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H₂O₂ activation of HSP25/27 protects desmin from calpain proteolysis in rat ventricular myocytes

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 15 March 2006 ; accepted in final form 17 March 2007

	ABSTRACT

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Ischemia-reperfusion-induced Ca²⁺ overload results in activation of calpain-1 in the heart. Calpain-dependent proteolysis contributes to myocardial dysfunction and cell death. Previously, preischemic treatment with low doses of H₂O₂ was shown to improve postischemic function and reduce myocardial infarct size. Our aim was to determine the mechanism by which H₂O₂ protects the heart. We hypothesized that H₂O₂ causes the activation of p38 MAPK which initiates translocation of heat shock protein 25/27 (HSP25/27) to the myofilament Z disk. We further hypothesized that HSP25/27 shields structural proteins, particularly desmin, from calpain-induced proteolysis. To address this hypothesis, we first determined that an ischemia-reperfusion-induced decrease in desmin content could be blocked by H₂O₂ pretreatment of hearts from rats. We next determined that ventricular myocytes that underwent Ca²⁺ overload also demonstrated a calpain-dependent disruption of desmin that could be reduced by H₂O₂/p38 MAPK activation. Furthermore, myocytes acutely treated with H₂O₂ exhibited a decrease in cleavage of desmin upon exposure to exogenous calpain-1 compared with myocytes not pretreated with H₂O₂. The H₂O₂-induced attenuation of desmin degradation by calpain-1 was blocked by inhibition of p38 MAPK. In a final series of experiments, we demonstrated that cardiac myofilaments exposed to recombinant phosphorylated HSP27, but not nonphosphorylated HSP27, had a significant reduction in the calpain-induced degradation of desmin compared with non-HSP27-treated myofilaments. These findings are consistent with the hypothesis that H₂O₂-induced activation of p38 MAPK and subsequent HSP25/27 translocation attenuates desmin degradation brought about by calpain-1 activation in ischemia-reperfused hearts.

ischemia-reperfusion; isolated heart; heat shock protein 25/27; p38 mitogen-activated protein kinase; actinin; troponin I

Studies into the role of reactive oxygen species and oxidative stress in ischemia-reperfusion injury have demonstrated that low doses of hydrogen peroxide (H₂O₂) are cardioprotective (19, 24). Sharma et al. (19) showed that acute preconditioning with 100 µM H₂O₂ before prolonged ischemia and reperfusion reduced lactate dehydrogenase and creatine kinase release into the coronary effluent and decreased infarct size in isolated hearts from rats. Similarly, Yaguchi et al. (24) observed improved recovery of postischemic left ventricular developed pressure and high-energy phosphate content with preischemic administration of 2 µM H₂O₂ in isolated hearts from rats. Low doses of H₂O₂ are known to activate p38 mitogen-activated protein kinase (p38 MAPK). Acute exposure to 30–100 µM H₂O₂ induces activation of p38 MAPK and leads to the phosphorylation of heat shock protein 25_rodent/27_human (HSP25/27) in ventricular myocytes (3, 5). Furthermore, p38 MAPK induces translocation of HSP25/27 from the cytosolic to the myofilament fraction in ventricular myocytes (2). Within myofilaments, HSP25/27 colocalizes with Z-disk proteins (10). Numerous studies have demonstrated that HSP25/27 can protect proteins from stress-induced damage. For example, overexpression of HSP27 protects from ischemia-reperfusion damage in both rat and canine myocardium (15, 23). These observations raise the possibility of a Z-disk protein(s) that is protected by H₂O₂-p38 MAPK-HSP25/27 activation.

Thus, for the present study, we hypothesized that the ischemia-reperfusion-induced activation of calpain and subsequent proteolysis of cytoskeletal proteins, such as desmin, can be reduced through localization of HSP25/27 to the Z disk. Preischemic treatment with H₂O₂ activates this protective pathway through p38 MAPK. Determining the mechanism(s) that H₂O₂ uses to protect the heart from ischemia-reperfusion will allow us to identify promising therapeutic targets for future research. Further, the findings of the present study provide background information for the consideration of clinical studies designed to determine whether activation of the H₂O₂ protective pathway might be of benefit when activated before procedures known to induce ischemia-reperfusion, i.e., coronary stent placement or thromobolysis.

