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Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression after acute myocardial infarction
Growth Hormone & IGF Research 2002, 12, 208–215 doi:10.1016/S1096-6374(02)00042-4, available online at http://www.idealibrary.com on
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression after acute myocardial infarction Hongkui Jin1, Renhui Yang1, Hsienwie Lu1, Annie K Ogasawara1, Wei Li1, Anne Ryan2, Franklin Peale2, and Nicholas F. Paoni1 1 Department of Cardiovascular Research, Genentech, Inc., South San Francisco, CA 94080, USA; and 2Department of Pathology, Genentech, Inc., South San Francisco, CA 94080, USA
Summary Objective. This study examined the effects of growth hormone (GH) on infarct size, survival, and cardiac gene expression in rats with acute myocardial infarction. Design. Animals randomly received sc injection of either saline vehicle (n ¼ 98) or GH (2 mg/kg/day, n ¼ 105) for 14 days commencing the day of left coronary artery ligation. Infarct size was determined by morphometric analysis at the time of death or at 52 weeks post-surgery. Gene expression was analyzed by real-time RT-PCR after 2week treatment. Results. GH decreased infarct size by18% ðP < 0:01) and increased survival by 36% at 52 weeks. GH also significantly reduced cardiac expression of atrial natriuretic factor, b-myosin heavy chain, a-smooth muscle actin, collagen I, collagen III, fibronectin, and pro-inflammatory cytokines. Conclusions. Treatment with GH for 2 weeks beginning on the day of myocardial infarction produced beneficial effects that were associated with reductions in cardiac gene expression symptomatic of pathological remodeling. ª 2002 Elsevier Science Ltd. All rights reserved.
Key words: gene expression, growth hormone, heart failure, infarct size, myocardial infarction
INTRODUCTION
It is known that growth hormone (GH) is essential to the control of linear growth, glucose, and homeostasis and for maintenance of skeletal muscle mass. Recent
Received 31 January 2002 Revised 12 March 2002 Accepted 28 March 2002 Correspondence to: Hongkui Jin, MD, Department of Cardiovascular Research, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Tel.: +1-650-225-1314; Fax: +1-650-225-6327; E-mail: [email protected]
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animal experiments and clinical studies have shown that GH also plays an important role in modulating cardiac structure and function.1;2 We3;4 and other investigators5–9 have demonstrated that administration of GH produces beneficial effects with improved cardiac function in animals with chronic heart failure post-myocardial infarction (MI) or with pacing-induced heart failure. Furthermore, some clinical studies suggest that treatment with GH results in a significant improvement in hemodynamics and clinical function in patients with chronic heart failure caused by ischemic10;11 or idiopathic dilated cardiomyopathy.12 The beneficial effects of GH on cardiovascular ª 2002 Elsevier Science Ltd. All rights reserved.
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression
function including an increase in cardiac index and a reduction in peripheral vascular resistant can be acutely observed after intravenous infusion of GH in these patients.13;14 Despite many investigations focused on treatment with GH for chronic heart failure, few studies have determined the effects of GH given early on acute MI. The reparative processes in cardiac muscle following experimental MI are stimulated with GH.15 It has been shown that GH given early after large experimental MI attenuates left ventricular remodeling and improves cardiac function.16;17 A recent study demonstrates that treatment with the GH secretagogue CP-424,391 reduces infarct size in a rabbit model of myocardial ischemia and reperfusion.18 However, little is known whether the beneficial effects of GH on cardiac remodeling and function accompany changes in the expression of maker genes associated with MI and whether the early therapeutic regimen prolongs survival. The present study was designed: (1) to determine effects of early treatment with GH on infarct size and long-term survival and (2) to test the hypothesis that GH may also attenuate the induction of cardiac genes in MI. Our results showed that early treatment with GH for 2 weeks resulted in beneficial effects in association with reductions in cardiac gene expression symptomatic of pathological remodeling following experimental MI. METHODS
All experimental procedures conformed with the Guide for the Care and Use of Laborotory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and were approved by Genentech’s Institutional Animal Care and Use Committee.
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suture. The chest was closed by suturing pectoral muscles and the skin. The incision of the trachea was sutured after withdrawal of the respirator. Growth hormone administration
Twenty minutes after surgery, animals with stable general condition were randomly treated with either saline vehicle or recombinant human GH (Genentech, Inc., South San Francisco, CA) at 1 mg/kg, twice a day by subcutaneous injection for 14 days. Our previous studies have demonstrated that GH at this dose for 2 weeks significantly improves cardiac performance without inducing cardiac hypertrophy in rats with heart failure post-MI.3 In rats post-MI, this dose of GH increases serum insulin-like growth factor-1 by 45% (528 29 vs. 366 31 ng/ml, P < 0:01Þ. The animals were followed up for 52 weeks after surgery. Infarct size measurements
Infarct size was determined by morphometric analysis at the time of death or when the surviving animals were killed at 52 weeks post-surgery. The right ventricular free wall was dissected from the left ventricle (LV). The LV was cut in four transverse slices from apex to base. Five micrometer sections were cut and stained with Massons’ trichrome stain and mounted.22;23 The endocardial and epicardial circumferences of the infarcted and non-infarcted regions were determined with a planimeter Digital Image Analyzer. The infarcted circumference and the total left ventricular circumference of all four slices for both endocardial and epicardial surfaces were summed and expressed as a percentage of infarcted circumference to total circumference for determination of infarct size. Measurement of infarct size was carried out by the same person, who did not know the sample from the GH or vehicle group before measurement.
Animal model
Male Sprague–Dawley (SD) rats (Charles River Breeding Laboratories, Inc., 8 weeks of age) were acclimated to the facility for at least 1 week before surgery. Rats were fed a pelleted rat chow and water ad libitum, and housed in a light and temperature controlled room. The procedure used for left coronary ligation has been described in detail elsewhere.19–21 In brief, the rats were anesthetized with ketamine hydrochloride (100 mg/kg, ip) and xylazine (10 mg/kg, ip). The trachea was intubated and ventilated with a respirator (Harvard Apparatus Model 683). After a left-sided thoracotomy, the left coronary artery was ligated approximately 2 mm from its origin with a 7-0 silk
Study on cardiac gene expression
In a parallel experiment, ligated rats with evident MI on ECG3;4 were treated with vehicle or GH at the dose as described above, and sham-operated rats at the same age were used without treatment as controls. At the end of the 2-week treatment period, the rats were killed, the heart was removed and dissected, and the left ventricle was weighed, fast frozen in liquid nitrogen, and stored at )70 °C for subsequent RNA analysis. Total RNA was isolated from the ventricular samples using the RNeasy Maxi Kit (Qiagen) according to the manufacturer’s instructions. Gene expression analysis was performed using real-time RT-PCR (Taq-
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Man) technology. RT-PCR was performed on 1 ng of total RNA per reaction using the TaqMan Model 7700 Sequence Detector (ABI–Perkin Elmer).24 Amplification reaction conditions (for 50 ll) were 1 TaqMan Buffer A, 300 lM dATP, dCTP, dGTP, and 600 lM dUTP, 10% glycerol, 5.5 mM MgCl2, 50 U MuLV reverse transcriptase, 20 U RNase Inhibitor, 1.25 U AmpliTaq Gold, 100 nM forward and reverse primers, and 100 nM fluorogenic probe. RT-PCR reagents and glycerol were purchased from Perkin Elmer and Sigma, respectively. Reactions were performed in MicoAmp Optical Tubes and Caps (ABI–Perkin Elmer). TaqMan primers and probes were designed according to guidelines determined by Perkin Elmer and synthesized at Genentech, except for those for rodent GAPDH which were a generous gift from Perkin Elmer. Reverse transcription was performed at 48 °C for 30 min followed by heat activation of AmpliTaq Gold at 95 °C for 10 min. Thermal cycling was at 95 °C for 30 s and 60 °C for 1.5 min for 40 cycles. Quantitation of the TaqMan results was performed as described by Heid et al.25 with modifications. Briefly, standard curves (1:5 serial dilution) for each target gene of interest were run in duplicate. The Ct was plotted on the Y-axis vs. the log of the total RNA concentration (X-axis), and the equation describing the line was determined. Experimental samples were analyzed using 3–5 replicates each, and the quantity of the mRNA for each target gene was determined from the appropriate standard curve by entering the Ct (Y value) and solving for the input mRNA (X). The value for the target gene was then normalized to GAPDH by solving the following equation: 10X1 =10X2 , where X1 is the target gene, and X2 is GAPDH.
