Yuehong Wang1, Yuwen Wang2, Fadong Li1, Xinying Zhang1, Hongzhu Li1, Guangdong Yang3, Changqing Xu1, Can Wei4. 1. Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin, 150081, China. 2. Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150000, China. 3. Departemnt of Chemistry and Biochemistry, Laurentian University, Sudbury, P3E 2C6, Canada. 4. Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin, 150081, China. canwei528@163.com.
Abstract
PURPOSE: To determine the mediation of spermine on energy metabolism disorder and diabetic cardiomyopathy (DCM) development as well as the underlying mechanisms. METHODS: An in vitro model of DCM was established by incubating primary cultured neonatal rat cardiomyocytes with high glucose (HG). Spermine content was assessed by RP-HPLC. The protein levels were detected by western blot. Mitochondrial functions were analyzed using the respiratory chain complex assay kit and immunofluorescence staining. RESULTS: The endogenous content of spermine was decreased in the HG group, and the protein levels of ornithine decarboxylase, respiratory chain complex (I-V), mitochondrial fusion-related protein (Mfn1, Mfn2), Cx43, N-cadherin, CaSR, and β-catenin (in cytomembrane) were also down-regulated by HG. In contrast, the protein levels of spermine-N1-acetyltransferase, gp78, Fis1, Drp1, and β-catenin were up-regulated by HG. Meanwhile, we observed that HG increased ubiquitination levels of Mfn1, Mfn2, and Cx43, decreased membrane potential (ΔΨm), and the opening of mitochondrial permeability transport pore (mPTP) followed by intracellular ATP leakage. The supplement of spermine or siRNA-mediated knockdown of gp78 significantly alleviated the detrimental effects of HG, while downregulation of CaSR aggravated the development of DCM. We further confirmed that the lower level of spermine by HG activates the gp78-ubiquitin-proteasome pathway via downregulation of CaSR protein level, which in turn damages mitochondrial gap junction intercellular communication and leads to reduced ATP level. CONCLUSION: The protective role of spermine on energy metabolism disorder is based on higher CaSR protein level and lower gp78 activation, pointing to the possibility that spermine can be a target for the prevention and treatment of DCM.
PURPOSE: To determine the mediation of spermine on energy metabolism disorder and diabetic cardiomyopathy (DCM) development as well as the underlying mechanisms. METHODS: An in vitro model of DCM was established by incubating primary cultured neonatal rat cardiomyocytes with high glucose (HG). Spermine content was assessed by RP-HPLC. The protein levels were detected by western blot. Mitochondrial functions were analyzed using the respiratory chain complex assay kit and immunofluorescence staining. RESULTS: The endogenous content of spermine was decreased in the HG group, and the protein levels of ornithine decarboxylase, respiratory chain complex (I-V), mitochondrial fusion-related protein (Mfn1, Mfn2), Cx43, N-cadherin, CaSR, and β-catenin (in cytomembrane) were also down-regulated by HG. In contrast, the protein levels of spermine-N1-acetyltransferase, gp78, Fis1, Drp1, and β-catenin were up-regulated by HG. Meanwhile, we observed that HG increased ubiquitination levels of Mfn1, Mfn2, and Cx43, decreased membrane potential (ΔΨm), and the opening of mitochondrial permeability transport pore (mPTP) followed by intracellular ATP leakage. The supplement of spermine or siRNA-mediated knockdown of gp78 significantly alleviated the detrimental effects of HG, while downregulation of CaSR aggravated the development of DCM. We further confirmed that the lower level of spermine by HG activates the gp78-ubiquitin-proteasome pathway via downregulation of CaSR protein level, which in turn damages mitochondrial gap junction intercellular communication and leads to reduced ATP level. CONCLUSION: The protective role of spermine on energy metabolism disorder is based on higher CaSR protein level and lower gp78 activation, pointing to the possibility that spermine can be a target for the prevention and treatment of DCM.
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