Hibernation during Hypoxia in Cardiomyocytes

During myocardial hibernation, decreases in coronary perfusion elicit inhibition of contraction, suggesting that energy demand is attenuated. We previously found an inhibition of contraction and O2 consumption during hypoxia (3% O2; PO2 = 20 torr for >2 h) in cardiomyocytes, which was reversible after reoxygenation. This study sought to determine whether mitochondria function as cellular O2 sensors mediating this response. Embryonic cardiomyocytes were studied under controlled O2 conditions. Hypoxia produced no acute decrease in mitochondrial potential as assessed using tetramethylrhodamine ethylester (TMRE). Cellular ATP was preserved throughout hypoxia, as assessed using the probe Magnesium Green. Thus, ATP synthesis and utilization remained closely coupled. Cells adapted to hypoxia for >2 h exhibited a 4% increase in mitochondrial potential upon reoxygenation, suggesting that a partial inhibition of cytochrome c oxidase had existed. To test whether the oxidase serves as an O2sensor, azide was administered (1 mm) to simulate the effects of hypoxia by lowering the V max of the oxidase. The effects of azide on contraction and mitochondrial potential mimicked the response to hypoxia. We conclude that partial inhibition of cytochrome oxidase during hypoxia allows mitochondria to function as the O2 sensor mediating the decreases in ATP utilization and O2 consumption during hypoxia. During myocardial hibernation, decreases in coronary perfusion elicit inhibition of contraction, suggesting that energy demand is attenuated. We previously found an inhibition of contraction and O2 consumption during hypoxia (3% O2; PO2 = 20 torr for >2 h) in cardiomyocytes, which was reversible after reoxygenation. This study sought to determine whether mitochondria function as cellular O2 sensors mediating this response. Embryonic cardiomyocytes were studied under controlled O2 conditions. Hypoxia produced no acute decrease in mitochondrial potential as assessed using tetramethylrhodamine ethylester (TMRE). Cellular ATP was preserved throughout hypoxia, as assessed using the probe Magnesium Green. Thus, ATP synthesis and utilization remained closely coupled. Cells adapted to hypoxia for >2 h exhibited a 4% increase in mitochondrial potential upon reoxygenation, suggesting that a partial inhibition of cytochrome c oxidase had existed. To test whether the oxidase serves as an O2sensor, azide was administered (1 mm) to simulate the effects of hypoxia by lowering the V max of the oxidase. The effects of azide on contraction and mitochondrial potential mimicked the response to hypoxia. We conclude that partial inhibition of cytochrome oxidase during hypoxia allows mitochondria to function as the O2 sensor mediating the decreases in ATP utilization and O2 consumption during hypoxia. Regional decreases in myocardial oxygen delivery have been shown to result in decreased contractile activity and O2consumption in a phenomenon termed hibernating myocardium. For example, Arai et al. (1Arai A.E. Pantely G.A. Anselone C.G. Bristow J. Bristow J.D. Circ. Res. 1991; 69: 1458-1469Crossref PubMed Scopus (116) Google Scholar) induced a 30% reduction in coronary blood flow in swine and observed an early depletion of ATP and phosphocreatine (PCr) 1The abbreviations used are: PCr, phosphocreatine; TMPD, N, N, N′, N′-tetramethyl-p-phenylenediamine; TMRE, tetramethylrhodamine ethylester; MgG, Magnesium Green; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; BDM, 2,3-butanedione monoxime. 1The abbreviations used are: PCr, phosphocreatine; TMPD, N, N, N′, N′-tetramethyl-p-phenylenediamine; TMRE, tetramethylrhodamine ethylester; MgG, Magnesium Green; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; BDM, 2,3-butanedione monoxime. and an increase in tissue lactate followed by a later recovery of PCr and lactate, despite continued hypoperfusion and impaired contraction. During progressive decreases in coronary blood flow, the same group found that regional decreases in contractile function occurred without a sustained decrease in the PCr to ATP ratio (2Arai A.E. Grauer S.E. Anselone C.G. Pantely G.A. Bristow J.D. Circulation. 1995; 92: 244-252Crossref PubMed Scopus (52) Google Scholar). Other investigators have also found evidence of myocardial hibernation in intact hearts (3Lee S.-C. Downey H.F. Cardiovasc. Res. 1993; 27: 1542-1550Crossref PubMed Scopus (15) Google Scholar, 4Ito B.R. Circulation. 1995; 91: 2058-2070Crossref PubMed Scopus (52) Google Scholar, 5Marban E. Circulation. 1991; 83: 681-688Crossref PubMed Scopus (151) Google Scholar). Collectively these results indicate that the myocardium can develop significant contractile inhibition during reductions in blood flow without apparent evidence of ischemia. This would appear to represent an adjustment in ATP demand in response to a decrease in regional O2 supply, which could protect the myocardium from ischemic injury in states where blood flow is reduced more severely (6Ross J.J. Circulation. 1991; 83: 1076-1082Crossref PubMed Scopus (376) Google Scholar). However, the mechanisms underlying this response are not fully known (5Marban E. Circulation. 