Tsalikakis Growth Search & Igf

Growth Hormone & IGF Research 16 (2006) 93–100 www.elsevier.com/locate/ghir

Early, intracoronary growth hormone administration attenuates ventricular remodeling in a porcine model of myocardial infarction A.C. Mitsi a,1,2, K.E. Hatzistergos b,1,2, D. Niokou c, L. Pappa b, G.G. Baltogiannis a, D.G. Tsalikakis d,2, A. Papalois e, Z.S. Kyriakides f, V. Malamou-Mitsi b, T.M. Kolettis a,* a

Department of Cardiology, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece Department of Pathology, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece c Department of Anesthesiology, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece Department of Computer Sciences, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece e ELPEN Research Laboratory, 21st km Marathon Avenue, 19009 Athens, Greece f Department of Cardiology, Red Cross Hospital, 27 Venizelou Street, 17123 Athens, Greece b

d

Received 15 August 2005; revised 9 February 2006; accepted 9 February 2006 Available online 18 April 2006

Abstract Objective: Ventricular remodeling is a common corollary of myocardial infarction. We hypothesized that this process may be attenuated by growth hormone, administered as a single high-dose, selectively in the infarct zone, early postmyocardial infarction. Design: In 35 pigs (29 ± 4 kg), myocardial infarction was generated by inflation of an over-the-wire angioplasty balloon in the circumflex artery for 60 min and 5 further pigs were sham-operated. Ten minutes after reperfusion, the pigs were randomized (2:1) to either growth hormone (1 IU/kg) (n = 23) or normal saline (n = 12), delivered via the balloon catheter. All survivors were treated with captopril and were sacrificed 4 weeks after myocardial infarction. Results: Compared to controls, growth hormone-treated animals displayed lower heart weight (4.1 ± 0.5 g/kg body weight, versus 3.4 ± 0.4 g/kg, respectively, p = 0.003) and dimensions (left ventricular short axis diameter 46 ± 7 mm versus 37 ± 6 mm, p = 0.01; right ventricular short axis diameter 38 ± 7 mm versus 30 ± 5 mm p = 0.001). Growth hormone increased wall thickness in the infarct (6.0 ± 1.8 in controls versus 9.9 ± 3.7 in treated animals, p = 0.004) and non-infarct zones (10.6 ± 1.8 in controls versus 15.5 ± 3.8 in treated animals, p = 0.0006) and produced higher (p < 0.05) microvascular density in both zones. Conclusion: Intracoronary administration of growth hormone attenuates left and right ventricular remodeling by inducing hypertrophy and by enhancing angiogenesis.  2006 Elsevier Ltd. All rights reserved. Keywords: Angiogenesis; Growth hormone; Hypertrophy; Myocardial infarction; Ventricular remodeling

1. Introduction * Corresponding author. Tel.: +30 265 1097227; fax: +30 265 1097053. E-mail address: [email protected] (T.M. Kolettis). 1 Both authors contributed equally to this study. 2 Supported by a grant from the National General Council for Research and Technology.

1096-6374/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2006.02.001

Myocardial infarction remains a leading cause of morbidity and mortality worldwide. The most important determinant of prognosis is the extent of myocardial injury and the subsequent development of heart failure [1]. The loss of contractile tissue elevates local

