Gene Therapy for Myocardial Protection
Transfection of Donor Hearts With Heat Shock Protein 70 Gene Protects Cardiac Function Against Ischemia-Reperfusion Injury
Background—Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. We investigated the effect of HSP70 gene transfection on mechanical and endothelial function in a protocol mimicking clinical heart preservation.
Methods and Results—Rat hearts were infused ex vivo with Hemagglutinating Virus of Japan–liposome complex containing HSP70 gene (HSP, n=8) or no gene (CON, n=8), and heterotopically transplanted into recipient rats. Four days after surgery, transfected hearts were perfused on a Langendorff apparatus for 45 minutes, arrested with St Thomas’ No. 1 cardioplegia for 4 hours at 4°C, and reperfused for 1 hour. Mechanical and endothelial function was studied before and after ischemia. Creatine kinase was measured in reperfusion effluent. Hearts underwent Western blotting and immunohistochemistry to confirm HSP70 overexpression. Postischemic recovery of mechanical function (% preischemic±SEM) was greater in HSP versus CON: Left ventricular developed pressure recovery was 76.7±3.9% versus 60.5±3.1% (P<0.05); dP/dtmax recovery was 79.4±4.9% versus 56.2±3.2% (P<0.05); dP/dtmin recovery was 74.8±4.6% versus 57.3±3.6% (P<0.05). Creatine kinase release was attenuated in HSP versus CON: 0.22±0.02 versus 0.32±0.04 IU/min/g wet wt. (P<0.05). Recovery of coronary flow was greater in HSP versus CON: 76.5±3.8% versus 59.2±3.2% (P<0.05). Recovery of coronary response to 5-hydroxytryptamine (5×10−5 mol/L) was 55.6±4.7% versus 23.9±3.2% (P<0.05); recovery of coronary response to glyceryltrinitrate (15 mg/L) was not different between HSP and CON: 87.4±6.9% versus 84.3±5.8% (NS).
Conclusions—In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function.
Heat shock proteins (HSPs) are a family of inducible and constitutively expressed intracellular proteins that have a major role in protecting cells from the effects of environmental stress.1 They act by playing an essential role in protein folding and translocation and as chaperones for intracellular proteins. The levels of HSPs are increased by a variety of environmental stresses, including heat stress2 and ischemia-reperfusion injury.3
Previous work has shown that a rise in levels of a particular 70-kDa HSP (HSP70), induced by heat stress, is associated with protection against ischemia-reperfusion injury.4 We have shown improved recovery of both ventricular and coronary endothelial function of rat hearts after heat stress, in a protocol involving prolonged cardioplegic arrest and reperfusion.5
The mechanisms by which heat stress leads to protection of ventricular and endothelial function after ischemia-reperfusion injury may involve many pathways. These include not only an increase in HSP70 levels6 but also induction of free-radical scavengers7 and attenuation of apoptosis.8 Recent studies from our laboratory have also indicated a role for beneficial changes in metabolic pathways after heat stress, in protocols involving normothermic9 and hypothermic ischemia.10
To study the role of individual HSPs rather than the many complex pathways induced by heat stress, techniques available include the use of transgenic animals overexpressing HSPs and gene transfection. We used an established in vivo gene transfection technique, which has been shown to provide high-level expression of protein in the whole heart, with intracoronary infusion of Hemagglutinating Virus of Japan (HVJ)-liposome complex11 to transfect rat hearts with the gene for HSP70.
In a clinically relevant model of donor heart preservation involving cardioplegic arrest, prolonged hypothermic ischemia, and reperfusion, we investigated if HSP70 gene transfection in a rat cardiac transplant model leads to preservation of ventricular and endothelial function.
Male Sprague-Dawley rats were used in all studies; donor rats weighed 225 to 250g, and recipient rats weighed 325 to 350g. Animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research; the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources (NIH publication No. 86-23, revised 1985); and the European Convention on Animal Care guide. The study was approved by the institutional ethics committee on animal research.
Construction of Expression Vector
Full-length human HSP70 cDNA12 (donated by Dr S. Fox and Dr R. Morimoto, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Ill) was cloned at the EcoRI/BamHI site of pcDNA3, which has a cytomegalovirus promoter (Invitrogen Corp).11
Preparation of HVJ-Liposome Complex
The preparation of the HVJ-liposome complex (donated by Prof Y. Kaneda, Osaka University, Japan) has been described previously.13 Briefly, 10 mg of lipid mixture (phosphatidylserine, phosphatidylcholine, and cholesterol) was deposited on the side of a flask by removing tetrahydrofuran in a rotary evaporator. The dried lipid was hydrated in 200 μL of balanced salt solution (137.0 mmol/L NaCl, 5.4 mmol/L KCl, 10.0 mmol/L Tris-HCl; pH 7.6) containing a DNA (200 μg)-HMG1 (high-mobility group 1 nuclear protein, 64 μg) complex.