1991; 83: 681-688Crossref PubMed Scopus (151) Google Scholar). The inference that intact myocardium can down-regulate energy requirements and ATP demand during hypoxia suggests that cardiac myocytes may behave similarly. In contracting embryonic cardiomyocytes, we previously observed decreases in contractile motion and in the rate of O2 uptake during prolonged moderate hypoxia (PO2 = 20–40 torr for 1–2 h) (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). Moreover, this inhibition was reversible within 3 h after return to normoxia, which suggests that cardiac myocytes can detect moderate hypoxia and initiate a suppression of ATP utilization in response. Recently, Silverman et al. (8Silverman H.S. Wei S. Haigney M.C.P. Ocampo C.J. Stern M.D. Circ. Res. 1997; 80: 699-707Crossref PubMed Scopus (71) Google Scholar) found decreases in extent of shortening in rat cardiac myocytes after incubation under 1% O2 for 48 h, which is consistent with our observations and reveals that this response is not unique to embryonic cells. Collectively, these findings suggest that cardiomyocytes can respond to moderate hypoxia by reversibly decreasing contractile activity, atPO2 levels that should have been sufficient to sustain mitochondrial respiration. Although hibernating myocardium is a phenomenon of intact hearts by definition, studies of cellular responses to hypoxia may provide insight into the mechanisms involved in the intact ventricle. A fundamental question in understanding the mechanism underlying the response to hypoxia relates to how cardiac myocytes detect changes inPO2. An ability to adjust cellular respiration in response to PO2 implies the existence of a sensor capable of detecting changes within the physiological range. It is conceivable that such an O2 sensor could then activate a signaling pathway leading to a down-regulation of contractile motion, energy utilization, and oxygen demand. Recent evidence points to the mitochondrial electron transport chain as a possible site of O2 transduction (9Chandel N. Budinger G.R.S. Kemp R.A. Schumacker P.T. Am. J. Physiol. 1995; 268: L918-L925Crossref PubMed Google Scholar). In this regard, we previously found a reversible inhibition in cytochrome c oxidaseV max during hypoxia, as evidenced byPO2-dependent decreases in TMPD-ascorbate respiration (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). Moreover, kinetic studies of isolated bovine heart cytochrome oxidase confirmed the existence ofPO2-dependent alterations inV max (10Chandel N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). APO2-dependent change in the kinetic activity of cytochrome oxidase could elicit changes in mitochondrial redox state, which could confer a sensitivity toPO2 and allow the mitochondria to act as the cellular O2 sensor. In the present study we assessed the effects of moderate hypoxia and reoxygenation on mitochondrial transmembrane potential to determine whether changes in the function of the oxidase were apparent in the intact myocyte, and whether under normoxic conditions the cytochrome c oxidase inhibitor sodium azide could mimic the reversible decrease in contraction observed during hypoxia. Myocytes were isolated using a method modified from Barry et al. (11Barry W.H. Pober J. Marsh J.D. Frankel S.R. Smith T.W. Am. J. Physiol. 1980; 239: H651-H657PubMed Google Scholar) and previously described (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). Briefly, hearts of 10–11-day-old chick embryos were removed and placed in Hanks' balanced salt solution without magnesium and without calcium (Life Technologies, Inc.). The ventricles were minced, and the cells were dissociated using four to six cycles of trypsin (0.025%, Life Technologies, Inc.) degradation at 37 °C with gentle agitation. Trypsin digestion was halted after 8 min by transferring dissociated cells to a trypsin inhibitor solution. After filtering (100 μm), the cells were centrifuged for 5 min at 1200 rpm at 4 °C and then resuspended in nutritive media (54% Barry's solution (in mm: NaCl (116), KCl (1.3), NaHCO3(22Monteiro H.P. Stern A. Free Radic. Biol. Med. 1996; 21: 323-333Crossref PubMed Scopus (218) Google Scholar), MgSO4 (0.8), NaH2PO4 (1.0), CaCl2 (0.87), glucose (5.6)), 40% M199 with Earle's salts (Life Technologies, Inc.), 6% heat-inactivated fetal bovine serum and penicillin (100 units/ml), and streptomycin (100 mg/ml)). These cells were then placed in a large Petri dish in a humidified incubator (5% CO2, 95% air at 37 °C) for 45 min to allow early adherence of fibroblasts. The nonadherent cells were then enumerated (hemacytometer), their viability confirmed at >85% (trypan blue) and between 0.6 and 1.5 × 106 cells were plated on glass coverslips (25 mm) in nutritive medium. Cell yield averaged 5–6 × 105 cells per embryo. Cells were maintained in a humidified incubator for 2–3 days, at which point synchronous contractions of the monolayer were noted. All experiments were performed on spontaneously contracting cells at day 3 4 after at which point viability