Simultaneous Overexpression of Two Stress Proteins in Rat Cardiomyocytes and Myogenic Cells Confers Protection Against Ischemia-Induced Injury
Background Mitochondria are known to be a major target during ischemic cardiac injury. Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which the heat shock or stress protein 70 is increased, there is a marked tolerance to ischemia/reperfusion injury. Two other heat shock proteins (HSP60 and HSP10) are known to form, within the mitochondria, a chaperonin complex that is important for mitochondrial protein folding and function. We were then interested in investigating whether increased expression of these two stress proteins is able to protect myogenic cells against ischemia/reperfusion injury.
Methods and Results We generated recombinant adenoviral vectors containing HSP60, HSP10, or a combination of the two genes. These adenoviral constructs overexpress significant amounts of these stress proteins in both rat neonatal cardiomyocytes and the myogenic H9 c2 cell line. Cells infected with an adenoviral construct overexpressing both HSP60 and HSP10 were found to be protected against simulated ischemia, whereas cells infected with adenoviral constructs overexpressing only HSP60 or HSP10 alone were not rendered tolerant to simulated ischemic injury.
Conclusions These results suggest that the simultaneous expression of these two proteins that form a chaperonin complex in the mitochondria plays an important role in the survival of myogenic cells after ischemia/reperfusion injury.
Myocardial infarction and the resulting ischemia produce a number of intracellular changes within the cardiomyocyte. These changes include, among others, increased cellular calcium levels, altered osmotic control, membrane damage, free radical production, decreased intracellular pH, depressed ATP levels, oxygen depletion, and decreased intracellular glucose levels.1 These events represent a form of stress that is known to result in protein denaturation within the cell. An increase in denatured proteins in the cell has previously been reported to trigger the heat shock response, which increases the synthesis of the so-called stress or heat shock proteins (hsps).2 This heat shock response produces a transient arrangement of cellular activities to cope with the stress period by protecting essential components within the cell so as to permit it to resume normal activity during recovery from the stress.3 This ability of the cell for self-preservation has attracted the attention of several investigators in the field of cardiovascular research. Up to the present, most of the research concerning myocardial protection has concentrated on hsp70, which is the most abundant of the hsps, but recent research suggests that other stress proteins may very well be involved in preserving heart tissue against ischemic damage. Two of the hsps that are nuclearly coded but reside in the mitochondria are HSP10 and HSP60, which are known to form the chaperonin complex. This chaperonin complex is the primary site for protein folding of multimeric enzymatic complexes4 and is therefore extremely important for the viability of the cell. The exact ratio of HSP60 to HSP10 needed to form a chaperonin complex within the mitochondria is believed to be two molecules of HSP60 to each molecule of HSP10. Hsp60 has been shown to form a cylindrical structure made up of 14 HSP60 molecules and requires, for the proper folding of mitochondrial proteins, the assistance of a cofactor made up of 7 HSP10 molecules in addition to ATP. There is presently some dispute by other investigators that actually two cofactor structures are needed in the chaperonin complex, which would bring the number of HSP10 molecules required to 14. It is obvious that the cardiomyocyte depends on mitochondrial respiratory functions to meet the high energy demands of the continuous contractile work of the heart. During ischemic stress, the induction of the mitochondrially located HSP60 and HSP10 is therefore likely to play an important cytoprotective role within the cardiomyocyte.
In the present study, we have used adenoviral vectors to overexpress HSP10 or HSP60 by itself or in combination. Our results show that while simultaneous overexpression of HSP10 and HSP60 confers protection against simulated ischemia “in vitro” to cardiomyocytes and H9 c2 cells, overexpression of each stress protein by itself is unable to confer protection.