	METHODS

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Isolated heart and ventricular myocyte preparations. All procedures were approved and carried out according to the guidelines set by the Animal Care and Use Committee of the University of Tennessee Health Science Center. Female Wistar rats (196–205 g) were anesthetized with 65 mg/kg pentobarbital sodium. For studies done in whole hearts, the heart was excised and cannulated in ice-cold modified Krebs-Henseleit buffer consisting of (in mM) 118 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 11 glucose, 25 NaHCO₃, and 1.3 CaCl₂ (pH 7.4). Hearts were then mounted on a Langendorff apparatus and perfused with oxygenated Krebs-Henseleit buffer at 37°C. A left atriotomy allowed a cellophane balloon attached to a pressure transducer (BLPR, World Precision Instruments, Sarasota, FL) to be inserted into the left ventricle, and the balloon was inflated until a maximum developed pressure was observed with an end-diastolic pressure of 5–10 mmHg. Hearts were paced with electrodes at 300 beats/min. Isolated hearts underwent 5 min of paced equilibration and four periods of 5 min vehicle or 100 µM H₂O₂ perfusion followed by 5 min washout. Nonischemic hearts were harvested immediately after perfusion/washout, whereas ischemic-reperfused hearts were further subjected to 60 min of global ischemia and 30 min of reperfusion before harvesting. This protocol was identical to that of Matsumura et al. (16). Upon completion of reperfusion, the left ventricle was homogenized in a solution containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100, and 1% protease inhibitor cocktail (Cat. No. P8340; Sigma, St. Louis, MO), and the homogenate was centrifuged at 13,000 g for 15 min. The pellet was washed twice in a solution containing 60 mM KCl, 30 mM imidazole (pH 7.2), 2 mM MgCl₂, and 1% protease inhibitor cocktail. The pellet was then resuspended in the wash solution, diluted with an equal volume of Laemmli sample buffer, and frozen for use in Western blot analysis. An analysis of desmin content was carried out by normalizing the desmin immunoreactive band to two separate proteins on silver-stained gels run with the same samples. This was done to confirm a change in desmin independent of the protein used for normalization.

Ventricular myocytes were isolated using procedures previously described (12). Cell yield was calculated from three separate photomicrograph fields for each isolation. Cells having a length-to-width ratio of 2:1 or greater were designated "rod shaped." Typically, cell yield, calculated as the percentage of rod-shaped myocytes in a Ringer solution containing 1.25 mM Ca²⁺ relative to total cell number, was 60–70%. Yields below 40% were not used.

For those protocols using demembranated/skinned cells, myocytes were exposed to standard relax solution containing 100 mM KCl, 10 mM imidazole (pH 7.2), 1 mM MgCl₂, 2 mM EDTA, 4 mM ATP, 5 mM dithiothreitol, and 0.3% Triton X-100 for 10 min at room temperature. The cells were then centrifuged at 2,000 g for 1 min and washed three times with Triton-free relax solution and resuspended in a pCa 5.6 solution.

Calpain-1 activity and purified desmin degradation. Calpain-1 (Cat. No. 208712; EMD/Calbiochem, San Diego, CA) was used in all experiments involving isolated myocytes or myofilaments. Specific activity of the calpain lots used ranged from 139–1,569 U/mg protein according to the manufacturer. Depending on the specific activity of each calpain lot, we used 3–13 µg of calpain-1 to give a typical calpain activity of 2 mU/µl solution.

Purified chicken desmin (Cat. No. D3221–13; US Biological, Swampscott, MA) was incubated with or without purified calpain-1 to determine the relative molecular masses and antibody reactivity of calpain-induced degradative products of desmin. Calpain-1 degradation of desmin was carried out in a solution containing (in mM) 100 NaCl, 4 CaCl₂, and 2 MgCl₂. Samples were diluted with an equal volume of urea sample buffer and frozen for Western blot analysis.

