Insulin deficiency downregulates HSP60 and IGF-I receptor signaling and disrupts intracellular signaling homeostasis in diabetic cardiac muscle. Our previous studies had shown that IGF-I receptor signaling can be modulated by the abundance of HSP60. Since HSP60 localizes to the cytoplasmic compartment and mitochondria, this study was carried out to determine the distribution of cytosolic and mitochondria HSP60 in diabetic myocardium and to explore whether cytosolic HSP60 can modulate IGF-I receptor signaling in cardiac muscle cells. In streptozotocin-induced diabetes, both the cytosolic and mitochondrial fractions of HSP60 were decreased in the myocardium. Incubating primary cardiomyocytes with insulin leads to increased abundance of HSP60 in the cytosolic and mitochondria compartments. To determine whether cytosolic HSP60 can modulate IGF-I receptor signaling, we used rhodamine 6G to deplete functional mitochondria in cardiomyocytes. In the mitochondria-depleted cells, overexpression of HSP60 with adenoviral vector increased the abundance of IGF-I receptor, enhanced IGF-I-activated receptor phosphorylation, and augmented IGF-I activation of Akt and ERK. Thus overexpressing HSP60 in the cytosolic compartment enhanced IGF-I receptor signaling through upregulation of IGF-I receptor protein. However, IGF-I receptor signaling was significantly reduced in the mitochondria-depleted cells, which suggested that maintaining normal IGF-I receptor signaling in cardiomyocytes required functioning mitochondria. The effect of cytosolic HSP60 involved suppression of ubiquitin conjugation to IGF-I receptor in cardiomyocytes. These data suggest two different mechanisms that can regulate IGF-I signaling, one via cytosolic HSP60 suppression of IGF-I receptor ubiquitination and the other via mitochondria modulation. These findings provide new insight into the regulation of IGF-I signaling in diabetic cardiomyopathy.
- insulin receptor
- insulin-like growth factor I
- heat shock protein-60
diabetic cardiomyopathy is associated with increased risk of heart failure and cardiac mortality (1, 6). The mechanisms of diabetic cardiomyopathy are not yet clear, but our recent studies (18) suggested that reduced IGF-I receptor signaling may contribute to decreased myocardial protection in diabetic animals. The etiology of IGF-I receptor downregulation in diabetic myocardium probably involves dysregulation of HSP60 expression. We (2, 18) had shown that insulin deficiency led to downregulation of HSP60 in diabetic myocardium, and HSP60 downregulation in turn contributed to increased IGF-I receptor degradation and, hence, attenuated IGF-I receptor signaling. Heat shock proteins (HSPs) are a group of molecular chaperones that help polypeptide chain folding and prevent protein damage and proteolysis. Eighty to eighty-five percent of HSP60 are located in the mitochondria and play an important role in maintaining normal mitochondrial function (11). Fifteen to twenty percent of HSP60 are localized in the cytosolic compartment, but the function of cytosolic HSP60 on cardiac muscle biology remains to be defined.
Both IGF-I and HSP60 can protect myocardium against injuries (10, 12, 19). We had suggested that downregulation of HSP60 and IGF-I receptor signaling may contribute to the development of diabetic cardiomyopathy (2, 18). Since HSP60 reduced the abundance of ubiquitinated IGF-I receptor and ubiquitination occurs in the cytoplasm, it seemed logical to speculate that the effect of HSP60 on IGF-I receptor degradation is mediated through the cytoplasmic pool of HSP60 in cardiomyocytes. However, whether diabetes downregulates HSP60 in the cytosolic compartment has never been studied. The goals of this study were to determine the distribution of cytosolic and mitochondria HSP60 in diabetic myocardium and to explore whether cytosolic HSP60 can modulate IGF-I receptor signaling in cardiac muscle cells.
RESEARCH DESIGN AND METHODS
Mouse anti-HSP60 monoclonal antibody was purchased from StressGen Biotechnologies (Victoria, BC, Canada). Other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). MitoTracker Green FM, anti-oxidative phosphorylation (OxPhos) complex V, Alexa Fluor 594 goat anti-mouse IgG2b, and Alexa Fluor 488 goat anti-mouse IgG1 antibodies were obtained from Molecular Probes (Eugene, OR). Protein A/G Plus-agarose beads were from Santa Cruz Biotechnology. IGF-I is from GroPep (Adelaide, Australia); other chemicals were purchased from Sigma (St. Louis, MO) or Fisher Scientific (Fairlawn, NJ).
