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Am J Physiol Heart Circ Physiol 294: H249-H256, 2008. First published November 2, 2007; doi:10.1152/ajpheart.00775.2007
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Mitochondria protection from hypoxia/reoxygenation injury with mitochondria heat shock protein 70 overexpression

Courtney L. Williamson,1 Erinne R. Dabkowski,1 Wolfgang H. Dillmann,2 and John M. Hollander1

1Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia; and 2Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, California

Submitted 5 July 2007 ; accepted in final form 30 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The majority of mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as preproteins containing a mitochondria import sequence. Preproteins traverse the outer mitochondrial membrane in an unfolded state and then translocate through the inner membrane into the matrix via import machinery that includes mitochondrial heat shock protein 70 (mtHSP70). Neonatal rat cardiac myocytes (NCM) infected with an adenoviral vector expressing mtHSP70 or an empty control (Adv–) for 48 h were submitted to 8 h of simulated ischemia (hypoxia) followed by 16 h of reperfusion (reoxygenation). Infection with mtHSP70 virus yielded an increase in mtHSP70 protein in NCM mitochondria compared with Adv– (P < 0.05). Cell viability after simulated ischemia/reperfusion (I/R) was decreased in both Adv– and mtHSP70 groups, relative to control (P < 0.05), but mtHSP70-infected NCM had enhanced viability after I/R relative to Adv-infected NCM (P < 0.05). Simulated I/R caused an increase in reactive oxygen species generation and lipid peroxidation in Adv-infected NCM (P < 0.05, for both) that was not observed in mtHSP70-infected NCM. Mitochondrial complex III and IV activities were greater in mtHSP70-infected NCM after simulated I/R compared with Adv– (P < 0.05 for both). After simulated I/R, ATP content increased in mtHSP70-infected NCM, compared with Adv– (P < 0.05). Apoptotic markers were decreased in mtHSP70-infected NCM compared with Adv– after simulated I/R (P < 0.05). These results indicate that overexpression of mtHSP70 protects the mitochondria against damage from simulated I/R that may be due to a decrease in reactive oxygen species leading to preservation of mitochondrial complex function activities and ATP formation.

ischemia; reperfusion; free radical scavenger

MYOCARDIAL ISCHEMIA/REPERFUSION (I/R) injury elicits cellular stress that can result in myocyte death, and reactive oxygen species (ROS) are central to this phenomenon. ROS generated during I/R participate in pathological, biochemical, and physiological responses that include membrane lipid peroxidation, cytoskeletal structure disruption, cellular redox status disturbance, enzyme inactivation, protein misfolding, deterioration of mitochondrial function, and excitation-contraction coupling impairment, all of which may play a role in myocyte loss (2, 17, 32, 36–38). Although many potential sources of ROS generation exist in myocardium, the mitochondrion is considered to be the primary site (7, 12, 30). Both direct evidence and indirect evidence exists indicating that ROS production is increased in heart mitochondria after I/R (6, 37). Several sites in the electron transport chain (ETC) are particularly prone to the formation of ROS and include oxidizable electron carriers in the inner mitochondrial membrane (7). This has implications for ETC proteins because a major constituent of these structures is their iron-sulphur centers (29), which can react with ROS and produce the highly reactive hydroxyl radical (OH·; Ref. 8). As a result, mitochondria are particularly prone to the oxidative environment present during I/R.

The vast majority of mitochondrial proteins (>90%) are encoded by nuclear genes and synthesized in the cytosol as preproteins containing a mitochondria import sequence (16, 21). Precursor proteins are guided by cytosolic chaperones to the mitochondrion where they interact with translocases of the outer membrane (Tom) to initiate mitochondrial import (16, 42). When the precursor protein reaches the outer membrane, it is imported into the inner membrane space by members of the Tom complex, where the presequence interacts with translocases of the inner membrane (Tim; Ref. 41). Tim44, a peripheral membrane protein that is bound to the matrix side of the inner membrane, serves to anchor mitochondrial heat shock protein 70 (mtHSP70), which pulls the presequence and the remainder of the protein into the matrix in an energy-dependent manner (41). MtHSP70 is a member of the HSP70 family of proteins and forms an ATP-dependent motor in cooperation with the Tim complex to help translocate proteins through a "trapping" and "pulling" mechanism (41). MtHSP70 presence is required for polypeptide translocation across the inner membrane and in subsequent protein folding reactions in the mitochondrial matrix (41).

