Adenoviral Gene Transfer of FGF-5 to Hibernating Myocardium Improves Function and Stimulates Myocytes to Hypertrophy and Reenter the Cell Cy
http://www.100md.com
循环研究杂志 2005年第4期
the VA WNY Health Care System and the Departments of Medicine and Physiology and Biophysics and the Center for Research in Cardiovascular Medicine at the University at Buffalo, Buffalo, NY.
Abstract
Fibroblast growth factors (FGFs) have diverse actions on the myocardium but the importance of stimulating angiogenesis versus direct effects of FGFs on cardiac myocytes is unclear. We used intracoronary injection of a replication-deficient adenoviral construct overexpressing FGF-5 (AdvFGF-5) to improve flow and function in swine with hibernating myocardium. Two-weeks after AdvFGF-5 (n=8), wall-thickening increased from 2.4±0.04 to 4.7±0.7 mm in hibernating LAD regions (P<0.05) whereas remote wall-thickening was unchanged (6.7±0.4 to 5.8±0.5 mm). This was associated with small increases in resting flow to dysfunctional myocardium, but flow during adenosine was unchanged (LAD 1.45±0.27 versus 1.46±0.23 mL/min per g and remote 4.84±0.23 versus 4.71±0.47 mL/min per g, P=NS). Unexpectedly, animals receiving AdvFGF-5 demonstrated a 29% increase in LV mass over the 2-week period (P<0.05 versus untreated animals with hibernating myocardium and normal shams). Histological analysis confirmed profound myocyte cellular hypertrophy in AdvFGF-5 treated myocardium (19.9±0.32 versus 15.2±0.92 e in untreated, P<0.001). Myocytes in the proliferative phase of the cell cycle (Ki-67 staining) increased 7-fold after AdvFGF-5 (2,904±405 versus 409±233 per 106 myocyte nuclei in untreated, P<0.05). Myocyte nuclei in the mitotic phase (phosphorylated histone H3 staining) also increased after AdvFGF-5 (127±24 versus 35±13 per 106 myocyte nuclei in untreated, P<0.05). Thus, rather than angiogenesis, stimulation of hypertrophy and reentry of a small number of myocytes into the mitotic phase of the cell cycle are responsible for the effects of AdvFGF-5 on function. Although additional mechanisms may contribute to the improvement in wall-thickening, overexpression of AdvFGF-5 may afford a way to restore function in hibernating myocardium and ameliorate heart failure in chronic ischemic cardiomyopathy.
Key Words: hibernating myocardium growth factors gene therapy
Introduction
Hibernating myocardium is common in patients with ischemic cardiomyopathy, and myocardial revascularization can improve function and ameliorate symptoms of heart failure. Unfortunately, many patients are not suitable candidates for surgical or percutaneous revascularization and developing nonsurgical approaches to reverse dysfunction and improve perfusion would be desirable. Administration of FGFs as recombinant proteins or overexpression using plasmid and adenoviral vectors elicits multiple effects that could favorably affect flow and function in viable chronically dysfunctional myocardium. Considerable enthusiasm for therapeutic angiogenesis has arisen from promising experimental animal studies using rapidly developing coronary collaterals and ameroid occluder models.1eC6 Unfortunately, FGF-mediated improvements in myocardial perfusion are small, and few laboratories have demonstrated objective changes in flow during pharmacological or metabolic stress. In addition, when administered to dogs with chronic well-developed coronary collaterals, FGF did not improve myocardial perfusion.5 This raises the possibility that FGFs accelerate collateral growth in animal models yet may not afford improvements when coronary flow reserve is chronically reduced. Such actions could be the basis for the inability to translate experimental findings to clinical studies in patients with chronic coronary disease.7
Despite the paucity of data demonstrating increases in coronary flow reserve, many studies show substantial effects of FGFs on myocardial function, raising the possibility that the major therapeutic actions of FGFs may be due to nonangiogenic mechanisms.8 For example, FGF has been shown to protect myocytes against irreversible injury1,9eC11 and reversible stunning,10,12 promote alterations in calcium handling that could improve contractility,13 attenuate ischemia-induced myocyte apoptosis, and stimulate myocyte hypertrophy in cell culture.14 Whereas the relative contribution of each of these mechanisms in models of chronic ischemia in vivo is unknown, they could improve function independently of changes in myocardial perfusion.
We performed the present study to evaluate the effects of FGF-5, a secreted fibroblast growth factor, in a swine model of chronic collateral-dependent hibernating myocardium where the physiological characteristics of hibernating myocardium, namely reduced resting flow and function with a critical impairment of subendocardial flow reserve, remain unchanged between 3 and 5 months after instrumentation.15,16 This contrasts with relatively short-term ameroid instrumented swine models of collateral-dependent myocardium, where transient upregulation of endogenous growth factors and subendocardial infarction near the time of ameroid occlusion could be important in modulating the effects of exogenous growth factors. We overexpressed FGF-5 with an adenovirus that has previously been demonstrated to transfect myocardium after intracoronary administration.17 A similar approach ameliorated stress-induced dysfunction in a porcine ameroid model where resting function was normal with initially promising results in phase II/III human trials.18,19 Our results demonstrate that the salutatory effects of AdvFGF-5 on function are dissociated from perfusion and related to stimulating myocyte hypertrophy and inducing a small population of myocytes to reenter the mitotic phase of the cell cycle.
Materials and Methods
Procedures and protocols conformed to institutional guidelines for the care and use of animals in research and are detailed in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Hibernating myocardium was produced as previously described.15
Serial Physiological Studies
Pigs began studies 4 months after instrumentation, at which time flow, function, and coronary flow reserve are critically reduced. Using a percutaneous approach under propofol sedation, we inserted a 5F multipurpose catheter into the left ventricle using the 6F introducer side port for pressure monitoring and arterial sampling. Regional wall-thickening was assessed with transthoracic echocardiography from a right parasternal approach, and myocardial perfusion was assessed with fluorescent microspheres.20 After an equilibration period, we assessed hemodynamics, flow, and regional wall thickening at rest, during submaximal epinephrine infusion, and after pharmacological vasodilation with adenosine (0.9 mg/kg per min IV) with phenylephrine infused to prevent adenosine-induced hypotension.
We administered a replication deficient adenovirus containing FGF-5 under control of the CMV promoter (AdvFGF-5, 1x1011 pfus, n=8)4 in equally divided doses into the LAD, circumflex, and right coronary arteries over 30 seconds, taking care to avoid reflux into the systemic circulation. First-pass myocardial uptake was enhanced using histamine (25 e/min IC). As controls, we assessed the effects of a similar dose of nuclear localizing EGFP adenovirus (AdvEGFP, n=2 hibernating and n=2 sham) after intracoronary administration.21
An identical physiological study was repeated 2 weeks after AdvFGF-5, after which animals were euthanized under anesthesia. The LV was weighed, sections incubated in TTC to assess infarction, and samples taken for flow and histological analyses.