Myocyte treatments. Short-term intracellular Ca²⁺ overload was induced in intact ventricular myocytes by exposing them to a 6 mM extracellular Ca²⁺ solution for 10 min. Before this high extracellular Ca²⁺ challenge, ventricular myocytes were pretreated with 10 µM SB-202190 (EMD/Calbiochem), a p38 MAPK inhibitor, or vehicle for 10 min, followed by an addition of 0.8 µM MDL-28170 (Sigma Chemical), a calpain inhibitor, or vehicle for 15 min. Cells were then exposed to 100 µM H₂O₂ or vehicle for 3 min. Finally, cells were challenged with an extracellular [Ca²⁺] of 6 mM or maintained at a normal extracellular [Ca²⁺] of 1.25 mM. All reactions were stopped by the addition of electrophoretic sample buffer containing SDS and 8 M urea, and samples were frozen for later Western blot analysis of desmin content. SB-202190 inhibits p38 MAPK with an IC₅₀ of 50–100 nM (4). A concentration of 10 µM SB-202190 has been shown to act as a potent inhibitor of both p38- and -isoforms while having no significant effect on any other MAPK or related kinase (4).

To characterize the effects of exogenous calpain-1 on myofilament proteins, skinned myocytes were exposed to vehicle or calpain-1 for 3, 5, 10, and 15 min. Following calpain exposure, cells were centrifuged at 1,000 g for 1 min and the supernatants were diluted with an equal volume of urea sample buffer and frozen for Western blot analysis of -actinin, desmin, and troponin I.

The extent and mechanism of H₂O₂-dependent protection from exogenous calpain were investigated using isolated ventricular myocytes with intact membranes that were 1) exposed to 0, 2, 30, and 100 µM H₂O₂ for 3 min; 2) exposed to 100 µM H₂O₂ for 0, 3, 5, and 15 min; or 3) treated with or without 5 µM SB-202190 for 30 min followed by treatment with or without 100 µM H₂O₂ for 3 min. Following drug treatment, membranes were removed as described, and skinned cells were incubated with calpain-1 for 5 min followed by centrifugation at 1,000–2,000 g for 1 min. The supernatants were diluted with an equal volume of urea sample buffer and frozen for Western blot analysis.

Exogenous phosphorylated HSP27 in myofilaments. Initially, we wished to determine whether phosphorylated HSP27 (pHSP27) and HSP27 associate to an equal extent with both normal and calpain-treated myofilaments. To do this, ventricular tissue was homogenized, sieved through a 250-µm polypropylene mesh, and incubated at pCa 5.6 with or without calpain-1 for 5 min at room temperature. The reaction was stopped by an addition of 1 mM calpastatin (Cat. No. 208902; EMD/Calbiochem). Samples were incubated with exogenous pHSP27, HSP27, or vehicle for 5 min and centrifuged at 5,000 g for 5 min. The pellet was then washed twice, diluted with urea sample buffer, and frozen for Western blot analysis.

To determine whether pHSP27 influences the extent of calpain-induced degradation of desmin, ventricular tissue homogenates were incubated with pHSP27, HSP27, MAPKAPK2 (MK2), or vehicle in a pCa 5.6 solution with or without calpain-1 for 30 min at 30°C. Samples were diluted with an equal volume of urea sample buffer and frozen for Western blot analysis.

pHSP27 was prepared by incubating human recombinant HSP27 (Cat. No. 385891; EMD/Calbiochem) with constitutively active, human recombinant MK2 (Cat. No. 14–337; Upstate, Lake Placid, NY) at 30°C for 45 min in the presence of Mg²⁺ and ATP. Exogenous pHSP27 and appropriate controls (vehicle, HSP27, and MK2) were made before experimental protocols. For each identical ventricular homogenate aliquot, 2 µg HSP27 or HSP27 vehicle and 4.5 U/µg protein MK2 or MK2 vehicle were added.

Immunoblotting. Proteins were separated via sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis was performed. Antibodies to desmin (Cat. No. D1033; Sigma), -actinin (Cat. No. A7811; Sigma), troponin-I (Cat. No. SC-8118; Santa Cruz Biotechnology, Santa Cruz, CA), HSP27 (Cat. No. SC-1049; Santa Cruz Biotechnology), and pHSP27-Ser82 (Cat. No. 07–646; Upstate) were used. Densitometry was carried out using NIH ImageJ software.