animals died before treatment with GH or vehicle. Of the 229 rats, 26 rats (13 GH-treated and 13 vehicletreated) were excluded from the present study. One rat had a large tumor in the abdominal cavity, 15 rats had no infarction, and in 10 other animals measurement of infarct size was not possible because of postmortem cannibalization or body discard. There was no difference in the baseline of BW before ligation or treatment between GH-treated (299:3 1:6 g, n ¼ 105) and vehicle-treated rats (300:7 1:6 g, n ¼ 98). Two weeks after treatment, an
Fig. 1 Effects of early treatment with GH for 2 weeks on BW in rats with MI. Data expressed as mean SEM. The number in the parenthesis is the animal number in each group. *P < 0:05, **P < 0:01, compared to the vehicle group.
Statistical analysis
Results are expressed as mean SEM. For analysis of body weight, infarct size, and gene expression, parameters between the GH and vehicle group were compared by an unpaired Student t test. Survival (%) between the GH-treated and vehicle-treated groups was compared by log rank (Mantel–Cox) and Breslow–Gehan–Wilcoxon tests. For the data of gene expression, one-way analysis of variance (ANOVA) was carried out for comparisons of each gene expression between groups. P < 0:05 was considered significant.
RESULTS Effects of GH on BW, infarct size, and survival
The 229 of the 235 rats receiving ligation of the left coronary artery entered into the study because six
Fig. 2 Effects of early treatment with GH for 2 weeks on infarct size in rats with MI. Data expressed as mean SEM. The number in the parenthesis is the animal number in each group. **P < 0:01, compared to the vehicle group.
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression
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the dose used, which was consistent with our previous observations.3 LV expression levels of 19 genes were used to examine the effect of GH on the molecular phenotypes of MI hearts. MI for 2 weeks substantially increased expression of atrial natriuretic factor (ANF) that was significantly attenuated by treatment with GH (top panel, Fig. 4). As compared to the sham group, the mRNA level of a-myosin heavy chain (aMHC) was significantly reduced in the MI + vehicle group but not in the
Fig. 3 Effects of treatment with GH for 2 weeks on 1-year survival (%) in rats with MI. The number in the parenthesis is the animal number in each group.
increase in BW was significantly greater in the GH group than the vehicle group (78:63:0 g, n ¼ 84 vs. 60:0 3:9 g, n ¼ 75; P < 0:001) indicating treatment with GH significantly increased BW growth (Fig. 1). After stopping GH administration, the increase in BW growth was maintained for at least 4 weeks (Fig. 1). Pathological studies revealed that infarct size was significantly reduced (P < 0:01) by 18% in GH-treated animals (30:8 1:6%, n ¼ 105) compared to vehicle controls (37:4 1:6%, n ¼ 98) (Fig. 2). There was a significant negative correlation between infarct size and survival days in both vehicle-treated rats (r ¼ 0:6664, P < 0:0001, n ¼ 98), and GH-treated animals (r ¼ 0:6283, P < 0:0001). Early treatment with GH for 2 weeks prolonged one-year (52-week) survival by 36% (30.48% and 22.45% in the GH-treated and vehicle-treated animals, respectively). Although the difference did not reach statistical significance (P ¼ 0:200 using the Mantel–Cox test, or 0.188 using the Breslow–Gehan– Wilcoxon test), there was a trend toward improved survival in rats receiving GH (Fig. 3). The GH-induced increase in survival appeared to occur as early as 2–3 weeks, and was then maintained. Effects of GH on cardiac gene expression
The ratio of LV weight to BW at 2 weeks after operation was significantly increased in the MI group compared to the sham controls (2:11 0:06 vs. 1:86 0:08, P < 0:05), but this ratio was similar in the MI + vehicle vs. MI + GH group (2:11 0:06 vs. 2:05 0:04, NS). This suggested that MI-induced LV hypertrophy was not altered by treatment with GH at
Fig. 4 Effects of early treatment with GH for 2 weeks on cardiac expression of seven genes in rats with MI. Data expressed as mean SEM (n ¼ 5–7 in each group). *P < 0:05, **P < 0:01, compared to the sham group. # P < 0:05; ## P < 0:01, compared to the MI + vehicle group. ANF, atrial natriuretic factor; aMHC, a-myosin heavy chain; bMHC, b-myosin heavy chain; MLC2, myosin light chan-2; ACA, a-cardiac actin; SKA, a-skeletal muscle actin; SMA, a-smooth muscle actin.
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MI + GH group (left of middle panel, Fig. 4). In contrast, MI induced a significant elevation in the expression level of b-myosin heavy chain that was normalized by GH (middle of middle panel, Fig. 4). aSmooth muscle actin was markedly increased by 6fold in the MI + vehicle group compared to the sham group but significantly attenuated by GH administration (right of bottom panel, Fig. 4). Compared to sham controls, MI rats exhibited a 1.7-, 8.3-, and 12.5-fold increase in expression of collagen I, collagen III, and fibronectin, respectively, whereas treatment with GH significantly inhibited the expression of these three genes (bottom panel, Fig. 5). MI was associated with a significant increase in the mRNA level of a-skeletal muscle actin (middle of bottom panel, Fig. 4) and isocitrate dehydrogenase (left of top panel, Fig. 5), but GH did not alter the expression of these genes significantly. Neither MI nor GH had effect on expression of myosin light chan-2 (right of middle panel, Fig. 4), acardiac actin (left of bottom panel, Fig. 4), mitochondrial ATP synthase (right of top panel, Fig. 5), phospholamban, and sarcoplasmic reticulum Ca2þ ATPase (middle panel, Fig. 5). LV gene expression of leukemia inhibitory factor, endothelin-1 (top panel, Fig. 6), tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) (middle panel, Fig. 6), and interleukin-6 (IL-6) (bottom panel, Fig. 6) was significantly increased in the MI + vehicle group compared to the sham group. Treatment with GH significantly attenuated the mRNA levels of leukemia inhibitory factor and IL-6, and tended to reduce endothelin-1, TNF-a, and IL-1b. DISCUSSION
In the present study, administration of GH for 2 weeks beginning on the day of MI resulted in a significant reduction in infarct size by 18%, an increase in survival by 36% at 52 weeks, and a attenuation of LV gene expression of ANF, b-myosin heavy chain, asmooth muscle actin, collagen I, collagen III, fibronectin, and pro-inflammatory cytokines. This is the first demonstration that early treatment with GH produces beneficial effects in association with reductions in cardiac gene expression symptomatic of pathological remodeling in experimental MI. It is known that GH promotes wound healing. It has been reported that the reparative processes in cardiac muscle following experimental MI are stimulated with anabolic hormones. The stimulation action is most effective during early phase of the reparative process. In the dog model of acute MI, treatment with GH increases protein synthesis in infarcted muscle by 168% 24 h after infarction.15 GH enhances glycin-2 14 C incorporation into the connective tissue of healing in-
Fig. 5 Effects of early treatment with growth hormone (GH) for 2 weeks on cardiac expression of seven genes in rats with MI. Data expressed as mean SEM (n ¼ 5–7 in each group). *P < 0:05, **P < 0:01, compared to the sham group. # P < 0:05, ## P < 0:01, compared to the MI + vehicle group. ICDH, isocitrate dehydrogenase; MTATP, mitochondrial ATP synthase; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca2þ -ATPase; COL1, collagen 1; COL3, collagen 3; FIB, fibronectin.