Apoptosis, Myocyte Hypertrophy, and Cell Cycle Markers
Details are provided in the expanded Materials and Methods. Briefly, we quantified myocyte nuclear density, diameter, and nuclear length to estimate myocyte volume as previously described.22 Myocyte diameter and volume from the AdvFGF-5 group were compared with matched animals with untreated hibernating myocardium (n=10), sham control groups (n=5), and animals receiving AdvEGFP (n=4) to exclude nonspecific effects of the adenovirus. Myocyte apoptosis was assessed using fluorescent TUNEL staining.22
Tissue sections were also incubated with antibodies for the nuclear cell cycle markers Ki-67 (a specific marker for cells that have reentered the growth phase of the cell cycle) and anti-phospho-histone H3 (a marker of mitosis). We quantified positive myocyte nuclei in relation to myocyte nuclear density using both light and fluorescent confocal multifluorescence microscopy (Bio-Rad MRC 1024). Cardiac myocytes were costained with antibodies to troponin I, and nuclei were costained with TO-PRO-3. Similar quantitative results were obtained with each approach (online data supplement).
Statistics
Data are expressed as mean±SE. Differences after treatment with AdvFGF-5 and comparisons between the hibernating and normally perfused remote regions of the same heart were assessed using paired t tests. Details of the piece-wise flow analysis are provided in the online data supplement. Differences among AdvFGF-5eCtreated animals, age-matched shams, and untreated animals with hibernating myocardium were assessed using a two-way ANOVA and the post-hoc Holm-Sidak test (Sigma Stat 3.0) with P<0.05 considered significant.
Results
Pigs were in good health at the time of study, and TTC staining showed no infarction. Initial physiological studies were performed 123±2 days after instrumentation and repeated 2 weeks after AdvFGF-5 (137±2 days).
Findings before AdvFGF-5 confirmed dysfunctional hibernating myocardium. There were reductions in resting perfusion (LAD 0.98±0.09 versus 1.38±0.15 mL/min per g in remote; P<0.01) with the greatest reduction in the subendocardium (0.79±0.05 versus 1.52±0.16 mL/min per g in remote; P<0.01). Full-thickness flow during epinephrine was attenuated (LAD 1.27±0.09 versus 1.65±0.08 mL/min per g in remote; P<0.01), and subendocardial flow did not increase significantly above resting levels (LAD 0.93±0.09 mL/min per g; P=NS versus rest). Likewise, the increase in full-thickness flow during adenosine was severely attenuated (LAD 1.45±0.27 versus 4.84±0.23 mL/min per g in remote; P<0.001), and subendocardial flow did not increase above the resting value (LAD 0.72±0.17 mL/min per g; P=NS versus rest).
Efficiency of Intracoronary AdvEGFP Gene Transfer in Swine
Figure 1 demonstrates the efficiency of intracoronary gene transfer after pretreatment with histamine to increase endothelial permeability. Fluorescence imaging of EGFP demonstrated a high frequency of cardiac nuclear colocalization. When this was quantified using TO-PRO-3 to assess cardiac nuclei, the EGFP was present in 39±2% of cells. Cytoplasmic staining of EGFP was weaker but still present 2 weeks after AdvEGFP administration. These data confirm a high transfection efficiency after histamine pretreatment with the intracoronary approach that is similar to that reported using LacZ transfection by other laboratories.17
Effects of AdvFGF-5 on Flow and Function in Hibernating Myocardium
Figure 2 summarizes the effects of AdvFGF-5 on flow and function with detailed transmural, circumferential, and relative perfusion analysis provided in the online data supplement. Hemodynamic measurements are summarized in the Table. Two-weeks after AdvFGF-5, LAD wall thickening (WT) increased from 2.4±0.4 to 4.7±0.7 mm (P<0.05 versus initial), whereas wall thickening in normally perfused regions was unchanged. Global LV function and the response to epinephrine were not affected by AdvFGF-5 (online data supplement).
Despite prominent effects on function, there were only small changes in myocardial perfusion when averaged among all samples in the region distal to the stenosis. As summarized in Figure 2, resting perfusion tended to increase in hibernating LAD regions while decreasing in remote regions, but the differences were not significant. There were no significant differences in absolute flow during adenosine vasodilation or epinephrine (online data supplement). When analyzed as paired measurements from LAD pieces pooled from all animals (n=147), there was a small increase in relative resting perfusion (LAD/Remote 0.70±0.02 to 0.75±0.02; P<0.0001) and analysis of the integrated flow deficit showed it to decrease from 18.9±2.9% to 12.5±4% (P<0.05) indicative of a reduction in the resting perfusion deficit size. There was no improvement in relative flow during adenosine (LAD/Remote 0.33±0.02 versus 0.31±0.02), but there was a small reduction in the vasodilated perfusion deficit size (46.7±3.5% versus 43.2±3.5%; P<0.05). Thus, the effects of AdvFGF-5 on flow were primarily restricted to the border regions between normally perfused and hibernating regions.
Effects of AdvFGF-5 on Myocyte Cellular Hypertrophy
Although body weight (96±3 to 104±3 kg) and LV end-diastolic dimension (50±2 to 54±2 mm) increased by only 8%, echocardiographic estimates of LV mass increased by 29% 2 weeks after AdvFGF-5 (177±19 to 228±15 g; P<0.05). Figure 3 shows that AdvFGF-5 produced myocyte cellular hypertrophy with quantitation summarized in Figure 4. Myocyte diameter was compared with similarly instrumented but untreated animals with hibernating myocardium (controls) and uninstrumented shams of a similar age (AdvFGF-5 134±2 days and untreated hibernating 137±2 days) and body weight (AdvFGF-5 104±3 kg, untreated hibernating 106±4 kg, and normal shams 115±4 kg). The LV mass/body weight ratio in animals receiving AdvFGF-5 was higher than untreated animals (2.5±0.1 versus 2.2±0.1 g/kg; P<0.05) as well as sham controls (1.8±0.2; P<0.05 versus both groups). AdvFGF-5 increased myocyte diameter in parallel with the changes in LV mass (Figure 4). In the LAD region, subendocardial myocyte diameter increased from 16.2±0.40 to 18.6±1.12 e (P<0.05) in AdvFGF-5eCtreated animals and both were significantly increased in comparison to sham controls (14.8±0.19 e; P<0.05). Changes in subepicardial myocyte diameter were even more pronounced (15.2±0.94 to 19.9±0.32 e after AdvFGF-5, P<0.05, and 13.0±0.83 e in shams, P<0.05). Changes in myocyte diameter in normally-perfused remote regions were similar to hibernating LAD regions and particularly prominent in the subepicardial layers (20.3±0.7 versus 15.2±0.39 e in untreated, P<0.05, and 13.7±0.81 e in shams, P<0.05). Animals receiving AdvEGFP showed no change in echocardiographic LV mass over 2 weeks, and myocyte diameter after AdvEGFP (remote zone myocardium 14.4±1.22 e) was no different from sham or untreated hibernating groups.