A liposome-DNA-HMG1 complex suspension was prepared by vortexing, sonication, and shaking to form liposome. The liposome suspension was incubated with 30 000 hemagglutinating units of HVJ, which was inactivated by ultraviolet irradiation, first at 4°C and then at 37°C. Finally, 4 mL of the sucrose gradient layer containing HVJ-liposome was collected for use.
Gene transfection was performed on hearts of Sprague-Dawley rats (225 to 250 g), as described previously.14 Donor rats were anesthetized with sodium pentobarbital (50 mg/kg), and sodium heparin (1000 IU/kg) was injected through the femoral vein. Their hearts were arrested with cold cardioplegia injected retrograde through the abdominal aorta [St Thomas’ Hospital cardioplegic solution No. 1, supplied as a concentrate (Martindale), was diluted (1:50) in Ringer’s solution (Travenol Labs) and filtered].
A thoracotomy was performed, and hearts were excised. Hearts from the group transfected with the HSP70 gene (HSP, n=8) were infused with 1 mL of HVJ-liposome containing pcDNA3 with human HSP70 cDNA through the coronary artery, with the venae cavae, pulmonary arteries, and veins ligated. The control hearts (CON, n=8) were infused with the same volume of HVJ-liposome containing pcDNA3 but without the HSP70 gene. After incubation on ice for 10 minutes, the hearts were then heterotopically transplanted into the abdomens of recipient rats (300 to 325 g) of the same strain.15
Recipient rats were killed on the fourth day after gene transfection, thus allowing the introduced gene to express proteins stably and providing adequate time for intrinsic HSP70 induced by surgical stress to decrease to preoperative levels.11
HSP70 and control gene–transfected hearts were studied to determine ventricular and endothelial function before, during, and after 4 hours of cardioplegic arrest at 4°C (Figure 1⇓).
Rats were anesthetized with diethyl ether, and sodium heparin (1000 IU/kg) was injected through the femoral vein. Transplanted hearts were rapidly excised, placed in ice-cold Krebs-Henseleit buffer, immediately attached to a Langendorff apparatus, and perfused with filtered Krebs-Henseleit buffer (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 24 mmol/L NaHCO3, 11 mmol/L glucose, 1.2 mmol/L CaCl2, pH 7.4) at a constant pressure of 100 cm H2O and continuously gassed with a 95% O2/5% CO2 mixture at 37°C, as described previously.10
After an initial stabilization period of 20 minutes of normoxic perfusion, preischemic mechanical function was evaluated with the use of an intraventricular balloon. Subsequently, hearts were arrested by infusion of 4°C cardioplegia at a constant pressure of 60 cm H2O for 2 minutes. Hearts were immersed in cardioplegia and maintained at 4°C with the aid of a temperature probe.
After 4 hours of cardioplegic arrest, hearts were reperfused with Krebs-Henseleit buffer at 37°C, and coronary effluent was collected during the first 15 minutes of reperfusion. After 30 minutes of reperfusion, postischemic mechanical function was evaluated. At the end of the experiments, hearts were freeze-clamped in liquid nitrogen for Western blot analysis.
Hearts were not paced during the entire protocol; preischemic and postischemic heart rates were recorded after 30 minutes of perfusion and 30 minutes of reperfusion, respectively.
Ventricular function was assessed with a balloon catheter inserted into the left ventricle, as previously described.16 The balloon was inflated to an end-diastolic pressure of 10 mm Hg. Peak systolic pressures were recorded and used to calculate developed pressure. Recovery of mechanical function was expressed as relative recovery of postischemic versus preischemic developed pressure (relative recovery of developed pressure) and time derivatives of pressure changes (+dP/dt and −dP/dt).
Coronary flow was recorded with an electromagnetic flowmeter (Scalar). Endothelial function was assessed through observations of preischemic and postischemic coronary flow responses to 5-hydroxytryptamine (5-HT) (10−7 mmol/L, 10−6 mmol/L, 10−5 mmol/L) and glyceryltrinitrate (GTN) (15 mg/L). For final calculations, the response to 10−5 mmol/L 5-HT was used. Our protocol for this test has been described in earlier studies.17 In the intact endothelium, 5-HT causes vasodilation through the release of endothelium-derived relaxing factor, whereas in the presence of endothelial damage, it causes vasoconstriction by a direct effect on smooth muscle. GTN causes vasodilation by an endothelial-independent effect on smooth muscle.
