Vol.
184,
No.
April
30,
1992
TUMOR
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1056-1059
NECROSIS FACTOR-o PRETREATMENT IS PROTECTIVE IN A RAT MODEL OF MYOCARDIAL ISCHEMIA-REPERFUSION INJURY Lynne J. Eddy! David V. Goeddel? and Grace H. W. Won$*
‘Institute for Toxicology,
University of Southern California, Los Angeles, CA 90033
*Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080
Received
March
19,
1992
SUMMARY: We have demonstrated that tumor necrosis factor-a (TNF-CX) pretreatment protected the rat heart from &hernia-reperfusion injury. This effect was monitored by assaying for lactate dehydrogenase (LDH) , an enzyme whose release correlates with loss of cell membrane integrity. Intact hearts removed from rats pretreated with TNF released significantly lower amounts of LDH compared to control hearts after 20 min. of total global ischemia followed by reperfusion. Hearts from TNF-ol-pretreated animals contained higher levels of manganous superoxide dismutase (MnSOD) mRNA than hearts from untreated rats. Because oxygen free radicals have been implicated as a major cause of reperfusion damage and the function of MnSOD is to detoxify superoxide anions in the mitochondria, a possible protective mechanism for TNF-(I! may be to induce expression of MnSOD in the heart and thus confer 4, 1992AcadrmrcPESb, 1°C. resistance to oxygen free radicals generated during reperfusion.
INTRODUCTION: immunoregulatory,
TNF-(r is a cytokine that has numerous biological activities, such as antimicrobial,
and anti-inflammatory
effects (l-3).
TNF-a! is produced
mainly by activated lymphoid and myeloid cells (l-3). However, nearly all cells express TNF-a receptors and respond to TNF-a in various ways, including induction of cellular genes, among them MnSOD (4). Overexpression of MnSOD can protect cells in culture from TNF-a, heat, and radiation (5, 6), and possibly, other insults associated with production of oxygen free radicals.
Similarly,
TNF-a pretreatment protects animals from radiation (7), hyperoxia (8),
chemotherapeutic drugs (9) and insults that involve the production of oxygen free radicals. Ischemia-reperfusion
injury occurs in the aftermath of an episode of oxygen deprivation
followed by a sudden influx of oxygen. For example, loss of blood supply to cardiac muscle
* To whom correspondence should be addressed.
Vol.
184,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
caused by coronary thrombosis may be suddenly reversed by the removal of the obstruction and restoration of the blood supply.
These events can be mimicked
perfused rat hearts. Investigation of ischemia-reperfusion
in vitro
using isolated and
injury *has led to the proposal that
much of the damage is caused not by prolonged oxygen deprivation, but rather by the sudden reappearance of oxygen and the accompanying production of oxygen free radicals (10). We therefore hypothesized that TNF-ol, whose association with oxygen free radical metabolism has been documented (see ll),
might also protect against ischemia-reperfusion injury.
MATERIALS AND METHODS: Healthy Sprague Dawley rats, 250-300 g, were injected intravenously through the lateral tail vein with 10 pg murine recombinant TNF-cx in 50 ~1. Control animals were injected with similar volumes of isotonic saline. Twenty-four hours later the rats were anesthetized with sodium pentobarbital, 30 mg/kg, intmperitoneally. The chests were opened and the hearts removed and perfused retrogradely through the aorta (Langendorff (12)) with Krebs-Henseleit buffer, aerated with 95 % 0, - 5 % CO,. Perfusion was maintained using a peristaltic pump at a flow rate of 10 ml/min. The complete perfusion system was maintained within a thermostatically controlled chamber maintained at 37” C. The hearts were perfused for 3-4 min. to remove blood. A non-recirculating system of 80 ml. total volume was used for the remainder of the experiment. After a 30 min. control period, global ischemia was initiated by stopping the pump and turning off the Oz. At the end of the 20 min. ischemic period, the pump was restarted and the buffer reoxygenated. Enzyme leakage from the heart was determined in effluent samples collected from the heart at specific time periods during the control period and during reperfusion. Lactate dehydrogenase (LDH, EC 1.1.1.27) activity was assayed by monitoring the oxidation of NADH, using pymvate as the substrate. NADH was monitored at 340 nm, using a Perkin Elmer Lambda 3 recording spectrophotometer. At the end of the experiments the hearts were frozen in liquid nitrogen. Levels of MnSOD and @-actin RNA were detected by northern blot hybridization. Total RNA was isolated from the rat hearts using guanidinium thiocyanate buffer (5 M guanidinium thiocyanate, 50 mM Tris-HCl, 10 mM EDTA, pH 7.5). Poly (A)+ RNA was enriched as described (13), separated by size using 1.2% formaldehyde-agarose vertical gel electrophoresis, and transferred to nitrocellulose fdters. The RNA filters were baked for 30 min. at 80” C in a vacuum oven and hybridized with human MnSOD synthetic DNA and P-actin probe as described (4).