Effects of AdvFGF-5 on Myocyte Nuclear Density, Cell Volume, and Apoptosis
Figure 5 summarizes the effects of AdvFGF-5 on myocyte nuclear density, apoptosis, and calculations of myocyte cellular volume from subendocardial LAD samples. Whereas LAD connective tissue was mildly increased in animals receiving AdvFGF-5 (LAD 6.6±1.1% versus 4.1±0.8% in remote; P<0.05), it was no different in untreated controls (LAD 6.9±0.7% versus 4.5±0.2% in remote; P<0.05). In addition, there were no inflammatory changes in AdvFGF-5eCtreated pigs.
Due to prominent cellular hypertrophy, AdvFGF-5 treatment produced significant reductions in myocyte nuclear density. In the LAD subendocardium, myocyte nuclear density was 746±26 versus 926±44 nuclei per mm2 in untreated (P<0.01) and 1212±36 nuclei per mm2 (P<0.01) in shams. Estimates of cell volume using morphometric approaches further confirmed hypertrophy with LAD myocyte cell volume increasing from 11 987±653 to 14 988±901 e3/nucleus (P<0.05) after AdvFGF-5. The differences in myocyte volume (30%) were proportional to the increase in LV mass (29%) and not indicative of hypertrophy from apoptosis-induced myocyte loss, which, if anything, was lower in animals receiving AdvFGF-5 than untreated hibernating myocardium.
Reentry of Myocytes Into the Growth Phase of the Cell Cycle After AdvFGF-5
Figure 6 illustrates confocal photomicrographs demonstrating myocyte nuclear Ki-67 staining in an animal treated with AdvFGF-5. The frequency of Ki-67 staining, a marker of myocytes in the growth phase of the cell-cycle, was expressed in relation to the number of myocyte nuclei. Myocytes are normally in G0 as confirmed by the low frequency of Ki-67 staining (0.028% of myocytes or 284±69 per 106 myocyte nuclei in shams). Untreated animals with hibernating myocardium and animals receiving AdvEGFP had similar low values of Ki-67 staining (447±212 and 213±69 per 106 myocyte nuclei, respectively). After AdvFGF-5, Ki-67 positivity increased significantly (LAD 2904±405 and remote 2066±326 per 106 myocyte nuclei; both P<0.05 versus sham and untreated), but the frequency of Ki-67 positive myocyte nuclei after AdvFGF-5 was still less than 1% of myocytes (LAD 0.29±0.04% and remote 0.21±0.03% versus 0.03±0.01% in shams). Increased Ki-67 positivity was not restricted to myocytes but was also detected in fibroblasts, endothelial cells, and smooth muscle cells. Nonmyocyte staining represented 51±7% of all nuclear Ki-67 staining.
Although Ki-67 reflects active cellular hypertrophy it could also indicate myocytes undergoing nuclear division.23 We therefore quantified phosphohistone H3 staining, a marker of DNA replication (Figure 7). Phosphohistone H3 staining was rare in normal shams (2.4±2.4 per 106 myocyte nuclei). It was higher in untreated hibernating myocardium (35±13 per 106 myocyte nuclei; P<0.05) and increased further after AdvFGF-5 (127±24 per 106 myocyte nuclei; P<0.05 versus untreated or shams). Frequencies of myocyte apoptosis were much lower (Figure 4) averaging 4.7±3.2 per 106 myocyte nuclei in AdvFGF-5eCtreated animals. Mitotic myocyte nuclei were also visualized by light microscopy but cytokinesis was not seen. Based on these results, approximately 4% of the Ki-67eCpositive myocytes (1 in 10 000) were in the mitotic phase of the cell cycle. The frequency distribution of Ki-67 positive myocyte diameters was similar to unstained cells in untreated hibernating myocardium (15.9±0.3 versus 15.1±0.1 e). In animals receiving AdvFGF-5, Ki-67 positive myocyte were smaller (17.4±0.4 versus 18.8±0.1 e; P<0.05) but a subpopulation of cells smaller than normal (potentially indicative of differentiating stem cells) was not identified (online data supplement).
Discussion
There are several important new findings from our study. First, intracoronary administration of AdvFGF-5 in chronic hibernating myocardium produces only small changes in resting perfusion that are predominantly restricted to regions bordering normal and hypoperfused myocardium. In contrast, AdvFGF-5 induced striking improvements in myocardial function that were limited to dysfunctional LAD regions and disproportionate to increases in perfusion. This was accompanied by increases in LV mass and cellular myocyte hypertrophy throughout the LV. Histological staining demonstrated that AdvFGF-5 induced a population of myocytes to reenter the growth phase of the cell cycle. Collectively, these results support the notion that favorable actions of exogenously administered FGFs are largely independent of functional collateral vessel growth and predominantly related to myocyte remodeling.
Limited Effects of AdvFGF-5 on Flow in Hibernating Myocardium
Our study provides a comprehensive assessment of myocardial perfusion after administration of growth factors that has previously been lacking in the majority of studies evaluating angiogenic interventions. AdvFGF-5 elicited small increases in resting perfusion that were largely restricted to the border regions. This small flow change was more difficult to ascertain during vasodilation but the reduction in the perfusion deficit size at rest and after vasodilation was similar (4% to 6% absolute difference in each condition). Importantly, our observations regarding the effects of AdvFGF-5 on the distribution of flow are strikingly similar to observations in many clinical trials of angiogenic growth factors where it has been difficult to measure objective increases in myocardial perfusion despite promising findings based on functional improvement in porcine ameroid models. The most demonstrable change in nuclear perfusion studies has been a reduction in the resting perfusion defect size.24 Recent studies evaluating intracoronary AdvFGF-4 have also demonstrated small reductions in defect size during adenosine vasodilation19 that are similar in magnitude to those we observed in hibernating myocardium.
Collectively, the results indicate that mature coronary collaterals supplying chronically dysfunctional hibernating myocardium have a limited ability to increase perfusion after exogenous FGF-5. The fact that this occurs in the setting of resting dysfunction and a critical impairment in flow indicates that factors present in rapidly developing collateral models such as upregulation of other elements of the angiogenic cascade25 and/or coexisting subendocardial infarction are required to effect an improvement in flow. Thus, the results in pigs with chronic hibernating myocardium are similar to the dog with well-developed chronic collaterals where interventions administered near the point of ameroid occlusion accelerate but were unable to increase collateral flow beyond that attainable with intrinsic angiogenic stimuli.5 Although speculative, this could be a consequence of intrinsic myocardial adaptations that limit the development of metabolic ischemia during stress in hibernating myocardium.26
Effects of AdvFGF-5 on Function in Hibernating Myocardium
In studies where flow and function have been evaluated after growth factor administration, their effect on function has greatly exceeded their effect on perfusion. Consistent with this is the fact that intracoronary AdvFGF-4 improved function in swine with pacing-induced heart failure without altering myocardial perfusion.27 Whereas findings of hibernating myocardium persisted after AdvFGF-5, there was an improvement in the myocardial flow-function relation. Importantly, the improvement in function occurred regionally and did not reflect altered loading conditions because function in normally perfused remote regions, global function, and systolic and end-diastolic LV pressure remained unchanged after AdvFGF-5. This suggests that AdvFGF-5 may have ameliorated a component of dysfunction that was due to acute myocardial stunning superimposed on chronic hibernating myocardium.28 The coexistence of stunning is consistent with the observation that reductions in wall thickening in chronic hibernating myocardium exceed the reduction in subendocardial perfusion.