Neonatal rat cardiomyocytes were cultured as previously described.5 The embryonic rat heart–derived cell line H9 c2(2-1) and the human embryonic kidney cell line 293 were both obtained from the American Type Culture Collection, Rockville, Md, and were maintained in DMEM supplemented with antibiotics (penicillin/streptomycin/fungizone) and 10% FCS. Cells were infected in 60-mm tissue culture plates at ≈80% confluency by addition of enough of the adenoviral infectious stock to 1 mL of DMEM containing 2% heat-inactivated FCS to obtain a multiplicity of infection (MOI) of 10:1 or 1:1. Cells were then incubated with viral constructs for 60 minutes with mild, constant shaking, after which 2 mL of DMEM/2% heat-inactive FCS was added, and the plates were incubated for 2 days in a 37°C CO2 incubator. Ischemia of the infected neonatal rat cardiomyocytes and H9 c2 plates was simulated as previously described.6 Briefly, ischemia was simulated by placing cells in hypotonic balanced salt solution (in mmol/L: CaCl2 1.3, KCl 5, KH2PO4 0.3, MgCl2 0.5, MgSO4 0.4, NaCl 69, NaHCO3 4, and Na2HPO4 0.3) without glucose or serum and made hypoxic for 12 hours at 37°C. Hypoxia was attained with an airtight jar from which the O2 was flushed with argon for 5 minutes to achieve <0.2% O2. Hypoxia was maintained with an O2-consuming GasPak System from BBL Microbiology Systems. At the end of the experiment, the plates were removed from the chamber, and the supernatant and pellet were separately assayed for both enzyme and protein content.
Construction of Replication-Deficient Adenoviral Vectors
The human HSP60 and HSP10 genes were inserted into the E1 region of an adenoviral vector construct by the general strategy previously described.7 Briefly, the human HSP60 and HSP10 genes were cloned into the multiple cloning site of the adenoviral shuttle plasmid pACCMVpLpASR− (kindly provided by Dr Robert D. Gerard, University of Texas Southwestern Medical Center, Dallas).8 This plasmid contains the 5′ end of the adenovirus serotype 5 genome (map units 0 to 17) in which the E1 region has been substituted with the human cytomegalovirus enhancer-promoter followed by the multiple cloning site from pAC19 and the polyadenylation region from SV40. The resulting plasmid was cotransfected with pJM17, a plasmid that contains the complete adenovirus 5 genome, into the human embryonic kidney cell line 293 by the calcium phosphate transfection method. Infectious viral particles containing the inserted HSP60, HSP10, or both were generated by in vivo recombination in the 293 cells and were isolated as single plaques 10 to 20 days later.
In addition, we also generated a control recombinant adenoviral construct that consists of the pACCMVpLASR− plasmid without any insert. The isolated plaques were propagated in 293 cells for several passages to obtain high titer stocks. Viral particles were purified by CsCl ultracentrifugation. The titer of viral stocks was determined by either plaque assay or deproteination of an aliquot of the viral stock, and the amount of DNA was determined by optical density.9
Total RNA was prepared by the guanidine thiocyanate method.10 Northern blot analysis was done on 10 μg of total RNA of each sample, which was fractionated on a 1% formaldehyde-agarose gel, blotted onto a nylon membrane (Nytran), and subsequently hybridized with a DNA fragment containing human HSP60, human HSP10, and rat β-actin genes by standard methods.11 The DNA probes were labeled with [α-32P]dCTP and the multiprime DNA labeling system (Amersham). Northern blots were hybridized at 42°C overnight and subsequently washed with 0.2×SSC/0.1% SDS at 55°C and exposed to x-ray film for 14 to 16 hours. The results shown are representative of three separate Northern analysis experiments, which yielded similar results. Values for the different mRNA levels were obtained by densitometric scanning of the autoradiograms.
Culture plates (6 cm) of rat neonatal cardiomyocytes and H9 c2 cells were infected with the different adenoviral constructs or heat shocked (sealed plates were placed in a 42°C water bath for 60 minutes) and then returned to 37°C for 2 hours. The cells were then metabolically labeled during the next 2 hours at 37°C in 1 mL of DMEM deficient in methionine and cysteine (ICN Biochemicals) containing 100 mCi of [35S]methionine (Trans [35S]-label; ICN Biochemicals). Cellular protein extracts were prepared by washing the cells twice with ice-cold PBS; cells were then scraped with a silicone rubber policeman in 1 mL of PBS and centrifuged at 1000g, and the pellet was resuspended in 100 mL of solution B12 containing 1% Triton X-100, 0.5% deoxycholate, and 1 mmol/L 2-mercaptoethanol. The suspension was then kept on ice for 15 minutes with occasional vortexing, after which the cell suspension was centrifuged at 12 000g, where the supernatant constituted our total protein fraction. Protein concentration was determined by the Bradford Assay (BioRad), and trichloroacetic acid–precipitable counts were determined as previously described.13 Protein samples (5×105 cpm/lane) were fractionated on a gradient 4% to 20% SDS-polyacrylamide gel, fixed, enhanced, dried, and exposed to x-ray film for 14 to 16 hours at −70°C. Immunoprecipitation was carried out as previously described,5 except that immunoprecipitated proteins were fractionated by 4% to 20% gradient SDS-PAGE. The antibody used was a rabbit polyclonal anti-human HSP10 kindly provided by Dr J. Chen, Tufts University, Boston, Mass.14