RESULTS AND DISCUSSION:
Reperfusion of isolated hearts following 20 min. of ischemia
resulted in release of LDH into the medium (Figure 1). After 10 min. of reperfusion there was a statistically significant increase in LDH release. By 35 min. post-reperfusion, LDH levels in the medium had increased to more than 5 times the initial level. Subsequent increases in LDH levels were less dramatic, suggesting that the damage to the heart had reached a plateau.
In
contrast, ischemic and reperfused hearts from animals that had received intravenous TNF-a (10 pg/rat) 24 hours previously released much less LDH after repetision.
After 35 min. of
reperfusion, LDH levels were only 2-fold higher than at the outset and no significant increase in LDH levels occurred later in the experiment. 1057
Thus, the margin of difference in damage
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
MnSOD4kb
-+.a /^
MnSOD
lkb
0 0
p-aclin
Minutes
2
of Reperfusion
3
w LDH release from Control (0) and TNP-pretreated (e) rat hearts during reperfusion subsequent to 20 min. of global no-flow ischemia. Each line represents the mean &- S.E. of 5 experiments, Fj,,,e2. Northern blot analysis of LDH mRNA in hearts from rats pretreated with or without TNF (10 pg/rat). Poly (A)+ RNA was hybridized with LDH synthetic DNA probe according to the published sequence from Genbank for LDH mRNA (RATLDH). Figure Northern blot analysis of MnSOD and fi-actin mRNA in hearts from control and W-treated rats. The results from four separate hearts are shown. Poly (A)+ RNA (3 pgilane) was hybridized with MnSOD and @actin probes as described (4).
between the hearts of control approximately 3-fold.
and TNF-&mated
rats, as judged by LDH release, was
One possible explanation for these results is that TNF-a! specifically inhibited the expression of LDH in the rat heart. However, the levels of LDH mRNA in the two groups of hearts used in these experiments were not noticeably different (Figure 2). This suggests that the release of LDH is a bonafide
signature of tissue damage and not a measure of TNF-a’s effects
on LDH expression. Another possible explanation for the ability of TNF-CY to protect animal hearts from damage caused by ischemia-reperfusion proteins, such as MnSOD.
lies in TNFw’s
effects on induction of protective
TNF-(Y is known to induce the expression of MnSOD in various
tissues, including spleen, kidney, bone marrow, thymus (4), and lung (14). These fmdings are supported by a variety of studies in cultured cells (4, 15). If induction of MnSOD plays a role in protection of ischemia-reperfusion injury, then the levels of MnSOD mRNA should be higher in hearts from rats pretreated with TNF-or. Indeed, these rat hearts contained approximately IOfold more MnSOD mFCNA compared with hearts from control rats (Figure 3). The hypothesis that MnSOD induction is an important component of TNF-a’s protective activity is consistent with several other findings. Inhibitors of xanthine oxidase which block the 1058
Vol.