was contracting cardiac myocytes on glass coverslips were placed in a This was into a °C) on an A glass °C) the was used to the to known oxygen The used during experiments of a salt solution (in mm: NaCl KCl J. Biol. Chem. Full Text PDF PubMed Google Scholar), NaH2PO4 (1.0), CaCl2 glucose that was with at O2 The used to and of the was by a flow A of was used to the to the to the of O2 into the In in the was confirmed using an method Inc.) In a to (5% was to the J. Biol. Chem. Full Text PDF PubMed Google Scholar, C.J. 1996; PubMed Scopus Google Scholar) and the was the glass of the In in the was from the a of by a The was for and a a a and and and were and using motion of cells was at on using a as described previously (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). Briefly, the cells were with at using Inc.). This to the changes in that were apparent during contraction. min of contractile motion was at for later were and were an from to For the change in was These changes in were for a of motion in the that described the motion that was by The contraction was as a of using the The mitochondrial transmembrane potential was using the tetramethylrhodamine ethylester This the cells and is in mitochondria to the Wei J. Full Text PDF PubMed Scopus Google Scholar, Wei J. Full Text PDF PubMed Scopus Google Scholar) and been used previously to changes in mitochondrial with we not of cellular with was into the cells. and were used To of the the was with a and were to with spontaneously contracting cells were for h with (100 in a humidified incubator at 37 The cells were then placed in the and with the salt solution After min for a was of a of cells using a the cells of cells were as of and was as an without cells with cellular were and the for of the and was for later is as the of the of for ATP a for and the magnesium during ATP The in can assessed using the Magnesium and the of this in cardiomyocytes been studied in by et al. A. J. Physiol. 1996; PubMed Scopus Google Scholar). We in contracting cardiomyocytes during prolonged hypoxia and reoxygenation to ATP Cells on coverslips were with in the for min at 37 °C in a humidified the cells were to the and with salt solution under controlled O2 and conditions. To the for mm) was to the with mm) to mitochondrial respiration. Cells continued to spontaneously under these conditions. were for of using was then for and as a of after of the of the the mitochondrial was to ATP to activity of the In experiments the cells were with the mitochondrial FCCP, the electron transport inhibitor the mitochondrial ATP inhibitor and 2,3-butanedione a of these were found to at at not experiments were using coverslips with cells. were using of on the from the and was at the are as S.E. The contractile response to moderate hypoxia and reoxygenation was studied in cardiomyocytes After h under normoxic conditions (PO2 = the was reduced to torr (3% for 3 on contraction was within 1–2 h a significant decrease in motion was the O2 was to no on contractile motion was noted. However, a progressive return of contraction 2–3 h, and no significant from levels was apparent at 3 These reversible decreases in contraction were to previously (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). studies of cardiomyocytes in that prolonged moderate hypoxia was with a decrease in oxygen consumption rate without a decrease in cellular ATP phosphocreatine (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). To determine whether ATP depletion during prolonged hypoxia in contracting was used to ATP A. J. Physiol. 1996; PubMed Scopus Google Scholar). To determine the of this to detect ATP cells with were during After 4 the mitochondrial was administered to ATP and an increase in was consistent with a in In cells the ATP inhibitor produced a increase in as To determine the effects of hypoxia on ATP cells with were 5 min during h of incubation at PO2 = 20 torr shown in a of was in which to a of the progressive of the However, no was between and cells. Moreover, the of an increase in during hypoxia that not increase as a result of ATP However, ATP synthesis was halted by at the of the a increase in an increase in was of mitochondrial ATP inhibition on in spontaneously contracting Cells with were under normoxic conditions (PO2 = and administered to The increase in suggests an increase in as a result of ATP of hypoxia on in spontaneously contracting cells with were with normoxic (PO2 = solution. = PO2 was reduced to 20 torr (3% in the hypoxia group remained = the mitochondrial was administered to elicit ATP between was = were to that the probe in a consistent with that for a mitochondrial of the mitochondrial would to the mitochondrial of to the of contracting cardiac myocytes at = produced a and decrease in a of potential 5 potential is by the electron transport which is to the of from the of electron transport should decrease in a decrease in the potential the mitochondrial the mitochondrial inhibitor was to the of contracting cardiac 5 This produced a significant decrease in consistent with a decrease in potential is by ATP