Animal model of diabetes.
Streptozotocin (STZ)-induced diabetes was produced by injecting STZ (80 mg/kg body wt ip) into Sprague-Dawley rats. Blood glucose levels were monitored by tail vein sampling. The diabetic rats were harvested 4 days after the onset of diabetes (random glucose >200 mg/dl). The animal experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of California, Irvine, CA.
Cardiomyocyte culture and transduction of adenoviral constructs.
Primary cultures of neonatal cardiomyocytes were prepared from Sprague-Dawley rats according to a protocol we described previously (22). The construction of recombinant adenovirus with HSP60 and the control adenovirus adenoviral vector (Ad-SR) had been described in our previous studies (18, 19). The adenoviruses were replicated in 293 cells and purified by Virakit (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% FBS and 1% penicillin/streptomycin. When indicated, the cells were infected with equal amounts of Ad-SR or adenoviral vector carrying HSP60 (Ad-HSP60) and incubated for 72 h at 37°C, 5% CO2.
Cardiomyocytes or pulverized myocardial tissues were washed twice with ice-cold PBS and scraped/mixed with mitochondria isolation buffer (20 mM HEPES-KOH, pH 7.2, 10 mM KCl, 1.5 mM MgCL2, 1.0 mM EDTA, 1.0 mM EGTA, 250 mM sucrose, 1.0 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mM each leupeptin and aprotinin). The mixtures were homogenized by being passed 15 times through a Dounce Homogenizer and then centrifuged at 750 g for 15 min to remove nuclei and unbroken cells. The supernatant was collected and subjected to further centrifugation for 15 min at 10,000 g to pellet the mitochondrial fraction. The supernatant was saved as cytosolic fractions. After determining the protein concentrations, the resulting cytosolic and mitochondrial extracts were used for immunoblotting.
Immunofluorescence study of subcellular HSP60.
Cardiomyocytes were grown on slides and permeabilized with 50% methanol-50% acetone for 30 min at −20°C, washed with PBS, and incubated with PBS blocking buffer containing 3% BSA and 0.2% Tween-20. OxPhos V antibody was diluted 1:100 in blocking buffer and incubated with slides for 1 h at room temperature, followed by three washes with PBS-Tween-20 buffer and incubation with Alexa Fluor 594 goat anti-mouse IgG2b antibodies for 30 min. Slides were again washed with PBS-Tween-20 buffer and incubated with HSP60 antibodies (1:100 dilution) in blocking buffer for 1 h, followed by three washes with PBS-Tween-20 buffer and incubation with Alexa Fluor 488 goat anti-mouse IgG1 antibodies for 30 min. The slides were viewed with a Zeiss Axiophot epifluorescence microscope. The images were recorded with a Sensys digital camera and captured with AxioVision imaging software.
MitoTracker Green stainning of mitochondria.
Cardiomyocyte mitochondria were stained with MitoTracker Green FM (Molecular Probes, Eugene, OR). After treatment with 5 μg/ml rhodamine 6G (R6G) or vehicles for 2 days, cardiomyocytes were incubated with 10 nM MitoTracker Green for 60 min, washed with PBS twice, and then visualized with a Zeiss Axiophot epifluorescence microscope.
The tissues and cells were lysed with a lysis buffer (137 mM NaCl, 20 mM Tris·HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 0.5% Nonidet P-40, 2 mM EDTA, pH 8.0, 3 μg/ml aprotinin, 3 μg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 10 mM NaPP, and 2 mM Na3VO4). Equal amounts of proteins from each sample were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membrane and incubated with a blocking buffer (5% nonfat milk or 3% BSA in 20 mM Tris·HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4°C, washed three times (20 mM Tris·HCl, pH 7.5, 137 mM NaCl, and 0. 1% Tween 20), incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000 to 1:10,000 dilution) for 1 h at room temperature, washed three times, and then detected with enhanced chemiluminescence (Pierce).
The protein lysates (1,000 μg of protein/ml) were preabsorbed with 20 μl of protein A/G-agarose beads (Santa Cruz Biotechnology) at 4°C for 30 min on a rocking platform and spun for 5 min at 10,000 g, and the supernatants were incubated with specific primary antibody at 4°C overnight. After incubation with 20 μl of 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 Na3VO4). The final products were briefly boiled and resolved by SDS-PAGE and immunoblotted with specific antibodies as indicated.
