Hsp10 and Hsp60 suppress ubiquitination of insulin-like growth factor-1 receptor and augment insulin-like growth factor-1 receptor signaling in cardiac muscle: implications on decreased myocardial protection in diabetic cardiomyopathy.

We have investigated the effects of two heat shock proteins, Hsp10 and Hsp60, on insulin-like growth factor-1 receptor (IGF-1R) signaling in cardiac muscle cells. Neonatal cardiomyocytes were transduced with Hsp10 or Hsp60 via adenoviral vector. Compared with the cells transduced with a control vector, overexpression of Hsp10 or Hsp60 increased the abundance of IGF-1R and IGF-1-stimulated receptor autophosphorylation. Thus, Hsp10 and Hsp60 overexpression increased the number of functioning receptors and amplified activation of IGF-1R signaling. IGF-1 stimulation of MEK, Erk, p90Rsk, and Akt were accordingly augmented. Transducing cardiomyocytes with antisense Hsp60 oligonucleotides reduced Hsp60 expression, decreased the abundance of IGF-1R, attenuated IGF-1R autophosphorylation, and suppressed the pro-survival action of IGF-1 in cardiomyocytes. Using cycloheximide to inhibit protein synthesis did not alter the effect of Hsp60 on IGF-1R signaling, and IGF-1R mRNA levels were not up-regulated by Hsp10 or Hsp60. Additional experiments showed that Hsp10 and Hsp60 suppressed polyubiquitination of IGF-1 receptor. These data indicate that Hsp10 and Hsp60 can modulate IGF-1R signaling through post-translational modification. In animal models of diabetes, diabetic myocardium is associated with decreased abundance of Hsp60, increased ubiquitination of IGF-1R, and lower level of IGF-1R protein. Declined myocardial protection is a major feature of diabetic cardiomyopathy. These data suggest that decreased Hsp60 expression and subsequent decline of IGF-1R signaling may be a fundamental mechanism underlying the development of diabetic cardiomyopathy.

The insulin-like growth factor 1 (IGF-1) 1 binds to the IGF-1 receptor, induces autophosphorylation of the receptor, acti-vates receptor tyrosine kinase, and triggers cascades of intracellular signaling events. IGF-1 receptor signaling is involved in the regulation of multiple aspects of biological actions (1). For example, IGF-1 activation of phosphatidylinositol 3 kinase and MAP kinase suppresses cardiac muscle apoptosis and enhances myocardial protection during myocardial injuries (2,3). In addition to phosphorylation and dephosphorylation, growth factor receptor signaling can be modulated through transcriptional and post-translational modification of ligands, receptors, and signaling intermediates. Recent studies (4,5) have shown that insulin and IGF-1 receptor signaling can be modulated through post-translational modification of signaling molecules such as arrestin-1 and insulin receptor substrate (IRS2). There was evidence suggested that IGF-1 receptor signaling might be modulated through degradation of receptor proteins as proteolysis inhibitors prevented degradation of IGF-1 receptor (6). However, whether post-translational modification of IGF-1 receptor contributed to the regulation of IGF-1 receptor signaling in cardiac muscle has not yet been investigated.
Heat shock proteins are a group of molecular chaperones that are capable of preventing protein damages and proteolysis. In cardiac muscle, Hsp60 and Hsp10 may form mitochondrial chaperoning complexes and are believed to play a role in the maintenance of normal mitochondria function (7). Hsp60 and Hsp10 also exist in the cytosolic compartment and interact with cytosolic proteins (8), which suggests that Hsp60 and Hsp10 may regulate proteins through post-translational modification. Whether Hsp10 and Hsp60 can modulate IGF-1 receptor signaling has not been studied. This study was carried out to determine whether Hsp10 and Hsp60 can modulate IGF-1 receptor signaling in cardiomyocytes. The results showed that IGF-1 receptor signaling can be modulated by Hsp60 and Hsp10 through post-translational modification in cardiac muscle cells. Further experiments on animal models of diabetes showed that the changes of Hsp60 paralleled the levels of IGF-1 receptor in various tissues. Moreover, diabetic myocardium exhibited reduced Hsp60 and increased ubiquitination of IGF-1 receptor. These findings may have significant implications in understanding the fundamental mechanisms that lead to a decreased myocardial protection during the development of diabetic cardiomyopathy.