farct. It is possible that the increased rate of protein synthesis in and around the infarcted area would add strength to the damaged cardiac muscle, increase its resistance against infarct expansion, and improve post-infarction healing. These effects of GH may contribute, in part, to a reduction of infarct size. The present study showed that early treatment with GH for 2 weeks tended to increase 1-year survival rate by 36% in MI rats. Since there is a significant negative correlation between infarct size and survival days in
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression
Fig. 6 Effects of early treatment with GH for 2 weeks on cardiac expression of five genes in rats with MI. Data expressed as mean SEM (n ¼ 5–7 in each group). *P < 0:05, **P < 0:01, compared to the sham group. # P < 0:05, ## P < 0:01, compared to the MI + vehicle group. LIF, leukemia inhibitory factor; ET-1, endothelin-1; TNF-a, tumor necrosis factor-a; IL-1b, interleukin-1; IL-6, interleukin-6.
both vehicle-treated and GH-treated animals, it is likely that a reduction in mortality in the GH-treated group is attributed to a decrease in infarct size. In addition, an improvement in cardiac function may also contribute to the lower mortality rate in GHtreated animals. Several lines of evidence indicate that GH given early after moderate and large MI significantly improves systolic and diastolic LV function in this rat model of MI.16;17 Our previous studies also demonstrate that GH at the same dose enhances contractile reserve in myocytes and improves LV performance in rats with heart failure post MI.3;26
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To evaluate the molecular effects of GH on the heart, LV gene expression of several markers of pathological remodeling and cytokines were analyzed after 2 weeks of MI or treatment. There are 3 major findings obtained from the gene analysis in the present study. First, MI was associated with a significant increase in LV gene expression of ANF, b-myosin heavy chain, a-skeletal actin, and a-smooth muscle. This is essentially consistent with recent studies that demonstrate the increased ventricular expression of these genes coding for the fetal phenotype during ventricular remodeling following MI in rats.27–30 It is noted in the present study that treatment with GH attenuated the expression ANF, b-myosin heavy chain, and a-smooth muscle actin. It is known that fetal gene expression may be one of the hallmarks of disease progression in LV remodeling and dysfunction. The reduction in LV fetal gene expression observed in the MI rats treated with GH might reflect an improvement of cardiac function and attenuation of LV remodeling. Second, consistent with previous observations that cardiac extracellular matrix genes were up-regulated 2 weeks after MI,29;31 we found that LV mRNA levels of collagen I, collagen III, and fibronectin were increased in MI rats compared to sham controls. Furthermore, the present study demonstrated that early treatment with GH significantly inhibited over-induction of these genes. This is in agreement with the report by Grimm et al., who demonstrate that GH given early after moderate and large MI substantially reduced the considerably increased depositions of fibronectin and collagen I in the rat LV.17 The effect of GH on cardiac extracellular matrix mRNA and proteins may have an important clinical impact, since experimental and clinical studies have demonstrated an increase in interstitial collagens of the LV or non-ischemic myocardium at a chronic or late stage post-MI, which may enhance cardiac stiffness and result in diastolic dysfunction, finally leading to heart failure.29;31–34 Third, there was LV induction of genes encoding pro-inflammatory cytokines or growth factors including TNF-a, IL-1b, IL-6, leukemia inhibitory factor, and endothelin-1 in rats with MI. This is consistent with recent findings that cardiac expression of mRNA of TNF-a, IL-1b, and IL-6 is up-regulated in the early phase of acute MI and the late stage post-MI35–38 Clinical and experimental studies have shown that the inflammatory response to MI is associated with induction of cytokines such as of TNF-a, IL-1b, and IL-6, which are thought to act as a ’’cascade fashion.’’39–42 As induced, cytokine gene expression is clearly observed before significant histological evidence for inflammation can be identified in rats with MI, cytokine
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gene expression may be primarily generated by intrinsic myocardial cells, instead of inflammatory cells, in response to ischemia.36 The present study demonstrated that early treatment with GH significantly attenuated the induction of IL-6 and leukemia inhibitory factor (one of IL-6 family members), and tended to reduce mRNA levels of TNF-a, IL-1b, and endothelin-1 in the MI hearts. It has been shown that pro-inflammatory cytokines exert a direct negative inotropic effect mediated through a myocardial nitric oxide synthase,43 which may depress myocardial function. For example, IL-6 may act as a nitric oxidedependent cardiac depressant and may be associated with stunned myocardium.39;43;44 IL-6 has also been shown to produce nitric oxide-mediated reduction in calcium flux and contractility in ventricular myocytes45 Our finding of inhibitory effect of GH on induction of the pro-inflammatory cytokines may contribute, in part, to improvement of cardiac function after early treatment with GH observed in rats with MI.16;17 In summary, early administration of GH following acute MI for 2 weeks resulted in a reduction in infarct size and an increase in survivals. The beneficial effects of GH were accompanied by attenuating cardiac expression of several marker genes of pathological remodeling and cytokines. Taken together with previous observations that GH attenuates early LV remodeling and improves cardiac function,16;17 the data in the present study implicate that GH might have the potential for early treatment of acute MI. Our study, however, cannot evaluate the effect of early treatment with GH for a long term, because administration of human GH for more than 15 days would produce the antibody against the human GH in rats. A further study is necessary to determine the effects of early, long-term therapy with recombinant rat GH in rats with MI. REFERENCES 1. Cittadini A, Longobardi S, Fazio S, Sacca L. Growth hormone and the heart. Miner Electrolyte Metab 1999; 25: 51–55. 2. Sacca L, Fazio S. Cardiac performance: growth hormone enters the race. Nat Med 1996; 2: 29–31. 3. Yang R, Bunting S, Gillett N, Clark R, Jin H. Growth hormone improves cardiac performance in experimental heart failure. Circulation 1995; 92: 262–267. 4. Jin H, Yang R, Gillett N, Clark RG, Ko A, Paoni NF. Beneficial effects of growth hormone and insulin-like growth factor-1 in experimental heart failure in rats treated with chronic ACE inhibition. J Cardiovasc Pharmacol 1995; 26: 420–425. 5. Duerr RL, McKirnan MD, Gim RD, Clark RG, Chien KR, Ross J Jr. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic ventricular failure in the rat. Circulation 1996; 93: 2188–2196.
