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Ann Thorac Surg 2004;77:2122-2129
© 2004 The Society of Thoracic Surgeons
a Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
b Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
Accepted for publication December 10, 2003.
* Address reprint requests to Dr Woo, Division of Cardiothoracic Surgery, University of Pennsylvania School of Medicine, 6 Silverstein Pavilion, 3400 Spruce St, Philadelphia, PA, USA 19104
e-mail: wooy{at}uphs.upenn.edu
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Abstract |
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METHODS: Rats underwent direct intramyocardial injection of adenovirus encoding either human growth hormone (n = 9) or empty null vector as control (n = 9) 3 weeks after ligation of the left anterior descending coronary artery. Analysis of the following was performed 3 weeks after delivery: hemodynamics, ventricular geometry, cardiomyocyte fiber size, and serum growth hormone levels.
RESULTS: The growth hormone group had significantly better systolic cardiac function as measured by maximum left ventricular pressure (73.6 ± 6.9 mm Hg versus control 63.7 ± 7.8 mm Hg, p < 0.05) and maximum dP/dt (2845 ± 453 mm Hg/s versus 1949 ± 605 mm Hg/s, p < 0.005), and diastolic function as measured by minimum dP/dt (–2,520 ± 402 mm Hg/s versus –1,500 ± 774 mm Hg/s, p < 0.01). Ventricular geometry was preserved in the growth hormone group (ventricular diameter 12.2 ± 0.7 mm versus control 13.1 ± 0.4 mm, p < 0.05; borderzone wall thickness 2.0 ± 0.2 mm versus 1.5 ± 0.1 mm, p < 0.001), and was associated with cardiomyocyte hypertrophy (6.09 ± 0.63 µm versus 4.66 ± 0.55 µm, p < 0.005). Local myocardial expression of growth hormone was confirmed, whereas serum levels were undetectable after 3 weeks.
CONCLUSIONS: Local myocardial overexpression of growth hormone after myocardial infarction resulted in cardiomyocyte hypertrophy, attenuated ventricular remodeling, and improved systolic and diastolic cardiac function. The induction of localized myocardial hypertrophy presents a novel therapeutic approach for the treatment of ischemic heart failure.
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Introduction |
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In clinical trials of post-MI patients, treatment with ACE inhibitors or ß-blockers resulted in inhibition or reversal of left ventricular (LV) remodeling, improved myocardial function, and decreased morbidity and mortality [5, 6]. Surgical approaches to improve ventricular geometry such as aneurysmectomy, infarct restraint, and cardiomyoplasty have resulted in reduced LV size, reduced wall stress, and in many cases improved contractile function in both animal models and clinical trials [7–13]. Taken together, these results suggest that improved ventricular geometry can result in better cardiac mechanics and function, with reduced morbidity and mortality.
Human growth hormone (GH) is a 191-amino acid polypeptide whose receptor is expressed on cardiomyocytes [14]. Sustained elevated serum levels of GH, as in acromegaly, result in significant cardiac hypertrophy. Systemic treatment with GH after MI can induce myocardial hypertrophy and may thereby attenuate or reverse ventricular remodeling by reducing wall stress [15]. This effect has been demonstrated in animal models, in which systemic GH administration results in modest preservation of LV systolic function and geometry [15–18]. To achieve this benefit, however, supraphysiologic serum levels of GH have been required for extended periods of time, and global cardiac hypertrophy has been observed. Because high serum levels of GH are associated with a diabetogenic state, hypertension, and increased risks of breast, prostate, and colon cancer, this strategy would not be desirable in clinical application [19, 20]. In addition, global LV hypertrophy, as seen in hypertension and aortic stenosis, can result in significant diastolic dysfunction with myocardial stiffening.
We therefore postulated that local myocardial overexpression of GH by adenoviral gene transfer could be targeted to the borderzone area surrounding the infarct, the region most susceptible to abnormally high wall strain and subsequent thinning and remodeling [21]. This technique could result in high levels of GH in the myocardial borderzone area with minimal systemic exposure and adverse effects on other organs. We hypothesized that this strategy for local GH overexpression would induce localized myocardial hypertrophy and thereby prevent the progression to dilated failure after a large MI in a rat model. We chose to initiate GH overexpression 3 weeks after an ischemic myocardial insult in rats that, both in our experience and as reported by others, is a time point that coincides with the transition to decompensated heart failure in this model [22]. It was our intention to model a clinically relevant scenario for patients with ischemic cardiomyopathy.