EXPERIMENTAL PROCEDURES
Materials-Mouse anti-Hsp60 monoclonal antibody and rabbit anti-Cpn10 (Hsp10) peptide polyclonal antibody were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Horseradish peroxidase-conjugated secondary antibodies to mouse and goat immunoglobulins were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Other antibodies were from Santa Cruz Biolabs (Santa Cruz, CA). Immobilon-P membranes were from Millipore Co. (Bedford, MA). DIG RNA labeling kit, Anti-digoxigenin-AP antibodies, DNase I, protector RNase inhibitor, ␤-actin RNA probe, DIG Easy Hyb, Wash and Block Buffer Set, and positively charged nylon membranes were from Roche. A Х0.71 kb fragment of IGF-1R cDNA (EcoRI insert from ATCC clone pIGF-1R.8, subcloned into pGEM-7Zf) was kindly provided by Dr. Murray Korc (University of California, Irvine, CA). Other chemicals were purchased from Sigma or Fisher Scientific.
Animal Models of Diabetes-Two different models of diabetes were used in this study. Streptozotocin (STZ)-induced diabetes was obtained by injecting STZ (75 mg/kg body weight, intravenous) to 5-week-old Sprague-Dawley rats. Zucker Fatty rats (ZDF fa/fa) and lean control rats were obtained from Charles River Laboratories (9). Blood glucose levels were monitored by tail vein sampling, the diabetic rats were harvested 10 days after the onset of diabetes (random glucose Ͼ 200 mg/dL). The animal experimental protocol was approved by the IACUC at University of California, Irvine.
Cell Culture and Transduction of Adenoviral Constructs-Primary cultures of neonatal cardiomyocytes were prepared from Sprague-Dawley rats according to a protocol we previously described (2). The construction of recombinant adenoviruses expressing Hsp10, Hsp60, and the control adenovirus Ad-SR was described previously (7,10). In brief, the human HSP60 and HSP10 genes were cloned into the multiple cloning site of the adenoviral shuttle plasmid pACCMVpLpASR. The viruses were replicated in 293 cells, purified by Virakit TM from Virapau (Carlsbad, CA), and the viral titers were determined by plaque assay in 293 cells. Cardiomyocytes were plated in 100-mm Petri dishes in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. When indicated, the cells were infected with adenoviruses of Ad-SR, Ad-Hsp10, or Ad-Hsp60 and incubated for 36 -48 h at 37°C, 5% CO 2 .
Cell Viability-Cell viability was performed by staining the cells with calcein AM (Molecular Probe, Eugene, OR) as previously described (2). Calcein AM hydrolyzes to calcein and retains only in live cells, thus serves as an indicator for cell viability. To measure cell viability, cardiomyocytes were plated in 48-well plates. After rinsing twice with 1ϫ PBS buffer (pH 7.4), 200 l of 1 M calcein AM (in 1ϫ PBS) were added to each well and incubated for 45 min at room temperature in the dark. The plates were analyzed with the Cytofluor 2300 Fluorescence Measurement System (Millipore) at excitation of 485 nm and emission of 530 nm.