Several additional mechanisms exist through which AdvFGF-5 could affect function disproportionately from perfusion.8 Previous studies have demonstrated that short-term administration of basic FGFs can protect the heart against reversible and irreversible ischemic injury and produce a "preconditioning like" effect.10eC12 These actions would also be consistent with the improvement in function restricted to myocardium with limited coronary flow reserve. Although acute administration of FGF can also alter myocyte calcium transients and directly increase contractility in vitro,13 this mechanism seems less likely because remote zone function, global function, and contractile reserve were not altered after AdvFGF-5. A final mechanism through which AdvFGF-5 could have altered systolic function is through an improvement in myocardial efficiency via activation of NOS.29 Activation of NOS could produce chronic preconditioning against stunning in hibernating myocardium as well as optimize myocardial efficiency if NO release was chronically downregulated in chronic hibernating myocardium.
AdvFGF-5 Mediated Myocyte Hypertrophy
An alternative explanation for the dissociation between flow and functional effects of AdvFGF-5 could relate to stimulation of myocyte hypertrophy and the resultant regional remodeling. Intracoronary administration of AdvFGF-5 led to significant myocardial hypertrophy with a 30% increase in LV mass within 2 weeks. Previous studies have established a role for endogenous FGFs in acquired hypertrophy produced by a variety of increased physiological loading conditions,14 and manipulating exogenous basic FGFs can stimulate myocyte cellular hypertrophy and mitosis in neonatal myocytes in vitro.8 Our study extends previous work to demonstrate significant FGF-5eCdependent myocyte plasticity in vivo. Both cellular and anatomic hypertrophy occurred over a brief time interval without changes in ventricular loading conditions. Although some cellular myocyte hypertrophy was apparent in untreated animals with hibernating myocardium versus sham controls, AdvFGF-5 led to substantially greater increases in LV mass and myocyte diameter.
Although myocyte hypertrophy was global and particularly prominent in the subepicardium, the improvement in function was restricted to the LAD region. The trend toward a reduction in function in the remote zone could be consistent with a hypertrophy mediated reduction in contractility but is more likely due to a resolution of moderate compensatory hyperkinesis as LAD function improved. Because transmural wall thickening is usually strongly related to subendocardial function, the prominent LAD subepicardial hypertrophy after AdvFGF-5 may have allowed hypertrophied myocytes in outer layers to compensate for subendocardial myocytes lost from apoptosis during the development of hibernating myocardium.22 Additional studies will be needed to determine the relative importance of replacing regional myofibrils via cellular hypertrophy versus other mechanisms.
Increased Cell Cycle Markers After AdvFGF-5
A surprising finding was the fact that AdvFGF-5 caused a small population of cardiac myocytes to initiate DNA synthesis. We were able to confirm this using phophohistone-H3 staining, which has previously been used to label myocyte nuclei in various phases of nuclear division and correlates with BrdU positivity.30 Although cytokinesis was not seen, there is considerable difficulty in identifying such short-lived events in histological tissue. Hein et al23 demonstrated that DNA synthesis increases during hypertrophy and heart failure. Increased Ki-67 was detected without mitosis with DNA replication potentially activated to maintain the DNA content/myocyte volume ratio in hypertrophied myocytes. Regardless of whether cell or nuclear division predominates, the impact of AdvFGF-5 on cellular hypertrophy seems more dominant based on several observations. First, myocytes in the growth phase of the cell cycle (Ki-67) were nearly 20-times higher than those positive for phosphohistone H3. Second, myocyte nuclear density was lower in animals treated with AdvFGF-5. The failure of nuclear density to increase after AdvFGF-5 indicates that cellular hypertrophy exceeded myocyte nuclear hyperplasia making quantitatively important changes in myocyte polyploidy unlikely. Directionally, similar effects were found by manipulating telomerase reverse transcriptase in mice.30 Finally, we cannot exclude the possibility that some of the Ki-67eCpositive myocytes reflect stem cell recruitment because the diameters were smaller than nonstained cells in AdvFGF-5eCtreated hearts and further studies will be required to address this possibility. Collectively, our results support that AdvFGF-5 contributes to functional remodeling through both myocyte cell growth and possibly cell division. This may represent a potentially new approach to reverse the effects of myocyte loss in ischemic cardiomyopathy.
Methodological Limitations
The effects of AdvFGF-5 cannot be directly extrapolated to other growth factors because they may have different physiological actions, and further studies are required to evaluate their effect on myocyte hypertrophy.7 Interestingly, plasmid VEGF has been reported to increase myocyte mitosis leading to nuclear hyperplasia in a porcine ameroid model.31 We did not colocalize phosphohistone H3 and Ki-67 staining, and it is possible that cellular hypertrophy and nuclear division are occurring in different myocyte populations. It is plausible that AdvFGF-5 is producing hypertrophy in adult myocytes and recruiting a smaller stem cell population responsible for phosphohistone H3 positivity. Although our studies were conducted in "adult" farm-bred swine (more than 100 kg) over 6 months old at the time of study, the animals were otherwise normal, and it is possible that intracoronary gene transfer would be insufficient in aged animals32 or when coexisting atherosclerosis and endothelial function is present. We injected two-orders of magnitude more viral particles than published clinical trials of intracoronary AdvFGF-4 and one-order higher than previous studies of AdvFGF-5 in swine. Further studies will be required to examine the dose-dependency of the effects we have reported. Finally, we have documented EGFP protein but have not confirmed the presence of FGF-5 protein production 2 weeks after transfection. In a previous study, Giordano4 confirmed FGF-5 in pigs using a similar approach. Even if the FGF-5 was only transiently expressed, it would not negate the fact that it had lead to substantial increases in LV mass and myocyte hypertrophy in the absence of any alterations in ventricular loading conditions. These were specific to the AdvFGF-5 construct because AdvEGFP did not produce similar effects.
Clinical Implications
Our study provides further support for the pleiotrophic effects of FGFs on the myocardium, and identifies additional mechanisms to explain the functional improvement after growth factor interventions. The increases in function and myocyte hypertrophy in hibernating myocardium are more prominent than effects related to angiogenesis, raising the possibility that administration of AdvFGF-5 may be more efficacious as an adjunctive therapy in patients with advanced ischemic cardiomyopathy and heart failure than chronic angina. Further studies evaluating AdvFGF-5 in animal models of heart failure as well as clinical trials will be required to test this possibility.