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Material and methods |
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Adenoviral vector construction
Replication-deficient (E1, E3 deleted) adenoviral vectors containing the human GH transgene driven by the cytomegalovirus promoter (Ad.GH) were made and obtained from the Institute of Human Gene Therapy, Vector Core, Dr. James M. Wilson, University of Pennsylvania. Human GH shares 75% homology with rat GH at the mRNA level, and 
80% homology at the amino acid level [23]. Empty replication-deficient adenovirus containing no transgene (Ad.Null) was obtained as a control from the University of Iowa Gene Transfer Vector core (supported in part by the National Institutes of Health and the Roy J. Carver Foundation).
In vivo transgene expression by immunoblotting and immunohistochemistry
Successful virus delivery using our method of direct intramyocardial injection was confirmed using a replication-deficient E1, E3-deleted adenovirus containing the ß-galactosidase gene under control of the cytomegalovirus promoter. X-gal staining was performed on histologic sections by fixation in 2% paraformaldehyde plus 0.1% glutaraldehyde followed by overnight incubation in X-gal reaction solution at 37°C (1 mg/mL X-gal, 4 mmol/L K-ferrocyanide, 2 mmol/L MgCl2 in phosphate-buffered saline, pH = 7.4). Sections were then counterstained with eosin.
In addition, in vivo expression of GH transgene was confirmed by performing direct intramyocardial injections of either Ad.GH or Ad.Null control virus into 2 rats using the methodology described below. The hearts were procured after 1 week, protein samples were prepared from ventricular biopsy specimens and 50 µg of each were electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel after 10 minutes of denaturation at 100°C. Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and immunoblotting was performed using a mouse anti-human GH monoclonal antibody (NeoMarkers, Fremont, CA). In addition, immunohistochemistry analysis was performed on the hearts of these animals. Histologic sectioning was performed as described below, and sections were incubated with mouse antihuman GH antibody (NeoMarkers, 1:500 in 5% bovine serum albumin [BSA]) followed by rhodamine-conjugated anti-mouse secondary antibody (1:200 in 5% BSA).
Animal surgery
Eighteen male Lewis inbred rats (250 to 300 g, Charles River Laboratories) were used for the main portion of this study. The Lewis strain of rat was chosen for the consistent infarct size, low mortality, and predictable development of post-MI heart failure after ligation of the left anterior descending coronary artery (LAD) [22].
Rats were anesthetized with intraperitoneal doses of ketamine (50 mg/kg) and xylazine (5 mg/kg), intubated, and mechanically ventilated with 0.5% isoflurane at the initial surgery. A left thoracotomy was performed through the fourth interspace and the LAD was ligated with a 7-0 polypropylene suture at the level of the left atrial appendage, a standardized location that is easily identified and reproducible. Myocardial ischemia was confirmed by blanching of the anterolateral left ventricle at the time of ligation. The animals were subsequently closed and allowed to recover. Three weeks after the infarction procedure, as heart failure ensued, repeat thoracotomies were performed. The animals were then randomly assigned to one of two experimental groups in a blinded fashion: delivery of Ad.GH (n = 9) (GH) or Ad.Null virus (n = 9) (control). All animals received direct intramyocardial injections of 5 x 109 plaque-forming units of virus into the infarction borderzone region through a 30-gauge needle. A total volume of 250 µL was injected into six separate areas. The animals were then closed and allowed to recover for an additional 3 weeks.
Hemodynamic measurements
Three weeks after the virus injection procedure the animals were once again anesthetized, intubated, mechanically ventilated, and a repeat thoracotomy was performed. A 2F pressure-volume conductance microcatheter was inserted into the left ventricle through the apex of the heart (Millar Instruments, Houston, TX). Hemodynamic measurements were analyzed using the ARIA 1 Pressure Volume Analysis software (Millar Instruments) in a blinded fashion. The relative volume unit (RVU) is a calculated estimation of ventricular volume based on the conductance of blood in the LV cavity as measured by the conductance microcatheter, a method that has been validated as an accurate assessment of volume [24]. The heart was then arrested in diastole by injection of 0.1 mL KCl (1 mEq/mL), then the LV cavity was filled with Optimum Cutting Temperature (OCT) embedding compound retrogradely through the transected aortic root. Hearts were filled with OCT in a blinded fashion using a standardized delivery system utilizing a 5-mL syringe and an 18-gauge angiocatheter. Hearts were then frozen in liquid nitrogen.