Immunoprecipitation-Immunoprecipitation was carried out as we previously reported (8). The cells were lysed and the lysates (500 g proteins in 1 ml) were pre-absorbed with 20 l protein A/G agarose beads (Santa Cruz Biotechnology) at 4°C for 30 min on a rocking platform, spun for 5 min at 10,000 rpm for 10 s, and the supernatant was incubated with specific primary antibody at 4°C overnight. After incubation with 20 l protein A/G agarose beads for 1.5 h at 4°C, the immunocomplexes were collected by centrifugation and washed three times with ice-cold washing buffer (137 mM NaCl, 20 mM Tris-HCl, pH 7.5; 1% Triton X-100, 2 mM EDTA, pH 8.0; 2 mM PMSF, and 2 mM Na 3 VO 4 ). The final products were briefly boiled and resolved with SDS-PAGE, and immunoblotted with specific antibodies as indicated.
Northern Blot Analysis-Total RNAs were isolated with TRIzol reagent (Invitrogen). The quality of RNA was verified by electrophoresis with 1% agarose gel containing 6% formaldehyde. Northern blots were performed with a non-radioactive method according to the manufacturer's instructions (Roche). In brief, equal amounts of total RNA were resolved with 1% agarose gel (6% formaldehyde), blotted, and cross-linked onto nylon membranes. IGF-1 receptor RNA probes were labeled with digoxigenin by in vitro transcription in the presence of digoxigenin-11-UTP. Hybridization was performed at 68°C overnight in DIG Easy Hyb solution (Roche) containing denatured digoxigenin-labeled RNA probes (100 ng/ml). After hybridization, the membranes were washed two times in 2ϫ SSC ϩ 0.1% SDS for 10 min at room temperature and two times in 0.1ϫ SSC ϩ 0.1% SDS at 60°C for 15 min. Membranes were sequentially incubated with a blocking solution for 30 min, anti-digoxigenin antibodies conjugated with alkaline phosphatase (1:10000) for 30 min at room temperature, and rinsed twice in the washing buffer (15 min each). After equilibrating for 5 min in the detection buffer, the blots were developed with the CDP-Star and exposed to film. ␤-actin probes were used as control probe in these blots.
Reducing Hsp60 Expression with Hsp60 Antisense Oligo-Hsp60 antisense oligonucleotides (AS) were introduced to cardiomyocytes to reduce the expression of endogenous Hsp60. Phosphorothioate oligonucleotides were obtained from TriLink BioTechnologies (San Diego, CA). The antisense Hsp60 (AS) sequence corresponds to bases 109 to 123 (5Ј-TAAGGCTCGAGCATC-3Ј) of rat Hsp60 and a scrambled oligo (SCR) (5Ј-GCTCGTGGTCAATAC-3Ј) was used as a control oligo as previously described (8). Cardiomyocytes were incubated with AS or SCR (25 g/ml) in serum-free medium for 24 h.
Statistical Analysis-The data were expressed as mean Ϯ S.E. based on data derived from three to six independent experiments. The intensity of bands from Western and Northern blots were scanned with densitometry and digitally analyzed. The statistical significance was tested by Student's t test or analysis of variance with post hoc analysis when appropriate. A p value below 0.05 was considered statistically significant.