Acknowledgments
This work was supported by the VA, AHA, NHLBI, Albert and Elizabeth Rekate Fund, and the Oishei Foundation. We thank Anne Coe, Deanna Gretka, Amy Johnson, and Rebeccah Young for their technical assistance and the University of Pittsburgh Vector Core Laboratory for supplying the AdvEGFP virus.
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Abstract
Fibroblast growth factors (FGFs) have diverse actions on the myocardium but the importance of stimulating angiogenesis versus direct effects of FGFs on cardiac myocytes is unclear. We used intracoronary injection of a replication-deficient adenoviral construct overexpressing FGF-5 (AdvFGF-5) to improve flow and function in swine with hibernating myocardium. Two-weeks after AdvFGF-5 (n=8), wall-thickening increased from 2.4±0.04 to 4.7±0.7 mm in hibernating LAD regions (P<0.05) whereas remote wall-thickening was unchanged (6.7±0.4 to 5.8±0.5 mm). This was associated with small increases in resting flow to dysfunctional myocardium, but flow during adenosine was unchanged (LAD 1.45±0.27 versus 1.46±0.23 mL/min per g and remote 4.84±0.23 versus 4.71±0.47 mL/min per g, P=NS). Unexpectedly, animals receiving AdvFGF-5 demonstrated a 29% increase in LV mass over the 2-week period (P<0.05 versus untreated animals with hibernating myocardium and normal shams). Histological analysis confirmed profound myocyte cellular hypertrophy in AdvFGF-5 treated myocardium (19.9±0.32 versus 15.2±0.92 e in untreated, P<0.001). Myocytes in the proliferative phase of the cell cycle (Ki-67 staining) increased 7-fold after AdvFGF-5 (2,904±405 versus 409±233 per 106 myocyte nuclei in untreated, P<0.05). Myocyte nuclei in the mitotic phase (phosphorylated histone H3 staining) also increased after AdvFGF-5 (127±24 versus 35±13 per 106 myocyte nuclei in untreated, P<0.05). Thus, rather than angiogenesis, stimulation of hypertrophy and reentry of a small number of myocytes into the mitotic phase of the cell cycle are responsible for the effects of AdvFGF-5 on function. Although additional mechanisms may contribute to the improvement in wall-thickening, overexpression of AdvFGF-5 may afford a way to restore function in hibernating myocardium and ameliorate heart failure in chronic ischemic cardiomyopathy.
Key Words: hibernating myocardium growth factors gene therapy
Introduction
Hibernating myocardium is common in patients with ischemic cardiomyopathy, and myocardial revascularization can improve function and ameliorate symptoms of heart failure. Unfortunately, many patients are not suitable candidates for surgical or percutaneous revascularization and developing nonsurgical approaches to reverse dysfunction and improve perfusion would be desirable. Administration of FGFs as recombinant proteins or overexpression using plasmid and adenoviral vectors elicits multiple effects that could favorably affect flow and function in viable chronically dysfunctional myocardium. Considerable enthusiasm for therapeutic angiogenesis has arisen from promising experimental animal studies using rapidly developing coronary collaterals and ameroid occluder models.1eC6 Unfortunately, FGF-mediated improvements in myocardial perfusion are small, and few laboratories have demonstrated objective changes in flow during pharmacological or metabolic stress. In addition, when administered to dogs with chronic well-developed coronary collaterals, FGF did not improve myocardial perfusion.5 This raises the possibility that FGFs accelerate collateral growth in animal models yet may not afford improvements when coronary flow reserve is chronically reduced. Such actions could be the basis for the inability to translate experimental findings to clinical studies in patients with chronic coronary disease.7
Despite the paucity of data demonstrating increases in coronary flow reserve, many studies show substantial effects of FGFs on myocardial function, raising the possibility that the major therapeutic actions of FGFs may be due to nonangiogenic mechanisms.8 For example, FGF has been shown to protect myocytes against irreversible injury1,9eC11 and reversible stunning,10,12 promote alterations in calcium handling that could improve contractility,13 attenuate ischemia-induced myocyte apoptosis, and stimulate myocyte hypertrophy in cell culture.14 Whereas the relative contribution of each of these mechanisms in models of chronic ischemia in vivo is unknown, they could improve function independently of changes in myocardial perfusion.
We performed the present study to evaluate the effects of FGF-5, a secreted fibroblast growth factor, in a swine model of chronic collateral-dependent hibernating myocardium where the physiological characteristics of hibernating myocardium, namely reduced resting flow and function with a critical impairment of subendocardial flow reserve, remain unchanged between 3 and 5 months after instrumentation.15,16 This contrasts with relatively short-term ameroid instrumented swine models of collateral-dependent myocardium, where transient upregulation of endogenous growth factors and subendocardial infarction near the time of ameroid occlusion could be important in modulating the effects of exogenous growth factors. We overexpressed FGF-5 with an adenovirus that has previously been demonstrated to transfect myocardium after intracoronary administration.17 A similar approach ameliorated stress-induced dysfunction in a porcine ameroid model where resting function was normal with initially promising results in phase II/III human trials.18,19 Our results demonstrate that the salutatory effects of AdvFGF-5 on function are dissociated from perfusion and related to stimulating myocyte hypertrophy and inducing a small population of myocytes to reenter the mitotic phase of the cell cycle.
Materials and Methods
Procedures and protocols conformed to institutional guidelines for the care and use of animals in research and are detailed in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Hibernating myocardium was produced as previously described.15
Serial Physiological Studies
Pigs began studies 4 months after instrumentation, at which time flow, function, and coronary flow reserve are critically reduced. Using a percutaneous approach under propofol sedation, we inserted a 5F multipurpose catheter into the left ventricle using the 6F introducer side port for pressure monitoring and arterial sampling. Regional wall-thickening was assessed with transthoracic echocardiography from a right parasternal approach, and myocardial perfusion was assessed with fluorescent microspheres.20 After an equilibration period, we assessed hemodynamics, flow, and regional wall thickening at rest, during submaximal epinephrine infusion, and after pharmacological vasodilation with adenosine (0.9 mg/kg per min IV) with phenylephrine infused to prevent adenosine-induced hypotension.
We administered a replication deficient adenovirus containing FGF-5 under control of the CMV promoter (AdvFGF-5, 1x1011 pfus, n=8)4 in equally divided doses into the LAD, circumflex, and right coronary arteries over 30 seconds, taking care to avoid reflux into the systemic circulation. First-pass myocardial uptake was enhanced using histamine (25 e/min IC). As controls, we assessed the effects of a similar dose of nuclear localizing EGFP adenovirus (AdvEGFP, n=2 hibernating and n=2 sham) after intracoronary administration.21
An identical physiological study was repeated 2 weeks after AdvFGF-5, after which animals were euthanized under anesthesia. The LV was weighed, sections incubated in TTC to assess infarction, and samples taken for flow and histological analyses.