184,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
production of superoxide anions attenuate damage caused by reperfusion (16), suggesting that superoxide anions may be important contributors to injury. cellular location of MnSOD,
Furthermore, mitochondria,
the
are extremely susceptible to damage following ischemia and
reperfusion (17). The activity of MnSOD isolated from rabbit hearts has been shown to be significantly reduced following ischemia and reperfusion (18). This may be due to degradation of MnSOD by proteases activated by oxygen radicals.
Even under normal conditions, the
myocardium contains lower SOD levels compared to other organs (19). Finally, interleukin-1 , which also dramatically
induces MnSOD (4), additionally protects animals from reperfusion
injury (20). ACKNOWLEDGMENTS: writing this manuscript,
G.H.W.
Wong thanks Dr. Sasha Kamb (Gua Jai) for help in
Louis Tamayo for art work, and Elizabeth van Genderen for help in
preparing the manuscript. REFERENCES 1. Goeddel, D.V., Aggarwal, B.B., Gray, P.W., Leung, D.W., Nedwin, G.E., Palladino, M. A., Patton, J.S., Petica, D., Shepard, H.M., Sugarman, B.J., and Wong, G.H. W. (1986) Cold Spring Harbor Symp. Quant. Biol. 51, 597-609. 2. Old, L.J. (1990) In: TNF: Structure, Mechanism of Action, Role in Disease and Therapy (Bonavida B. and Gmnger G. ed) New York: Karger, l-30. 3. Fiers, W. (1991) FEBS L&t. 285, 199-212. 4. Wong, G.H.W., and Goeddel, D.V. (1988) Science 242, 941-944. 5. Wong, G.H. W., Elwell, J.H., Oberley, L.W., and Goeddel, D.V. (1989) Cell 58,923- 932. 6. Wong, G.H.W., McHugh, T., Weber, R., and Goeddel, D.V. (1991) Proc. Natl. Acad. Sci. USA 88, 4372-4376. 7. Neta, R., Gppenheim, J.J., and Douches, S.D. (1988) J. Immunol. 140, 108-111. 8. Tsan, M.-F., White, J.E., Santana, T.A., and Lee, C.Y. (1990) J. Appl. Physiol. 68, 1211-1219. 9. Slordal, L., Warren, D.J., and Moore, M.A.S. (1990) Cancer Res. 50, 4216-4220. 10. Garlick, P.B., Davies, M.J., Hearse, D.J., and Slater, T.F. (1987) Circ. Res. 61, 757- 760. 11. Wong, G.H.W., Kamb, A., Elwell, J.H., Oberley, L.W., and Goeddel, D.V. (1992) In: MnSOD Induction by TNF and its Protective Role. Tumor Necrosis Factors: the Molecules and their Emerging Roles in Medicine (Beutler, B. ed.) New York: Raven Press, 473-484. 12. Langendorff, 0. (1895) Pflugers Arch. 61, 291-331. 13. Wong, G.H.W., Krowka, J.F., Stites, D.P., and Goeddel, D.V. (1988) J. Immunol. 140, 120-124. 14. Tsan, M.-F., White, J.E., Treanor, C., and Shaffer, J.B. (1990) Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L506-L512. 15. Shaffer, J.B., Treanor, C.P., and Del Vecchio, P.J. (1990) Free Rad. Biol. Med. 8, 1206. 16. Manning, A.S., Coltart, D.J., Hearse, D.J. (1984) Circ. Res. 55, 545-548. 17. Sugiyama, S., Ozawa, T., Kato, T., and Suzuki, S. (1980) Am. Heart J. 100, 829-837. 18. Shlafer, M., Myers, C.L., and Adkins, S. (1987) J. Mol. Cell. Cardiol. 19,1195-497-502 19. Doroshow, J.H., Locker, G.Y., and Myers, C.E. (1980) J. Clin. Invest. 65, 128-135. 20. Brown, J.M., White, C.W., Terada, L.S., Grosso, M.A., Shanley, P.F., Mulvin, D.W., Banerjee, A., Whitman, G.J.R., Harken, A.H., and Repine, J.E. (1990) Proc. Natl. Acad. Sci. USA 87, 5026-5030. 1059
184,
No.