IGF-1 Signaling in the Cardiomyocytes Overexpressing
Hsp10 or Hsp60 -If Hsp10 or Hsp60 can modulate IGF-1 receptor signaling, overexpression of Hsp10, and Hsp60 should change IGF-1 receptor autophosphorylation. To this end, neonatal cardiomyocytes were transduced with adenoviral vectors carrying Hsp10 or Hsp60. As shown in Fig 1a, the abundance of Hsp10 and Hsp60 respectively increased (Hsp60, 321 Ϯ 31%; Hsp10, 401 Ϯ 29%; p Ͻ 0.001 versus Ad-SR) in the cardiomyocytes infected with Ad-Hsp10 and Ad-Hsp60. Autophosphorylation of IGF-1 receptor was accordingly enhanced in the cardiomyocytes transduced with Hsp10 or Hsp60 (Fig. 1b). Immunoblots with anti-IGF-1 receptor ␤ subunits antibodies showed that the abundance of IGF-1 receptor protein was increased in the cells overexpressing Hsp10 or Hsp60 (Fig. 1b). Further analysis on receptor phosphorylation showed an increased stoichiometry of phosphorylation/receptor protein in the cells overexpressing Hsp10 or Hsp60 (p Ͻ 0.01, Ad-Hsp10 or Ad-Hsp60 versus Ad-SR), which suggests there were more functioning IGF-1 receptor available in response to IGF-1 activation (Fig. 1c). In these studies, the effect of Hsp60 appeared more robust than the effect of Hsp10 (p Ͻ 0.03). Further experiments showed that IGF-1-stimulated phosphorylation of MEK, Erk, Akt, and p90Rsk were accordingly increased in the cells overexpressing Hsp10 or Hsp60 (Fig. 1d). The effect of Hsp10 and Hsp60 on IGF-1 signaling occurred at pharmacological as well as physiological concentrations of IGF-1 (Fig.  1e). The effects of Hsp60 and Hsp10 on signaling were more significant at physiological concentrations of IGF-1. At maximal concentration of IGF-1 (10 Ϫ8 M) the augmentative effect of Hsp10 and Hsp60 were moderate. Interestingly, at physiological concentrations of IGF-1, phosphorylation of Akt were more robust than phosphorylation of MEK and p90RSK in the cells overexpressing Hsp60.
The Effect of Hsp10 and Hsp60 Involves Post-translational Modification of IGF-1 Receptor-To determine whether the effect of Hsp10 and Hsp60 on IGF-1 receptor involves increased synthesis IGF-1 receptor, cycloheximide or vehicles were added to the culture medium in the cells overexpressing Hsp10 or Hsp60 (Fig. 2a). In these cells, cycloheximide reduced the abun-dance of IGF-1 receptor; the relative elevation of IGF-1R in the Hsp10/60-transduced cells treated with cycloheximide was the same as in the controls. However, the level of IGF-1 receptor remained higher in the cells overexpressing Hsp10 or Hsp60. This suggests that the effect of Hsp10 and Hsp60 on IGF-1 receptor did not involve protein synthesis. To further confirm that Hsp10 or Hsp60 did not increase IGF-1 receptor synthesis, we had analyzed the abundance of IGF-1 receptor mRNA with Northern blots (Fig. 2b). The results showed that IGF-1 receptor mRNA level was not up-regulated by Hsp10 or Hsp60. Cardiomyocytes were transduced with Ad-SR (control vector), Ad-Hsp10, or Ad-Hsp60 for 48 h. The cell lysates were immunoblotted with antibodies against Hsp10 or Hsp60. b, tyrosine phosphorylation of IGF-1R. After overnight serum deprivation, cardiomyocytes were stimulated with IGF-1 for 2 min. The cell lysates were immunoprecipitated (IP) with anti-IGF-1 receptor antibody and then immunoblotted (IB) with anti-phosphotyrosine or anti-IGF-1R antibodies. c, comparing the effect of Hsp10 and Hsp60 on IGF-1R protein and IGF-1R phosphorylation. These data derived from four independent experiments. The effect of Hsp60 on receptor phosphorylation is more potent than on receptor protein (*, p Ͻ 0.04). d, downstream IGF-1R signaling pathways. After overnight serum deprivation, cardiomyocytes were stimulated with IGF-1 as indicated. The cell lysates were immunoblotted with phospho-specific antibodies or antibodies against specific signaling kinase. e, dose-response effects of IGF-1 on Akt, Mek, and p90RSK. The data represent summarized results from four to six independent experiments. *, p Ͻ 0.01, Ad-Hsp60 versus Ad-SR.