Immunocytochemistry to visualize the expression of the human HSP60 and HSP10 was done on neonatal cardiomyocytes that were plated on chamber slides (Lab Tek). Chamber slides were infected with the adenoviral constructs containing HSP60, HSP10, and the control adenoviral construct AdSR−. Myocytes were washed with PBS and fixed with cold methanol (−20°C) for 2 minutes. Slides were then rehydrated with PBS containing 0.1% BSA fraction V (Sigma) and 1% goat whole serum for 15 minutes at room temperature. Slides were reacted with the monoclonal antibody MA3-012 (Affinity Bioreagents), which binds specifically to HSP60 or the rabbit polyclonal anti-HSP10, provided by Dr J. Chen, for 60 minutes.14 Slides were washed 3 times and further developed with a biotinylated goat anti-mouse IgG (for the HSP60 mouse monoclonal antibody) or goat anti-rabbit IgG (for the HSP10 rabbit polyclonal antibody), streptavidin-conjugated alkaline phosphatase system (Vectastain ABC kit), and VectorRed substrate kit (Vector Laboratories).
Creatine kinase activity released from simulated ischemic-stressed and control cardiomyocytes as well as that remaining in the myocytes was determined with a CPK kit (Sigma Diagnostics). Lactate dehydrogenase activity released from simulated ischemic-stressed and control H9 c2 cells as well as that remaining in the cells was determined with an LD kit (Sigma Diagnostics). In both cases, cytoplasmic enzyme released is shown as a percent released over that of control cells after normalization by the total protein content of each individual plate. Cell survival was also measured with an in vitro toxicology assay kit (Sigma, No. TOX-2), which is based on the function of mitochondrial activity in vital cells. The assay was carried out as recommended by the manufacturer’s instructions in 96-well multiwell plates in which 105 cardiomyocytes per well were plated. The multiwell plates were spectrophotometrically measured at 450 nm with a microplate reader.
Results are expressed as mean±SEM. The statistical analysis was carried out with a Student’s t test, and results were interpreted to be significantly different when P<.05.
Recent studies have shown that the sole expression of exogenous copies of hsp70 in cardiac tissue is sufficient to render the heart tolerant to ischemic injury.15 16 17 Hsp70 is mainly a cytosolic protein, although different isoforms have been identified in all the different compartments of the cell. Two other heat shock or stress proteins are HSP60 and HSP10. These two hsps are nuclearly encoded but are located primarily in the mitochondria of the cell. HSP60 and HSP10 have been shown to form a chaperonin complex that is thought to be responsible for the assembly of the different mitochondrial protein complexes within the mitochondria. Our interest in the present study was to investigate whether these mitochondrially located hsps were also able to confer protection against ischemic damage.
For this purpose, we constructed a replication-deficient recombinant adenoviral vector containing human HSP60,18 HSP10,14 and a combination of the two genes. The general strategy used to introduce a foreign gene into the E1 region of the replication-deficient adenoviral vector is shown schematically in Fig 1A⇓ (see “Methods” section). In addition, we also generated a control adenoviral construct using the same scheme with the exception that it lacks an insert. Fig 1B⇓ shows the organization of the different inserts within our adenoviral constructs, including our control adenoviral vector (AdSR), the constructs containing HSP10 (Adhsp10) and HSP60 (Adhsp60) under the transcriptional control of the human CMV enhancer-promoter, followed by the SV40 polyadenylation signals, and the construct containing both HSP10 and HSP60 (Adhsp10/60).
To characterize the levels of infection and expression achieved with our adenoviral constructs, we initially prepared total RNA from H9 c2 cells and rat neonatal cardiomyocytes that had been infected 48 hours previously with our adenoviral constructs. Fig 2A⇓ shows a representative Northern blot analysis of total RNA from H9 c2 cells infected with the different adenoviral constructs. Northern blots were hybridized and rehybridized consecutively with DNA probes to human HSP60 and HSP10 and rat β-actin. Fig 2B⇓ shows the results of densitometric scanning of the resulting Northern blots to quantify the levels of expression obtained from the different adenoviral constructs. As can be observed, significant expression of the infected transgene is obtained with all three of our adenoviral constructs (Adhsp60, Adhsp10, and Adhsp10/60). Surprisingly, the adenoviral construct containing both hsps (Adhsp10/60) consistently expressed higher levels of HSP10 than the construct containing HSP10 alone (Adhsp10) but at the same time expressed lower levels of HSP60 than the construct containing HSP60 alone (Adhsp60).