April
30,
1992
TUMOR
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1056-1059
NECROSIS FACTOR-o PRETREATMENT IS PROTECTIVE IN A RAT MODEL OF MYOCARDIAL ISCHEMIA-REPERFUSION INJURY Lynne J. Eddy! David V. Goeddel? and Grace H. W. Won$*
‘Institute for Toxicology,
University of Southern California, Los Angeles, CA 90033
*Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080
Received
March
19,
1992
SUMMARY: We have demonstrated that tumor necrosis factor-a (TNF-CX) pretreatment protected the rat heart from &hernia-reperfusion injury. This effect was monitored by assaying for lactate dehydrogenase (LDH) , an enzyme whose release correlates with loss of cell membrane integrity. Intact hearts removed from rats pretreated with TNF released significantly lower amounts of LDH compared to control hearts after 20 min. of total global ischemia followed by reperfusion. Hearts from TNF-ol-pretreated animals contained higher levels of manganous superoxide dismutase (MnSOD) mRNA than hearts from untreated rats. Because oxygen free radicals have been implicated as a major cause of reperfusion damage and the function of MnSOD is to detoxify superoxide anions in the mitochondria, a possible protective mechanism for TNF-(I! may be to induce expression of MnSOD in the heart and thus confer 4, 1992AcadrmrcPESb, 1°C. resistance to oxygen free radicals generated during reperfusion.
INTRODUCTION: immunoregulatory,
TNF-(r is a cytokine that has numerous biological activities, such as antimicrobial,
and anti-inflammatory
effects (l-3).
TNF-a! is produced
mainly by activated lymphoid and myeloid cells (l-3). However, nearly all cells express TNF-a receptors and respond to TNF-a in various ways, including induction of cellular genes, among them MnSOD (4). Overexpression of MnSOD can protect cells in culture from TNF-a, heat, and radiation (5, 6), and possibly, other insults associated with production of oxygen free radicals.
Similarly,
TNF-a pretreatment protects animals from radiation (7), hyperoxia (8),
chemotherapeutic drugs (9) and insults that involve the production of oxygen free radicals. Ischemia-reperfusion
injury occurs in the aftermath of an episode of oxygen deprivation
followed by a sudden influx of oxygen. For example, loss of blood supply to cardiac muscle
* To whom correspondence should be addressed.
Vol.
184,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
caused by coronary thrombosis may be suddenly reversed by the removal of the obstruction and restoration of the blood supply.
These events can be mimicked
perfused rat hearts. Investigation of ischemia-reperfusion
in vitro
using isolated and
injury *has led to the proposal that
much of the damage is caused not by prolonged oxygen deprivation, but rather by the sudden reappearance of oxygen and the accompanying production of oxygen free radicals (10). We therefore hypothesized that TNF-ol, whose association with oxygen free radical metabolism has been documented (see ll),
might also protect against ischemia-reperfusion injury.
MATERIALS AND METHODS: Healthy Sprague Dawley rats, 250-300 g, were injected intravenously through the lateral tail vein with 10 pg murine recombinant TNF-cx in 50 ~1. Control animals were injected with similar volumes of isotonic saline. Twenty-four hours later the rats were anesthetized with sodium pentobarbital, 30 mg/kg, intmperitoneally. The chests were opened and the hearts removed and perfused retrogradely through the aorta (Langendorff (12)) with Krebs-Henseleit buffer, aerated with 95 % 0, - 5 % CO,. Perfusion was maintained using a peristaltic pump at a flow rate of 10 ml/min. The complete perfusion system was maintained within a thermostatically controlled chamber maintained at 37” C. The hearts were perfused for 3-4 min. to remove blood. A non-recirculating system of 80 ml. total volume was used for the remainder of the experiment. After a 30 min. control period, global ischemia was initiated by stopping the pump and turning off the Oz. At the end of the 20 min. ischemic period, the pump was restarted and the buffer reoxygenated. Enzyme leakage from the heart was determined in effluent samples collected from the heart at specific time periods during the control period and during reperfusion. Lactate dehydrogenase (LDH, EC 1.1.1.27) activity was assayed by monitoring the oxidation of NADH, using pymvate as the substrate. NADH was monitored at 340 nm, using a Perkin Elmer Lambda 3 recording spectrophotometer. At the end of the experiments the hearts were frozen in liquid nitrogen. Levels of MnSOD and @-actin RNA were detected by northern blot hybridization. Total RNA was isolated from the rat hearts using guanidinium thiocyanate buffer (5 M guanidinium thiocyanate, 50 mM Tris-HCl, 10 mM EDTA, pH 7.5). Poly (A)+ RNA was enriched as described (13), separated by size using 1.2% formaldehyde-agarose vertical gel electrophoresis, and transferred to nitrocellulose fdters. The RNA filters were baked for 30 min. at 80” C in a vacuum oven and hybridized with human MnSOD synthetic DNA and P-actin probe as described (4).
