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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-0348fje. |
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,2
* Division of Medical Engineering Research, National Health Research Institutes, Taipei, Taiwan; and
Department of Medicine, University of California, San Diego, La Jolla, California, USA
2Correspondence: Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0618 USA. E-mail: wdillmann{at}ucsd.edu
SPECIFIC AIMS
Mitochondrial heat shock proteins (hsp) hsp60 and hsp10 are involved in the folding of newly imported mitochondrial matrix proteins and refolding of denatured proteins after acute stress. In this study, we examined whether protection from SI/RO injury afforded by hsp10 is due, in part, to a cytoplasmic mechanism independent of its mitochondrial chaperonin function, and whether this function involves interaction with the Ras-GTPase signaling cascade.
PRINCIPAL FINDINGS
1. Hsp10 expression reduces NCM death in multimodalities of ischemia injury
The 10 kd heat shock protein (hsp10) together with the 60 kd heat shock protein (hsp60) form a ring-like structure that is a major site of protein folding in mitochondria. Increased expression of hsp10 and/or hsp60 provides protection to myocytes submitted to simulated ischemia/reoxygenation (SI/RO) insult. Because hsp60 is rate-limiting in many folding studies, it is unlikely that protection afforded to myocytes by hsp10 during SI/RO is due solely to preservation of mitochondrial proteins. In this study, we determined if increased expression of hsp10 protects against hypoxia and reoxygenation related injuries, by examining three separate models: H2O2 treatment, metabolic ischemia (NaCN) and recovery, and SI/RO. Rat neonatal cardiac myocytes (NCM) were infected with adenoviral vectors expressing wild-type hsp10 or an empty control vector (AdvSR–). Lactate dehydrogenase (LDH) release was used as an assessment of cell death. Overexpression of hsp10 decreased H2O2-induced cell death to 81% of the level in control virus infected myocytes. Hsp10 expression decreased NaCN-induced cell death to 64% of the level of control infected myocytes and decreased SI/RO induced cell death to 45% of control. These results indicate that increased hsp10 expression provided protection against injuries involving both hypoxia (metabolic ischemia or simulated ischemia) and reoxygenation. Hsp10 provided protection against H2O2 insult, due mainly to excessive oxidative stress.
2. Modification of the hsp10 mobile loop abolishes MDH folding, mitochondrial ETC capacity, and protection against SI/RO
The mechanism by which hsp10 binds with hsp60 and facilitates movement of substrates in and out of the chaperonin complex is due in part to a mobile loop in the hsp10 protein. Mutations in this loop can substantially reduce hsp10’s binding affinity for hsp60. Based on this information, we engineered an hsp10 mutant construct (mutant hsp10(P34H)), in which proline 34 was replaced by a histidine residue. We examined the ability of this mutant protein to assist in folding of denatured malate dehydrogenase (MDH). At 37°C, mutant hsp10 was less effective at assisting MDH folding, and may have competed with wild-type hsp10 for hsp60, thus hampering hsp60’s chaperonin function.
Because hsp10 may assist in folding of newly imported mitochondrial matrix proteins, we speculated that proteins in the mitochondrial respiratory chain may be modulated by overexpression of wild-type or mutant hsp10(P34H). Activities of individual components of the mitochondrial electron transport chain (ETC) were measured by polarographic analysis. Hsp10 expression caused a significant increase in NADH:ubiquinone oxidoreductase (complex I) capacity, compared with that of control cells in the basal state, and mutant hsp10(P34H) significantly reduced complex II (succinate dehydrogenase) capacity in the basal state (Fig. 1
A). Hsp10 overexpression preserved both complex I and II function after SI/RO, while hsp10 (P34H) had no significant effect on either (Fig. 1B
). Expression of wild-type or mutant hsp10 (P34H) did not significantly alter the capacity of complex III or IV in either the basal state or following SI/RO. Impaired protein folding assistance and decreased oxidative phosphorylation capacity may be detrimental to cells. We examined whether mutant hsp10(P34H) was less effective than hsp10 at protecting myocytes from cell death following SI/RO. Cell death by SI/RO in cells infected with control virus was taken as 100%, and overexpression of mutant hsp10(P34H) increased cell death to 120%. Combination of mutant hsp10(P34H) and wild-type hsp10 abolished the protection afforded by hsp10 overexpression.