H9 c2 cells that had been infected 48 hours previously with our adenoviral constructs were also metabolically labeled with [35S]methionine. Total protein extracts were prepared from these cardiomyocytes and fractionated by 4% to 20% gradient SDS-PAGE, enhanced, dried, and exposed to x-ray film (see “Methods” section). Fig 3A⇓ and 3B⇓ shows representative results of such an analysis. As can be seen, a significant amount of HSP60 protein is synthesized by H9 c2 cells infected with the Adhsp60 construct (Fig 3A⇓). H9 c2 cells infected with the Adhsp10/60 construct also exhibit increased expression of HSP60 as well as HSP10 (Fig 3B⇓), although to a lesser extent. Unfortunately, the level of HSP10 expressed by cells infected with Adhsp10 was barely detectable (Fig 3B⇓). One explanation for this result may be the lower level of expression of HSP10 achieved by the Adhsp10 construct compared with the Adhsp10/60 construct at the messenger RNA level (Fig 2B⇑). Furthermore, the small molecular size of HSP10 results in weaker labeling, mainly because of the smaller number of amino acids available to be labeled. To elucidate whether our Adhsp10 construct was truly overexpressing any HSP10 at the protein level, we obtained a rabbit polyclonal antibody raised against the human HSP10 and kindly provided by Dr J. Chen, Tufts University, Boston, Mass.14 Although in our hands, this antibody did not produce adequate Western blots because of technical problems (data not shown), it was found to be very useful for immunoprecipitation. Fig 3C⇓ shows the result of our immunoprecipitation analysis on metabolically labeled proteins from H9 c2 cells infected with our adenoviral constructs as well as cells that were heat shocked and untreated (control). Approximately 106 cpm per protein sample was immunoprecipitated with the polyclonal antibody against HSP10, and the resulting immunoprecipitated proteins were fractionated by 4% to 20% gradient SDS-PAGE, enhanced, dried, and exposed to x-ray film. As can be observed, heat shock induces the expression of a significant amount of HSP10; also, one can observe the expression of HSP10 originating from the adenoviral constructs Adhsp10 and Adhsp10/60.
To directly assess the level of overexpression of HSP10 and HSP60 achieved with our adenoviral constructs, we performed immunocytochemistry on cardiomyocytes infected with the different constructs. Fig 4⇓ shows the results of such an analysis. Fig 4A⇓ presents a rat neonatal cardiomyocyte infected with our control adenoviral construct (AdSR) and reacted with a monoclonal antibody against HSP60 and subsequently developed with VectorRed, which produces the red coloration at the sites at which the antibody has detected the antigen. As can be seen, the normal level of HSP60 present in the cardiomyocyte is concentrated in the perinuclear area of the myocyte, an area occupied primarily by the mitochondria. Fig 4B⇓ shows a cardiomyocyte infected with the Adhsp60 construct and reacted with the anti-HSP60 antibody. There is an increase in the intensity and in the area of the signal due to the increased expression of HSP60 from the Adhsp60 construct. Fig 4C⇓ shows again a myocyte infected with AdSR but this time reacted with the polyclonal antibody against HSP10. We can observe a level of intensity and distribution of the endogenous HSP10 similar to that seen for HSP60 in panel A. Fig 4D⇓ shows a cardiomyocyte infected with the Adhsp10 construct and subsequently reacted with the anti-HSP10 antibody. A marked increase in the intensity and distribution is noticeable because of the overexpression of HSP10 from the Adhsp10 construct. A similar analysis was done on cardiomyocytes infected with the Adhsp10/60 construct, which produced identical results (data not shown).