HSP70 concentration was assessed at the end of reperfusion in both groups (n=5/group) by Western immunoblotting, as previously described.18 Whole-heart homogenates were solubilized in 1% wt/vol SDS, assayed for total protein with the Bradford assay, denatured by heating at 100°C in Laemmli buffer, and separated on 10% SDS gels until the bromophenol blue tracking dye reached the end of the gel. The gels were equilibrated for 30 minutes in transfer buffer before protein transfer at 500 mA for 1 hour. Western blots were blocked for 1 hour with 3% wt/vol skimmed milk powder (Marvel) in PBS (0.15 mol/L NaCl, 0.05 mol/L phosphate buffer, pH 7.2) containing 0.05% wt/vol Tween-20; this blocks nonspecific binding sites. Blots were then probed with monoclonal mouse antibody to inducible HSP70 (SPA-810; Stress Gen Biotechnologies Corp) diluted to a final concentration of 1:1000, for 1 hour. Blots were washed 3 times and incubated with secondary horseradish peroxidase–conjugated rabbit anti-mouse antibody for 1 hour.
Blots were visualized with the use of an enhanced chemiluminescence (ECL) detection system (Amersham). Hyperfilm MP (myoperoxidase) was exposed to blots treated with ECL for 30 seconds and developed in an automatic film processor; after ECL exposure, antibodies were removed from blots by incubation in a solution of 2% wt/vol SDS, 6.25% vol/vol 1 mol/L Tris-HCL, pH 6.8, and 0.7% vol/vol 2-mercaptoethanol. Proteins were then visualized by staining with 0.01% amido black in a solution of methanol, water, and acetic acid (45:45:10 vol/vol/vol ratio). Amido black–stained blots and ECL films were scanned with a Molecular Dynamics 300A laser densitometer, and HSP70 levels were determined as a proportion of total protein loaded with the use of Quantity One software (PDI).
Hearts from both groups (n=3 from each group) were removed from the Langendorff apparatus at the end of reperfusion and quickly divided into 2 parts. One part was immediately frozen in embedding medium, OCT compound (Miles Inc, Diagnostics Division) with liquid nitrogen. The samples were cut into thin sections (5 μm). After blocking with 5% FBS, the sections were incubated first with a 1:1000 dilution of monoclonal mouse antibody to inducible HSP70 (SPA-810) followed by incubation with a 1:180 dilution of FITC-conjugated goat anti-mouse IgG monoclonal antibody. The sections were observed with a fluorescence microscope. Immunohistochemical analysis was also performed on additional hearts 4 days after transfection (HSP, n=3; CON, n=3), which did not undergo perfusion, to compare the effects of perfusion on HSP expression.
Values are presented as mean±SEM. Statistical comparison was performed by an unpaired Student’s t test. A value of P<0.05 was considered a significant difference.
Western blot analysis indicated stronger expression of HSP70 in HSP versus CON (Figure 2⇓). Tubulin expression was constant throughout the lanes, indicating equal protein loading on the blot. According to semiquantitative analysis with computed densitometry, the mean levels of HSP70 in HSP and CON were 4.12±0.55 (range 1.11 to 6.44) and 0.60±0.54 (range 0.02 to 4.93), respectively. This represented nearly a 7-fold increase in HSP70 expression in HSP versus CON (P<0.05).
Immunohistochemical examination showed apparent and extensive overexpression of HSP70 in the cytoplasm of cardiomyocytes in HSP as well as cytoplasm of coronary endothelial cells, as compared with those from CON. Approximately 60% of the cardiomyocytes in hearts from HSP were shown to overexpress HSP70.
Also, there were no appreciable differences in immunostaining between the perfused and nonperfused hearts from both HSP and CON. These results correlated well with previous immunohistochemical studies in a similar protocol of HSP70 or sham gene transfection and subsequent perfusion.11
Postischemic recovery of mechanical function (%preischemic baseline mean values±SEM; at 10 mm Hg left ventricular end-diastolic pressure) was greater in HSP versus CON (Figure 3⇓). Left ventricular developed pressure recovery was significantly higher in HSP versus CON: 76.7±3.9% versus 60.5±3.1% (P<0.05). The maximum dP/dt recovery was significantly higher in HSP versus CON: 79.4±4.9% versus 56.2±3.2% (P<0.05); likewise the minimum dP/dt recovery was also significantly higher in HSP versus CON: 74.8±4.6% versus 57.3±3.6% (P<0.05).