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3. Hsp10 protection involves attenuation of the cytoplasmic Ras GTP-ase pathway
The Ras, Raf, ERK signaling pathway has been studied in various cardiac myopathy models. We explored the potential association of hsp10 and subsequent activity of the Ras pathway during SI/RO insult. Adenoviral vectors expressing a constitutively active form of Ras (AdvRasV12) and a dominant negative mutant form of Ras (AdvRasN17) were used as models to determine whether Ras activation or inactivation resulted in myocyte preservation after SI/RO. We found that constitutively activating Ras in myocytes for two days resulted in an increase in cell number and cell size with increased cell death after SI/RO (124% vs. 100%) compared with control virus infected myocytes. Overexpressing RasN17 resulted in fewer cells after two days of culture, and NCM were better preserved (56% vs. 100%) after the same SI/RO treatment as compared with control virus infected myocytes. To test whether wild-type hsp10 or mutant hsp10(P34H) could modulate protective or detrimental effects by either RasN17 or RasV12, respectively, we co-infected myocytes and subjected them to SI/RO. Overexpression with either wild-type hsp10 or mutant hsp10(P34H) in addition to RasV12 did not result in significant differences in cell death as compared with RasV12 alone (Fig. 2
). In contrast, coexpression of wild-type hsp10 and RasN17 attenuated cell death as compared with RasN17 alone (Fig. 2)
. Coexpression of mutant hsp10(P34H) with RasN17 abolished this effect (Fig. 2)
. These results suggest that both protection by hsp10 and damage by mutant hsp10(P34H) required Ras inactivation, and as such, RasV12 (activation) expression was able to abolish both effects. Conversely, effects of wild-type hsp10 or mutant hsp10(P34H) on myocyte cell death appeared to be independent of Ras activation. We examined phosphorylation status of Raf, RafC, phosphorylated ERK, ERK1/2, and phosphorylated p90ribosomal kinase (p90RSK), on myocytes infected with hsp10 or an empty control virus and subjected to SI/RO. We observed decreases in phosphorylation level of Raf, ERK, and p90RSK in myocytes overexpressing hsp10, before and after SI/RO. We also found hsp10 overexpression led to a slight decrease in the amount of phosphorylated p90RSK. In contrast, RasV12 and RasN17 overexpression, led to a strong increase and decrease of p90RSK phosphorylation, respectively. Our results suggest a possible role for hsp10 in cardiac protection during SI/RO, independent of its chaperonin function.
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CONCLUSIONS AND SIGNIFICANCE
Mitochondrial chaperonins play a crucial role in preserving cardiac viability during myocardial ischemia due to folding of mitochondrial proteins, preservation of oxidative phosphorylation, and ATP generation which are essential to cardiac contractile function. Involvement of the Ras signaling cascade in cardiac myopathy has been extensively discussed in the context of dilated cardiac myopathy and activation of Ras leads to hypertrophy and heart failure. Our results suggest that Ras inactivation is required for the beneficial effects of hsp10. The mechanism for hsp10 protection is hypothesized in Fig. 3
. Ras inactivation attenuates myocyte death during SI/RO (cell protection). Hsp10 and mutant hsp10(P34H) both require deactivation of the Ras pathway to exercise their biological effects. Ras-independent mechanisms also exist because additional benefits and damage were found in cells expressing hsp10 or mutant hsp10(P34H) in addition to RasN17. The protein folding and mitochondrial ETC results we have observed are probably due to a Ras-independent mechanism. In our polarographic analysis, hsp10(P34H) caused a reduction in complex II capacity. In contrast, overexpression of wild-type hsp10 increased complex I capacity. Complex I is comprised of the most enzyme subunits in the ETC and is considered the rate-limiting step during state 3 respiration. The beneficial effect of hsp10 overexpression during ischemic injury may be related to increased efficiency of respiration by increased complex I capacity (Fig. 1B
). Decrease in SDH capacity by hsp10(P34H) suggests that either expression of SDH or assembly of SDH was affected. The detrimental effect of hsp10(P34H) overexpression in myocytes may be the result of defects other than impaired substrate folding. We also examined the phosphorylation state (presumably activity) of Raf, ERK, and p90RSK, which all belong to one of many cascades that can be activated by Ras. Our data indicate that hsp10 leads to down-regulation of this pathway in control and SI/RO myocytes, but that the level of deactivation is not as significant as that by RasN17. The potential effecter of this cascade is the sodium proton exchanger (NHE), a substrate of p90RSK. Activation of NHE and subsequent calcium overload is implicated as a major determinant of myocyte death in I/R injury. It has been proposed that inhibition of NHE activity is a potential therapeutic for treating ischemic cardiomyopathy. Our findings are in agreement with this strategy.
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Results indicate that increased hsp10 expression protects myocytes against SI/RO, metabolic ischemia (NaCN), and H2O2 injury. Mutation of the mobile loop of hsp10 impairs chaperonin function and abolishes cardiac protection by hsp10. Protection by hsp10 requires down-regulation of Ras and also involves a Ras-independent mechanism that may be related to preservation of the mitochondrial ETC, particularly complex I. Our data suggest that a novel mechanism independent of a chaperonin function may contribute to beneficial effects afforded by hsp10 during SI/RO in myocytes. Our findings indicate a potential alternate locus for hsp10 action.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0348fje; 
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