To test whether the adenovirally transferred HSP10 and HSP60 are able to protect against simulated ischemia, we first infected the rat embryonic heart–derived cell line H9 c2, which exhibits morphological, electrophysiological, and biochemical characteristics similar to those of cardiomyocytes19 20 but is nonetheless a skeletal muscle cell line. H9 c2 cells were infected with three of our adenoviral constructs: Adhsp60, Adhsp10, and Adhsp10/60 (MOI of 1:1), as well as with Adhsp70 (MOI of 1:1), an adenoviral construct containing the inducible hsp70 gene, which we have previously shown to be protective against simulated ischemia21 ; some cells were left uninfected as controls. Two days after infection, to permit sufficient expression of the adenoviral-transferred gene, cells were submitted to simulated ischemia. Subsequently, cellular damage was assessed by measurement of the percentage of cytosolic lactate dehydrogenase released from the total present in each plate. Fig 5⇓ shows the results obtained from six independent experiments. A similar series of experiments was performed in primary cultures of rat neonatal cardiomyocytes that were infected with the same constructs and at the same MOI (1:1) and submitted 48 hours later to simulated ischemia. Creatine kinase activity released and remaining after simulated ischemia was measured to assess cellular damage in the cardiomyocytes. Fig 6⇓ shows the results obtained in six independent experiments. In both of these sets of experiments, it can be observed that the simultaneous expression of HSP10 and HSP60 seems to render the cardiomyocyte and H9 c2 cells more tolerant to cellular damage due to simulated ischemia, whereas expression of either HSP10 or HSP60 alone does not confer protection.
Since both HSP10 and HSP60 are mitochondrial proteins and their function is directly related to the proper assembly and folding of the protein complexes within the mitochondria, one would expect that the main cellular organelle protected by the overexpression of HSP10 and HSP60 will be the mitochondria. Therefore, we used a cell survival assay kit based on measuring mitochondrial function; more specifically, the activity of the mitochondrial dehydrogenases in viable cells (in vitro toxicology assay kit, Sigma No. TOX-2). Rat neonatal cardiomyocytes were plated in 96-well multiwell plates, infected with our three adenoviral constructs, and submitted to simulated ischemia 2 days later. The multiwell plates were tested with the survival assay kit by addition of the tetrazolium substrate to each well, followed by incubation at 37°C for 4 hours, and plates were then read in a multiwell plate reader at 450 nm. Fig 7⇓ shows the results obtained in eight independent experiments. As can be noted, cardiomyocytes infected with HSP10 or HSP60 alone did not survive any better or exhibit any more mitochondrial dehydrogenase activity than uninfected cardiomyocytes after simulated ischemia. In contrast, cardiomyocytes infected with the Adhsp10/60 adenoviral construct exhibited a significant increase in percent survival (20% to 25%) or mitochondrial dehydrogenase activity after simulated ischemia compared with uninfected cardiomyocytes. This indicates that the simultaneous overexpression of both HSP10 and HSP60 seems to preserve mitochondrial dehydrogenase activity during ischemic stress.
Recent studies have shown that hsps play an important role in preserving myocardial tissue against ischemia/reperfusion injury.6 15 16 17 22 23 Our present challenge is to understand the precise mechanism in which hsps confer myocardial protection. In recent studies, most of the attention has been concentrated on the ability of hsp70 to protect against ischemia-induced damage. Studies have previously shown that hsp70 performs a diversity of functions in the cell. Hsp70 has been shown to bind to nascent polypeptides during translation,24 to be involved in assisting in the translocation of proteins through the different membranes of the cell,25 26 and to bind to malfolded or denatured proteins during periods of stress, to mention just a few of its many reported functions within the cell. The result of these many functions of hsp70 is that pinpointing a specific function responsible for its observed protective role during ischemia/reperfusion injury becomes a challenging enterprise. In contrast, other hsps are known to perform very specific functions in the cell. Such is the case for HSP60 and HSP10, which, although nuclearly encoded, reside in the mitochondria. These two stress proteins have been shown to form a chaperonin complex that is involved in the folding and assembly of the protein complexes within the mitochondria.27 Because heart tissue possesses a high number of mitochondria on which it depends for the generation of energy to allow for cardiac contraction, it is then obvious that preservation of the mitochondrial structure and function during ischemia/reperfusion stress would be beneficial to ensure the survival of the cardiomyocyte. Therefore, we were interested in investigating the possible protective role that HSP60 and HSP10 might play during ischemia-induced injury.
