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. 2017 May 18;8(5):e2799.
doi: 10.1038/cddis.2017.193.

Calcium Sensing Receptor Protects High Glucose-Induced Energy Metabolism Disorder via Blocking gp78-ubiquitin Proteasome Pathway

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Free PMC article

Calcium Sensing Receptor Protects High Glucose-Induced Energy Metabolism Disorder via Blocking gp78-ubiquitin Proteasome Pathway

Yuehong Wang et al. Cell Death Dis. .
Free PMC article

Abstract

Diabetic cardiomyopathy (DCM) is a major complication and fatal cause of the patients with diabetes. The calcium sensing receptor (CaSR) is a G protein-coupled receptor, which is involved in maintaining calcium homeostasis, regulating cell proliferation and apoptosis, and so on. In our previous study, we found that CaSR expression, intracellular calcium levels and cardiac function were all significantly decreased in DCM rats; however, the exact mechanism are not clear yet. The present study revealed the protective role of CaSR in myocardial energy metabolism disorder induced by high glucose (HG) as well as the underlying mechanism. Here, we demonstrated that HG decreased the expression of CaSR, mitochondrial fusion proteins (Mfn1, Mfn2), cell gap junction related proteins (Cx43, β-catenin, N-cadherin), and intracellular ATP concentration. In contrast, HG increased extracellular ATP concentration, the expression of gp78, mitochondrial fission proteins (Fis1, Drp1), and the ubiquitination levels of Mfn1, Mfn2 and Cx43. Moreover, CaSR agonist and gp78-siRNA significantly reduced the above changes. Taken together, these results suggest that HG induces myocardial energy metabolism disorder via decrease of CaSR expression, and activation of gp78-ubiquitin proteasome system. In turn, these effects disrupt the structure and function of the mitochondria and the cell gap junction, result in the reduced ATP synthesis and the increased ATP leakage. Stimulation of CaSR significantly attenuates HG-induced abnormal myocardial energy metabolism, suggesting CaSR would be a promising potential therapeutic target for DCM.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effect of HG on CaSR expression in primary cultured neonatal rat cardiomyocytes. The cardiomyocytes cultured in Control group (5.5 mM) and HG group (40 mM) with or without 5 μM NPS R568 or 3 μM Calhex231 for 48 h were collected for immunoblotting analysis of CaSR expression. (a) Representative western blot of CaSR expression in cardiomyocytes exposed to HG in the presence of 5 μM NPS R568 or 3 μM Calhex231; (b) The CaSR protein levels normalized by β-actin. All data expressed as means±S.E. (n=3). *P<0.05 versus Control group; #P<0.05 versus HG group
Figure 2
Figure 2
Mitochondrial dysfunction caused by HG was attenuated through CaSR activation. The cardiomyocytes were cultured in control group (5.5 mM) and HG group (40 mM) with or without 5 μM NPS R568 or 3 μM Calhex231 for 48 h. (a) The intracellular ATP of each group was detected by chemiluminescence (n=3). (b) Representative western blot of UQCRQ, ND1, ATP5F1, COX5A, SDHA and Hsp70 in comparison with VDAC expression in cardiomyocytes exposed to HG in the presence of 5 μM NPS R568 or 3 μM Calhex231 (n=3). (c) Activities of complex (IV) were detected by UV Spectrophotometry (n=4). (d) Δψm was measured by JC-1 staining and the images were obtained by fluorescent microscopy. Scale bars=200 μm. The average fluorescence intensities are expressed as the ratio of red to green (n=10). (e) Calcein-AM was used to measure the changes of mPTP opening. Cells in different groups were stained by the calcein-AM. The images were obtained by fluorescent microscopy. Scale bars=200 μm (n=10). The changes in fluorescence intensity are varies inversely with the opening degree of mPTP. *P<0.05 versus control; #P<0.05 versus HG
Figure 3
Figure 3