These data render evidence that Hsp10 and Hsp60 might have modulated the abundance of IGF-1 receptor through posttranslational modification. Proteolysis can be enhanced through ubiquitination of proteins. To explore whether Hsp10 and Hsp60 can regulate ubiquitination of IGF-1 receptor, we next analyzed the abundance of ubiquitinated IGF-1 receptor ␤ subunits. As shown in Fig. 2c, the abundance of ubiquinated IGF-1 receptor ␤ subunits was significantly reduced in the cells transduced with Hsp10 and Hsp60. The ubiquitinated IGF-1 receptors migrated around 180 K d and around 130 -140 K d , suggesting that these receptors were polyubiquitinated. Thus, the effect of Hsp10 and Hsp60 on IGF-1 receptor degradation might have involved inhibition of IGF-1 receptor polyubiquitination. In consistent with the effect of Hsp10 and Hsp60 on IGF-1 receptor signaling, the effect of Hsp60 on receptor ubiquitination is more robust than the effect of Hsp10. These findings suggest that the effect of Hsp10 and Hsp60 on IGF-1 receptor signaling involves post-translational modification of receptor.
Antisense Hsp60 Reduced IGF-1 Receptor Signaling-If Hsp60 plays a major role in the modulation of IGF-1 receptor signaling in cardiac muscle, reduced expression of Hsp60 should lead to a reduction in IGF-1 receptor protein and receptor signaling. To this end, an antisense Hsp60 was transfected to the cardiomyocytes to reduce endogenous Hsp60 expression as shown in Fig. 3a. The control oligo (SCR) did not affect the expression of Hsp60. The reduction of Hsp60 was accompanied by a decrease of IGF-1 receptor protein in the cells transfected with antisense Hsp60. Using this approach, we have characterized the abundance of IGF-1 receptor protein and receptor tyrosine phosphorylation in the control and IGF-1-stimulated cardiomyocytes (Fig. 3b). IGF-1-stimulated receptor tyrosine phosphorylation was significantly reduced in the cells transfected with antisense Hsp60, and was accompanied by decreased activation of Akt, MEK, Erk, and p90Rsk. Thus, reducing Hsp60 expression lead to attenuation of IGF-1 receptor signaling. To determine whether down-regulation of Hsp60 attenuated survival action of IGF-1, cardiomyocytes were subjected to serum withdrawal in the presence or absence of IGF-1 (Fig. 3c). Serum withdrawal reduced cell survival in the cardiomyocytes. As expected, IGF-1 rescued cardiomyocytes from the effect of serum withdrawal in the un-transfected cells and the control-oligo-transfected cells. However, the pro-survival effect of IGF-1 was inhibited in the cells transfected with the antisense Hsp60.
The Relationship of Hsp60 and IGF-1 Receptor Protein in Diabetic Myocardium-Our findings thus far suggest that Hsp60 is involved in the regulation of IGF-1 receptor signaling in cardiac muscle cells. Because IGF-1 signaling enhances myocardial protection, Hsp60 may modulate myocardial survival through modulation of IGF-1 receptor signaling. An important feature of diabetic cardiomyopathy is decreased myocardial protection against myocardial injuries. To investigate whether the occurrence of diabetes lead to perturbation of Hsp60 and IGF-1 receptor, we first analyzed the abundance of myocardial Hsp60 and IGF-1 receptor protein in STZ-induced diabetes (Fig. 4a). The abundance of Hsp60 and IGF-1 receptor was concomitantly reduced in the STZ-diabetic myocardium. In contrast, the abundance of insulin receptor was up-regulated as expected (11). In addition to Hsp60, we also investigated Hsp10 in the normal and diabetic myocardium. Unfortunately, the levels of myocardial Hsp10 were quite low and could not be reliably detected with immunoblot. To further confirm the changes of Hsp60 and IGF-1 receptor in diabetic state, we next studied the ZDF rats and the lean controls. The levels of Hsp60 and IGF-1 receptor protein were reduced in the ZDF myocardium (Fig. 4, b and c). The content of Hsp60 and IGF-1 receptor proteins were both decreased in the myocardium and adipose tissue (Fig 4c). Hsp60 and IGF-1 receptor were unchanged in the liver, but Hsp60 and IGF-1 receptor were simultaneously increased in the kidney (Fig. 4c). These observations support FIG. 2. Hsp10 and Hsp60 regulated IGF-1 receptor protein through post-translational modification. a, cycloheximide did not block up-regulation of IGF-1R by Hsp10 and Hsp60. Immediately after the cardiomyocytes were transduced with Ad-SR (control vector), Ad-Hsp10, or Hs-Hsp60, the cells were then incubated with vehicles (Control) or cycloheximide (CHX) for 24 h. The abundance of IGF-1 receptor protein and ␤-actin was analyzed with immunoblot (IB). b, Northern blots of IGF-1R and ␤-actin mRNA. The cells were transduced with Ad-SR, Ad-Hsp10, or Hsp60 for 12, 24, or 48 h, and then the RNA was extracted for Northern blots. c, Hsp10 and Hsp60 attenuated ubiquitination of IGF-1R. The cell lysates were first immunoprecipitated with anti-ubiquitin (IP: ␣-Ubiquitin) antibodies and then immunoblotted with anti-IGF-1R antibodies (IB: ␣-IGF-1R␤).