RESULTS AND DISCUSSION:
Reperfusion of isolated hearts following 20 min. of ischemia
resulted in release of LDH into the medium (Figure 1). After 10 min. of reperfusion there was a statistically significant increase in LDH release. By 35 min. post-reperfusion, LDH levels in the medium had increased to more than 5 times the initial level. Subsequent increases in LDH levels were less dramatic, suggesting that the damage to the heart had reached a plateau.
In
contrast, ischemic and reperfused hearts from animals that had received intravenous TNF-a (10 pg/rat) 24 hours previously released much less LDH after repetision.
After 35 min. of
reperfusion, LDH levels were only 2-fold higher than at the outset and no significant increase in LDH levels occurred later in the experiment. 1057
Thus, the margin of difference in damage
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
MnSOD4kb
-+.a /^
MnSOD
lkb
0 0
p-aclin
Minutes
2
of Reperfusion
3
w LDH release from Control (0) and TNP-pretreated (e) rat hearts during reperfusion subsequent to 20 min. of global no-flow ischemia. Each line represents the mean &- S.E. of 5 experiments, Fj,,,e2. Northern blot analysis of LDH mRNA in hearts from rats pretreated with or without TNF (10 pg/rat). Poly (A)+ RNA was hybridized with LDH synthetic DNA probe according to the published sequence from Genbank for LDH mRNA (RATLDH). Figure Northern blot analysis of MnSOD and fi-actin mRNA in hearts from control and W-treated rats. The results from four separate hearts are shown. Poly (A)+ RNA (3 pgilane) was hybridized with MnSOD and @actin probes as described (4).
between the hearts of control approximately 3-fold.
and TNF-&mated
rats, as judged by LDH release, was
One possible explanation for these results is that TNF-a! specifically inhibited the expression of LDH in the rat heart. However, the levels of LDH mRNA in the two groups of hearts used in these experiments were not noticeably different (Figure 2). This suggests that the release of LDH is a bonafide
signature of tissue damage and not a measure of TNF-a’s effects
on LDH expression. Another possible explanation for the ability of TNF-CY to protect animal hearts from damage caused by ischemia-reperfusion proteins, such as MnSOD.
lies in TNFw’s
effects on induction of protective
TNF-(Y is known to induce the expression of MnSOD in various
tissues, including spleen, kidney, bone marrow, thymus (4), and lung (14). These fmdings are supported by a variety of studies in cultured cells (4, 15). If induction of MnSOD plays a role in protection of ischemia-reperfusion injury, then the levels of MnSOD mRNA should be higher in hearts from rats pretreated with TNF-or. Indeed, these rat hearts contained approximately IOfold more MnSOD mFCNA compared with hearts from control rats (Figure 3). The hypothesis that MnSOD induction is an important component of TNF-a’s protective activity is consistent with several other findings. Inhibitors of xanthine oxidase which block the 1058
Vol.