The allows to their as the in the of to ATP using the energy of ATP should of the mitochondrial shown in of to the of contracting cardiac myocytes in a significant increase in an inhibition of ATP utilization should a decrease in the rate of ATP synthesis by the of to the which should of the mitochondrial To mm) was to the of contracting cardiac myocytes = an inhibitor of produced an and significant increase in which was by an of contraction. Collectively, these results that serves as an of mitochondrial of decreased ATP utilization on in spontaneously contracting in cardiac in a decrease in to The decrease in ATP utilization should result in a of the mitochondrial mm) at = an of contractile motion, and a sustained increase in in the cells in the cells remained = potential was assessed during hypoxia under In the in contracting cells was in as the O2 was decreased from to torr within min shown in acute hypoxia produced no acute change in which suggests that potential was In the mitochondrial potential was in contracting cells that had been for h atPO2 = as the from to torr a that during reoxygenation, which suggests that an increase in potential had This is consistent with an increase the rate of electron transport during acute reoxygenation, with the rate of ATP the change in potential not min after the was to In the PO2 within the was assessed under conditions of reoxygenation using a method J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the PO2 within the a of torr at 4 which that the in response was by the response in the in spontaneously contracting cardiomyocytes with media (PO2 = for = the in the was to PO2 = torr maintained at torr was for A significant increase in was in the reoxygenation group with change in PO2 within the after the to = To test whether a partial inhibition of cytochrome oxidase function could elicit the same response as moderate hypoxia, sodium azide (1 mm) was to the in cells maintained under normoxic conditions. In studies this of azide was found to sufficient to the V max of cytochrome to respiration in cells. azide was no was a progressive suppression in contractile motion h of the azide min and was with a progressive return of contractile motion that mimicked the response to hypoxia. To the effects of azide (1 mm) on mitochondrial cells with were for 5 at which point sodium azide (1 mm) was to the acute decrease in potential was suggesting that potential was In cells with and with azide for h, of the azide was with an acute increase in suggesting that a of the had These responses were to during and recovery from moderate in of spontaneously contracting cardiomyocytes with normoxic (PO2 = solution. were for In group sodium azide (1 mm) was to the at = A group of cells had been with azide (1 mm) for h to = azide was removed from the as the cells continued to A significant increase in was after of the azide hibernation is a of contractile function with coronary hypoperfusion J. 1996; Full Text PDF PubMed Scopus Google Scholar). This is coronary blood flow is which suggests that the is not a of ischemic cellular using suggest levels are not during hibernation, which the that the of contraction is not a of a reduction in energy (5Marban E. Circulation. 1991; 83: 681-688Crossref PubMed Scopus (151) Google Scholar). However, the mechanisms underlying myocardial hibernation are not fully We previously that spontaneously contracting cardiomyocytes down-regulate contractile motion and O2 consumption during prolonged h) moderate hypoxia (PO2 = (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). Cells under conditions for h no in suggesting that the inhibition was not a of findings of decreased contractile motion with sustained viability have been in rat cardiomyocytes under 1% O2 for 48 h (8Silverman H.S. Wei S. Haigney M.C.P. Ocampo C.J. Stern M.D. Circ. Res. 1997; 80: 699-707Crossref PubMed Scopus (71) Google Scholar). study that our results are not unique to embryonic cells. Moreover, our by that hibernating cardiac myocytes were more to acute in of the to elicit These studies suggest that a phenomenon to myocardial hibernation in cardiac myocytes during prolonged hypoxia, which from and is reversible within after recovery to normoxic conditions. In this cardiomyocytes at PO2 = 20 torr progressive decreases in contractile motion that 40% of levels within to PO2 = torr was with a recovery of contraction, were to We previously observed that cardiomyocytes maintained under normoxic conditions for h no significant changes in contractile motion, the observed response to hypoxia by the effects of During to hypoxia, et al. J. Biol. Chem. Full Text PDF PubMed Google Scholar) found that rat cardiac myocytes could of O2 consumption the to torr J. Biol. Chem. Full Text PDF PubMed Google Scholar). Moreover, the observations that cells continued to function during the of hypoxia and that the decreases in contraction were reversible suggest that the effects of hypoxia we found could not by an O2 of mitochondrial ATP to test whether hypoxia ATP we used Magnesium to ATP in contracting cells during moderate hypoxia. The of this with the which during ATP ATP a for A. J. Physiol. 