our hypothesis that Hsp60 modulates the abundance of IGF-1 receptor. If the reduction of myocardial IGF-1 receptor in diabetic myocardium involves post-translational modification, ubiquitination of IGF-1 receptor should have increased in the diabetic myocardium. To this end, we have analyzed ubiquitination of IGF-1 receptor in the ZDF myocardium. As shown in Fig. 4c, there was increased polyubiquitination of IGF-1 receptor in the diabetic myocardium. These observations suggest that the mechanisms underlying the reduction of IGF-1 receptor in diabetic myocardium may involve down-regulation of Hsp60 and subsequent alterations in post-translational modification. DISCUSSION IGF-1 plays an important role in myocardial protection and remodeling (12). The biological actions of IGF-1 are mediated through activation of IGF-1 receptors on cell surface. Previous studies (1,13,14) have shown that IGF-1 receptor signaling can be modulated in a number of ways, such as increased synthesis of IGF-1 receptors, formation of hybrid IGF-1/insulin receptors, different IGF-1 receptor isoforms, and interactions between IGF-1 receptors and secondary signaling/adaptor proteins. In this study we have identified Hsp10 and Hsp60 as novel modulators of IGF-1 receptor signaling in cardiac muscle. This is the first report showing that members of the heat shock There are growing interests in the role of heat shock proteins in cardiac muscle biology (15). Heat shock proteins were originally discovered as a set of proteins induced by temperature increase. A wide array of biological actions of heat shock proteins have been identified, such as protein folding/unfolding, protein degradation, anti-oxidative stress, and anti-apoptosis. During the last two years, several studies showed important function of Hsp60 in the heart (7,16,17). The expression of myocardial Hsp60 is increased under ischemic distress (17), and overexpression of Hsp60 lead to inhibition of apoptosis in the cardiomyocytes underwent ischemic and reperfusion injuries (10). These findings, along with studies showed protective role of Hsp60 in tissues other than cardiac muscle (18), emphasized the protect effect of Hsp60. The present study showed a causal relationship between the abundance of Hsp60 and the magnitude of IGF-1 receptor signaling, which suggests the pro-survival action of Hsp60 may involve augmentation of IGF-1 receptor signaling. Although both Hsp10 and Hsp60 modulated IGF-1 receptor signaling, the effects of Hsp10 were less profound than that of Hsp60. Furthermore, the abundance of myocardial Hsp10 is low and cannot be reliably detected with Western blots in the two lines of normal/diabetic rats we studied (data not shown). Thus, between these two heat shock proteins, it appears that Hsp60 is the major modulator of IGF-1 receptor signaling in myocardium.