Effect of HG and CaSR activation on the expression of mitochondrial fusion and fission-related proteins. The inhibition of mitochondrial fusion by HG were attenuated by NPS R568. Expression of Mfn1, Mfn2, Fis1 and Drp1 in cultured cardiomyocytes in Control group (5.5 mM) and HG group (40 mM) after various treatment for 48 h. (a) Representative western blot of Mfn1, Mfn2, Fis1 and Drp1 in comparison with VDAC expression in cardiomyocytes exposed to HG in the presence of 5 μM NPS R568 or 3 μM Calhex231. (b) Morphology of mitochondria were detected by Mito-Tracker and photo were taken by fluorescence microscopy. Scale bars=10 μm (n=12). (c) The average length of the mitochondria in each group was quantified (n=12). *P<0.05 versus control; #P<0.05 versus HG
Figure 4
Figure 4
Activation of CaSR blocks HG-induced Cx43 degradation and stabilizes function of cell gap junction. HG-induced degradation of β-catenin, N-cadherin and Cx43. NPS R568 could decrease the effect of HG. (a) Representative western blot of β-catenin, N-cadherin and Cx43 in comparison with Na+, K+-ATPase expression in cardiomyocytes exposed to HG in the presence of 5 μM NPS R568 or 3 μM Calhex231 (n=3). (b) The expression of β-catenin and N-cadherin complex were detected (n=3). A typical blot is shown. (c) The function of Cx43 was measured by scrape-loading dye transfer technique (SLDT) and photoed by fluorescence microscopy. Scale bars=10 μm (n=3). The yellow arrow represents the scratch. (d) The extracellular ATP content of each group was detected by chemiluminescence (n=3). *P<0.05 versus control; #P<0.05 versus HG
Figure 5
Figure 5
Activation of CaSR inhibits the phosphorylation of GSK-3β and the nuclear translocation of β-catenin induced by HG. HG promoted the nuclear translocation of β-catenin, which was attenuated by NPS R568 pretreatment. (a) The whole-cell proteins from primary neonatal rat cardiomyocytes in control group (5.5 mM) and HG group (40 mM) after various treatment conditions for 48 h were detected by immunoblotting with antibodies against N-β-catenin, Cyto-β-catenin, p-β-catenin, p-GSK-3β and total GSK-3β. A representative blot was shown. (b) The protein levels of N-β-catenin were quantified as a ratio against Histone H3. The expression of Cyto-β-catenin and p-β-catenin were quantified as a ratio against β-actin. The expression of p-GSK-3β were quantified as a ratio against total GSK-3β (n=3). *P<0.05 versus control; #P<0.05 versus HG
Figure 6
Figure 6
Activation of CaSR can attenuate the degradation of Mfn1, Mfn2 and Cx43 by HG-gp78-ubiquitin proteasome pathway. The expression of gp78 and the ubiquitination level of Mfn1, Mfn2, Cx43 were detected in cultured cardiomyocytes in control group (5.5 mM) and HG group (40 mM) after various treatment for 48 h. (a) Representative western blot of gp78 in comparison with β-actin expression in cardiomyocytes exposed to HG in the presence of 5 μM NPS R568 or 3 μM Calhex231 (n=3); (b) Representative western blot of gp78 in cardiomyocytes which were transfected with gp78-siRNA or Con-siRNA (n=3). (c) The ubiquitination level of Mfn1, Mfn2 and Cx43 in cardiomyocytes which were transfected with gp78-siRNA or Con-siRNA and treated by 5 μM NPS R568 or 3 μM Calhex231 respectively by immunoprecipitation (n=3). A representative blot is shown. *P<0.05 versus control; #P<0.05 versus HG
Figure 7
Figure 7
Schematic diagram on the mechanism of CaSR cardioprotection via attenuating high glucose-induced myocardial energy metabolism disorder. The downregulation of CaSR expression in cardiomyocytes induced by HG have a crucial role in myocardial energy metabolism disorder. The decreased CaSR expression causes upregulation of gp78 expression and activation of the ubiquitin proteasome system, which then degrades Mfn1, Mfn2, respiratory chain complex protein, Cx43, N-cadherin and β-catenin protein, and results in the decrease of ATP synthesis and the increase of ATP leakage through damaged gap junction. Therefore, CaSR agonists are expected to be a new target for the prevention and treatment of DCM

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