Ubiquitin conjugation and pull-down assay.
Glutathione S-transferase (GST)-ubiquitin fusion proteins were transformed into E. coli, and expression was induced with isopropyl β-d-thiogalactoside for 16 h at 26°C. Recombinant proteins were purified on glutathione-Sepharose. Bacterially-expressed GST fusion proteins were purified and bound to agarose-GSH beads as described in the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ). Cardiomyocytes were lysed with a lysis buffer (137 mM NaCl, 20 mM Tris·HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 0.5% Nonidet P-40, 3 μg/ml aprotinin, 3 μg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4), and 1,000 μg of cell lysates were incubated with GSH-agarose-bound GST-ubiquitin (GST-Ub) overnight, centrifuged, washed four times with cell lysis buffer, and boiled in sample buffer for immunoblotting.
The data were expressed as means ± SE on the basis of data derived from three to six independent experiments. The intensity of bands from Western blots were scanned with densitometry and digitally analyzed. The statistical significance was tested by Student’s t-test or ANOVA with post hoc analysis when appropriate. A P value <0.05 was considered statistically significant.
Cytosolic and mitochondria HSP60 were reduced in diabetic myocardium.
Insulin increased the expression of cardiac HSP60, and diabetes led to decreased HSP60 expression in myocardium because of relative insulin deficiency (2, 18). The first series of experiments were to determine the effect of diabetes on subcellular distribution of HSP60. Diabetes was induced with STZ injection, and myocardial proteins were subfractionated and resolved by SDS-PAGE and immunoblotted with specific antibodies (Fig. 1). In the diabetic myocardium, the abundance of cytosolic HSP60 was significantly decreased in the STZ-diabetes myocardium. Interestingly, more significant reduction of mitochondria HSP60 was observed in the diabetic myocardium (P < 0.02, cytosolic vs. mitochondria fractions). Concurrent immunblotting with anti-OxPhos V suggested that the cytosolic and mitochondria fractions were successfully separated. In the diabetic myocardium, cytosolic HSP60 was reduced by 35% and mitochondria HSP60 by 81%. These data indicate that both cytosolic and mitochondria HSP60 were reduced in the diabetic myocardium.
Insulin upregulated cytosolic and mitochondria HSP60 in cardiomyocytes.
Diabetes is associated with inadequate insulin action (insulin deficiency/resistance). To determine the effect of insulin on cytosolic and mitochondria HSP60 in cardiac muscle cells, we incubated neonatal cardiomyocytes with insulin after overnight serum deprivation. The abundance of HSP60 in the subfractionated cell lysates was determined with immunoblots. Results are shown in Fig. 2; insulin increased cytosolic HSP60 by 240% and mitochondria HSP60 by 36% in neonatal cardiomyocytes. To further confirm the effect of insulin on subcellular localization of HSP60, mitochondria were stained with anti-OxPhos complex V antibodies (red) and HSP60 was stained with antibodies conjugated with Alexa Fluor 488 (green) (Fig. 2, bottom). In neonatal cardiomyocytes, after serum deprivation (Basal), nearly all HSP60 colocalized with mitochondria. Insulin treatment increased HSP60 in the cytosolic compartment as well as the mitochondria compartment. These results indicate that insulin upregulated HSP60 in both cytosolic and mitochondria compartments in cardiac muscle cells.
Overexpression of cytosolic HSP60 increased IGF-I receptor abundance and signaling in cardiomyocytes.
To study whether cytosolic HSP60 can modulate IGF-I receptor, neonatal cardiomyocytes were treated with R6G. R6G is a fluorescent dye that selectively accumulates in the inner mitochondrial membrane and biochemically destroys mitochondria permanently (4, 23). Treatment with R6G renders cells without functioning mitochondria (21). MitoTracker Green staining showed that treating cardiomyocytes with R6G 5 μg/ml for 2 days depleted functional mitochondria in cardiomyocytes (Fig. 3A). If cytosolic HSP60 could increase IGF-I receptor signaling, overexpressing HSP60 in the mitochondria-depleted cells should increase IGF-I signaling. To this end, we have characterized IGF-I receptor abundance, receptor phosphorylation, and Akt and ERK activation in the mitochondria-depleted cardiomyocytes transduced with Ad-SR or Ad-HSP60. The results are shown in Fig. 3B; the abundance of IGF-I receptor was increased in the mitochondria-depleted cells overexpressing HSP60. Autophosphorylation of IGF-I receptor and activation of ERK and Akt were accordingly increased in these cells upon IGF-I stimulation (Fig. 3, B and C). These data indicate that overexpressing cytosolic HSP60 in the mitochondria-depleted cells increased IGF-I receptor abundance and enhanced receptor signaling in cardiomyocytes.