6. Ross J Jr., Hongo M. The role of hypertrophy and growth factor in heart failure. J Cardiac Failure 1996; 2(4 Suppl): S121–S128. 7. Isgaard J, Kujacic V, Jennische E, et al. Growth hormone improves cardiac function in rats with experimental myocardial infarction. Eur J Clin Invest 1997; 27: 517–525. 8. Hongo M, Sentianin EM, Tanaka N, et al. Angiotensin II blockade followed by growth hormone as adjunctive therapy after experimental myocardial infarction. J Cardiac Failure 1998; 4: 213–224. 9. Houck WV, Pan LC, Kribbs SB, et al. Effects of growth hormone supplementation of left ventricular morphology and myocyte function with the development of congestive heart failure. Circulation 1999; 100: 2003–2009. 10. Genth-Zotz S, Zotz R, Geil S, Voigtlander T, Meyer J, Darius H. Recombinant growth hormone therapy in patients with ischemic cardiomyopathy: effects on hemodynamics, left ventricular function, and cardiopulmonary exercise capacity. Circulation 1999; 99: 18–21. 11. O’Driscoll JG, Green DJ, Ireland M, Kerr D, Larbalesiter RJ. Treatment of end-stage cardiac failure with growth hormone. Lancet 1997; 349: 1068. 12. Fazio S, Sabatini D, Capaldo B, et al. A preliminary study of growth hormone in the treatment of heart failure in patients with dilated cardiomyopathy. N Engl J Med 1996; 334: 809–814. 13. Voterrani M, Desenzani P, Lorusso R, D’Aloria A, Manelli F, Giustina A. Haemodynamic effects of intravenous growth hormone in congestive heart failure. Lancet 1997; 349: 1067–1068. 14. Giustina A, Volterrani M, Manelli F, Desenzani P, Poiesi C, Lorusso R. Endocrine predictors of acute hemodynamic effects of growth hormone in congestive heart failure. Am Heart J 1999; 137: 1035–1043. 15. Gudbjarnason S, Fenton JC, Wolf PL, Bing RJ. Stimulation of reparative processes following experimental myocardial infarction. Arch Intern Med 1996; 118: 33–40. 16. Cittadini A, Grossman JD, Napoli R, et al. Growth hormone attenuates early left ventricular remodeling and improves cardiac function in rats with large myocardial infarction. J Am Coll Cardiol 1997; 29: 1109–1116. 17. Grimm D, Cameron D, Griese DP, Riegger GAJ, Kromer EP. Differential effects of growth hormone on cardiomyocyte and extracellular matrix protein remodeling following experimental myocardial infarction. Cardiovasc Res 1998; 40: 297–306. 18. MacAndrew JT, Ellery SS, Parry MA, Pan LC, Black SC. Efficacy of a growth hormone-releasing peptide mimetic in cardiac ischemia/ reperfusion injury. Eur J Pharmacol 2001; 432: 195–202. 19. Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res 1979; 44: 503. 20. Fletcher PJ, Preffer JM, Preffer MA, Branuwald E. Left ventricular diastolic pressure-volume relations in rats with healed myocardial infarction. Effects on systolic function. Circ Res 1981; 49: 618. 21. Green DL, White TP, Lampman RM. Papillary mechanics and cardiac morphology of infarcted rat hearts after training. J Appl Physiol 1991; 63: 92–96. 22. Fishbein MC, Maclean D, Maroko PR. Experimental myocardial infarction in the rat. Am J Pathol 1978; 90: 57–70. 23. Drexler H, Depenbusch JW, Truog AG, Zelis R, Flaim SF. Effects of diltiazem on cardiac function and regional blood flow at rest and during exercise in a conscious rat preparation of chronic heart failure (myocardial infarction). Circulation 1985; 71: 1262–1270. 24. Gibson UEM, Heid CA, Williams PW. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: 995–1001.
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25. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986–994. 26. Tajima M, Weinberg EO, Bartunek JB, et al. Treatment with growth hormone enhances contractile reserve and intracellular calcium transients in myocytes from rats with postinfarction heart failure. Circulation 1999; 99: 127–134. 27. Yue P, Long CS, Austin R, Chang KC, Simpson PC, Massie BM. Post-infarction heart failure in the rat is associated with distinct alterations in cardiac myocyte molecular phenotype. J Mol Cell Cardiol 1998; 30: 1615–1630. 28. Oie E, Bjonerheim R, Grogaard HK, Kongshaug H, Smiseth OA, Attramadal H. ET-receptor antagonism, myocardial gene expression, and ventricular remodeling during CHF in rats. Am J Physiol 1998; 275: H868–H877. 29. Hanatani A, Yoshiyama M, Kim S, et al. Inhibition by angiotensin II type 1 receptor antagonist of cardiac phenotypic modulation after myocardial infarction. J Mol Cell Cardiol 1995; 27: 1905–1914. 30. Yoshiyama M, Takeuchi K, Hanatani A, et al. Differences in expression of sarcoplasmic reticulum Ca2þ -ATPase and Naþ –Ca2þ exchanger genes between adjacent and remote noninfarcted myocardium after myocardial infarction. J Mol Cell Cardiol 1997; 29: 255–264. 31. Dixon IMC, Ju H, Jassal DS, Peterson DJ. Effects of ramipril and iosartan on collagen expression in right and left heart after myocardial infarction. Mol Cell Biochem 1996; 165: 31–45. 32. Volders PGA, Williams IEMG, Cleutjens JPM, Arends J-W, Havenith MG, Daemen MJAP. Interstitial collagen is increased in the non-infarcted human myocardium after myocardial infarction. J Mol Cell Cardiol 1993; 25: 1317–1323. 33. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstium. Fibrosis and renin-angiotensin-aldosterone system. Circulation 1991; 83: 1849–1865. 34. Weber KT, Sun Y, Tyagi SC, Cleutjens JPM. Collagen network of the myocardium: function, structure remodeling and regulatory mechanisms. J Mol Cell Cardiol 1994; 26: 279–292.
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35. Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts. Circulation 1998; 98: 149–156. 36. Herskowitz A, Choi S, Ansari A, Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 1995; 146: 419–428. 37. Jacobs M, Staufenberger S, Gergs U, et al. Tumor necrosis factor-a at acute myocardial infarction in rats and effects on cardiac fibroblasts. J Mol Cell Cardiol 1999; 31: 1949–1959. 38. Irwin MW, Mak S, Mann DL, et al. Tissue expression and immunolocalization of tumor necrosis factor-a in postinfarction dysfunction myocardium. Circulation 1999; 99: 1492– 1498. 39. Gwechenberger M, Mendoza LH, Youker KA, et al. Cardiac myocytes produce interleukin-6 in culture and in viable border zone of reperfused infarctions. Circulation 1999; 99: 546–551. 40. Guillen I, Blanes M, Gomez-Lechon MJ, Castell JV. Cytokine signaling during myocardial infarction: sequential appearance of IL-1 beta and IL-6. Am J Physiol 1995; 269: R229–R235. 41. Ikeda U, Ohkawa F, Seino Y, et al. Serum interleukin-6 becomes elevated in acute myocardial infarction. J Mol Cell Cardiol 1992; 24: 579–584. 42. Neumann FJ, Ott I, Gawaz M, et al. Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. Circulation 1995; 92: 748–755. 43. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 1992; 257: 387–389. 44. Finkel MS, Hoffman RA, Shen L, Oddis CV, Simmons RL, Hattler BG. Interleukin-6 (IL-6) as a mediator of stunned myocardium. Am J Cardiol 1993; 71: 1231–1323. 45. Kinugawa K, Takahashi T, Kohmoto O, et al. Nitric oxide mediated effects of interleukin-6 on ½Ca2þ i , and cell contraction in cultured chick ventricular myocytes. Circ Res 1994; 75: 285–295.