184,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
production of superoxide anions attenuate damage caused by reperfusion (16), suggesting that superoxide anions may be important contributors to injury. cellular location of MnSOD,
Furthermore, mitochondria,
the
are extremely susceptible to damage following ischemia and
reperfusion (17). The activity of MnSOD isolated from rabbit hearts has been shown to be significantly reduced following ischemia and reperfusion (18). This may be due to degradation of MnSOD by proteases activated by oxygen radicals.
Even under normal conditions, the
myocardium contains lower SOD levels compared to other organs (19). Finally, interleukin-1 , which also dramatically
induces MnSOD (4), additionally protects animals from reperfusion
injury (20). ACKNOWLEDGMENTS: writing this manuscript,
G.H.W.
Wong thanks Dr. Sasha Kamb (Gua Jai) for help in
Louis Tamayo for art work, and Elizabeth van Genderen for help in
preparing the manuscript. REFERENCES 1. Goeddel, D.V., Aggarwal, B.B., Gray, P.W., Leung, D.W., Nedwin, G.E., Palladino, M. A., Patton, J.S., Petica, D., Shepard, H.M., Sugarman, B.J., and Wong, G.H. W. (1986) Cold Spring Harbor Symp. Quant. Biol. 51, 597-609. 2. Old, L.J. (1990) In: TNF: Structure, Mechanism of Action, Role in Disease and Therapy (Bonavida B. and Gmnger G. ed) New York: Karger, l-30. 3. Fiers, W. (1991) FEBS L&t. 285, 199-212. 4. Wong, G.H.W., and Goeddel, D.V. (1988) Science 242, 941-944. 5. Wong, G.H. W., Elwell, J.H., Oberley, L.W., and Goeddel, D.V. (1989) Cell 58,923- 932. 6. Wong, G.H.W., McHugh, T., Weber, R., and Goeddel, D.V. (1991) Proc. Natl. Acad. Sci. USA 88, 4372-4376. 7. Neta, R., Gppenheim, J.J., and Douches, S.D. (1988) J. Immunol. 140, 108-111. 8. Tsan, M.-F., White, J.E., Santana, T.A., and Lee, C.Y. (1990) J. Appl. Physiol. 68, 1211-1219. 9. Slordal, L., Warren, D.J., and Moore, M.A.S. (1990) Cancer Res. 50, 4216-4220. 10. Garlick, P.B., Davies, M.J., Hearse, D.J., and Slater, T.F. (1987) Circ. Res. 61, 757- 760. 11. Wong, G.H.W., Kamb, A., Elwell, J.H., Oberley, L.W., and Goeddel, D.V. (1992) In: MnSOD Induction by TNF and its Protective Role. Tumor Necrosis Factors: the Molecules and their Emerging Roles in Medicine (Beutler, B. ed.) New York: Raven Press, 473-484. 12. Langendorff, 0. (1895) Pflugers Arch. 61, 291-331. 13. Wong, G.H.W., Krowka, J.F., Stites, D.P., and Goeddel, D.V. (1988) J. Immunol. 140, 120-124. 14. Tsan, M.-F., White, J.E., Treanor, C., and Shaffer, J.B. (1990) Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L506-L512. 15. Shaffer, J.B., Treanor, C.P., and Del Vecchio, P.J. (1990) Free Rad. Biol. Med. 8, 1206. 16. Manning, A.S., Coltart, D.J., Hearse, D.J. (1984) Circ. Res. 55, 545-548. 17. Sugiyama, S., Ozawa, T., Kato, T., and Suzuki, S. (1980) Am. Heart J. 100, 829-837. 18. Shlafer, M., Myers, C.L., and Adkins, S. (1987) J. Mol. Cell. Cardiol. 19,1195-497-502 19. Doroshow, J.H., Locker, G.Y., and Myers, C.E. (1980) J. Clin. Invest. 65, 128-135. 20. Brown, J.M., White, C.W., Terada, L.S., Grosso, M.A., Shanley, P.F., Mulvin, D.W., Banerjee, A., Whitman, G.J.R., Harken, A.H., and Repine, J.E. (1990) Proc. Natl. Acad. Sci. USA 87, 5026-5030. 1059
