PGRMC1 Ablation Protects from Energy-Starved Heart Failure by Promoting Fatty Acid/Pyruvate Oxidation

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  Figure 1. Progesterone receptor membrane component 1 (PGRMC1) expression is associated with energy-starved heart failure. Public Gene Expression Omnibus (GEO) datasets were used for analysis. Patients data for analyses were from 12 non-failing hearts and 14 dilated cardiomyopathy cases in one dataset (GSE29819) and from 39 normal and 106 hypertrophic cardiomyopathy cases in another dataset (GSE36961). (A) mRNA expression levels of PGRMC1 (GSE29819 and GSE36961) and metabolic enzymes (GSE36961) were analyzed. (B) Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) immunostaining of H9C2 cells in a growth medium (Con; glucose 4500 mg/L, 5% fetal bovine serum (FBS)) and starvation medium (glucose 0 mg/L, 1% FBS). Notably, 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus control. (C) Western blotting analysis and quantification of the expression levels of PGRMC1, poly(ADP ribose) polymerase (PARP), cleaved PARP (Cl. PARP), and C/EBP homologous protein (CHOP) in hearts of resting and 72-h-starved mice. Ribosomal protein lateral stalk subunit P0 (RPLP0) was used as an internal control. Five mice from each group were used for the experiment. Student’s…

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  Figure 2. Pgrmc1 knockout (PKO) protects the heart from energy starvation-induced metabolic suppression. (A) Oxygen consumption (VO2; l/kg/h) and carbon dioxide production (VCO2; l/kg/h). (B) Respiratory exchange ratio (RER) was calculated as VCO2/VO2. (C) Respiratory quotient (RQ) was calculated as the proportion of VCO2 to VO2. (D,E) Heat generation and activity measurements. Comprehensive lab animal monitoring system (CLAMS) was adopted for all assessments. Mice (wild-type (WT); n = 6, PKO; n = 8) were fasted during the tests. Student’s t-test was used for analysis. (F) Survival rate of WT (n = 9) and PKO (n = 5) mice during fasting. Mice under fasting died spontaneously. Values represent the mean ± SD. * p < 0.05.

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  Figure 3. PKO increases fatty acid/pyruvate oxidation and restrains cardiac injury under energy starvation. (A) Levels of blood glucose (mg/dL), plasma free fatty acids (FFAs; µM), plasma triglycerides (TGs; U/I), and plasma total cholesterol (TCHO; U/I) in starved WT and PKO mice. (B) Heart weight (HW), body weight (BW), and HW/BW ratio in starved WT and PKO mice. (C) Western blotting analysis and quantification of the expression levels of PGRMC1, PARP, and Cl. PARP in the starved hearts of WT and PKO mice. β-Actin was used for an internal control. (D) mRNA expression levels of fatty acid oxidation enzymes in the starved hearts of WT and PKO mice. Rplp0 was used as an internal control. (E) Western blotting analysis and quantification of the levels of glycolysis and pyruvate oxidation enzymes in the starved hearts of WT and PKO mice. β-Actin was used as an internal control. Mice used for the experiments: 8 (WT) and 4 (PKO). Student’s t-test was used for analysis. Values represent the mean ± SD. * p < 0.05.

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  Figure 4. Pgrmc1 knockdown in cardiomyocytes reduces cardiac damage with induction of fatty acid/glucose oxidation in a low-glucose medium supplemented with fatty acids. (A) Western blotting analysis and quantification of the levels of PGRMC1, PARP, and cleaved PARP in H9c2 cells treated with the control small interfering RNA (siRNA) (control knockdown, CK) and Pgrmc1 siRNA (Pgrmc1 knockdown, PK). β-Actin was used as an internal control. (B) mRNA expression levels of fatty acid oxidation enzymes in H9c2 cells treated with the control siRNA (CK) and Pgrmc1 siRNA (PK). Rplp0 was used as an internal control. (C) Western blotting analysis and quantification of the levels of hexokinase (HK)-1, HK2, pyruvate kinase M2 (PKM2), and pyruvate dehydrogenase (PDH) in H9c2 cells treated with the control siRNA (CK) and Pgrmc1 siRNA (PK). β-Actin was used as an internal control. (D) Oxygen consumption rate (OCR) of CK and PK cells during mitochondrial stress test. ATP production and maximal respiration rates were calculated by changing the OCR after oligomycin and rotenone/antimycin (Rot/Ant) treatments, respectively. Cells were incubated in a medium containing low glucose (500 mg/L) and fatty acids…

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  Figure 5. Loss of Pgrmc1 increases the phosphorylation of AMP-activated protein kinase (AMPK) in the heart under energy starvation. (A) Western blotting analysis and quantification of the levels of pAMPK, tAMPK, and p/t AMPK in starved hearts of WT and PKO mice and H9c2 cells treated with the control siRNA (CK) and Pgrmc1 siRNA (PK). β-Actin was used as an internal control. Mice used for experiments were eight (WT) and four (PKO) in number. Cells were incubated in a medium containing low glucose (500 mg/L) and FAs (palmitic acid (110 µM)/oleic acid (220 µM)). (B) Western blotting analysis and quantification of the levels of PGRMC1, pAMPK, tAMPK, p/t AMPK, HK1, HK2, PKM2, and PDH in H9c2 cells treated with AICAR (200 µM) and Com C (5 µM). β-Actin was used for an internal control. (C) mRNA expression levels of fatty acids oxidation enzymes in H9c2 cells treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 200 µM) and compound C (Com C; 5 µM). Rplp0 was used as an internal control. All experiments were repeated at least three times. Student’s t-test was used for analysis. Values represent the mean ± SD. * p < 0.05.

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  Figure 6. PKO protects against cardiac injury induced by isoproterenol treatment. (A) Experimental scheme for isoproterenol-induced cardiac injury. Isoproterenol was injected five times (total 230 mg/kg) via a subcutaneous injection for two weeks. (B) Masson’s Trichrome staining of isoproterenol-injected WT and PKO hearts. Cardiac fibrosis was calculated by the ratio of blue area to brown area. Scale bar: 600 µm. (C) mRNA expression levels of transforming growth factor (Tgf)-β in isoproterenol-injected WT and PKO hearts. Rplp0 was used as an internal control. (D) mRNA expression levels of heart failure markers, i.e., actin alpha 1 (Acta1) and brain natriuretic peptide (Bnp), in isoproterenol-injected WT and PKO hearts. Rplp0 was used as an internal control. (E) mRNA expression levels of fatty acid oxidation enzymes, carnitine palmitoyltransferase 2 (Cpt2), very long-chain acyl-CoA dehydrogenase (Vlcad), and acyl-CoA oxidase 1 (Acox1), in isoproterenol-injected WT and PKO hearts. Rplp0 was used as an internal control. (F) Western blotting analysis and quantification of the levels of pAMPK, tAMPK, p/t AMPK, HK1, HK2, PKM2, and PDH in isoproterenol-injected hearts of WT and PKO mice. β-Actin…