Statistical analysis. All values were reported as means ± SE. Statistical significance was assumed for P < 0.05. Data were analyzed by a standard analysis of variance followed by a Student t-test or, for those data normalized to a paired control, an ANOVA with complete block design followed by Fisher least significant difference post hoc test.

	RESULTS

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Calpain-induced degradation of purified desmin. Desmin was incubated with or without calpain-1 in the presence of Ca²⁺ to identify the pattern of desmin degradation (Fig. 1). Full-length desmin has a predicted molecular mass of 52 kDa. In the absence of calpain, commercially purified chicken desmin had two distinct immunoreactive bands at 50 and 45 kDa. In the presence of calpain, there was a progressive loss of these higher molecular mass desmin immunoreactive proteins concomitant with the appearance of a band at 32 kDa. Thus anti-desmin immunoreactive proteins at 45 and 32 kDa were designated desmin degradative products A and B, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Representative Western blot illustrating calpain-1 degradation of desmin. Equal amounts of purified chicken desmin were incubated with (times of 10 and 60 sec) or without (time 0) calpain-1. A and B indicate desmin degradative products. Additional calpain-1 exposure causes disappearance of both A and B products (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Typical Western blot and cumulative analysis of desmin content in nonischemic (Non-Isch), vehicle-pretreated ischemic/reperfused (vehicle + Isch/Rep), and H2O2-pretreated ischemic/reperfused (H2O2 + Isch/Rep) hearts. Isolated hearts were subjected to 4 times 5 min vehicle or 100 µM H2O2 followed by 5-min wash and then 60 min of global ischemia and 30 min of reperfusion. Left ventricular homogenates were examined by Western blot analysis for the full length desmin product (50 kDa). Densities from anti-desmin Western blots were normalized to the densities of 2 separate protein bands on silver-stained SDS-polyacrylamide gels. Values are expressed as means ± SE; n = 3 hearts/group. *P < 0.05 compared with nonischemic hearts.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Typical Western blot and cumulative effect on cellular desmin content due to an extracellular Ca2+ challenge (Ca2+) in myocytes pretreated with a calpain inhibitor MDL-28170 (MDL), H2O2, a p38 MAPK inhibitor SB-202190 (SB), or cells not challenged with a high extracellular Ca2+ and pretreated with vehicle (Con). Ventricular myocytes were pretreated with combinations of 5 µM SB, 0.8 µM MDL, 100 µM H2O2 or vehicle and challenged with a high extracellular [Ca2+] of 6 mM (light bars) or maintained at a normal extracellular [Ca2+] of 1.25 mM (darker bars) for 10 min. Cells were then examined by Western blot analysis for the full-length desmin product with an apparent molecular mass of 50 kDa. In addition, aliquots from identical samples were electrophoresed and Coomassie stained to establish equality of protein load per isolation. Cumulative data are expressed as desmin density relative to the signal from vehicle-treated cells from that isolation. Cumulative analysis is from 4–6 isolations, and *P < 0.05 relative to the corresponding non-Ca2+ overloaded group. MDL treatment alone did not significantly decrease desmin as compared with Con (P = 0.13).

Exogenous calpain-induced degradation of myofilament protein: effects of H₂O₂ and p38 MAPK inhibition. Ventricular myocytes were skinned and exposed to low levels of exogenous calpain-1 in the presence of Ca²⁺. Calpain treatment resulted in myofilament proteins being cleaved and/or released as full-length products into the supernatant (Fig. 4). Calpain increased the desmin degradative product B in the myofilament supernatant at all time points (Fig. 4A). Troponin I, as the full-length product and a slightly lower molecular mass-cleaved product, was increased in the myofilament supernatant after 3, 5, and 10 min of calpain exposure (Fig. 4B). Troponin I was also present in the supernatant from vehicle-treated myofilaments. The origin of this troponin I is likely the large precursor pool of troponin I that is loosely associated with the myofilaments (14). Calpain also increased the -actinin full-length protein released into the myofilament supernatant after 5, 10, and 15 min of exposure (Fig. 4C). A time-dependent increase in -actinin and troponin I release and cleavage were also observed for myofilaments not exposed to exogenous calpain (Con in Fig. 4). These changes were not as great or as consistent as that seen with calpain addition. Activation of endogenous calpain, induced by the increase in bathing [Ca²⁺] of the vehicle solution, could account for these effects.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Representative Western blots of desmin (A), troponin I (B), and -actinin (C) reactive proteins in the myofilament supernatant (lanes 1–8) following exposure to calpain-1 (Calp) or vehicle (Con) for 3, 5, 10, and 15 min. Also shown is the reactivity of the myofilament fraction (Pellet) after 15 min of vehicle exposure. Two additional experiments gave similar results.