Like many other HSPs, mtHSP70 has been reported to be responsive to cellular insult including thyroid hormone treatment (34), glucose deprivation (24), and myocardial I/R (18). Using PC12 cells, Liu et al. (24) demonstrated that transfection with a mtHSP70 cDNA provided protection against glucose deprivation by attenuating ROS accumulation. Using an in vivo model of myocardial infarction, Kilgore et al. (18) demonstrated decreases in mtHSP70 protein content in at-risk (margin) areas bordering the infarcted zone. These authors reported that loss of mtHSP70 protein content in the margin zone may lead to decreased functional mitochondria. They go on to suggest that treatments designed to increase mtHSP70 presence before myocardial infarction may provide therapeutic potential for cardiac viability (18). Studies examining the effect of ischemia on other mitochondrial protein import constituents have come to similar conclusions. Preservation of the mitochondrial import protein Tom20 may contribute to improved mitochondrial function after ischemia and may be involved in the enhanced cardiac protection observed with ischemic preconditioning (3). Bowers and Ardehali (4) have hypothesized that adjustments in mitochondrial activity during ischemia may be dependent on protein transport machinery, suggesting that preservation and/or enhancement of this process may provide the cell with an increase in ischemic tolerance.

In the current study, we sought to determine whether increased mtHSP70 presence protects cardiac myocytes from simulated I/R insult. To test this question, we infected rat neonatal cardiac myocytes (NCM) with an adenoviral vector expressing mtHSP70 and submitted them to a hypoxia-reoxygenation protocol. Since the mitochondrion is centrally involved in ROS generation during I/R, we determined whether mitochondrial function, oxidative stress, and apoptosis were influenced by mtHSP70 overexpression. Because mtHSP70 is a crucial participant in the process of mitochondrial protein import, we hypothesized that its overexpression would provide protection to myocytes during hypoxia-reoxygenation insult, perhaps by providing increases in mitochondrial proteins that are subjected to oxidative damage during I/R. Our results indicate that mtHSP70 preserves mitochondrial function and lessens oxidative stress and apoptosis associated with hypoxia-reoxygenation insult.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. NCM were isolated, cultured, and infected with adenoviral vectors expressing mtHSP70 or an empty vector (Adv–), as previously described (15, 23, 27).

Construction of adenoviral vectors. A rat mtHSP70 cDNA (a gift from S. Massa, University of California, San Francisco, CA) was inserted into the E1 region of an adenoviral vector construct as previously described (9, 15). Briefly, the rat mtHSP70 cDNA was cloned into the multiple cloning site of the adenoviral plasmid pACCMVpLpASR– (a gift from R. Gerard, University of Texas Southwestern Medical Center, Dallas, TX). Additionally, a recombinant adenoviral construct containing no insert (Adv–) was generated in parallel and used as a control for viral infection of NCM. Use of an adenoviral vector containing no insert as a control enabled us to account for the affect of viral infection. To date, none of our previous studies (2, 15, 22, 23, 25, 27, 28) have indicated increased expression of proteins of interest with infection of our Adv– control. All viral constructs were plaque purified and propagated in 293 cells and then cesium chloride purified by ultracentrifugation. Viral stock titers were determined by plaque assay as previously described (9). Multiplicity of infection (MOI) for all treatments was 50.

Western blot analyses. SDS-PAGE was run as described by Laemmli (19) with equal amounts of protein loaded for each study treatment (19). Protein content was assessed using the Bradford method using bovine serum albumin as a standard (5). Relative amounts of mtHSP70, {alpha}-actin, voltage-dependent anion channel (VDAC), translocase of the outer membrane 20 (Tom20), translocase of the inner membrane 44 (Tim44), catalase, and manganese superoxide dismutase (MnSOD) proteins were determined using specific antibodies; anti-mtHSP70 goat polyclonal (Santa Cruz, Santa Cruz, CA), anti-{alpha}-actin monoclonal (Sigma, St. Louis, MO), anti-VDAC monoclonal (EMD, San Diego, CA), anti-Tom20, anti-Tim44 monoclonals (BD Biosciences, San Jose, CA), anti-catalase polyclonal (AB Cam, Cambridge, MA), and anti-MnSOD polyclonal (Bethyl Laboratories, Montgomery, TX). The secondary antibody was either an anti-rabbit (Cayman Chemicals, Ann Arbor, MI), anti-goat, or anti-mouse IgG horseradish peroxidase conjugate (Sigma), and detection of signal was performed according to the NEN Renaissance ECL detection system manufacturer's directions (NEN Life Sciences, Boston, MA). Blots were then exposed to film, and autoradiographic signals were assessed. Quantification of autoradiographic signals was performed using ImageJ 1.37v (National Institutes of Health, Bethesda, MD), and data are expressed as arbitrary optical density units.