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression after acute myocardial infarction Hongkui Jin1, Renhui Yang1, Hsienwie Lu1, Annie K Ogasawara1, Wei Li1, Anne Ryan2, Franklin Peale2, and Nicholas F. Paoni1 1 Department of Cardiovascular Research, Genentech, Inc., South San Francisco, CA 94080, USA; and 2Department of Pathology, Genentech, Inc., South San Francisco, CA 94080, USA
Summary Objective. This study examined the effects of growth hormone (GH) on infarct size, survival, and cardiac gene expression in rats with acute myocardial infarction. Design. Animals randomly received sc injection of either saline vehicle (n ¼ 98) or GH (2 mg/kg/day, n ¼ 105) for 14 days commencing the day of left coronary artery ligation. Infarct size was determined by morphometric analysis at the time of death or at 52 weeks post-surgery. Gene expression was analyzed by real-time RT-PCR after 2week treatment. Results. GH decreased infarct size by18% ðP < 0:01) and increased survival by 36% at 52 weeks. GH also significantly reduced cardiac expression of atrial natriuretic factor, b-myosin heavy chain, a-smooth muscle actin, collagen I, collagen III, fibronectin, and pro-inflammatory cytokines. Conclusions. Treatment with GH for 2 weeks beginning on the day of myocardial infarction produced beneficial effects that were associated with reductions in cardiac gene expression symptomatic of pathological remodeling. ª 2002 Elsevier Science Ltd. All rights reserved.
Key words: gene expression, growth hormone, heart failure, infarct size, myocardial infarction
INTRODUCTION
It is known that growth hormone (GH) is essential to the control of linear growth, glucose, and homeostasis and for maintenance of skeletal muscle mass. Recent
Received 31 January 2002 Revised 12 March 2002 Accepted 28 March 2002 Correspondence to: Hongkui Jin, MD, Department of Cardiovascular Research, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Tel.: +1-650-225-1314; Fax: +1-650-225-6327; E-mail: [email protected]
1096-6374/02/$ - see front matter
animal experiments and clinical studies have shown that GH also plays an important role in modulating cardiac structure and function.1;2 We3;4 and other investigators5–9 have demonstrated that administration of GH produces beneficial effects with improved cardiac function in animals with chronic heart failure post-myocardial infarction (MI) or with pacing-induced heart failure. Furthermore, some clinical studies suggest that treatment with GH results in a significant improvement in hemodynamics and clinical function in patients with chronic heart failure caused by ischemic10;11 or idiopathic dilated cardiomyopathy.12 The beneficial effects of GH on cardiovascular ª 2002 Elsevier Science Ltd. All rights reserved.
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression
function including an increase in cardiac index and a reduction in peripheral vascular resistant can be acutely observed after intravenous infusion of GH in these patients.13;14 Despite many investigations focused on treatment with GH for chronic heart failure, few studies have determined the effects of GH given early on acute MI. The reparative processes in cardiac muscle following experimental MI are stimulated with GH.15 It has been shown that GH given early after large experimental MI attenuates left ventricular remodeling and improves cardiac function.16;17 A recent study demonstrates that treatment with the GH secretagogue CP-424,391 reduces infarct size in a rabbit model of myocardial ischemia and reperfusion.18 However, little is known whether the beneficial effects of GH on cardiac remodeling and function accompany changes in the expression of maker genes associated with MI and whether the early therapeutic regimen prolongs survival. The present study was designed: (1) to determine effects of early treatment with GH on infarct size and long-term survival and (2) to test the hypothesis that GH may also attenuate the induction of cardiac genes in MI. Our results showed that early treatment with GH for 2 weeks resulted in beneficial effects in association with reductions in cardiac gene expression symptomatic of pathological remodeling following experimental MI. METHODS
All experimental procedures conformed with the Guide for the Care and Use of Laborotory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and were approved by Genentech’s Institutional Animal Care and Use Committee.
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suture. The chest was closed by suturing pectoral muscles and the skin. The incision of the trachea was sutured after withdrawal of the respirator. Growth hormone administration
Twenty minutes after surgery, animals with stable general condition were randomly treated with either saline vehicle or recombinant human GH (Genentech, Inc., South San Francisco, CA) at 1 mg/kg, twice a day by subcutaneous injection for 14 days. Our previous studies have demonstrated that GH at this dose for 2 weeks significantly improves cardiac performance without inducing cardiac hypertrophy in rats with heart failure post-MI.3 In rats post-MI, this dose of GH increases serum insulin-like growth factor-1 by 45% (528 29 vs. 366 31 ng/ml, P < 0:01Þ. The animals were followed up for 52 weeks after surgery. Infarct size measurements
Infarct size was determined by morphometric analysis at the time of death or when the surviving animals were killed at 52 weeks post-surgery. The right ventricular free wall was dissected from the left ventricle (LV). The LV was cut in four transverse slices from apex to base. Five micrometer sections were cut and stained with Massons’ trichrome stain and mounted.22;23 The endocardial and epicardial circumferences of the infarcted and non-infarcted regions were determined with a planimeter Digital Image Analyzer. The infarcted circumference and the total left ventricular circumference of all four slices for both endocardial and epicardial surfaces were summed and expressed as a percentage of infarcted circumference to total circumference for determination of infarct size. Measurement of infarct size was carried out by the same person, who did not know the sample from the GH or vehicle group before measurement.
Animal model
Male Sprague–Dawley (SD) rats (Charles River Breeding Laboratories, Inc., 8 weeks of age) were acclimated to the facility for at least 1 week before surgery. Rats were fed a pelleted rat chow and water ad libitum, and housed in a light and temperature controlled room. The procedure used for left coronary ligation has been described in detail elsewhere.19–21 In brief, the rats were anesthetized with ketamine hydrochloride (100 mg/kg, ip) and xylazine (10 mg/kg, ip). The trachea was intubated and ventilated with a respirator (Harvard Apparatus Model 683). After a left-sided thoracotomy, the left coronary artery was ligated approximately 2 mm from its origin with a 7-0 silk
Study on cardiac gene expression
In a parallel experiment, ligated rats with evident MI on ECG3;4 were treated with vehicle or GH at the dose as described above, and sham-operated rats at the same age were used without treatment as controls. At the end of the 2-week treatment period, the rats were killed, the heart was removed and dissected, and the left ventricle was weighed, fast frozen in liquid nitrogen, and stored at )70 °C for subsequent RNA analysis. Total RNA was isolated from the ventricular samples using the RNeasy Maxi Kit (Qiagen) according to the manufacturer’s instructions. Gene expression analysis was performed using real-time RT-PCR (Taq-
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Man) technology. RT-PCR was performed on 1 ng of total RNA per reaction using the TaqMan Model 7700 Sequence Detector (ABI–Perkin Elmer).24 Amplification reaction conditions (for 50 ll) were 1 TaqMan Buffer A, 300 lM dATP, dCTP, dGTP, and 600 lM dUTP, 10% glycerol, 5.5 mM MgCl2, 50 U MuLV reverse transcriptase, 20 U RNase Inhibitor, 1.25 U AmpliTaq Gold, 100 nM forward and reverse primers, and 100 nM fluorogenic probe. RT-PCR reagents and glycerol were purchased from Perkin Elmer and Sigma, respectively. Reactions were performed in MicoAmp Optical Tubes and Caps (ABI–Perkin Elmer). TaqMan primers and probes were designed according to guidelines determined by Perkin Elmer and synthesized at Genentech, except for those for rodent GAPDH which were a generous gift from Perkin Elmer. Reverse transcription was performed at 48 °C for 30 min followed by heat activation of AmpliTaq Gold at 95 °C for 10 min. Thermal cycling was at 95 °C for 30 s and 60 °C for 1.5 min for 40 cycles. Quantitation of the TaqMan results was performed as described by Heid et al.25 with modifications. Briefly, standard curves (1:5 serial dilution) for each target gene of interest were run in duplicate. The Ct was plotted on the Y-axis vs. the log of the total RNA concentration (X-axis), and the equation describing the line was determined. Experimental samples were analyzed using 3–5 replicates each, and the quantity of the mRNA for each target gene was determined from the appropriate standard curve by entering the Ct (Y value) and solving for the input mRNA (X). The value for the target gene was then normalized to GAPDH by solving the following equation: 10X1 =10X2 , where X1 is the target gene, and X2 is GAPDH.