Apoptosis, Myocyte Hypertrophy, and Cell Cycle Markers
Details are provided in the expanded Materials and Methods. Briefly, we quantified myocyte nuclear density, diameter, and nuclear length to estimate myocyte volume as previously described.22 Myocyte diameter and volume from the AdvFGF-5 group were compared with matched animals with untreated hibernating myocardium (n=10), sham control groups (n=5), and animals receiving AdvEGFP (n=4) to exclude nonspecific effects of the adenovirus. Myocyte apoptosis was assessed using fluorescent TUNEL staining.22
Tissue sections were also incubated with antibodies for the nuclear cell cycle markers Ki-67 (a specific marker for cells that have reentered the growth phase of the cell cycle) and anti-phospho-histone H3 (a marker of mitosis). We quantified positive myocyte nuclei in relation to myocyte nuclear density using both light and fluorescent confocal multifluorescence microscopy (Bio-Rad MRC 1024). Cardiac myocytes were costained with antibodies to troponin I, and nuclei were costained with TO-PRO-3. Similar quantitative results were obtained with each approach (online data supplement).
Statistics
Data are expressed as mean±SE. Differences after treatment with AdvFGF-5 and comparisons between the hibernating and normally perfused remote regions of the same heart were assessed using paired t tests. Details of the piece-wise flow analysis are provided in the online data supplement. Differences among AdvFGF-5eCtreated animals, age-matched shams, and untreated animals with hibernating myocardium were assessed using a two-way ANOVA and the post-hoc Holm-Sidak test (Sigma Stat 3.0) with P<0.05 considered significant.
Results
Pigs were in good health at the time of study, and TTC staining showed no infarction. Initial physiological studies were performed 123±2 days after instrumentation and repeated 2 weeks after AdvFGF-5 (137±2 days).
Findings before AdvFGF-5 confirmed dysfunctional hibernating myocardium. There were reductions in resting perfusion (LAD 0.98±0.09 versus 1.38±0.15 mL/min per g in remote; P<0.01) with the greatest reduction in the subendocardium (0.79±0.05 versus 1.52±0.16 mL/min per g in remote; P<0.01). Full-thickness flow during epinephrine was attenuated (LAD 1.27±0.09 versus 1.65±0.08 mL/min per g in remote; P<0.01), and subendocardial flow did not increase significantly above resting levels (LAD 0.93±0.09 mL/min per g; P=NS versus rest). Likewise, the increase in full-thickness flow during adenosine was severely attenuated (LAD 1.45±0.27 versus 4.84±0.23 mL/min per g in remote; P<0.001), and subendocardial flow did not increase above the resting value (LAD 0.72±0.17 mL/min per g; P=NS versus rest).
Efficiency of Intracoronary AdvEGFP Gene Transfer in Swine
Figure 1 demonstrates the efficiency of intracoronary gene transfer after pretreatment with histamine to increase endothelial permeability. Fluorescence imaging of EGFP demonstrated a high frequency of cardiac nuclear colocalization. When this was quantified using TO-PRO-3 to assess cardiac nuclei, the EGFP was present in 39±2% of cells. Cytoplasmic staining of EGFP was weaker but still present 2 weeks after AdvEGFP administration. These data confirm a high transfection efficiency after histamine pretreatment with the intracoronary approach that is similar to that reported using LacZ transfection by other laboratories.17
Effects of AdvFGF-5 on Flow and Function in Hibernating Myocardium
Figure 2 summarizes the effects of AdvFGF-5 on flow and function with detailed transmural, circumferential, and relative perfusion analysis provided in the online data supplement. Hemodynamic measurements are summarized in the Table. Two-weeks after AdvFGF-5, LAD wall thickening (WT) increased from 2.4±0.4 to 4.7±0.7 mm (P<0.05 versus initial), whereas wall thickening in normally perfused regions was unchanged. Global LV function and the response to epinephrine were not affected by AdvFGF-5 (online data supplement).
Despite prominent effects on function, there were only small changes in myocardial perfusion when averaged among all samples in the region distal to the stenosis. As summarized in Figure 2, resting perfusion tended to increase in hibernating LAD regions while decreasing in remote regions, but the differences were not significant. There were no significant differences in absolute flow during adenosine vasodilation or epinephrine (online data supplement). When analyzed as paired measurements from LAD pieces pooled from all animals (n=147), there was a small increase in relative resting perfusion (LAD/Remote 0.70±0.02 to 0.75±0.02; P<0.0001) and analysis of the integrated flow deficit showed it to decrease from 18.9±2.9% to 12.5±4% (P<0.05) indicative of a reduction in the resting perfusion deficit size. There was no improvement in relative flow during adenosine (LAD/Remote 0.33±0.02 versus 0.31±0.02), but there was a small reduction in the vasodilated perfusion deficit size (46.7±3.5% versus 43.2±3.5%; P<0.05). Thus, the effects of AdvFGF-5 on flow were primarily restricted to the border regions between normally perfused and hibernating regions.
Effects of AdvFGF-5 on Myocyte Cellular Hypertrophy
Although body weight (96±3 to 104±3 kg) and LV end-diastolic dimension (50±2 to 54±2 mm) increased by only 8%, echocardiographic estimates of LV mass increased by 29% 2 weeks after AdvFGF-5 (177±19 to 228±15 g; P<0.05). Figure 3 shows that AdvFGF-5 produced myocyte cellular hypertrophy with quantitation summarized in Figure 4. Myocyte diameter was compared with similarly instrumented but untreated animals with hibernating myocardium (controls) and uninstrumented shams of a similar age (AdvFGF-5 134±2 days and untreated hibernating 137±2 days) and body weight (AdvFGF-5 104±3 kg, untreated hibernating 106±4 kg, and normal shams 115±4 kg). The LV mass/body weight ratio in animals receiving AdvFGF-5 was higher than untreated animals (2.5±0.1 versus 2.2±0.1 g/kg; P<0.05) as well as sham controls (1.8±0.2; P<0.05 versus both groups). AdvFGF-5 increased myocyte diameter in parallel with the changes in LV mass (Figure 4). In the LAD region, subendocardial myocyte diameter increased from 16.2±0.40 to 18.6±1.12 e (P<0.05) in AdvFGF-5eCtreated animals and both were significantly increased in comparison to sham controls (14.8±0.19 e; P<0.05). Changes in subepicardial myocyte diameter were even more pronounced (15.2±0.94 to 19.9±0.32 e after AdvFGF-5, P<0.05, and 13.0±0.83 e in shams, P<0.05). Changes in myocyte diameter in normally-perfused remote regions were similar to hibernating LAD regions and particularly prominent in the subepicardial layers (20.3±0.7 versus 15.2±0.39 e in untreated, P<0.05, and 13.7±0.81 e in shams, P<0.05). Animals receiving AdvEGFP showed no change in echocardiographic LV mass over 2 weeks, and myocyte diameter after AdvEGFP (remote zone myocardium 14.4±1.22 e) was no different from sham or untreated hibernating groups.