Simulated ischemia-reoxygenation protocol. Simulated ischemia/reoxygenation was performed using the BBL GasPak System from Becton-Dickinson (Cockeysville, MD), as previously described (15, 27, 35).

Cellular viability. MTT reduction was used as a determinant of cellular viability (Sigma). Briefly, the MTT solution was added in an equal amount to 10% of the culture medium of each plate and then was returned to the incubator for 2 h. After incubation, an equal volume of solubilization solution (10% Triton X-100 and 0.1 N HCl in isopropanol) was added to each plate and gently mixed on an orbital shaker. Absorbance was measured spectrophotometrically at 570 nm on a Biotek Synergy HT spectrophotometer (Biotek, Winooski, VT), and results are expressed as a percentage of respective preischemic values.

ROS generation. ROS generation was measured in NCM using 2',7'-dichlorofluorescin diacetate (DCFH-DA) as a probe according to LeBel and Bondy (20) with modifications (1). Briefly, NCM were incubated in an assay buffer containing 5.0 µM DCFH-DA dissolved in 1.25 mM methanol, for 15 min at 37°C. DCF fluorescence was followed at an excitation wavelength of 488 nm and an emission wavelength of 525 nm for 30 min on a Biotek Synergy HT fluorometric plate reader (Biotek) and compared with a standard curve containing various amounts of DCF. The rate of DCFH conversion to DCF was linear for at least 60 min and was corrected with the autooxidation rate of DCFH without protein. Values were expressed per protein concentration. All assays were carried out in triplicate, and protein content was assessed using the Bradford method and bovine serum albumin as a standard (5).

Lipid peroxidation. Peroxidation of biomembrane lipids was assessed using the ratiometric fluorescent dye BODIPY C11(581/591) (Molecular Probes, Eugene, OR). BODIPY contains a lipophilic moiety causing it to distribute into membranes, and upon oxidation by ROS, it undergoes a red-to-green shift in fluorescence. By examining the ratio of green and red emissions, subcellular heterogeneity in dye loading can be minimized and quantitative index of lipid peroxidation can be obtained (31). NCM were loaded with 5 uM of BODIPY C11(581/591) and incubated at 37°C for 30 min. NCM were washed, and fresh maintenance media were added. Samples were analyzed immediately on a Biotek Synergy HT fluorometric plate reader (Biotek) at 484/510 and 581/610 nm. Protein content was assessed using the Bradford method and bovine serum albumin as a standard (5).

ETC complex function. Respiratory capacity was performed in digitonin-permeabilized cultured cells according to the method of Hofhaus et al. (14) with modifications (15). Briefly, adenovirus-infected cells were trypsinized and permeabilized by incubating with digitonin (10 µg/ml) for 10 min or until ≥95% of the cells were permeabilized. Equal volumes of cells were loaded into a YSI 5300 biological oxygen monitor (YSI, Yellow Springs, OH), and activity of complex I, complex III, and complex IV was determined. Activity was defined as the rate of oxygen consumed in the presence of the specific substrates glutamate/malate (complex I), succinate (complex III), and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (complex IV), and was calculated as the fraction that was sensitive to the specific inhibitors rotenone (complex I), anti-mycin (complex III), and sodium cyanide (complex IV). Protein content was assessed using the Bradford method and bovine serum albumin as a standard (5), and data are expressed as femtamoles of O2 consumed per min per mg protein.

ATP content. Bioluminescent determination of ATP was performed as per the manufacturer's instructions, using a kit (Sigma). Briefly, cellular ATP was determined on a Biotek Synergy HT luminescence plate reader (Biotek) and normalized to cell number using a coulter counter (Coulter Electronics, Hialeah, FL). Results are reported as a percentage of preischemic values.

Caspase-3 activation. Caspase-3 activation was determined colorimetrically at 405 nm (BioVision, Mountain View, CA). Briefly, cells were trypsinized, pelleted, and resuspended in chilled lysis buffer for 10 min. After lysis, cells were centrifuged and the supernatant (cytosolic extract) was placed in a fresh tube. Two hundred micrograms of cytosolic protein were assayed in the presence of substrate (DEVD-p-nitroanilide) by incubating at 37°C for 2 h. Samples were read spectrophotometrically at 405 nm on a Biotek Synergy HT plate reader (Biotek), and results were subtracted from associated control samples. Results are expressed as optical density units per 200 µg cytosolic protein.