Our approach in the present study was to overexpress exogenous copies of human HSP60 and HSP10 in both rat neonatal cardiomyocytes and the myogenic cell line H9 c2 using recombinant adenoviral vectors. We have previously used this approach successfully to overexpress rat hsp70 in these same cells and found high levels of expression of the exogenous gene accompanied by protection against ischemic injury.21 We then generated recombinant adenoviral constructs containing the human HSP60 (Adhsp60) and the human HSP10 (Adhsp10) genes by themselves as well as a third construct containing both genes (Adhsp10/60) (Fig 1⇑). These adenoviral constructs, once infected into cardiomyocytes and H9 c2 cells, were found to express high levels of the exogenous HSP60 and HSP10 genes (Figs 2⇑, 3⇑, and 4⇑). It is important to point out that the high levels of expression of these two hsps alone were not found to induce, in themselves, stress to the cell. As can be observed in Fig 3A⇑ and 3B⇑, although high levels of HSP60 and HSP10 were achieved, there was no indication of the induction of the endogenous hsp70, a sure hallmark of stress to the cell (compare lane Adhsp70i with lanes AdSR, Adhsp10, and Adhsp60 in Fig 3B⇑). Therefore, any protection against simulated ischemia observed in cardiomyocytes or H9 c2 cells infected with these adenoviral constructs containing HSP60 or HSP10 cannot be due to the induction of the endogenous hsp70 gene.
Our results with these recombinant adenoviral constructs containing HSP60 and HSP10 show that both in H9 c2 cells (Fig 5⇑) and in rat neonatal cardiomyocytes (Fig 6⇑), overexpression of either stress protein by itself does not confer protection against simulated ischemia as measured by the release of cytosolic enzymes (lactate dehydrogenase and creatine kinase). In contrast, simultaneous expression of both HSP60 and HSP10 renders H9 c2 cells (Fig 5⇑) and cardiomyocytes (Fig 6⇑) tolerant to ischemia-induced injury, as does our positive control adenoviral construct Adhsp70 (Figs 5⇑ and 6⇑ and Reference 2121 ). It is important to note that the level of protection obtained by infection by either Adhsp10/60 or Adhsp70 in H9 c2 cells (≈20%) is less than in the rat neonatal cardiomyocytes (≈50%) (Figs 5⇑ and 6⇑). One probable explanation for this difference in the level of protection may be found in the nature of these two cells. Whereas rat neonatal cardiomyocytes are nondividing cells, the H9 c2 cells are an established proliferating cell line. Therefore, 2 days after infection (the time needed to obtain sufficient expression of the exogenous gene), the number of adenovirally infected H9 c2 cells will have been diluted out to a certain extent, resulting in a lower number of cells protected against simulated ischemia. It is also important to point out that a pre–heat shock treatment of both H9 c2 cells and rat neonatal cardiomyocytes confers a higher level of protection against simulated ischemia (≈75%) in the same experimental setup.28 This higher level of protection by a pre–heat shock treatment is obviously a consequence of the induction of all hsps and any other proteins or changes in the cell that then contribute to the protective effect.
Because our interest was in the possible protection by these two stress proteins against ischemic damage to the mitochondria, we also used a cell survival kit based on the measurement of the mitochondrial dehydrogenases. Our results show that overexpression of HSP60 and HSP10 from our adenoviral construct Adhsp10/60 preserves mitochondrial dehydrogenase activity during simulated ischemia (Fig 7⇑). As shown in Fig 7⇑, simultaneous expression of both HSP60 and HSP10 in cardiomyocytes by Adhsp10/60 resulted in an improvement in cell survival, as measured by the preservation of mitochondrial dehydrogenase activity compared with cardiomyocytes that were either uninfected or infected with Adhsp60 or Adhsp10.
It should be pointed out that our present results have been obtained in a muscle cell line (H9 c2) and a neonatal cardiomyocyte model, which obviously does not represent a differentiated cardiomyocyte. Nonetheless, our laboratory has recently achieved high levels of adenovirus-mediated expression of other hsps in rat adult cardiomyocytes and found that protective effects against simulated ischemia obtained in rat adult cardiomyocytes are similar to those obtained in rat neonatal cardiomyocytes.29 We are now engaged in validating our present results using the Adhsp10/60 adenoviral construct in H9 c2 cells and rat neonatal cardiomyocytes in a differentiated cardiomyocyte model (rat adult cardiomyocytes). In summary, we believe that our results show that the increased expression of HSP60 and HSP10, which form the chaperonin complex within mitochondria, is able to protect against ischemia/reperfusion injury. In addition, we believe that the most probable mechanism by which HSP60 and HSP10 protect the cardiomyocyte during ischemic injury is by the preservation of mitochondrial function. Therefore, overexpression of additional copies of HSP60 and HSP10 results in an increase in chaperonin complexes that then enable the mitochondria to refold proteins at a faster pace after simulated ischemia. Obviously, additional work will be needed to elucidate exactly how this observed protective role of HSP60 and HSP10 is mediated during ischemia/reperfusion injury.