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wall stress, resulting in expansion of the infarct zone and dilatation of the non-infarcted myocardium. This remodeling leads to significant alterations in ventricular dimensions, shape and function and, ultimately, to congestive heart failure [2]. Despite optimal management with early reperfusion, nitrates, beta-blockers and angiotensin converting enzyme inhibitors, the number of patients with left ventricular dysfunction after myocardial infarction remains high [1,2]. Ventricular remodeling is a complex and dynamic process, which starts during the first hours after coronary occlusion and is intertwined with healing of the damaged area. Increased levels of growth hormone and its mediator, insulin-like growth factor-1, have been found in the infarcted area of experimental animals [3] and in the plasma of patients with acute myocardial infarction [4]. Amidst other actions, they increase myocardial contractility and they promote hypertrophy [3,4]. In addition, these substances have been shown to augment angiogenesis in regenerating skeletal muscle [5] and in ageing rat myocardium [6], but their possible angiogenic effects on the infarcted myocardium are not known. In the present study, we evaluated a novel antiremodeling strategy, intervening during the acute phase of myocardial infarction. We hypothesized that a single, high-dosage of growth hormone, administered acutely and selectively in the infarcted area, may enhance local cardiac repair and, thereby, attenuate ventricular remodeling examined four weeks after infarction. To provide further insight into the possible beneficial mechanisms of such intervention, we examined the effects of acute growth hormone administration on angiogenesis.

2. Materials and methods The study was conducted in 35 domestic landrace pigs, of both sexes, weighing 25–40 kg. The animals were housed in individual cages in a climate controlled environment and received humane care. The study protocol was approved by the Central Health Council of the National Ministry of Health and is in accordance with the ‘Position of the American Heart Association on Research Animal Use’. The animals underwent a closed-chest induction of myocardial infarction under general anesthesia, as previously described [7]. After an overnight fast, the pigs were pre-anesthetized by intramuscular injection of azeperone (4 mg/kg) and ketamine (5 mg/kg). Anesthesia was initiated with intravenous pentothal (5–7 mg/kg). After intubation, the animals were ventilated using room air enriched with oxygen and anesthesia was maintained with inhaled sevoflurane. Oxygen saturation, aortic blood pressure and a three-lead electrocardiogram were monitored continuously throughout the procedure.

2.1. Myocardial infarction generation The femoral artery was surgically exposed under sterile conditions, ligated distally and cannulated. Under fluoroscopic guidance, the left coronary ostium was engaged and an over-the-wire angioplasty balloon catheter (balloon:artery ratio:1.0) was inflated in the proximal circumflex artery for 60 min. Total occlusion of the vessel was confirmed angiographically and by the presence of ST-segment elevation. In a separate group of sham-operated animals, the balloon was advanced in the circumflex artery, but it was not inflated. Because pigs are susceptible to ischemia-induced ventricular fibrillation, we evaluated various anti-arrhythmic regimens in a separate series of experiments (unpublished data). The regimen found to reduce the incidence of ventricular arrhythmias was used in the present study and consisted of: (a) 4 doses of 100 mg lidocaine, (b) 1.25 mg atenolol over the 60 min of occlusion, (c) 300 mg amiodarone over 120 min, beginning 5 min before reperfusion and (d) procainamide (10 mg/kg) for 60 min, beginning 30 min before reperfusion. In case of ventricular fibrillation despite this regimen, a direct current shock was delivered immediately. After balloon deflation, patency was confirmed angiographically. To ensure comparability in the size of myocardial infarction in the two groups, arterial blood was drawn from the guiding catheter, before drug delivery. The blood was centrifuged immediately and the serum was stored at 20 C for creatine kinase measurement. 2.2. Growth hormone administration Ten minutes after balloon deflation, the animals were randomized to either growth hormone or normal saline in a 2:1 fashion. The medication was administrated by a slow intracoronary infusion via the lumen of the balloon catheter, selectively in the infarcted area. The infusion duration was 20 min at a total dose of 1 IU/kg growth hormone (purified porcine somatotropin, S-8648, Sigma– Aldrich Corporation, St Louis, MO, USA) diluted in 20 cc normal saline (growth hormone group) or 20 cc normal saline (control group). The lumen of the balloon was thoroughly flushed to ensure delivery of the substance. Intravenous nitroglycerin was administered for 1 h, aiming at a systolic blood pressure drop of approximately 5%. The animals were observed until complete recovery and were then transferred to their cage. On the day following the experiment, all survivors received 6.25 mg captopril dissolved in drinking water, followed by 12.5 mg captopril daily. 2.3. Macroscopic measurements Four weeks after the infarction, the animals were sedated (as described above), weighed and euthanized