Effects of AdvFGF-5 on Myocyte Nuclear Density, Cell Volume, and Apoptosis
Figure 5 summarizes the effects of AdvFGF-5 on myocyte nuclear density, apoptosis, and calculations of myocyte cellular volume from subendocardial LAD samples. Whereas LAD connective tissue was mildly increased in animals receiving AdvFGF-5 (LAD 6.6±1.1% versus 4.1±0.8% in remote; P<0.05), it was no different in untreated controls (LAD 6.9±0.7% versus 4.5±0.2% in remote; P<0.05). In addition, there were no inflammatory changes in AdvFGF-5eCtreated pigs.
Due to prominent cellular hypertrophy, AdvFGF-5 treatment produced significant reductions in myocyte nuclear density. In the LAD subendocardium, myocyte nuclear density was 746±26 versus 926±44 nuclei per mm2 in untreated (P<0.01) and 1212±36 nuclei per mm2 (P<0.01) in shams. Estimates of cell volume using morphometric approaches further confirmed hypertrophy with LAD myocyte cell volume increasing from 11 987±653 to 14 988±901 e3/nucleus (P<0.05) after AdvFGF-5. The differences in myocyte volume (30%) were proportional to the increase in LV mass (29%) and not indicative of hypertrophy from apoptosis-induced myocyte loss, which, if anything, was lower in animals receiving AdvFGF-5 than untreated hibernating myocardium.
Reentry of Myocytes Into the Growth Phase of the Cell Cycle After AdvFGF-5
Figure 6 illustrates confocal photomicrographs demonstrating myocyte nuclear Ki-67 staining in an animal treated with AdvFGF-5. The frequency of Ki-67 staining, a marker of myocytes in the growth phase of the cell-cycle, was expressed in relation to the number of myocyte nuclei. Myocytes are normally in G0 as confirmed by the low frequency of Ki-67 staining (0.028% of myocytes or 284±69 per 106 myocyte nuclei in shams). Untreated animals with hibernating myocardium and animals receiving AdvEGFP had similar low values of Ki-67 staining (447±212 and 213±69 per 106 myocyte nuclei, respectively). After AdvFGF-5, Ki-67 positivity increased significantly (LAD 2904±405 and remote 2066±326 per 106 myocyte nuclei; both P<0.05 versus sham and untreated), but the frequency of Ki-67 positive myocyte nuclei after AdvFGF-5 was still less than 1% of myocytes (LAD 0.29±0.04% and remote 0.21±0.03% versus 0.03±0.01% in shams). Increased Ki-67 positivity was not restricted to myocytes but was also detected in fibroblasts, endothelial cells, and smooth muscle cells. Nonmyocyte staining represented 51±7% of all nuclear Ki-67 staining.
Although Ki-67 reflects active cellular hypertrophy it could also indicate myocytes undergoing nuclear division.23 We therefore quantified phosphohistone H3 staining, a marker of DNA replication (Figure 7). Phosphohistone H3 staining was rare in normal shams (2.4±2.4 per 106 myocyte nuclei). It was higher in untreated hibernating myocardium (35±13 per 106 myocyte nuclei; P<0.05) and increased further after AdvFGF-5 (127±24 per 106 myocyte nuclei; P<0.05 versus untreated or shams). Frequencies of myocyte apoptosis were much lower (Figure 4) averaging 4.7±3.2 per 106 myocyte nuclei in AdvFGF-5eCtreated animals. Mitotic myocyte nuclei were also visualized by light microscopy but cytokinesis was not seen. Based on these results, approximately 4% of the Ki-67eCpositive myocytes (1 in 10 000) were in the mitotic phase of the cell cycle. The frequency distribution of Ki-67 positive myocyte diameters was similar to unstained cells in untreated hibernating myocardium (15.9±0.3 versus 15.1±0.1 e). In animals receiving AdvFGF-5, Ki-67 positive myocyte were smaller (17.4±0.4 versus 18.8±0.1 e; P<0.05) but a subpopulation of cells smaller than normal (potentially indicative of differentiating stem cells) was not identified (online data supplement).
Discussion
There are several important new findings from our study. First, intracoronary administration of AdvFGF-5 in chronic hibernating myocardium produces only small changes in resting perfusion that are predominantly restricted to regions bordering normal and hypoperfused myocardium. In contrast, AdvFGF-5 induced striking improvements in myocardial function that were limited to dysfunctional LAD regions and disproportionate to increases in perfusion. This was accompanied by increases in LV mass and cellular myocyte hypertrophy throughout the LV. Histological staining demonstrated that AdvFGF-5 induced a population of myocytes to reenter the growth phase of the cell cycle. Collectively, these results support the notion that favorable actions of exogenously administered FGFs are largely independent of functional collateral vessel growth and predominantly related to myocyte remodeling.
Limited Effects of AdvFGF-5 on Flow in Hibernating Myocardium
Our study provides a comprehensive assessment of myocardial perfusion after administration of growth factors that has previously been lacking in the majority of studies evaluating angiogenic interventions. AdvFGF-5 elicited small increases in resting perfusion that were largely restricted to the border regions. This small flow change was more difficult to ascertain during vasodilation but the reduction in the perfusion deficit size at rest and after vasodilation was similar (4% to 6% absolute difference in each condition). Importantly, our observations regarding the effects of AdvFGF-5 on the distribution of flow are strikingly similar to observations in many clinical trials of angiogenic growth factors where it has been difficult to measure objective increases in myocardial perfusion despite promising findings based on functional improvement in porcine ameroid models. The most demonstrable change in nuclear perfusion studies has been a reduction in the resting perfusion defect size.24 Recent studies evaluating intracoronary AdvFGF-4 have also demonstrated small reductions in defect size during adenosine vasodilation19 that are similar in magnitude to those we observed in hibernating myocardium.
Collectively, the results indicate that mature coronary collaterals supplying chronically dysfunctional hibernating myocardium have a limited ability to increase perfusion after exogenous FGF-5. The fact that this occurs in the setting of resting dysfunction and a critical impairment in flow indicates that factors present in rapidly developing collateral models such as upregulation of other elements of the angiogenic cascade25 and/or coexisting subendocardial infarction are required to effect an improvement in flow. Thus, the results in pigs with chronic hibernating myocardium are similar to the dog with well-developed chronic collaterals where interventions administered near the point of ameroid occlusion accelerate but were unable to increase collateral flow beyond that attainable with intrinsic angiogenic stimuli.5 Although speculative, this could be a consequence of intrinsic myocardial adaptations that limit the development of metabolic ischemia during stress in hibernating myocardium.26
Effects of AdvFGF-5 on Function in Hibernating Myocardium
In studies where flow and function have been evaluated after growth factor administration, their effect on function has greatly exceeded their effect on perfusion. Consistent with this is the fact that intracoronary AdvFGF-4 improved function in swine with pacing-induced heart failure without altering myocardial perfusion.27 Whereas findings of hibernating myocardium persisted after AdvFGF-5, there was an improvement in the myocardial flow-function relation. Importantly, the improvement in function occurred regionally and did not reflect altered loading conditions because function in normally perfused remote regions, global function, and systolic and end-diastolic LV pressure remained unchanged after AdvFGF-5. This suggests that AdvFGF-5 may have ameliorated a component of dysfunction that was due to acute myocardial stunning superimposed on chronic hibernating myocardium.28 The coexistence of stunning is consistent with the observation that reductions in wall thickening in chronic hibernating myocardium exceed the reduction in subendocardial perfusion.