animals died before treatment with GH or vehicle. Of the 229 rats, 26 rats (13 GH-treated and 13 vehicletreated) were excluded from the present study. One rat had a large tumor in the abdominal cavity, 15 rats had no infarction, and in 10 other animals measurement of infarct size was not possible because of postmortem cannibalization or body discard. There was no difference in the baseline of BW before ligation or treatment between GH-treated (299:3 1:6 g, n ¼ 105) and vehicle-treated rats (300:7 1:6 g, n ¼ 98). Two weeks after treatment, an
Fig. 1 Effects of early treatment with GH for 2 weeks on BW in rats with MI. Data expressed as mean SEM. The number in the parenthesis is the animal number in each group. *P < 0:05, **P < 0:01, compared to the vehicle group.
Statistical analysis
Results are expressed as mean SEM. For analysis of body weight, infarct size, and gene expression, parameters between the GH and vehicle group were compared by an unpaired Student t test. Survival (%) between the GH-treated and vehicle-treated groups was compared by log rank (Mantel–Cox) and Breslow–Gehan–Wilcoxon tests. For the data of gene expression, one-way analysis of variance (ANOVA) was carried out for comparisons of each gene expression between groups. P < 0:05 was considered significant.
RESULTS Effects of GH on BW, infarct size, and survival
The 229 of the 235 rats receiving ligation of the left coronary artery entered into the study because six
Fig. 2 Effects of early treatment with GH for 2 weeks on infarct size in rats with MI. Data expressed as mean SEM. The number in the parenthesis is the animal number in each group. **P < 0:01, compared to the vehicle group.
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression
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the dose used, which was consistent with our previous observations.3 LV expression levels of 19 genes were used to examine the effect of GH on the molecular phenotypes of MI hearts. MI for 2 weeks substantially increased expression of atrial natriuretic factor (ANF) that was significantly attenuated by treatment with GH (top panel, Fig. 4). As compared to the sham group, the mRNA level of a-myosin heavy chain (aMHC) was significantly reduced in the MI + vehicle group but not in the
Fig. 3 Effects of treatment with GH for 2 weeks on 1-year survival (%) in rats with MI. The number in the parenthesis is the animal number in each group.
increase in BW was significantly greater in the GH group than the vehicle group (78:63:0 g, n ¼ 84 vs. 60:0 3:9 g, n ¼ 75; P < 0:001) indicating treatment with GH significantly increased BW growth (Fig. 1). After stopping GH administration, the increase in BW growth was maintained for at least 4 weeks (Fig. 1). Pathological studies revealed that infarct size was significantly reduced (P < 0:01) by 18% in GH-treated animals (30:8 1:6%, n ¼ 105) compared to vehicle controls (37:4 1:6%, n ¼ 98) (Fig. 2). There was a significant negative correlation between infarct size and survival days in both vehicle-treated rats (r ¼ 0:6664, P < 0:0001, n ¼ 98), and GH-treated animals (r ¼ 0:6283, P < 0:0001). Early treatment with GH for 2 weeks prolonged one-year (52-week) survival by 36% (30.48% and 22.45% in the GH-treated and vehicle-treated animals, respectively). Although the difference did not reach statistical significance (P ¼ 0:200 using the Mantel–Cox test, or 0.188 using the Breslow–Gehan– Wilcoxon test), there was a trend toward improved survival in rats receiving GH (Fig. 3). The GH-induced increase in survival appeared to occur as early as 2–3 weeks, and was then maintained. Effects of GH on cardiac gene expression
The ratio of LV weight to BW at 2 weeks after operation was significantly increased in the MI group compared to the sham controls (2:11 0:06 vs. 1:86 0:08, P < 0:05), but this ratio was similar in the MI + vehicle vs. MI + GH group (2:11 0:06 vs. 2:05 0:04, NS). This suggested that MI-induced LV hypertrophy was not altered by treatment with GH at
Fig. 4 Effects of early treatment with GH for 2 weeks on cardiac expression of seven genes in rats with MI. Data expressed as mean SEM (n ¼ 5–7 in each group). *P < 0:05, **P < 0:01, compared to the sham group. # P < 0:05; ## P < 0:01, compared to the MI + vehicle group. ANF, atrial natriuretic factor; aMHC, a-myosin heavy chain; bMHC, b-myosin heavy chain; MLC2, myosin light chan-2; ACA, a-cardiac actin; SKA, a-skeletal muscle actin; SMA, a-smooth muscle actin.
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MI + GH group (left of middle panel, Fig. 4). In contrast, MI induced a significant elevation in the expression level of b-myosin heavy chain that was normalized by GH (middle of middle panel, Fig. 4). aSmooth muscle actin was markedly increased by 6fold in the MI + vehicle group compared to the sham group but significantly attenuated by GH administration (right of bottom panel, Fig. 4). Compared to sham controls, MI rats exhibited a 1.7-, 8.3-, and 12.5-fold increase in expression of collagen I, collagen III, and fibronectin, respectively, whereas treatment with GH significantly inhibited the expression of these three genes (bottom panel, Fig. 5). MI was associated with a significant increase in the mRNA level of a-skeletal muscle actin (middle of bottom panel, Fig. 4) and isocitrate dehydrogenase (left of top panel, Fig. 5), but GH did not alter the expression of these genes significantly. Neither MI nor GH had effect on expression of myosin light chan-2 (right of middle panel, Fig. 4), acardiac actin (left of bottom panel, Fig. 4), mitochondrial ATP synthase (right of top panel, Fig. 5), phospholamban, and sarcoplasmic reticulum Ca2þ ATPase (middle panel, Fig. 5). LV gene expression of leukemia inhibitory factor, endothelin-1 (top panel, Fig. 6), tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) (middle panel, Fig. 6), and interleukin-6 (IL-6) (bottom panel, Fig. 6) was significantly increased in the MI + vehicle group compared to the sham group. Treatment with GH significantly attenuated the mRNA levels of leukemia inhibitory factor and IL-6, and tended to reduce endothelin-1, TNF-a, and IL-1b. DISCUSSION
In the present study, administration of GH for 2 weeks beginning on the day of MI resulted in a significant reduction in infarct size by 18%, an increase in survival by 36% at 52 weeks, and a attenuation of LV gene expression of ANF, b-myosin heavy chain, asmooth muscle actin, collagen I, collagen III, fibronectin, and pro-inflammatory cytokines. This is the first demonstration that early treatment with GH produces beneficial effects in association with reductions in cardiac gene expression symptomatic of pathological remodeling in experimental MI. It is known that GH promotes wound healing. It has been reported that the reparative processes in cardiac muscle following experimental MI are stimulated with anabolic hormones. The stimulation action is most effective during early phase of the reparative process. In the dog model of acute MI, treatment with GH increases protein synthesis in infarcted muscle by 168% 24 h after infarction.15 GH enhances glycin-2 14 C incorporation into the connective tissue of healing in-
Fig. 5 Effects of early treatment with growth hormone (GH) for 2 weeks on cardiac expression of seven genes in rats with MI. Data expressed as mean SEM (n ¼ 5–7 in each group). *P < 0:05, **P < 0:01, compared to the sham group. # P < 0:05, ## P < 0:01, compared to the MI + vehicle group. ICDH, isocitrate dehydrogenase; MTATP, mitochondrial ATP synthase; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca2þ -ATPase; COL1, collagen 1; COL3, collagen 3; FIB, fibronectin.