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by an intracardiac injection of potassium chloride (arresting the heart in diastole) mixed with a lethal dose of pentobarbital. The heart was rapidly excised and weighed after removal of the right and left atria. To ensure comparable measurements, heart weight was normalized to body weight at the time of euthanasia. Right and left ventricular diameters were measured across the short and long axes using high-precision hand-calipers. Sphericity index was defined as the ratio of the short axis diameter divided by the long axis diameter. The ventricles were cut in four transverse slices from apex to base, photographed and the perimeters as well as the crosssectional areas of the infarcted and non-infarcted myocardium were measured, using a previously validated software program (Image Tool, University of Texas, USA). Expansion index was defined as the ratio of the perimeter of the infarcted area divided by the sum of infarct and non-infarct perimeters. Myocardial wall thickness of the non-infarcted myocardium was measured at a distance from the papillary muscles. Myocardial wall thickness of the infarcted zone included measurement of: (a) the thinnest point and (b) the mean value of three measurements, namely the thinnest point and two other measurements, 0.5–1 cm adjacently to the thinnest point. The volume of the infarcted tissue was measured by multiplying the infarcted cross-sectional area (averaged from both sides of each slice) times the height of the slice(s). Measurements are shown in Fig. 1. 2.4. Histological analysis and immunohistochemistry The method used in this study has been described previously [8]. In brief, multiple tissue specimens were obtained from the infarcted area, the remote noninfarcted myocardium and the border-zone between the two areas. The samples were immersion-fixed for 24 h in formalin, embedded in paraffin and cut in 4 lm sections. Hematoxylin–eosin stained sections were used for the initial histological evaluation. Immunohistochemistry was performed with the use of the EnVision Detection Kit (DakoCytomation, Glostrup, Denmark). After drying for 60 min at 60 C, paraffin was removed in three changes of xylene. The tissue was rehydrated

Fig. 1. Macroscopic measurements. BZ: border zone; h1: minimum infarct wall thickness; (h1 + h2 + h3)/3: mean infarct wall thickness; hniz: non-infarct zone thickness; l1: infarct zone perimeter, l2: noninfarct zone perimeter; dl: long axis diameter, ds: short axis diameter.

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and subjected to epitope retrieval in a microwave oven. A 0.3% hydrogen peroxide solution was applied for 30 min to remove any endogenous peroxidase activity. The primary antibodies against vascular endothelial growth factor at a dilution of 1:100 (JH121, NeoMarkers Inc., Fremont, CA, USA) and alphasmooth muscle actin (Actin SM, AM128-10M, Biogenex, San Ramon, CA, USA) were applied for overnight incubation in a humified chamber. The sections were counterstained with hematoxylin, rinsed in distilled water, dehydrated, cleared in xylene and mounted. 2.5. Immunohistochemical evaluation The staining assessment was made by three independent observers (K.E.H., L.P., V.M.M.), blinded to treatment assignment. Blood vessels were identified by tubular morphologic features with a single layer of endothelial cells with positive alpha-smooth muscle actin staining, as previously described [8]. Care was taken to exclude from counting myofibroblasts or other sources of alpha-smooth muscle actin staining. Vessels with obvious lumen and a diameter less than 20 lm were defined as neo-vessels. After low-power examination, areas of interest were identified as those displaying the most prominent vascular density. These areas were located: (a) at the infarct zone and adjacent subendocardium, (b) the non-infarcted zone and adjacent subendocardium and (c) the border zone between the two aforementioned zones. Three representative high-power (·400) fields in each zone were randomly chosen and the number of neo-vessels in each field was counted. The average number of counts was recorded. To evaluate the immunoexpression of vascular endothelial growth factor in cardiac myocytes, a semi-quantifiable method was used, based on the staining intensity (0 = negative, 1 = weak or moderate, 2 = strong). 2.6. Statistical analysis All results are given as mean ± one standard deviation. Differences in categorical variables were assessed with the Yates’ corrected chi-square. Differences in continuous variables were assessed using Student’s t-test for independent variables, or one-way analysis of variance (followed by Tukey’s multiple comparisons test), as appropriate. All statistics were performed using the ‘Statistica’ software system (version 6.0, StatSoft Inc., Tulsa, OK, USA). Statistical significance was defined at an alpha value of 0.05.