View larger version (17K): [in this window] [in a new window]   Fig. 5. H2O2 concentration (A) and time-dependent (B) attenuation of the calpain-induced appearance of desmin degradative product B in the myofilament supernatant. A: myocytes were pretreated with vehicle (no H2O2) or with 2, 30, or 100 µM H2O2 for 3 min. Myofilaments were isolated, and all samples were exposed to calpain-1 for 5 min. B: myocytes were pretreated with vehicle (no H2O2) or with 100 µM H2O2 for 3, 5, or 15 min. Myofilaments were isolated, and all samples were exposed to calpain-1 for 5 min. Densities from anti-desmin Western blots were normalized to protein load based on the density of a calpain-resistant supernatant protein (20 kDa) visualized on silver-stained SDS-polyacrylamide gels and normalized to the no H2O2 group response. Values are expressed as means ± SE; n = 3–6 myocyte isolations. *P < 0.05 compared with no H2O2.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Representative Western blots and cumulative analysis of the appearance of desmin degradative product B showing the effect of p38 MAPK inhibition with SB on the H2O2-induced attenuation of desmin degradation by calpain-1. Myocytes were pretreated with 1 µM SB or vehicle for 30 min followed by 100 µM H2O2 or vehicle for 3 min. Myofilaments were then isolated, and all samples were exposed to calpain-1 for 5 min. Densities from anti-desmin Western blots were normalized to protein load based on the density of a calpain-resistant supernatant protein (20 kDa) visualized on silver-stained SDS-polyacrylamide gels and normalized to the no H2O2 or SB group response. Values are expressed as means ± SE; n = 7 myocyte isolations. *P < 0.05 compared with no H2O2.

View larger version (8K): [in this window] [in a new window]   Fig. 7. Western blot to phosphorylated HSP25/27 (pHSP25/27) demonstrating recombinant human HSP27 is phosphorylated when incubation with MAPKAPK2 (MK2), and calpain exposure (+Calp) does not alter the level of phosphorylation. For these experiments, MK2, HSP27, or HSP27 preincubated with MK2 (see METHODS) were added to myofilaments in the presence (+) and absence (–) of calpain-1 for 5 min. Endogenous levels of pHSP25 were undetectable.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Typical Western blot and cumulative analysis showing equal association of exogenous pHSP27 and HSP27 with myofilaments. Myofilaments were incubated with (+) or without (–) calpain for 5 min followed by incubation with exogenous pHSP27, HSP27, or vehicle for 5 min. Myofilaments were then twice washed in buffer not containing HSP27, and Western blot analysis was completed. HSP27 immunoreactivity exhibited by the vehicle groups was attributed to endogenous levels of the rodent homologue HSP25. Values are expressed as means ± SE; n = 3 myofilament isolations. *P < 0.05 compared with corresponding vehicle.

View larger version (26K): [in this window] [in a new window]   Fig. 9. The effect of exogenous HSP27 on calpain-induced degradation of desmin. Ventricular myofilaments were exposed to pHSP27, HSP27, MK2, or vehicle followed by incubation with calpain for 30 min at 30°C. Values are expressed as means ± SE; n = 7 myofilament isolations. *P < 0.05 compared with vehicle.

	DISCUSSION

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View larger version (10K): [in this window] [in a new window]   Fig. 10. Diagram of the proposed signaling pathway.

Disruption of cytoskeletal proteins such as desmin may contribute to or be a consequence of cell death induced by ischemia-reperfusion. In a study by Kido et al. (9), ischemia-reperfusion-induced changes in dystrophin preceded a disruption of the cell membrane and necrosis. Desmin disruption has been demonstrated in ischemia-reperfused whole hearts with maintained Z-band structure and cell membranes (16). These data support the possibility that cytoskeletal proteins contribute to reperfusion injury rather than being a consequence of cell death. Furthermore, from our Ca²⁺-overload studies, we established that desmin disruption due to an acute Ca²⁺ overload occurs without a decrease in the number of rod-shaped, membrane-intact ventricular myocytes. Thus the observed changes in desmin precede morphological changes consistent with irreversible Ca²⁺ overload, hypercontracture, and cell death.

