57 ± 0.21

3.51 ± 0.11

74.7 ± 0.7

77.2 ± 2.2

0.87 ± 0.05

1.10 ± 0.16

GH

5.08 ± 0.39

3.60 ± 0.31

72.1 ± 1.7

75.0 ± 0.9

0.82 ± 0.05

1.24 ± 0.26

Mean values ± SEM. RV: right ventricle; LV: left ventricle; Pyr: pyridinoline.

predominant collagen types, but types IV, V and VI collagen are also found27–29. The function of this matrix is to align and tether myocytes and capillaries30, and through its uncoiling and tensile strength collagen perimysial fibres may protect the sarcomeres from being overstretched during the diastole31. The concentration of collagen has been shown to increase in the rat myocardium in various models of hypertrophy: banding of aorta, renovascular or genetically-based hypertension. In hypertrophy caused by volume-overload27,32 and training33, the collagen concentration of the left ventricle was unchanged. Several studies suggest that the changes in concentration and structure of fibrillar collagen may influence the stiffness of the myocardium34–36. In the GH-injected group, we found the total amount of collagen to be increased by 63 and 52% in the RV and LV, respectively, but the concentration of collagen to be reduced by 17% in both ventricles. No differences concerning the area fraction of collagen relative to myocytes were found between the groups. This is consistent with histological studies on ventricles from rats submitted to GH excess for 4 weeks by either injections5,6 or bearing GH-producing tumours37, where no fibrosis was found. These results suggest that the growth of the heart in the GH-injected group was proportionate in relation to body weight, and that the increase in the total amount of collagen is accompanied by a similar growth of the myocytes. Further, the composition of collagen did not differ from the collagen found in the controls as regards collagen types I and III and pyridinoline cross-links. These results are in contrast with results on aorta, where we found that GH increased the collagen concentration and the relative amounts of type I collagen. The area fraction of collagen relative to myocytes of the ventricles was slightly higher compared with the collagen concentration measured biochemically, because collagen was related to the fractional area of the myocytes and not the total fractional area. The SEM-values of the histomorphometric measurements were larger compared with the biochemical measurements. This may explain why the decrease in the collagen concentration of the ventricles of the GH-injected group is not reflected in the histomorphometric measurements.

In humans, impairment of the diastolic function is found in both GH deficiency and acromegaly38,39 and long-term human acromegaly is associated with fibrosis of the heart39–41. In the present experiment, no fibrosis was found; however, in contrast with human acromegaly42, no observations of either hypertension or diabetes were found in this animal model of GH excess, conditions which may by themselves induce fibrosis. Another explanation could be that the GH excess period of the present experiment may mimic short-term acromegaly rather than long-term acromegaly, considering that the normal lifespan of a rat is 2–2.5 year. It has been shown that GH, injected in rats for 4 weeks, induced a hypertrophic growth of the heart and increased both systolic and diastolic performance5. This is consistent with an abstract by Fazio et al. reporting LV hypertrophy and increased cardiac output but no impairment of the diastolic function in acromegalic humans with a duration of disease less than 5 years43. This is in agreement with the hypothesis that early-stage acromegaly is characterized by increased cardiac output followed by a gradual myocardial hypertrophy with increasing interstitial fibrosis and impairment of the diastolic function in the intermediate stage. In the end-stage disease, cardiac output decreases and eventually congestive heart failure and ventricular dilatation are seen44. The anabolic effect of GH was shown by a 75% body weight increase in the GH-injected group. The increase in body weight was accompanied by a two-fold increase in serum IGF-I. When injecting rats with human GH, production of antibodies against GH can be demonstrated after 3–4 weeks. However, this does not seem to influence the effect of GH evaluated from the growth curves. A minor part of the increase in body weight may be caused by fluid retention, as GH has an antidiuretic and antinatriuretic effect on rats45. In female rats treated with GH, 5 mg/kg/day, for 90 days, the body weight was reduced by 9% after 1 week without GH-treatment46. The following weeks, still without treatment, the body weight remained constant. The peak levels of endogenous GH of female rats reach 300–600 µg/l serum, and between peaks the levels are 20–100 µg/l serum47. Subcutaneous injections of 2 and 4

The effect of GH on rat myocardial collagen 129

mg of GH per kg rat result in a broad peak of GH with a maximum GH concentration of approximately 600 and 1200 µg/l serum, respectively, 2 h after injection48. Consequently, the dosage of 2 × 2.5 mg/kg/day (~7.33 IU/kg/day) of GH used in this study is a pharmacological dosage resulting in very broad peaks twice a day. However, this dosage has been shown to affect the content and composition of aortic collagen and was therefore used in the present study. In summary, this study shows that GH administration for 80 days in pharmacological dosages induced a substantial, but proportionate growth of the rat myocardium. GH did not induce myocardial fibrosis, as the collagen concentration was actually decreased by 17% in the GHinjected group. The increase in the total amount of collagen by more than 50% was accompanied by a similar growth of the cardiac myocytes. The composition of the ventricular collagen in the GH-injected group did not differ from that of the control group concerning the relative amounts of collagen types I and III or pyridinoline content, a mature collagen cross-link.

7.

8.

9.

10.

11.

12.

13.

ACKNOWLEDGEMENTS This investigation was supported by The Danish Health Research Council, grants no. 9500922 and 9600822 (Aarhus University – Novo Nordisk Centre for Research in Growth and Regeneration), P. Carl Petersens Foundation, Novo Nordisk A/S and The Danish Heart Society. The authors thank E. Mikkelsen and C. Knæhus for technical assistance and M. Fischer for linguistic revision.

14. 15. 16.

17.

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