3. Results A total of 51 pigs were initially included in the study. Eleven pigs [7 pigs (out of 30, 23%) allocated growth

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hormone and 4 pigs (out of 16, 25%) allocated normal saline, p = 0.8] died within 24 h after infarct generation. Thus, the final study population consisted of 35 pigs (29 ± 4 kg), of which 23 (21 male, 30 ± 4 kg) received growth hormone and 12 (11 male, 29 ± 3 kg) received normal saline. Five further pigs (4 male, 28 ± 5 kg) were sham-operated. Creatine kinase was 2030 ± 749 IU in the growth hormone group and 1834 ± 645 IU in the control group (p = 0.6). During growth hormone administration, no heart rhythm disturbances were noted and no significant changes in the blood pressure or heart rate were found. During the four-week follow-up period, the increase in body weight was comparable in the three groups (48 ± 22% for the control group versus 50 ± 22% for the growth hormone group, versus 45 ± 12% for the sham operated animals, F = 0.3, p = 0.6). 3.1. Infarct size, heart weight, dimensions and geometry The volume of the infarcted tissue was comparable between the two groups (5714 ± 2507 mm3 in the control group, versus 6232 ± 3948 mm3 in the growth hormone group, p = 0.7). A significant variance in heart weight was present (F = 7.0, p = 0.003), that was due to significantly (p = 0.002) lower values in the growth hormone group (3.4 ± 0.4 g/kg) compared to the control group (4.1 ± 0.5 g/kg). Significant variances were found in the right ventricular long axis (F = 9.0, p = 0.0009), short axis (F = 9.7, p = 0.0006), left ventricular long axis (F = 4.4, p = 0.02) and short axis (F = 8.3, p = 0.001). The control group displayed significantly higher ventricular dimensions, when compared to either the growth hormone or the sham operated groups (Table 1). The sphericity index displayed a significant variance between the three groups (F = 3.4, p = 0.04). This index was significantly higher (p = 0.03) in the control group, compared to the growth hormone group, but was comparable in the growth hormone and sham operated groups. The expansion index tended to be lower in the growth hormone group (0.17 ± 0.08) compared to the control group (0.21 ± 0.07), although this difference did not reach statistical significance (p = 0.2). 3.2. Myocardial wall thickness Compared to controls, the minimum and mean infarct wall thicknesses were significantly higher in the growth hormone group (p = 0.017 and p = 0.004, respectively). Myocardial wall thickness at the remote, healthy myocardium displayed a significant variance between the three groups (F = 11.8, p = 0.0001). This was due to a significantly higher myocardial thickness in the growth hormone group compared to either the control group, or the sham operated group. Further-

Table 1 Macroscopic measurements Control Weight increase (%) Heart weight/body weight (g/kg) LV long axis (mm) LV short axis (mm) RV long axis (mm) RV short axis (mm) Expansion index Min infarct wall thickness (mm) Mean infarct wall thickness (mm) Non-infarct wall thickness (mm) Non-infarct wall area (slice 1) (mm) Sphericity index

Growth hormone

Sham

48 ± 22 4.1 ± 0.5

50 ± 22 3.4 ± 0.4*

45 ± 12 3.6 ± 0.3

73 ± 9 46 ± 7 52 ± 4 38 ± 7 0.21 ± 0.07 5.5 ± 2.0

67 ± 5 37 ± 6* 45 ± 5* 30 ± 5* 0.17 ± 0.08 8.6 ± 3.6*

63 ± 3 35 ± 3 43 ± 2 27 ± 3 – –

6.0 ± 1.8

9.9 ± 3.7*

–

10.6 ± 1.8

15.5 ± 3.8*

10.3 ± 0.4

794 ± 298

1264 ± 588*

–

64 ± 10

54 ± 9*

56 ± 6

LV: left ventricle; RV: right ventricle; min: minimum. * p < 0.05 between the growth hormone and control groups.