Heart rates were not significantly different between HSP versus CON after 30 minutes of perfusion: 254±11 versus 248±9 bpm (NS), nor after 30 minutes of reperfusion: 212±10 versus 206±14 bpm (NS). Creatine kinase release was significantly lower in HSP versus CON [0.22±0.02 versus 0.32±0.04 IU/min/g wet wt (P<0.05)].
Recovery of basal coronary flow was significantly higher in HSP versus CON: 76.5±3.8% versus 59.2±3.2% (P<0.05) (Figure 4⇓). Recovery of coronary response to 5-HT (5×10−5 mol/L) was also significantly higher in HSP versus CON: 55.6±4.7% versus 23.9±3.2% (P<0.05).
However, recovery of coronary response to GTN (15 mg/L) was not significantly different between HSP versus CON: 87.4±6.9% versus 84.3±5.8% (NS).
This study demonstrated significant improvement of postischemic recovery of mechanical function in HSP70 gene–transfected hearts compared with control gene–transfected hearts, following a protocol mimicking conditions of preservation for heart transplantation. This was accompanied by improved preservation of cardiac endothelial function.
The important role of HSP70 in protecting hearts against detrimental effects of ischemia-reperfusion injury has been clearly shown by experiments with transgenic mice overexpressing HSP70.19 20 These HSP70 overexpressing mice had better postischemic recovery of ventricular function after a period of ischemia and reperfusion; however, endothelial function was not formally assessed. Also, these protocols used a short period of normothermic ischemia, whereas our protocol used a prolonged period of hypothermic ischemia after cardioplegic arrest, which mimics clinical donor heart preservation protocols. Previous work has also demonstrated the beneficial effects of HSP70 gene transfection against various noxious stimuli, including thermal stress21 and ischemia-reperfusion injury.22 However, these experiments used cell culture models, whereas our results were derived from a whole-heart model, which parallels the clinical situation more closely.
To study the effects of HSP70 gene transfection in vivo, we used the HVJ-liposome technique, which has previously been described in a rat heart transplant model, with coronary infusion used as the route of gene delivery.14 Using this method, we were able to obtain a high level of HSP70 transfection into whole rat hearts.
Our results indicate that HSP70 may exert its beneficial effects not only on the myocardium but also on endothelial function. This is in keeping with our previous work, which showed that heat stress leads to induction of the 70-kDa heat shock protein in the heart.23 This was associated with attenuation of both postischemic mechanical and endothelial dysfunctions. Furthermore, we showed the relative importance of the endothelium in mediating the beneficial effects of HSP70 induction.
Advantages of the present protocol include use of a highly efficient in vivo gene transfection technique and established methods for assessment of ventricular and endothelial function. Limitations of our protocol include the use of rats and the use of crystalloid fluid for perfusion. HSP70 mRNA levels have been shown to increase in Langendorff-perfused hearts compared with unperfused hearts18 ; however, total HSP70 levels remained similar because of the longer period required for protein synthesis. Comparison of immunohistochemistry results from perfused and nonperfused gene-transfected hearts revealed no significant difference in HSP70 expression; thus, we conclude that Langendorff perfusion does not significantly alter HSP70 levels.
Gene transfection 4 days before assessment of cardiac function was required in our protocol. This was partly designed to reduce the transplant-associated HSP70 induction in CON (stress-induced rise in HSP70 levels return to prestress levels by 4 days).18 Furthermore, this interval allows optimal level of HSP70 expression resulting from gene transfection.11 Thus, our protocol is not directly applicable to the clinical situation; nevertheless, it provides a reliable experimental model for investigating gene therapy for myocardial protection. Advances in transfection techniques may allow a more rapid induction of protein expression, especially if genes can be introduced into the heart by catheter techniques before organ donation.24 Furthermore, patients with high initial myocardial levels of inducible HSP70 had better cardioprotection during cardiac surgery; heat shock proteins may thus have a role in clinical gene therapy for myocardial protection.25
In summary, this study demonstrates improved preservation of ventricular and endothelial function in HSP70 gene–transfected hearts, in a protocol mimicking conditions for heart preservation; gene therapy may provide a novel approach for myocardial protection in the setting of clinical transplantation.
This study was supported by The British Heart Foundation.
- Copyright © 2000 by American Heart Association
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