Emerging evidence suggest that heat shock proteins are involved in the regulation of intracellular signaling. Hsp90 chaperones STAT3 signaling at plasma membrane and during the formation of cytosolic STAT3 signaling complexes (19). Hsp90 also chaperones steroid receptor, Raf, Akt, and cdk4 (20,21,22). The role of Hsp60 on intracellular signaling is rarely explored, but Hsp60 was recently identified as a protein associated with integrin and is involved in the activation of ␣ 3 ␤ 1 integrin in breast cancer cells (23). As shown in the present study, Hsp60 likely modulates intracellular signaling at more than one signaling step. Some heat shock proteins, such as Hsp90, are known for their inhibitory effect on protein ubiquitination and degradation. Ubiquitin/proteasome pathway enables rapid protein degradation. Sepp-Lorenzino et al. (6) had shown that chemical inhibitors of the 20S proteasome can prevent degradation of IGF-1 receptor, which suggests ubiquitination of IGF-1 receptor play a major role in the regulation of IGF-1 receptor degradation.
Diabetic myocardium is different from other forms of cardiomyopathy regarding the expression of Hsp60. As discussed FIG. 4. Changes of Hsp60 and IGF-1 receptor in diabetic myocardium. a, the abundance of Hsp60 and IGF-1R in STZ-DM myocardium. The myocardium was harvested from the diabetic and control rats 10 days after the induction of diabetes. Myocardial proteins were immunoblotted for Hsp60, IGF-1R, and insulin receptor. The results from multiple experiments were analyzed with densitometry and presented in the bar graph. b, the changes of Hsp60 and IGF-1R in the myocardium of ZDF rats. This is a representative Western blot showing reduced Hsp60 and IGF-1 receptor in the cardiac muscle of ZDF-DM rats. Equal amounts of proteins were loaded in each lane. c, the changes of Hsp60 and IGF-1 receptor in various tissues in ZDF-DM rats. Bar graph represents summarized results from multiple experiments. d, polyubiquitination of IGF-1 receptor in the cardiac muscle of ZDF-DM rats. Myocardial proteins were immunoprecipitated with anti-ubiquitin antibodies (IP: Ubiquitin) and then immunoblotted with anti-IGF-1R␤ subunit antibodies (IB: ␣-IGF-1R␤). Bar graph represents summarized results from multiple experiments. earlier, Hsp60 expression is increased during ischemic/reperfusion injury, probably representing a myocardial self-defense mechanism (17). But we have found that Hsp60 expression was reduced in diabetic myocardium. Down-regulation of myocardial Hsp60 and its functional implications in the context of diabetic cardiomyopathy have not been investigated previously. The changes of myocardial Hsp60 and IGF-1 receptor were consistent in both STZ-DM (type 1 DM model) and ZDF-DM (type 2 DM model) rats, which indicates down-regulation of Hsp60 and IGF-1 receptor is a common feature of diabetic myocardium. These findings provide evidence that reduced Hsp60 proteins may contribute to an escalation of IGF-1 receptor ubiquitination and subsequent decline in the number of functional IGF-1 receptor in diabetic myocardium. Using antisense approach (Fig. 3), our results showed that the pro-survival effect of IGF-1 was suppressed when Hsp60 was decreased and IGF-1 signaling reduced. IGF-1 receptor signaling protects myocardium from injuries through suppression of oxidative stress, inhibition of cardiac muscle apoptosis, enhancement of contractile function, and modulation of cardiacspecific genes (2,3,24). Reduced IGF-1 receptor abundance can lead to decreased myocardial protection during myocardial ischemia and thus may play a fundamental role during the development of diabetic cardiomyopathy.
Our data not only raise the possibility that dysregulation of Hsp60-IGF-1 receptor axis represents a new paradigm contributing to the development of diabetic cardiomyopathy, but also corroborate and extend previous reports that augmentation of IGF-1 signaling will likely lead to better myocardial protection in diabetic cardiomyopathy (25). Further insight into the mechanisms responsible for the effect of Hsp10 and, especially, Hsp60 on ubiquitination of cardiac IGF-1 receptor may provide new opportunities to design novel therapeutic strategies for diabetic cardiomyopathy.