more, the cross-sectional area of the non-infarcted myocardium was 1264 ± 588 mm2 in the growth hormone group, which was significantly (p = 0.02) higher compared to the 794 ± 298 mm2 in the control group. All values are shown in Table 1 and a representative example is shown in Fig. 2. 3.3. Microvascular density and vascular endothelial growth factor immunostaining A significantly higher microvascular density was found in the growth hormone group in the infarct and non-infarct zones (Fig. 3, Table 2). In contrast, no significant differences were found in the protein expression of vascular endothelial growth factor (infarct zone: 1.85 ± 0.37 in the growth hormone group, versus 1.70 ± 0.48 in the control group, border zone: 1.60 ± 0.51 versus 1.57 ± 0.53, non-infarct zone: 1.40 ± 0.51 versus 1.14 ± 0.37, respectively, all p > 0.1).

4. Discussion Ventricular remodeling after myocardial infarction consists of ventricular dilatation and dysfunction, associated with increased morbidity and mortality [1,2]. In the early post-myocardial infarction period, significant cellular and extracellular alterations occur in the infarct zone, resulting in local wall thinning and elongation. Late remodeling involves the left ventricle globally and is associated with dilatation and distortion of its geometry [1,2]. During the past decade, a significant addition to our knowledge on the pathophysiology of myocardial infarction is a better understanding of the process of cardiac

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Fig. 2. Representative examples of macroscopic findings after: (A) normal saline and (B) growth hormone administration. Note the marked infarct expansion in (A), in contrast with the normal ventricular thickness and dimensions in (B).

Fig. 3. Angiogenesis in the infarct zone after: (A) normal saline and (B) growth hormone administration. Note the markedly increased number of neo-vessels in (B).

Table 2 Microvascular densities

IZ SIZ BZ NIZ SNIZ

Control

Growth hormone

Sham

66.2 ± 27.5 64.6 ± 46.8 68.9 ± 56.5 78.1 ± 66.1 33.9 ± 12.9

91.5 ± 23.1* 178.3 ± 59.8* 129.8 ± 47.8* 196.8 ± 70.9* 172.8 ± 69.9*

– – – 53.8 ± 18.3 48.2 ± 9.2

IZ: infarct zone; SIZ: subendocardium in the infarct zone; BZ: border zone; NIZ: non-infarct zone; SNIZ: subendocardium in the non-infarct zone. * p < 0.05 between the growth hormone and the control groups.

repair [2]. Amidst the plethora of substances involved, activation of the growth hormone/insulin-like growth factor-1 axis appears to play a central role, especially during the early phase of myocardial infarction [2–4]. Growth hormone has been shown to restore myocardial mass and function in patients with growth hormone deficiency [9], but the effects of growth hormone/insulin-like growth factor-1 axis stimulation postmyocardial infarction are diverse and not fully understood. They include actions on ventricular myocytes and extracellular matrix and on neo-vessel formation [3,4].