1996; PubMed Scopus Google Scholar). in were in normoxic cells with the mitochondrial with the ATP inhibitor of which should and ATP However, no increase in was during prolonged to PO2 = 20 suggesting that ATP was We previously ATP and in cardiomyocytes and found that cellular levels were preserved during prolonged moderate hypoxia. The present study results by that ATP levels are preserved in contracting cells during prolonged hypoxia. To test whether hypoxia ATP we assessed mitochondrial potential using the potential the for ATP The potential is by the of the and electron electron transport The ATP the energy from mitochondrial potential to ATP from In state, the mitochondrial potential a between the rate of electron transport and the rate of ATP utilization by the An inhibition within the electron transport chain during hypoxia would result in a decrease in electron and a of the a inhibition of ATP utilization an inhibition of the ATP should a these responses were observed electron transport was with the ATP was with hypoxia ATP synthesis by mitochondrial electron then a decrease in potential should have been at the of hypoxia in the mitochondrial potential was maintained during acute hypoxia, which suggests that ATP utilization and ATP synthesis remained closely This is consistent with the of ATP by the Collectively, these findings suggest that hypoxia decreases respiration in cardiomyocytes by a signaling pathway that a reduction in contraction and ATP by ATP Although mitochondrial potential was not during hypoxia, in potential were at reoxygenation. This suggests that was a increase in electron transport without a increase in ATP an increase would a partial inhibition of cytochrome oxidase were removed at reoxygenation. In studies of the we have observed an increase in V max upon reoxygenation after prolonged hypoxia (9Chandel N. Budinger G.R.S. Kemp R.A. Schumacker P.T. Am. J. Physiol. 1995; 268: L918-L925Crossref PubMed Google Scholar, N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We conclude that the changes in mitochondrial potential at reoxygenation changes in the of the oxidase that develop during prolonged hypoxia. These observations indicate that mitochondria can respond to changes in O2 within the physiological which could allow to function as an O2 sensor. azide is a inhibitor of oxidase PubMed Scopus Google Scholar). mm) can respiration by in an of contraction and depletion of mitochondrial mm) the V max of the without respiration. studies that prolonged hypoxia a reversible decrease inV max of the oxidase (7Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar, N. Budinger G.R.S. Kemp R.A. Schumacker P.T. Am. J. Physiol. 1995; 268: L918-L925Crossref PubMed Google Scholar, N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), which is not sufficient to respiration. Thus, of azide could mimic the effects of hypoxia on the that azide during produced the same changes in contractile function and mitochondrial potential during moderate hypoxia cytochrome oxidase as the oxygen sensor underlying the response to hypoxia. The that cells respond to physiological levels of O2 suggests the existence of a transduction pathway the sensor to the response. investigators have that cytochrome oxidase is to function as an O2 sensor apparent for O2 is (8Silverman H.S. Wei S. Haigney M.C.P. Ocampo C.J. Stern M.D. Circ. Res. 1997; 80: 699-707Crossref PubMed Scopus (71) Google Scholar, H.F. Physiol. 1996; PubMed Scopus Google Scholar). at physiological O2 the of such an would of O2 conditions were ability to changes in the physiological range. However, changes in max of the oxidase produced with hypoxia azide (1 mm) should increase the reduction of mitochondrial electron from the oxidase N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). It is conceivable that of H.P. Stern A. Free Radic. Biol. Med. 1996; 21: 323-333Crossref PubMed Scopus (218) Google Scholar, Circ. Res. 1997; 80: PubMed Scopus Google Scholar) could then to an inhibition of ATP utilization within the Although not the would consistent with the previously observed changes in cytochrome oxidase V max during hypoxia, the observed changes in mitochondrial potential during hypoxia and reoxygenation, the possible between of hypoxia reoxygenation and the changes in motion and ATP and the sustained between cellular respiration and ATP demand as by potential and However, study to fully the signaling mediating the response. A of between the response to hypoxia in cardiomyocytes and that in hibernating myocardium should noted. appear to a between ATP utilization and O2 without evidence of ischemia. conditions are fully reversible upon of without of viability is that contractile function in hibernating myocardium upon of flow J. 1996; Full Text PDF PubMed Scopus Google Scholar), recovery of cardiomyocytes 2–3 h after of normoxic conditions. Although the present study insight into the mechanisms to the response to hypoxia in cardiomyocytes, to the of these findings to the in the intact hibernating myocardium.