(Circulation. 2000;102:III-302.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Gene Therapy for Myocardial Protection
Transfection of Donor Hearts With Heat Shock Protein 70 Gene Protects Cardiac Function Against Ischemia-Reperfusion Injury
From the Department of Cardiothoracic Surgery, National Heart and Lung Institute, Harefield Hospital, Harefield, Middlesex, UK.
Correspondence to Professor Sir Magdi H. Yacoub, DSc, FRS, Department of Cardiothoracic Surgery, National Heart and Lung Institute, Harefield Hospital, Harefield, Middlesex, UB9 6JH, UK. E-mail jay.jayakumar{at}harefield.nthames.nhs.uk
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Abstract |
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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 (5x10-5 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.
Key Words: genes • proteins • ischemia • reperfusion • transplantation
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Introduction |
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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.
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Methods |
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Animals
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
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
Functional Assessment
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
).
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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
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).
Endothelial Function
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.
Western Blotting
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).
Immunohistochemistry
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.
Statistics
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.
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Results |
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Western Blotting
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).
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Immunohistochemistry
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
Ventricular Function
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).
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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)].
Endothelial Function
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 (5x10-5
mol/L) was also significantly higher in HSP versus CON: 55.6±4.7%
versus 23.9±3.2% (P<0.05).
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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).
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Discussion |
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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.
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Acknowledgments |
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This study was supported by The British Heart Foundation.
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