APO-BrdU TUNEL assay kit. The APO-BrdU TUNEL assay kit (Molecular Probes) was performed according to manufacturer's protocol. Briefly, NCM were trypsinized, pelleted, and resuspended in PBS. After addition of 1% paraformaldehyde, NCM were placed on ice for 15 min, washed with buffer, resuspended in 70% ethanol, and incubated at –20°C overnight. NCM were washed with buffer, then resuspended in DNA-labeling solution, and incubated at 37°C for 1 h. After incubation, NCM were rinsed with buffer, and Fluor 488 dye-labeled anti-BrdU antibody was added for a duration of 30 min. A minimum of 20,000 myocytes (events) were examined per treatment by flow cytometry (West Virginia University Flow Cytometry Core Facility), and experimentation was performed on four independent sets of experiments.

Statistics. Means ± SE were calculated for all data sets. Data were analyzed with a one-way ANOVA method to evaluate the main treatment effect, simulated I/R (Systat; version 5.03, Evanston, IL). Fisher's least significant difference post hoc tests were performed to determine the significant differences among means. When appropriate a Student's t-test was employed. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
mtHSP70 overexpression in neonatal rat cardiac myocytes. As indicated above, we utilized an empty viral vector (Adv–) as a control for our experimentation, and we observed no increase in mtHSP70 expression with Adv– infection compared with uninfected control NCM (Fig. 1). To determine whether infection with an adenoviral vector expressing mtHSP70 could increase mtHSP70 protein expression, NCM were infected for 48 h at an MOI of 50 with adenoviral vectors encoding either mtHSP70 or an empty control (Adv–). After 48 h, NCM were harvested, and equal amounts of total protein were probed for mtHSP70, {alpha}-actin, or VDAC protein content. Infection with a mtHSP70-encoded viral vector significantly increased mtHSP70 protein content (Fig. 2A; P < 0.05). When we attempted to normalize these results for loading, we observed a significant decrease in {alpha}-actin protein in mtHSP70-infected cells (Fig. 2B; P < 0.05). Analysis of VDAC protein content revealed a significant increase in mtHSP70-infected NCM (Fig. 2C; P < 0.05). Normalization of mtHSP70 protein content with either {alpha}-actin or VDAC indicated that mtHSP70 protein content was significantly increased in cells infected with an adenoviral vector expressing mtHSP70 (Table 1). These data indicate that infection of NCM with an adenoviral vector encoding mtHSP70 is effective at increasing mtHSP70 protein content.


Figure 1
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  		Fig. 1. Representative Western blot for mitochondrial heat shock protein 70 (mtHSP70) expression in uninfected neonatal cardiac myocytes (NCM) and empty viral vector (Adv–) infected NCM. Adv– were infected for 48 h with empty viral vector, and mtHSP70 protein levels were compared with uninfected NCM.

 

Figure 2
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  		Fig. 2. A: representative Western blot for mtHSP70 expression in NCM. NCM were infected at a multiplicity of infection (MOI) of 50 for 48 h with a viral vector encoding for mtHSP70 or an empty control (Adv–). Optical density scans are expressed as arbitrary units. B: representative Western blot for {alpha}-actin expression in NCM. NCM were infected at an MOI of 50 for 48 h with a viral vector encoding for mtHSP70 or an empty control (Adv–). Optical density scans expressed as arbitrary units. C: representative Western blot for voltage-dependent anion channel (VDAC) expression in NCM. NCM were infected at an MOI of 50 for 48 h with a viral vector encoding for mtHSP70 or an empty control (Adv–). Optical density scans expressed as arbitrary units. All lanes are loaded with equal amounts of protein, and graphs are mean ± SE; n = 5 for each treatment. *P < 0.05 for mtHSP70 vs. Adv–.

 

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  		Table 1. Normalization of mtHSP70 protein content with either {alpha}-actin or VDAC

 
Western blot analysis of neonatal rat cardiac myocytes. To gain some insight into specific proteins enhanced by mtHSP70 overexpression, we determined expression levels of two mitochondrial proteins that participate in protein import into the mitochondrion. NCM were infected for 48 h at an MOI of 50, with adenoviral vectors encoding either mtHSP70 or an empty control (Adv– ). Western blots were performed to probe for the outer mitochondrial membrane translocase Tom20 and the inner mitochondrial membrane translocase Tim44. We observed an increase in Tom20 expression (2-fold), with mtHSP70 overexpression, indicating that this translocase was upregulated as a result of mtHSP70 overexpression (Fig. 3). In contrast, no differences were observed in Tim44 expression with mtHSP70 overexpression (Fig. 3). Because antioxidant defense is crucial during I/R injury, we made examination of antioxidant enzyme defense constituents. NCM infected with a viral vector encoding mtHSP70 possessed greater MnSOD protein content (2-fold) than Adv-infected controls (Fig. 3). In contrast, no differences were observed in catalase protein content (Fig. 3). These data indicate that mtHSP70 expression may enhance expression of specific proteins involved in mitochondrial protein import and antioxidant defense.