Ventricular geometry and infarct size
Ten-micrometer sections were prepared from all hearts with a cryostat from the midway point between the site of LAD ligation and the apex of the heart, an easily identified, standardized, and reproducible location. Staining with hematoxylin and eosin was then performed. Geometry measurements were performed on digitized photomicrographs using Openlab image processing software (Improvision, Lexington, MA) with standards of known length and were obtained on two representative sections for each animal. For chamber size, LV diameter was recorded in both vertical and horizontal axes and averaged. For borderzone wall thickness, measurements were obtained on two separate areas for each section and averaged. The wall thickness of remote areas of the LV free wall was also measured in two separate areas in a similar manner. Infarct size was determined as the percent of infarct scar relative to the outer circumference of the LV free wall in all animals. A single investigator blinded to the treatment groups performed all measurements.
Laminin immunohistochemistry and fiber size measurements
To measure cardiomyocyte fiber width, immunohistochemical staining for laminin, an extracellular matrix and basal lamina marker, was performed on histologic sections with a primary mouse anti-laminin antibody (NeoMarkers, 1:200 in 5% BSA) and a secondary rhodamine-conjugated anti-mouse antibody (1:200 in 5% BSA). Myocyte fiber width was measured in four representative microscopic fields in the borderzone area for each animal. Fiber width was measured at the level of the myocyte nucleus, and approximately 100 fibers in the borderzone area were counted in each heart and the average width was calculated. All measurements were performed by a single investigator blinded to the treatment group.
Serum assays
To determine the early time course of systemic exposure to the human GH transgene, a separate group of 8 rats underwent direct intramyocardial injections with either Ad.GH (n = 4) or Ad.Null (n = 4), and blood was collected after 1 week. Blood was also collected from the experimental animals in the primary study groups before procurement of the heart at the time of sacrifice. Serum levels of human GH were measured using a human-specific ELISA kit that does not detect rat GH (ICN Pharmaceuticals, Orangeburg, NY).
Statistical analysis
All values are expressed as mean ± standard deviation (SD). The unpaired Student's t test was used to calculate the statistical significance between the means of groups. A p value of less than 0.05 was considered to be significant.
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Results |
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Hemodynamics and cardiac function
The GH group had significant preservation of LV systolic function as measured by maximum LV pressure (GH 73.6 ± 6.9 mm Hg versus control 63.7 ± 7.8 mm Hg, p < 0.05) and maximum dP/dt (2845 ± 453 mm Hg/s versus 1949 ± 605 mm Hg/s, p < 0.005) (Fig 2).
A sensitive measure of the contractile state of the heart is the slope of the maximum dP/dt versus end-diastolic volume curve, which was significantly better in the GH group compared with control animals (1018 ± 276 mm Hg · s–1RVU–1 versus 675 ± 192 mm Hg · s–1RVU–1, p < 0.01) (Fig 2). No significant difference was noted in LV end-diastolic pressure between the groups (GH 5.7 ± 1.6 mm Hg versus control 7.1 ± 2.6 mm Hg, p > 0.05).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Ventricular remodeling is a central component of ischemic heart failure, and LV size is a predictor of clinical outcome after MI [2–4]. Successful pharmacologic treatment of CHF is associated with attenuation or reversal of the remodeling process, in addition to improved long-term outcomes [5, 6]. Surgical maneuvers to return the LV to a more normal geometric configuration have included passive epicardial restraint [11, 12], cardiomyoplasty [8, 9], and aneurysm plication or resection [7, 10]. These treatment strategies have resulted in improvement of end-diastolic volume, end-systolic volume, ejection fraction, and LV diameter to varying degrees. However, many patients continue to experience a progressive decline in cardiac function despite optimal medical management, and surgical modalities involve additional risks for operative morbidity and mortality. Therefore, alternative methods to improve ventricular geometry and function in ischemic heart failure are needed.
Growth hormone is a potent stimulus for cardiomyocyte hypertrophy when secreted in excess [25]. In addition, receptor levels for GH may be higher in the heart than in other tissues [26]. We hypothesized that local overexpression of GH targeted to the borderzone area could result in preserved myocardial geometry with reduced dilation and improved wall thickness, and thereby significantly reduced LV wall stress and improved cardiac performance. Because diastolic function can be significantly impaired by myocardial hypertrophy and the resultant decrease in LV compliance, an important finding in this study is that Ad.GH treatment actually improved diastolic function as measured by both minimum dP/dt and tau. In contrast to the pathologic hypertrophy seen in hypertension and aortic stenosis, it appears that local hypertrophy induced by GH overexpression only in the infarct borderzone targeted the area most susceptible to mechanical stress and progressive thinning after an acute MI. The borderzone areas adjacent to infarcts are subject to supraphysiologic stress because they act as a bridge between normally contracting myocardium and noncontractile infarct. The targeted hypertrophy induced by GH overexpression was sufficient to attenuate the remodeling process and preserve cardiac function after MI.