Ca²⁺-dependent activation of calpain-1, due to ischemia-reperfusion, has been shown to degrade cytoskeletal and regulatory myocardial proteins (16, 17, 21). In the present study, using intact cells, we demonstrated that endogenous calpain inhibition blocks desmin degradation due to Ca²⁺ overload (Fig. 3). Furthermore, using myofilaments exposed to exogenous calpain-1, we demonstrated a time-dependent increase in the appearance of immunoreactive desmin, -actinin, and troponin I in the myofilament supernatant (Fig. 4). The calpain-dependent appearance of a lower molecular mass desmin product suggests that calpain-1 causes proteolysis and release of desmin into the supernatant. Calpain also causes an increased rate of release of a lower molecular mass troponin I product along with the intact troponin I compared with untreated controls. -Actinin was only found as the full-length molecular mass in the supernatant of calpain-treated myofilaments, a finding consistent with work by Goll et al. (6). Thus, it is likely that calpain-1 cleaves a protein necessary for -actinin to associate with the myofilaments.

Acute 100 µM H₂O₂ exposure attenuates the disruption of desmin from intact cells and myofilaments (Figs. 3 and 4). One explanation for this effect is that H₂O₂ reduces calpain activity. However, this seems unlikely given that myofilaments were washed free of H₂O₂ before exogenous calpain exposure (Figs. 5 and 6), and myocytes pretreated with H₂O₂ continued to have a calpain-dependent disruption of -actinin or troponin I in myofilaments (data not shown). Another explanation of the ability of H₂O₂ to block desmin degradation is that it is able to activate a pathway that leads to the protection of specific protein(s). Consistent with this idea are our observations that the protective effect of H₂O₂ on desmin disruption could be blocked by an administration of the p38 MAPK inhibitor SB-202190 (Figs. 3 and 6). This suggests that low doses of H₂O₂ stimulate the p38 MAPK signaling pathway to limit the ability of calpain-1 to degrade desmin.

The mechanism by which p38 MAPK acts may involve a decrease in calpain activation or an effect on the target of calpain activation. However, past studies have linked p38 MAPK activation with an increase in in vivo calpain activity (8, 20). Thus it seems unlikely that p38 MAPK activation decreased exogenous calpain activity. Another possible mechanism of p38 MAPK action is through HSP25/27. Previous studies have demonstrated that stimulation of p38 MAPK results in the phosphorylation and translocation of HSP25/27 from the cytosolic to the Z-disk region of myofilaments (2, 3, 10, 11). Ischemic preconditioning has also been shown to induce a translocation of HSP25/27 in a p38 MAPK-dependent manner, and the activation of p38 MAPK-HSP25/27 correlates with an increase in postischemic myocardial function (18). Furthermore, overexpression of HSP27 has been shown to decrease lactate dehydrogenase release and to increase the number of ventricular myocytes that exclude trypan blue upon ischemic stress (15, 23). In the present study, cardiac myofilaments were incubated with recombinant HSP27 or pHSP27 before treatment with calpain-1 (Fig. 9). Exposure to exogenous pHSP27, but not HSP27, resulted in decreased calpain-induced desmin degradation. This lack of effect of the nonphosphorylated form of HSP27 was not due to a decrease in HSP27 associated with the myofilaments compared with pHSP27 (Fig. 8). These data lend further support to the hypothesis that H₂O₂ protection from ischemia-reperfusion damage is due, at least in part, to the p38 MAPK activation of HSP25/27 and the ability of pHSP25/27 to shield desmin from calpain-1 degradation in the heart.

	GRANTS

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This study was supported by the National Heart, Lung, and Blood Institute Training Fellowship HL-07641 (to B.C. Blunt) and Grant HL-48839 (to P. A. Hofmann) and American Heart Association Grant-In-Aid 0655584 (to P. A. Hofmann).

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. Hofmann, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: phofmann{at}physio1.utmem.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.

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