Figure 3
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  		Fig. 3. Representative Western blot for Tim44, Tom20, MnSOD, and catalase protein levels in NCM. NCM were infected at an MOI of 50 for 48 h with a viral vector encoding for mtHSP70 or an empty control (Adv–) and then probed for Tim44, Tom20, MnSOD, and catalase.

 
Preservation of cell viability after simulated I/R. To determine whether overexpression of mtHSP70 preserves cell viability after simulated I/R, NCM were infected with an adenovirus expressing either mtHSP70 or an empty control (Adv–) for 48 h at an MOI of 50 and then submitted to 8 h of hypoxia and 16 h of reoxygenation. Assessment of cell viability was performed spectrophotometrically using MTT. After simulated I/R, NCM cell viability was significantly decreased in both Adv– and mtHSP70 groups (Fig. 4; P < 0.05 for both). Overexpression of mtHSP70 enhanced cell viability after simulated I/R compared with Adv-infected NCM as indicated by an increase in postischemic cell viability (Fig. 4; P < 0.05 for Adv-I/R vs. mtHSP70-I/R). These data suggest that our simulated I/R protocol caused a significant decrease in cell viability that was attenuated by mtHSP70 overexpression.


Figure 4
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  		Fig. 4. Cell viability after simulated hypoxia/reoxygenation. NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–). NCM were subjected to 8 h of ischemia followed by 16 h of reoxygenation. Cell viability was determined using MTT, and values are a percentage of preischemic values. Results are mean ± SE; n = 8 for each treatment. *P < 0.05 for mtHSP70 vs. Adv–; {dagger}P < 0.05 for mtHSP70 ischemia/reperfusion (I/R) vs. Adv– I/R.

 
Decreased ROS generation after simulated I/R. ROS generation is a central component of myocardial I/R injury and has been shown to elicit oxidative damage leading to biochemical and physiological dysfunction (17, 32, 37). To index ROS content, we chose the nonspecific fluorometric probe DCFH-DA and monitored DCF fluorescence over time. NCM infected with either an adenoviral vector encoding mtHSP70 or control (Adv–) showed no significant differences in ROS content before simulated I/R (Fig. 5). Eight hours of hypoxia, followed by 16 h of reoxygenation, elicited a significant increase in ROS content in Adv-infected myocytes that was not observed in mtHSP70-infected myocytes (Fig. 5; P < 0.05), indicating that mtHSP70 overexpression was associated with a decrease in ROS generation resulting from simulated I/R.


Figure 5
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  		Fig. 5. Reactive oxygen species generation after simulated hypoxia/reoxygenation, indexed using 2',7'-dichlorofluorescin diacetate (DCFH-DA). NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–) and then were subjected to 8 h of ischemia, followed by 16 h of reoxygenation. Reactive oxygen species generation was determined by using dichlorofluorescein (DCF) fluorescence over time and comparing it to a standard curve of known DCF concentrations. Data are normalized for total protein content. Values are mean ± SE; n = 8 for each treatment. *P < 0.05 for Adv–-I/R vs. all other groups.

 
Increased lipid peroxidation generation after simulated I/R. Peroxidation of biomembrane lipids was assessed using the ratiometric dye BODIPY C11(581/591), which distributes into cellular membranes. When oxidized by ROS, BODIPY C11(581/591) undergoes a red-to-green shift that can be quantitatively indexed fluorometrically (green/red + green). NCM infected with either an adenoviral vector encoding mtHSP70 or control (Adv–) showed no significant differences in lipid peroxidation before simulated I/R (Fig. 6). Eight hours of hypoxia, followed by 16 h of reoxygenation, elicited a significant increase in ROS content in Adv-infected myocytes (Fig. 6; P < 0.05) that was not observed in mtHSP70-infected myocytes. These results, taken together with the observed increase in ROS (Fig. 5), indicate that mtHSP70 may play a role in attenuating oxidative stress associated with simulated I/R.


Figure 6
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  		Fig. 6. Peroxidation of biomembrane lipid generation after simulated hypoxia/reoxygenation, indexed using BODIPY C11(581/591). NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–) and then were subjected to 8 h of ischemia, followed by 16 h of reoxygenation. Lipid peroxidation was determined by examining the ratio of green (oxidized) and red (nonoxidized) emissions using a fluorometric plate reader. Data are a percentage of preischemic values. Values are mean ± SE; n = 8 for each treatment. *P < 0.05 for Adv–-I/R vs. all other groups.