Several additional mechanisms exist through which AdvFGF-5 could affect function disproportionately from perfusion.8 Previous studies have demonstrated that short-term administration of basic FGFs can protect the heart against reversible and irreversible ischemic injury and produce a "preconditioning like" effect.10eC12 These actions would also be consistent with the improvement in function restricted to myocardium with limited coronary flow reserve. Although acute administration of FGF can also alter myocyte calcium transients and directly increase contractility in vitro,13 this mechanism seems less likely because remote zone function, global function, and contractile reserve were not altered after AdvFGF-5. A final mechanism through which AdvFGF-5 could have altered systolic function is through an improvement in myocardial efficiency via activation of NOS.29 Activation of NOS could produce chronic preconditioning against stunning in hibernating myocardium as well as optimize myocardial efficiency if NO release was chronically downregulated in chronic hibernating myocardium.
AdvFGF-5 Mediated Myocyte Hypertrophy
An alternative explanation for the dissociation between flow and functional effects of AdvFGF-5 could relate to stimulation of myocyte hypertrophy and the resultant regional remodeling. Intracoronary administration of AdvFGF-5 led to significant myocardial hypertrophy with a 30% increase in LV mass within 2 weeks. Previous studies have established a role for endogenous FGFs in acquired hypertrophy produced by a variety of increased physiological loading conditions,14 and manipulating exogenous basic FGFs can stimulate myocyte cellular hypertrophy and mitosis in neonatal myocytes in vitro.8 Our study extends previous work to demonstrate significant FGF-5eCdependent myocyte plasticity in vivo. Both cellular and anatomic hypertrophy occurred over a brief time interval without changes in ventricular loading conditions. Although some cellular myocyte hypertrophy was apparent in untreated animals with hibernating myocardium versus sham controls, AdvFGF-5 led to substantially greater increases in LV mass and myocyte diameter.
Although myocyte hypertrophy was global and particularly prominent in the subepicardium, the improvement in function was restricted to the LAD region. The trend toward a reduction in function in the remote zone could be consistent with a hypertrophy mediated reduction in contractility but is more likely due to a resolution of moderate compensatory hyperkinesis as LAD function improved. Because transmural wall thickening is usually strongly related to subendocardial function, the prominent LAD subepicardial hypertrophy after AdvFGF-5 may have allowed hypertrophied myocytes in outer layers to compensate for subendocardial myocytes lost from apoptosis during the development of hibernating myocardium.22 Additional studies will be needed to determine the relative importance of replacing regional myofibrils via cellular hypertrophy versus other mechanisms.
Increased Cell Cycle Markers After AdvFGF-5
A surprising finding was the fact that AdvFGF-5 caused a small population of cardiac myocytes to initiate DNA synthesis. We were able to confirm this using phophohistone-H3 staining, which has previously been used to label myocyte nuclei in various phases of nuclear division and correlates with BrdU positivity.30 Although cytokinesis was not seen, there is considerable difficulty in identifying such short-lived events in histological tissue. Hein et al23 demonstrated that DNA synthesis increases during hypertrophy and heart failure. Increased Ki-67 was detected without mitosis with DNA replication potentially activated to maintain the DNA content/myocyte volume ratio in hypertrophied myocytes. Regardless of whether cell or nuclear division predominates, the impact of AdvFGF-5 on cellular hypertrophy seems more dominant based on several observations. First, myocytes in the growth phase of the cell cycle (Ki-67) were nearly 20-times higher than those positive for phosphohistone H3. Second, myocyte nuclear density was lower in animals treated with AdvFGF-5. The failure of nuclear density to increase after AdvFGF-5 indicates that cellular hypertrophy exceeded myocyte nuclear hyperplasia making quantitatively important changes in myocyte polyploidy unlikely. Directionally, similar effects were found by manipulating telomerase reverse transcriptase in mice.30 Finally, we cannot exclude the possibility that some of the Ki-67eCpositive myocytes reflect stem cell recruitment because the diameters were smaller than nonstained cells in AdvFGF-5eCtreated hearts and further studies will be required to address this possibility. Collectively, our results support that AdvFGF-5 contributes to functional remodeling through both myocyte cell growth and possibly cell division. This may represent a potentially new approach to reverse the effects of myocyte loss in ischemic cardiomyopathy.
Methodological Limitations
The effects of AdvFGF-5 cannot be directly extrapolated to other growth factors because they may have different physiological actions, and further studies are required to evaluate their effect on myocyte hypertrophy.7 Interestingly, plasmid VEGF has been reported to increase myocyte mitosis leading to nuclear hyperplasia in a porcine ameroid model.31 We did not colocalize phosphohistone H3 and Ki-67 staining, and it is possible that cellular hypertrophy and nuclear division are occurring in different myocyte populations. It is plausible that AdvFGF-5 is producing hypertrophy in adult myocytes and recruiting a smaller stem cell population responsible for phosphohistone H3 positivity. Although our studies were conducted in "adult" farm-bred swine (more than 100 kg) over 6 months old at the time of study, the animals were otherwise normal, and it is possible that intracoronary gene transfer would be insufficient in aged animals32 or when coexisting atherosclerosis and endothelial function is present. We injected two-orders of magnitude more viral particles than published clinical trials of intracoronary AdvFGF-4 and one-order higher than previous studies of AdvFGF-5 in swine. Further studies will be required to examine the dose-dependency of the effects we have reported. Finally, we have documented EGFP protein but have not confirmed the presence of FGF-5 protein production 2 weeks after transfection. In a previous study, Giordano4 confirmed FGF-5 in pigs using a similar approach. Even if the FGF-5 was only transiently expressed, it would not negate the fact that it had lead to substantial increases in LV mass and myocyte hypertrophy in the absence of any alterations in ventricular loading conditions. These were specific to the AdvFGF-5 construct because AdvEGFP did not produce similar effects.
Clinical Implications
Our study provides further support for the pleiotrophic effects of FGFs on the myocardium, and identifies additional mechanisms to explain the functional improvement after growth factor interventions. The increases in function and myocyte hypertrophy in hibernating myocardium are more prominent than effects related to angiogenesis, raising the possibility that administration of AdvFGF-5 may be more efficacious as an adjunctive therapy in patients with advanced ischemic cardiomyopathy and heart failure than chronic angina. Further studies evaluating AdvFGF-5 in animal models of heart failure as well as clinical trials will be required to test this possibility.
Acknowledgments
This work was supported by the VA, AHA, NHLBI, Albert and Elizabeth Rekate Fund, and the Oishei Foundation. We thank Anne Coe, Deanna Gretka, Amy Johnson, and Rebeccah Young for their technical assistance and the University of Pittsburgh Vector Core Laboratory for supplying the AdvEGFP virus.
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