This work was supported by NIH grant HL-17682 (K14-HL-03150-01) and an American Heart Association Grant-in-Aid (94/1564) to Dr Mestril. We would like to thank Roslyn Lara for secretarial assistance.
Reprint requests to Ruben Mestril, PhD, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0618.
- Received March 5, 1997.
- Revision received May 14, 1997.
- Accepted May 22, 1997.
- Copyright © 1997 by American Heart Association
Ananthan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic signals and trigger the activation of heat shock genes. Science. 1986;232:522-524.
Hendrick JP, Hartl AU. The role of molecular chaperones in protein folding. FASEB J. 1995;9:1559-1569.
Iwaki K, Chi S-H, Dillmann WH, Mestril R. Induction of HSP70 in cultured rat neonatal cardiomyocytes by hypoxia and metabolic stress. Circulation. 1993;87:2023-2032.
Mestril R, Chi S-H, Sayen MR, O’Reilly K, Dillmann WH. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia induced injury. J Clin Invest. 1994;93:759-767.
Graham FL, Prevec L. Manipulation of adenovirus vectors. In: Murray EJ, ed. Methods in Molecular Biology. Vol 7. Clifton, NJ: Humana Press; 1991:109-128.
Gomez-Foix AM, Coats WS, Baque S, Alam T, Gerard RD, Newgard CB. Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism. J Biol Chem. 1992;267:25129-25134.
Sambrook J, Fritsch EF, Maniatis T. In: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989:7.6-7.52.
Welch WJ, Feramisco JR. Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol. 1985;5:1229-1237.
Pontecorvi A, Tata JR, Phyillaier M, Robbins J. Selective degradation of mRNA: the role of short-lived proteins in differential destabilization of insulin-induced creatine phosphokinase and myosin heavy chain mRNAs during rat skeletal muscle L6 cell differentiation. EMBO J. 1988;7:1489-1495.
Marber MS, Mestril R, Chi S-H, Sayen MR, Yellon DM, Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest. 1995;95:1446-1456.
Plumier JCL, Ross BM, Currie RW, Angelides CE, Kazlaris H, Kollias G, Pagoulatos GN. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest. 1995;95:1854-1860.
Radford NB, Fina M, Benjamin IJ, Moreadith RW, Graves KH, Zhao P, Gavva S, Wiethoff A, Sherry AD, Malloy CR, Williams RS. Cardioprotective effects of 70-kDa heat shock protein in transgenic mice. Proc Natl Acad Sci U S A. 1996;93:2339-2342.
Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, Schultz G. Morphological, biochemical and electrophysiological characterization of a clonal cell H9 c2 line from rat heart. Circ Res. 1991;69:1476-1486.
Sipido KR, Marban E. L-type calcium channels, potassium channels, and novel nonspecific cation channels in a clonal muscle cell line derived from embryonic rat ventricle. Circ Res. 1991;69:1487-1499.
Williams RS, Thomas JA, Fina M, German Z, Benjamin IJ. Human heat shock protein 70 (HSP70) protects murine cells from injury during metabolic stress. J Clin Invest. 1993;92:503-508.
Hutter JJ, Mestril R, Tam EKW, Sievers RE, Dillmann WH, Wolfe CL. Overexpression of inducible heat shock protein 72 in transgenic mice decreases infarct size in vivo. Circulation. 1996;94:1408-1411.
Beckmann RP, Mizzen LA, Welch WJ. Interaction of hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science. 1990;248:850-854.
Hohfeld J, Hartl FU. Role of the chaperonin cofactor HSP10 in protein folding and sorting in yeast mitochondria. J Cell Biol. 1994;126:305-315.
Conde AG, Lau SS, Dillmann WH, Mestril R. Induction of heat shock proteins by tyrosine kinase inhibitors in rat cardiomyocytes and myogenic cells confers protection against simulated ischemia. J Mol Cell Cardiol. 1997;26:1927-1938.
Martin JL, Mestril R, Hilal-Dandan R, Brunton LL, Dillman WH. Small heat shock proteins and protection against ischemic injury in cardiac myocytes. Circulation. In press.