 
Preservation of mitochondrial function and ATP generation after simulated I/R. Because the mitochondrion is centrally involved in the damaging effects of cardiac I/R, we hypothesized that mtHSP70 overexpression would preserve mitochondrial function after simulated I/R. To test this hypothesis, we used polarographic assessment of ETC complex function after simulated I/R by measuring the activities of the individual complexes that consist of NADH dehydrogenase (complex I), ubiquinone dehydrogenase (complex III), and cytochrome c oxidase (complex IV). NCM were infected with an adenoviral vector encoding either mtHSP70 or an empty control (Adv–) and then submitted to 8 h of hypoxia, followed by 16 h of reoxygenation. After simulated I/R, both complex III and complex IV activities were significantly greater in mtHSP70-infected NCM compared with Adv-infected control NCM (Fig. 7; P < 0.05 for both). No differences in complex I activities were observed between mtHSP70 and Adv-infected NCM after simulated I/R (Fig. 7). To gain insight into whether ATP formation was affected by mtHSP70 overexpression after simulated I/R, we measured total ATP content in NCM. MtHSP70 overexpression elicited a significant increase in ATP content after simulated I/R, compared with Adv-infected NCM when expressed as a percentage of preischemic values (Fig. 8; P < 0.05).


Figure 7
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  		Fig. 7. Electron transport chain complex activities after simulated hypoxia/reoxygenation. NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–) and then were subjected to 8 h of ischemia, followed by 16 h of reoxygenation. Activity is defined as the rate of oxygen consumed in the presence of specific substrates, glutamate/malate (complex I), succinate (complex III), and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (complex IV) and is calculated as the fraction that is sensitive to the specific inhibitors rotenone (complex I), anti-mycin (complex III), and sodium cyanide (complex IV). Results are from 4 independent experiments and are expressed as femtamoles O2 consumed per minute per milligram of protein. Values are mean ± SE. *P < 0.05 for mtHSP70 vs. Adv–.

 

Figure 8
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  		Fig. 8. ATP levels after simulated hypoxia/reoxygenation. NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–) and then were subjected to 8 h of ischemia, followed by 16 h of reoxygenation. ATP levels were measured using a modified luciferase assay and compared against a standard curve of known ATP concentration. Data are a percentage of preischemic values. Values are mean ± SE; n = 6 for each group. *P < 0.05 for Adv-I/R vs. all other groups.

 
Attenuation of apoptosis after simulated I/R. To determine whether overexpression of mtHSP70 was protective against apoptosis, we examined caspase-3 activity and TUNEL-positive staining resulting from 8 h of ischemia followed by 16 h of reoxygenation. NCM overexpressing mtHSP70 showed significantly less caspase-3 activity compared with Adv-infected NCM (Fig. 9; P < 0.05), indicating that the apoptotic program was limited in NCM overexpressing mtHSP70. In addition, myocytes overexpressing mtHSP70 showed less TUNEL-positive cells compared with Adv-infected NCM (Fig. 10; P < 0.05), indicating that overexpression of mtHSP70 protects cells from the apoptotic program.


Figure 9
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  		Fig. 9. Caspase-3 activities after simulated hypoxia/reoxygenation. NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–) and then were subjected to 8 h of ischemia, followed by 16 h of reoxygenation. Caspase-3 activities were measured fluorometrically using Ac-DEVD-AFC as a substrate. Data are normalized to preischemic values and expressed as optical density units (o.d.u.) per 200 µg cytosolic protein. Values are mean ± SE; n = 6 for each group. *P < 0.05 for mtHSP70 vs. Adv–.

 