Decline of IGF-I receptor signaling in mitochondria-depleted cardiomyocytes.
We also investigated the effect of mitochondria depletion on IGF-I receptor. To determine whether mitochondria can modulate IGF-I receptor signaling in cardiomyocytes, we again utilized R6G to deplete functional mitochondria in cardiomyocytes. We have characterized the abundance of IGF-I receptor protein and receptor tyrosine phosphorylation in response to IGF-I stimulation. The abundance of IGF-I receptor was decreased in the R6G-treated cardiomyocytes (Fig. 4A), and IGF-I-stimulated receptor tyrosine phosphorylation, Akt, and ERK were accordingly reduced in the mitochondria-depleted cells. These results suggested that normal IGF-I receptor signaling requires functioning mitochondria in cardiomyocytes. To ensure that our experimental protocol with R6G did not confound our interpretation of the effect of cytosolic HSP60 on IGF-I receptor, we have characterized the time course effect of R6G in the cardiomyocytes infected with Ad-SR or Ad-HSP60 (Fig. 4B). Cardiomyocytes infected with Ad-HSP60 showed higher levels of HSP60 during the first three days of R6G treatment, and IGF-I receptor abundance was accordingly increased during the same time frame. However, IGF-I receptor, HSP60, and actin were all reduced to very low levels after 5 days of mitochondria depletion. Our results presented in Figs. 3 and 4A were obtained after 2-day R6G treatment. Therefore, under our experimental protocol, we were able to differentiate the effect of cytosolic HSP60 from the effect of mitochondria on IGF-I receptor and confirmed that overexpession of cytoplasmic HSP60 indeed increased IGF-I receptor abundance.
The effect of cytosolic HSP60 involves ubiquitination of IGF-I receptor.
Ubiquitination predisposes proteins for proteolysis in proteosome. Our previous studies (18) had shown that global overexpression of HSP60 reduced the abundance of ubiquitinated receptor and inhibited receptor degradation. However, we do not yet know whether overexpession of HSP60 in the cytosolic compartment can modulate ubiquitination of IGF-I receptor in cardiomyocytes. To this end, we have studied the effect of HSP60 overexpression on IGF-I receptor ubiquitination in the mitochondria-depleted cardiomyocytes. The abundance of ubiquitinated IGF-I receptor was significantly reduced in the mitochondria-depleted cells transduced with Ad-HSP60 (Fig. 5A). The ubiquitinated IGF-I receptors migrated ≥250 kDa on SDS-PAGE, suggesting that these receptors were polyubiquitinated. These results showed that cytosolic HSP60 might inhibit IGF-I receptor ubiquitination. R6G treatment under this protocol did not increase ubiquitination of IGF-I receptor (Fig. 5B). To further examine whether overexpression of HSP60 could reduce ubiquitination of IGF-I receptor, we carried out in vitro ubiquitination experiments in total cell lysates using GST-Ub. Cell lysates from cardiomyocytes infected with Ad-SR or Ad-HSP60 were incubated with GST-Ub, and proteins conjugated with GST-Ub were pulled down and resolved by SDS-PAGE. Overexpression of HSP60 inhibited IGF-I receptor conjugation with ubiquitin in vitro (Fig. 5C).
This is the first report to show that diabetic myocardium was associated with reduced cytosolic and mitochondria HSP60 and that cytosolic HSP60 modulated IGF-I receptor signaling in cardiomyocytes. The effect of cytosolic HSP60 on IGF-I receptor involved inhibition of receptor ubiquitination. In addition to the effect of cytosolic HSP60 on IGF-I receptor, mitochondria played a role in the regulation of IGF-I receptor signaling in cardiac muscle cells. This study suggests that two different mechanisms, in the cytosolic compartment and the mitochondria, could modulate IGF-I receptor signaling in cardiac muscle cells. These findings shed new light on the mechanisms leading to impaired IGF-I receptor signaling in diabetic myocardium.