farct. It is possible that the increased rate of protein synthesis in and around the infarcted area would add strength to the damaged cardiac muscle, increase its resistance against infarct expansion, and improve post-infarction healing. These effects of GH may contribute, in part, to a reduction of infarct size. The present study showed that early treatment with GH for 2 weeks tended to increase 1-year survival rate by 36% in MI rats. Since there is a significant negative correlation between infarct size and survival days in
Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression
Fig. 6 Effects of early treatment with GH for 2 weeks on cardiac expression of five genes in rats with MI. Data expressed as mean SEM (n ¼ 5–7 in each group). *P < 0:05, **P < 0:01, compared to the sham group. # P < 0:05, ## P < 0:01, compared to the MI + vehicle group. LIF, leukemia inhibitory factor; ET-1, endothelin-1; TNF-a, tumor necrosis factor-a; IL-1b, interleukin-1; IL-6, interleukin-6.
both vehicle-treated and GH-treated animals, it is likely that a reduction in mortality in the GH-treated group is attributed to a decrease in infarct size. In addition, an improvement in cardiac function may also contribute to the lower mortality rate in GHtreated animals. Several lines of evidence indicate that GH given early after moderate and large MI significantly improves systolic and diastolic LV function in this rat model of MI.16;17 Our previous studies also demonstrate that GH at the same dose enhances contractile reserve in myocytes and improves LV performance in rats with heart failure post MI.3;26
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To evaluate the molecular effects of GH on the heart, LV gene expression of several markers of pathological remodeling and cytokines were analyzed after 2 weeks of MI or treatment. There are 3 major findings obtained from the gene analysis in the present study. First, MI was associated with a significant increase in LV gene expression of ANF, b-myosin heavy chain, a-skeletal actin, and a-smooth muscle. This is essentially consistent with recent studies that demonstrate the increased ventricular expression of these genes coding for the fetal phenotype during ventricular remodeling following MI in rats.27–30 It is noted in the present study that treatment with GH attenuated the expression ANF, b-myosin heavy chain, and a-smooth muscle actin. It is known that fetal gene expression may be one of the hallmarks of disease progression in LV remodeling and dysfunction. The reduction in LV fetal gene expression observed in the MI rats treated with GH might reflect an improvement of cardiac function and attenuation of LV remodeling. Second, consistent with previous observations that cardiac extracellular matrix genes were up-regulated 2 weeks after MI,29;31 we found that LV mRNA levels of collagen I, collagen III, and fibronectin were increased in MI rats compared to sham controls. Furthermore, the present study demonstrated that early treatment with GH significantly inhibited over-induction of these genes. This is in agreement with the report by Grimm et al., who demonstrate that GH given early after moderate and large MI substantially reduced the considerably increased depositions of fibronectin and collagen I in the rat LV.17 The effect of GH on cardiac extracellular matrix mRNA and proteins may have an important clinical impact, since experimental and clinical studies have demonstrated an increase in interstitial collagens of the LV or non-ischemic myocardium at a chronic or late stage post-MI, which may enhance cardiac stiffness and result in diastolic dysfunction, finally leading to heart failure.29;31–34 Third, there was LV induction of genes encoding pro-inflammatory cytokines or growth factors including TNF-a, IL-1b, IL-6, leukemia inhibitory factor, and endothelin-1 in rats with MI. This is consistent with recent findings that cardiac expression of mRNA of TNF-a, IL-1b, and IL-6 is up-regulated in the early phase of acute MI and the late stage post-MI35–38 Clinical and experimental studies have shown that the inflammatory response to MI is associated with induction of cytokines such as of TNF-a, IL-1b, and IL-6, which are thought to act as a ’’cascade fashion.’’39–42 As induced, cytokine gene expression is clearly observed before significant histological evidence for inflammation can be identified in rats with MI, cytokine
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gene expression may be primarily generated by intrinsic myocardial cells, instead of inflammatory cells, in response to ischemia.36 The present study demonstrated that early treatment with GH significantly attenuated the induction of IL-6 and leukemia inhibitory factor (one of IL-6 family members), and tended to reduce mRNA levels of TNF-a, IL-1b, and endothelin-1 in the MI hearts. It has been shown that pro-inflammatory cytokines exert a direct negative inotropic effect mediated through a myocardial nitric oxide synthase,43 which may depress myocardial function. For example, IL-6 may act as a nitric oxidedependent cardiac depressant and may be associated with stunned myocardium.39;43;44 IL-6 has also been shown to produce nitric oxide-mediated reduction in calcium flux and contractility in ventricular myocytes45 Our finding of inhibitory effect of GH on induction of the pro-inflammatory cytokines may contribute, in part, to improvement of cardiac function after early treatment with GH observed in rats with MI.16;17 In summary, early administration of GH following acute MI for 2 weeks resulted in a reduction in infarct size and an increase in survivals. The beneficial effects of GH were accompanied by attenuating cardiac expression of several marker genes of pathological remodeling and cytokines. Taken together with previous observations that GH attenuates early LV remodeling and improves cardiac function,16;17 the data in the present study implicate that GH might have the potential for early treatment of acute MI. Our study, however, cannot evaluate the effect of early treatment with GH for a long term, because administration of human GH for more than 15 days would produce the antibody against the human GH in rats. A further study is necessary to determine the effects of early, long-term therapy with recombinant rat GH in rats with MI. REFERENCES 1. Cittadini A, Longobardi S, Fazio S, Sacca L. Growth hormone and the heart. Miner Electrolyte Metab 1999; 25: 51–55. 2. Sacca L, Fazio S. Cardiac performance: growth hormone enters the race. Nat Med 1996; 2: 29–31. 3. Yang R, Bunting S, Gillett N, Clark R, Jin H. Growth hormone improves cardiac performance in experimental heart failure. Circulation 1995; 92: 262–267. 4. Jin H, Yang R, Gillett N, Clark RG, Ko A, Paoni NF. Beneficial effects of growth hormone and insulin-like growth factor-1 in experimental heart failure in rats treated with chronic ACE inhibition. J Cardiovasc Pharmacol 1995; 26: 420–425. 5. Duerr RL, McKirnan MD, Gim RD, Clark RG, Chien KR, Ross J Jr. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic ventricular failure in the rat. Circulation 1996; 93: 2188–2196.