Figure 10
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  		Fig. 10. TUNEL-positive cells after simulated hypoxia/reoxygenation. NCM were infected at an MOI of 50 for 48 h with adenoviral vectors expressing mtHSP70 or an empty control (Adv–) and were then subjected to 8 h of ischemia, followed by 16 h of reoxygenation. TUNEL-positive cells were measured by flow cytometry using the APO-BrdU TUNEL assay kit (Molecular Probes) as per manufacturer's instructions. Data are expressed as percent TUNEL-positive cells per treatment as a percentage of control. For each treatment, 20,000 myocytes were examined and 4 independent sets of experiments were performed. *P < 0.05 for Adv–-I/R vs. all other groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ROS generated from myocardial I/R injury cause damage within the cell and have been implicated in disturbance of cellular homeostasis via membrane lipid peroxidation, cellular redox status disruption, and mitochondrial function deterioration (2, 17, 32, 36–38). Although many sources of ROS production have been identified, mitochondria are considered the primary site (7, 12, 30). Submitochondrial targets are particularly prone to damage from enhanced ROS due to their close proximity to the source of generation. As a result, mitochondrial protein import has a crucial effect on the viability of mitochondria during cellular stress by providing the means for preserving function and integrity through replacement and/or enhanced delivery of mitochondrial proteins. Mitochondrial protein transport has been shown to increase during times of enhanced contractile activity in skeletal muscle and has been associated with increased expression of mitochondrial protein import machinery such as Tom20 (10, 39). Further, Tom20 has been shown to be decreased after 90 min of ischemia but preserved during ischemic preconditioning, lending indirect evidence that protein import machinery components may be involved in cardiac adaptation to ischemic preconditioning (3). In the current study, we determined whether enhanced expression of mtHSP70, a central component of the mitochondrial protein import machinery, could protect cardiac myocytes from simulated I/R insult. By utilizing adenoviral vectors encoding for mtHSP70, we effectively overexpressed mtHSP70 in neonatal cardiac myocytes (Fig. 2A). Interestingly, when normalizing for protein loading, we observed a decrease in the cytosolic protein {alpha}-actin and an increase in the mitochondrial protein VDAC (Fig. 2, B and C). However, mtHSP70 protein content was increased relative to Adv– control when normalized to either {alpha}-actin or VDAC (Table 1). The results suggest that overexpression of mtHSP70 may be contributing to increased import of other mitochondrial proteins as hypothesized.

Because we observed a change in the mitochondrial protein VDAC, we sought to identify other proteins that were influenced by mtHSP70 overexpression. A number of proteins are involved in the mitochondrial protein import process, one of which is mtHSP70; thus we examined changes in two other proteins involved in the mitochondrial protein import process, Tim44 and Tom20. Our findings indicated that Tom20 expression was increased as a result of mtHSP70 overexpression, while Tim44 expression was unaffected (Fig. 3). Although it is not entirely clear as to why we observed an increase only in Tom20, the results are interesting and suggest that mtHSP70 overexpression influences proteins involved in mitochondrial protein import in a specific manner that may affect outer membrane translocases to a greater extent. Further experimentation is required to substantiate these findings. In addition to proteins involved in mitochondrial protein import, we examined expression levels of antioxidant enzymes, which provide protection against ROS generated during simulated I/R. Our results indicate that MnSOD, the primary antioxidant defense against superoxide radical (O2.–) formation within the mitochondrion, was increased as a result of mtHSP70 overexpression (Fig. 3). In contrast, no change in catalase protein expression was observed (Fig. 3). Because MnSOD resides in the mitochondrion and catalase is located primarily in the peroxisome (13), our findings are consistent with the hypothesis that mtHSP70 is involved in increasing mitochondrially specific antioxidant defense components. These results suggest that mtHSP70 enhances import of antioxidant defense constituents into the mitochondrion providing one potential mechanism for the cardioprotection observed with mtHSP70 overexpression.

We have previously shown that our simulated I/R protocol decreases cell viability, which may be due to increased ROS generation, oxidative stress, and apoptosis (15, 22, 23, 27). In the current study, simulated I/R decreased cell viability by 30% as evidenced by MTT reduction (Fig. 4), which is consistent with our previous studies using this model (15, 22, 23, 27). Overexpression of mtHSP70 using adenoviral vectors attenuated this response and enhanced cell viability by ~60% of control values after simulated I/R (Fig. 4). Our results are similar to those of Liu et al. (24) who demonstrated that overexpression of mtHSP70 via transfection enhanced cell viability in PC12 cells submitted to glucose deprivation. Taken together, these results suggest that the protection provided by mtHSP70 overexpression may be due to an enhanced preservation of cell viability during numerous stress conditions and may not be specific to ischemia or glucose deprivation, per se. Rather, enhanced mtHSP70 content may be associated with a general response that serves to attenuate cell mortality during a multitude of cellular stresses, including I/R and glucose deprivation.