Dysregulation of ubiquitin-proteasome system is a newly recognized paradigm underlying the development of heart failure (5). Our previous studies (2) indicated that insulin deficiency leads to downregulation of HSP60 in diabetic myocardium and, hence, downregulation of IGF-I receptor and its signaling. HSP60 does not modulate synthesis of IGF-I receptor in cardiomyocytes (18). Overexpression of HSP60 inhibited IGF-I receptor degradation by reducing the abundance of ubiquitinated receptor and augmented IGF-I signaling in cardiomyocytes (18). Whether the effect of HSP60 in cardiomyocytes involves cytoplasmic HSP60 and whether cytoplasmic HSP60 inhibits ubiquitin conjugation to IGF-I receptor were not known. The present study not only provided new evidence that cytoplasmic HSP60 upregulates IGF-I receptor abundance and IGF-I signaling but also confirmed that cytosolic HSP60 can inhibit conjugation of ubiquitin to IGF-I receptor in cardiomyocytes.
HSPs are a family of proteins that function as molecular chaperones and were originally discovered as proteins induced by temperature increase (20). HSP60 assists folding of proteins imported into mitochondria and has been recognized as a mitochondria chaperone (15). Seventy-five to eighty percent of HSP60 are in the mitochondria, and 15 to 20% are in the cytosolic compartment (9). In the mammalian cell, cytoplasmic HSP60 has a 26-amino acid signal sequence at the NH2 terminus of the protein, which is capable of folding into a positively charged amphiphilic helix. Mitochondria HSP60 does not have this sequence. Recent studies (9, 19) have shown that cytosolic HSP60 interacted with proapoptotic bak and bax and has a key antiapoptotic role in cardiomyocytes. In vitro experiments also showed a potential chaperoning effect of cytoplasmic HSP60 on immunophilin and thermal protein aggregation (8). The results of our study add another piece of evidence corroborating the chaperoning effect of cytoplasmic HSP60.
Mitochondria are the most abundant organelles in cardiac muscle and play pivotal roles in energy production, oxidative stress, and regulation of apoptosis. Myocardial mitochondria dysfunction has been described in human and animal models of diabetes (3, 14, 16, 17). Our data showed that adequate IGF-I receptor signaling could not be maintained without functional mitochondria. The effect of mitochondria depletion on IGF-I receptor signaling was mediated mainly through downregulation of receptor abundance. However, we do not know exactly how mitochondria modulate IGF-I receptor ubiquitination. To the best of our knowledge, this is the first time mitochondria were found to modulate membrane receptor signaling.
HSP60 was significantly reduced in the mitochondria of diabetic myocardium. Exactly how downregulation of HSP60 alters myocardial mitochondria function is not known. But the dramatic reduction of mitochondria HSP60 suggests that HSP60 transport into the mitochondria might have been impaired in diabetic myocardium. HSP60 is an important component of defense mechanisms against myocardial injury. There is ample evidence to suggest that HSP60 is involved in the regulation of mitochondria function. HSP60 showed antioxidative properties during myocardial ischemic distress (13). HSP60 modulated Bcl-2 protein family, helped maintain mitochondria cross-membrane electrochemical gradient, and increased resistance to apoptosis in cardiomyocytes (7, 11, 19). Therefore, downregulation of mitochondria HSP60 might have contributed to mitochondria dysfunction in diabetic myocardium. The findings presented in this study suggest the existence of a complex interplay among HSP60, mitochondria function, and IGF-I receptor signaling in cardiac muscle.
We have described a new paradigm in diabetic myocardium involving the cytoplasmic and mitochondria pool of HSP60, mitochondria, and the cell surface IGF-I receptor. Further investigation into how cytoplasmic HSP60 inhibits IGF-I receptor ubiquitination, how HSP60 modulates mitochondria function, and how mitochondria modulates IGF-I and other peptide growth factor signaling may provide new opportunities to understand a fundamental aspect of myocardial biology that had not been explored in the past.
This work is supported by grants from National Heart, Lung, and Blood Institute (HL-55533) and the University of California Irvine (to P. H. Wang), National Institute of General Medical Sciences (GM-66164) and the California Breast Cancer Research Program (to P. Kaiser), and National Institute of Diabetes and Digestive and Kidney Diseases (DK-073691, to D. Wallace).
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.
- Copyright © 2007 by American Physiological Society