6. Ross J Jr., Hongo M. The role of hypertrophy and growth factor in heart failure. J Cardiac Failure 1996; 2(4 Suppl): S121–S128. 7. Isgaard J, Kujacic V, Jennische E, et al. Growth hormone improves cardiac function in rats with experimental myocardial infarction. Eur J Clin Invest 1997; 27: 517–525. 8. Hongo M, Sentianin EM, Tanaka N, et al. Angiotensin II blockade followed by growth hormone as adjunctive therapy after experimental myocardial infarction. J Cardiac Failure 1998; 4: 213–224. 9. Houck WV, Pan LC, Kribbs SB, et al. Effects of growth hormone supplementation of left ventricular morphology and myocyte function with the development of congestive heart failure. Circulation 1999; 100: 2003–2009. 10. Genth-Zotz S, Zotz R, Geil S, Voigtlander T, Meyer J, Darius H. Recombinant growth hormone therapy in patients with ischemic cardiomyopathy: effects on hemodynamics, left ventricular function, and cardiopulmonary exercise capacity. Circulation 1999; 99: 18–21. 11. O’Driscoll JG, Green DJ, Ireland M, Kerr D, Larbalesiter RJ. Treatment of end-stage cardiac failure with growth hormone. Lancet 1997; 349: 1068. 12. Fazio S, Sabatini D, Capaldo B, et al. A preliminary study of growth hormone in the treatment of heart failure in patients with dilated cardiomyopathy. N Engl J Med 1996; 334: 809–814. 13. Voterrani M, Desenzani P, Lorusso R, D’Aloria A, Manelli F, Giustina A. Haemodynamic effects of intravenous growth hormone in congestive heart failure. Lancet 1997; 349: 1067–1068. 14. Giustina A, Volterrani M, Manelli F, Desenzani P, Poiesi C, Lorusso R. Endocrine predictors of acute hemodynamic effects of growth hormone in congestive heart failure. Am Heart J 1999; 137: 1035–1043. 15. Gudbjarnason S, Fenton JC, Wolf PL, Bing RJ. Stimulation of reparative processes following experimental myocardial infarction. Arch Intern Med 1996; 118: 33–40. 16. Cittadini A, Grossman JD, Napoli R, et al. Growth hormone attenuates early left ventricular remodeling and improves cardiac function in rats with large myocardial infarction. J Am Coll Cardiol 1997; 29: 1109–1116. 17. Grimm D, Cameron D, Griese DP, Riegger GAJ, Kromer EP. Differential effects of growth hormone on cardiomyocyte and extracellular matrix protein remodeling following experimental myocardial infarction. Cardiovasc Res 1998; 40: 297–306. 18. MacAndrew JT, Ellery SS, Parry MA, Pan LC, Black SC. Efficacy of a growth hormone-releasing peptide mimetic in cardiac ischemia/ reperfusion injury. Eur J Pharmacol 2001; 432: 195–202. 19. Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res 1979; 44: 503. 20. Fletcher PJ, Preffer JM, Preffer MA, Branuwald E. Left ventricular diastolic pressure-volume relations in rats with healed myocardial infarction. Effects on systolic function. Circ Res 1981; 49: 618. 21. Green DL, White TP, Lampman RM. Papillary mechanics and cardiac morphology of infarcted rat hearts after training. J Appl Physiol 1991; 63: 92–96. 22. Fishbein MC, Maclean D, Maroko PR. Experimental myocardial infarction in the rat. Am J Pathol 1978; 90: 57–70. 23. Drexler H, Depenbusch JW, Truog AG, Zelis R, Flaim SF. Effects of diltiazem on cardiac function and regional blood flow at rest and during exercise in a conscious rat preparation of chronic heart failure (myocardial infarction). Circulation 1985; 71: 1262–1270. 24. Gibson UEM, Heid CA, Williams PW. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: 995–1001.
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25. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986–994. 26. Tajima M, Weinberg EO, Bartunek JB, et al. Treatment with growth hormone enhances contractile reserve and intracellular calcium transients in myocytes from rats with postinfarction heart failure. Circulation 1999; 99: 127–134. 27. Yue P, Long CS, Austin R, Chang KC, Simpson PC, Massie BM. Post-infarction heart failure in the rat is associated with distinct alterations in cardiac myocyte molecular phenotype. J Mol Cell Cardiol 1998; 30: 1615–1630. 28. Oie E, Bjonerheim R, Grogaard HK, Kongshaug H, Smiseth OA, Attramadal H. ET-receptor antagonism, myocardial gene expression, and ventricular remodeling during CHF in rats. Am J Physiol 1998; 275: H868–H877. 29. Hanatani A, Yoshiyama M, Kim S, et al. Inhibition by angiotensin II type 1 receptor antagonist of cardiac phenotypic modulation after myocardial infarction. J Mol Cell Cardiol 1995; 27: 1905–1914. 30. Yoshiyama M, Takeuchi K, Hanatani A, et al. Differences in expression of sarcoplasmic reticulum Ca2þ -ATPase and Naþ –Ca2þ exchanger genes between adjacent and remote noninfarcted myocardium after myocardial infarction. J Mol Cell Cardiol 1997; 29: 255–264. 31. Dixon IMC, Ju H, Jassal DS, Peterson DJ. Effects of ramipril and iosartan on collagen expression in right and left heart after myocardial infarction. Mol Cell Biochem 1996; 165: 31–45. 32. Volders PGA, Williams IEMG, Cleutjens JPM, Arends J-W, Havenith MG, Daemen MJAP. Interstitial collagen is increased in the non-infarcted human myocardium after myocardial infarction. J Mol Cell Cardiol 1993; 25: 1317–1323. 33. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstium. Fibrosis and renin-angiotensin-aldosterone system. Circulation 1991; 83: 1849–1865. 34. Weber KT, Sun Y, Tyagi SC, Cleutjens JPM. Collagen network of the myocardium: function, structure remodeling and regulatory mechanisms. J Mol Cell Cardiol 1994; 26: 279–292.
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35. Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts. Circulation 1998; 98: 149–156. 36. Herskowitz A, Choi S, Ansari A, Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 1995; 146: 419–428. 37. Jacobs M, Staufenberger S, Gergs U, et al. Tumor necrosis factor-a at acute myocardial infarction in rats and effects on cardiac fibroblasts. J Mol Cell Cardiol 1999; 31: 1949–1959. 38. Irwin MW, Mak S, Mann DL, et al. Tissue expression and immunolocalization of tumor necrosis factor-a in postinfarction dysfunction myocardium. Circulation 1999; 99: 1492– 1498. 39. Gwechenberger M, Mendoza LH, Youker KA, et al. Cardiac myocytes produce interleukin-6 in culture and in viable border zone of reperfused infarctions. Circulation 1999; 99: 546–551. 40. Guillen I, Blanes M, Gomez-Lechon MJ, Castell JV. Cytokine signaling during myocardial infarction: sequential appearance of IL-1 beta and IL-6. Am J Physiol 1995; 269: R229–R235. 41. Ikeda U, Ohkawa F, Seino Y, et al. Serum interleukin-6 becomes elevated in acute myocardial infarction. J Mol Cell Cardiol 1992; 24: 579–584. 42. Neumann FJ, Ott I, Gawaz M, et al. Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. Circulation 1995; 92: 748–755. 43. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 1992; 257: 387–389. 44. Finkel MS, Hoffman RA, Shen L, Oddis CV, Simmons RL, Hattler BG. Interleukin-6 (IL-6) as a mediator of stunned myocardium. Am J Cardiol 1993; 71: 1231–1323. 45. Kinugawa K, Takahashi T, Kohmoto O, et al. Nitric oxide mediated effects of interleukin-6 on ½Ca2þ i , and cell contraction in cultured chick ventricular myocytes. Circ Res 1994; 75: 285–295.