To determine whether the protection provided by mtHSP70 during simulated I/R was due in part to an attenuation of oxidative stress, we examined ROS formation and lipid peroxidation. Previous studies (32) have found that in response to hypoxia changes relating to oxidative processes occur in heart and mitochondria. ROS generation plays an important role in cellular dysfunction after myocardial I/R injury and includes altered cellular respiratory activity and decreased ATP production, both of which favor lipid peroxidation (36). Because the mitochondrion is central to the generation of ROS during I/R, we hypothesized that maintenance of mitochondrial viability, presumably through enhanced mitochondria protein import, may provide protection against oxidative stress. Using DCF fluorescence, we observed a twofold increase in ROS formation in Adv-infected control myocytes after simulated I/R, which was attenuated by mtHSP70 overexpression (Fig. 5). Because DCF fluorescence can be influenced by a number of different reactive oxygen intermediates, it is not entirely clear what specific species are attenuated by mtHSP70 overexpression after simulated I/R (1, 20). Our results are in agreement with others examining cellular response to glucose deprivation (24). To verify whether the increase in ROS generation observed was associated with membrane lipid peroxidation, we examined BODIPY C11(581/591) fluorescence. Simulated I/R enhanced lipid peroxidation as evidenced by an increase in the red-to-green shift in BODIPY C11(581/591) fluorescence (Fig. 6). Further, this increase in lipid peroxidation was not observed in mtHSP70-infected cardiac myocytes (Fig. 6). The production of ROS and lipid peroxidation after simulated I/R is consistent with previous studies (11, 17, 40, 44). These results are interesting and suggest that mtHSP70 may play a role in lessening oxidative stress associated with simulated I/R, although the specific mechanism by which this occurs is not clear. One could speculate that mtHSP70 may facilitate the import of proteins involved in the scavenging of ROS, such as MnSOD, which was observed in the current study (Fig. 3). Such a finding would be consistent with others who have observed increases in MnSOD and glutathione peroxidase (GPx) protein content in mitochondria fractions, resulting from transfection of the mitochondrial import protein Tim44 (26, 43). Because oxidative processes may be involved in initiation of the apoptotic program, we measured caspase-3 activity and observed a significant decrease in mtHSP70-infected cardiac myocytes after simulated I/R (Fig. 9). This was confirmed by observing an attenuation of TUNEL-stained NCM after simulated I/R, with mtHSP70 overexpression (Fig. 10). These findings indicate that overexpression of mtHSP70 during simulated I/R inhibits the apoptotic program, although it is unclear at what specific point or points this occurred. Taken together, one could speculate that increased expression of individual mitochondrial protein import constituents may provide similarly protective effects from I/R insult by enhancing import of antioxidant proteins and/or anti-apoptotic proteins.

To evaluate the role of mitochondrial dysfunction and the influence of mtHSP70 overexpression after simulated I/R injury, we examined ETC activities and ATP content. Simulated I/R has been shown to decrease ETC activities and ATP content (15, 23, 33). In the current study, overexpression of mtHSP70 preserved complex III and IV activities after simulated I/R compared with control (Fig. 7). In addition, mtHSP70 overexpression preserved ATP levels after simulated I/R compared with control (Fig. 8). These results are consistent with previous studies (15, 23) in which overexpression of the mitochondrial chaperones HSP60 and HSP10 enhanced ETC activities and ATP content after simulated I/R. Examining the role of HSP60 and HSP10 overexpression in a simulated I/R model, the Lin et al. (23) observed enhanced protection at complex III and IV, similar to our findings in the current study. The results are interesting in that the functional roles of HSP60 and HSP10 are distinctly different from that of mtHSP70. HSP60 and HSP10 act in concert with one another as mitochondrial chaperones during cellular stress and have not been identified as having a central role in the mitochondrial protein import process. These results suggest that components of ETC complex III and complex IV may either be particularly prone to the deleterious effects of simulated I/R and/or selective submitochondrial loci of mitochondrial HSP protection.

In conclusion, these results indicate for the first time that adenoviral-mediated overexpression of mtHSP70 in neonatal rat cardiac myocytes provides protection from simulated I/R insult by preserving cellular viability, decreasing ROS, attenuating oxidative stress, decreasing the apoptotic program, and enhancing ETC complex function. These findings suggest that adjustments in mitochondrial protein import machinery may provide the basis for enhanced cardiac protection and implicate mtHSP70 as a therapeutic protectant against cellular stress associated with I/R injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by American Heart Association Beginning Grant-In-Aid #0665237B (to J. M. Hollander). The flow cytometry studies were supported in part by Division of Research Resources Grants RR-020866 and RR-16440.


   ACKNOWLEDGMENTS
 
We thank C. Cuff and the contributions from the West Virginia University Flow Cytometry Core Facility and R. Mestril for generation of the mtHSP70 adenoviral vector.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Hollander, West Virginia Univ. School of Medicine, Division of Exercise Physiology, Center for Interdisciplinary Research in Cardiovascular Sciences, 1 Medical Center Drive, Morgantown, WV 26506 (e-mail: jhollander{at}hsc.wvu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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