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A number of experimental studies [10–13] examined the effects of exogenous growth hormone administration on left ventricular function and remodeling postmyocardial infarction. In these studies, growth hormone was administered systemically one to three weeks after infarction and resulted in attenuation of remodeling and in improvement of systolic function [10–13]. Various mechanisms have been implicated, including attenuation of extracellular matrix remodeling and apoptosis [13]. However, two small studies in patients with ischemic cardiomyopathy produced inferior results [14,15]. This discrepancy may be attributed to a number of reasons: First, small animals have a significantly higher metabolism and turn over of hormones than humans. Second, the existing human studies are small, and, therefore, under-powered to detect significant differences. Third, human studies have short treatment and followup duration. Lastly, the doses used in human studies were significantly lower than those in animals. However, high-dosage systemic administration of growth hormone in humans is limited by the potential to induce diabetes, hypertension or various malignancies [16]. More importantly, reversing ventricular remodeling is a difficult task and treatments delivered at a time period when myocardial thinning and ventricular dilatation are advanced, have been generally associated with poor results [17]. In the present study, we evaluated a novel treatment approach, aiming at preventing ventricular remodeling, intervening: (a) during the acute phase of myocardial infarction and (b) locally in the infarct area. Current evidence suggests that prevention of infarct expansion during the highly vulnerable period of the very early stages after coronary occlusion may abort the whole process of remodeling [18]. We report an attenuated left ventricular remodeling by acute, intracoronary growth hormone administration, as evidenced by lower heart weight and size and a more normal ventricular geometry in treated animals. These actions were over and above the established beneficial effects of reperfusion, acute beta-blockade and nitrate administration and long-term angiotensin converting enzyme inhibition. As expected from the short-term nature of treatment, no adverse systemic effects of growth hormone were noted, as evidenced by the similar growth pattern between treated and control animals. The present study confirms the beneficial effects of growth hormone treatment in the prevention of left ventricular remodeling post-myocardial infarction, reported in previous experimental studies [10–13]. Moreover, this is the first study demonstrating the long-term potential of an acute and short-term treatment delivered locally in the infarct area. Our results compare favorably with those reported in two previous experimental studies evaluating a similar treatment strategy [19,20]. The first study examined the long-term effects of systemic growth hormone adminis-

tration after myocardial infarction in rats [19]. Treatment was initiated on the day of the infarction, but, at variance with our protocol, it was continued for two weeks. Growth hormone reduced infarct size, down-regulated the expression of fetal genes regarded as markers of heart failure progression and increased long-term survival [19]. The second study evaluated the effects of direct intramyocardial injection of adenovirus encoding growth hormone selectively in the infarct area of rats [20]. This treatment resulted in the preservation of chamber size, systolic and diastolic ventricular function. Our study indicates that these favorable actions can be reproduced with a single, intracoronary administration of growth hormone, delivered very early post-myocardial infarction. In our experimental setting, no arrhythmias or significant hemodynamic changes were noted after such administration and similar findings were reported in the isolated blood-perfused rabbit heart [21]. 4.1. Myocardial hypertrophy in preventing ventricular remodeling In our study, acute growth hormone administration attenuated infarct thinning and elongation, as evidenced by an increased infarct wall thickness and a trend towards a decreased infarct perimeter. It is well established that the growth hormone/insulin-like growth factor-1 axis stimulates the synthesis of cardiac myosin heavy chain and actin, resulting in the increased myocyte cell mass and volume [22]. The increase in wall thickness leads, by LaPlace’s law, to a reduction of wall stress, which, in turn, may prevent myocardial slippage and progressive ventricular dilatation [23]. 4.2. Angiogenic actions of the growth hormone/insulin-like growth factor-1 axis Stimulation of the growth hormone/insulin-like growth factor-1 axis has been shown to induce angiogenesis in a variety of tissues in vitro and in vivo [5,6], but data supporting a similar effect on the infarcted myocardium are very scarce [24]. We report enhanced angiogenesis after acute growth hormone administration in the porcine model, as evidenced by an increased microvascular density in treated, compared to shamoperated or to control animals. Improvement in local tissue perfusion may promote healing and may have contributed to the attenuated remodeling process observed in our study. Activation of the growth hormone/insulin-like growth factor-1 axis has been shown to induce vascular endothelial growth factor expression and may act as an upstream regulator in the angiogenic cascade [5,6]. Although no significant differences in vascular endothelial growth factor expression were found in our experiments four weeks after treatment, this mechanism cannot be excluded. Since the