Systemic GH administration after acute MI is associated with preserved LV function in rodent studies [15–18]. However, supraphysiologic serum levels exceeding 1,700 ng/mL (normally less than 7 ng/mL) have been required to demonstrate positive effects. Systemic GH administration in human trials for cardiomyopathy have resulted in only marginal benefits with regard to improving LV geometry or function [27–30]. These clinical studies likely suffered from substantial underdosing, however, because serum GH was not significantly increased with the dosing regimens used (2 to 4 IU GH subcutaneous daily). Clinical dosing of GH is limited by its potential to induce a diabetogenic state or hypertension with prolonged exposure [20] and associated increased rates of breast, prostate, and in particular, colon cancer [19]. Therefore, our strategy of local overexpression through a gene transfer technique produces a high local level of GH within the targeted myocardial territory adjacent to the infarcts, while minimizing systemic exposure and potential morbidity. Serum levels of human GH were very low 1 week after virus administration (4.0 ± 0.8 ng/mL) and undetectable at the time of sacrifice 3 weeks after injection. Transgene expression was limited to the targeted borderzone areas by immunohistochemistry, and no evidence of human GH staining was seen in remote myocardial territories.
In our study virus delivery was performed 3 weeks after creation of a large MI. This time point was specifically chosen to maximize the clinical relevance while minimizing animal mortality. Previous work by our laboratory (unpublished data) and others using the Lewis rat model has shown that cardiac function remains stable between 2 and 4 weeks from infarction, but declines rapidly between 4 and 8 weeks [22]. Therefore, we injected the virus during a period of relative stability of cardiac function, with maximal virus transgene expression, which occurs between 1 and 2 weeks after delivery [31], during the period of greatest decline in cardiac function. Our intent was to model a clinical scenario in which a patient presents with symptomatic heart failure and declining cardiac function and undergoes a specific therapy.
To provide a reliable and reproducible means of achieving high local levels of cardiac transgene expression, we chose an adenoviral gene transfer strategy. Alternative methods of local overexpression include transcoronary delivery of adenoviral vectors, injection of naked plasmid DNA, myocardial implantation of drug-eluting beads, or catheter-based myocardial injections. It has been shown that adenoviral vectors provide that highest level of gene transfer and expression compared with naked DNA and liposomal strategies [32]. We have found that local adenoviral injection provides a means to achieve consistently high local levels of growth factor in our model, and thus serves as an important investigative tool. As a control injection we chose to utilize Ad.Null virus to introduce the same viral construct as in the GH group but without the actual transgene, and thereby maximize the similarities in treatment as much as possible between the two experimental groups. One potential concern with this strategy is that the control virus may actually enhance the progression into heart failure and thereby confound analysis of any beneficial effects of Ad.GH. However, we have performed experiments with saline injections as an alternative control after LAD ligation, and found no difference in cardiac function or geometry between the Ad.Null and saline groups (unpublished data). Therefore, we believe that the differences between the experimental and control groups in our study are due solely to the overexpression of GH.
Several important limitations to this study are worth noting. Although using blinded and standardized techniques we have shown an improvement in ex vivo cardiac geometry, an in vivo assessment using echocardiography or MRI would be helpful to demonstrate differences under physiologic loading conditions. In addition, these noninvasive modalities offer the opportunity to track postinfarction remodeling over the course of weeks and may allow further insight into the effects of local GH overexpression. Measurement of ejection fraction by echocardiography would have provided closed-chest data on cardiac performance and may therefore be a better method to assess myocardial function.
In conclusion, we have demonstrated in this study that targeted borderzone overexpression of GH in a rat model of postinfarction heart failure resulted in regional cardiomyocyte hypertrophy, diminished LV remodeling, preserved systolic and diastolic function, and minimal systemic exposure to GH protein. These results underscore the importance of normal ventricular geometry to cardiac function, and demonstrate that attenuation of remodeling can prevent functional decline. The induction of targeted regional myocardial hypertrophy is a novel approach to treating heart failure.
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p < 0.005. (GH = growth hormone; LV = left ventricular; MI = myocardial infarction; RVU = relative volume unit.)