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induction of vascular endothelial growth factor expression occurs during the early phase of the remodeling process [25], it can be postulated that acute growth hormone administration in our experiments resulted in an augmented, but not prolonged, production of vascular endothelial growth factor. An interesting, albeit unexpected, finding in our study was the fact that both ventricular hypertrophy and enhanced angiogenesis were evident not only in the infarct and border zones, but also in the remote, healthy myocardium. Although no data exist on the pharmacokinetics of intracoronary growth hormone infusion, we hypothesize that first-pass uptake occurs for part of the substance that binds with growth hormone receptors within and around the infarct area. These receptors are abundant on the myocyte membrane [26], and very early substance administration postmyocardial infarction may have facilitated such binding, before membrane disruption and cell death. The remaining substance may have reached the remote, non-infarcted myocardium via the systemic circulation.

hormone. Second, we did not examine the effects of acute growth hormone administration on arrhythmogenesis. The complex anti-arrhythmic regimen used, necessary to decrease periprocedural mortality in our experimental setting, precluded such evaluation. Third, apart from blood pressure and heart rate measurements, no hemodynamic studies were performed in our experiments; furthermore, our study did not include assessment of left ventricular function at various time intervals post-myocardial infarction. Although this issue has been addressed in previous reports [10,12,14,20], further studies incorporating functional in vivo data after growth hormone administration are necessary. Lastly, infarct size was evaluated solely by total creatine kinase measurements prior to treatment initiation. Nonetheless, we feel that measurement of creatine kinase at precisely the same time interval after reperfusion, together with the adequate size of our animal population, ensures comparable infarct size in both groups. The finding of comparable volume of infarcted tissue in the growth hormone and control groups reinforces this statement.

4.3. Right ventricular remodeling

4.5. Clinical implications

A significant finding of our study was the attenuation of right (in addition to left) ventricular remodeling after growth hormone treatment. Very few data exist on alterations in right ventricular dimensions and function that occur after left ventricular myocardial necrosis [27]. Recently, Nahrendorf et al. [28] reported significant right ventricular remodeling, detected with cinemagnetic resonance imaging, after ligation of the left anterior descending artery in rats. Although the pathophysiological mechanisms remain obscure, a decrease in the right ventricular pressure and/or volume overload, as well as local neurohormonal changes after growth hormone treatment may explain our findings [28].

At present, two anti-remodeling treatment strategies have been evaluated, focusing on two different time periods after acute coronary occlusion. The first strategy consists of acute unloading of the left ventricle, within the first 24 h of myocardial necrosis [2,17]. Similarly, acute beta-blockade, apart from its anti-ischemic and anti-arrhythmic effects, may also decrease regional wall stress [29]. The second strategy consists of chronic angiotensin-converting-enzyme inhibition; in addition to the hemodynamic benefits, such treatment inhibits several adverse neurohormonal aspects and has been shown to be the most effective anti-remodeling treatment [30]. The present study reinforces the hypothesis previously brought up by other investigators [19,20] and by our group [31] and points towards a third anti-remodeling treatment strategy. The proposed strategy consists of acute growth hormone administration targeted at the infarct area. We feel that our findings may pave the way for a clinical trial in patients with acute myocardial infarction, undergoing primary percutaneous coronary interventions. The ease of intracoronary administration of growth hormone, evaluated in our study, clearly potentates the possible clinical applications of such treatment.

4.4. Strengths and limitations of the study We feel that our study adds to the current understanding of anti-remodeling treatments post-myocardial infarction. The most important novelty is the introduction and evaluation of the ‘early-single infusion-local intracoronary delivery-concept’ of growth hormone administration. This concept was investigated in a large animal model, thus making inferences on potential clinical use more appealing. Furthermore, our study examined the angiogenic actions of growth hormone on the infarcted myocardium. This issue has not been systematically addressed in the past. Despite these considerations, a few limitations may be apparent. First, our study did not directly compare the proposed novel treatment strategy with the previously examined systemic administration of growth

Acknowledgement This work was supported by the national General Council for Research and Technology (Project: PENED-01ED511).

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