Progesterone receptor membrane component 1 reduces cardiac steatosis and lipotoxicity via activation of fatty acid oxidation and mitochondrial respiration

Abstract

Obesity is implicated in cardiovascular disease and heart failure. When fatty acids are transported to and not adequately oxidized in cardiac cells, they accumulate, causing lipotoxicity in the heart. Since hepatic progesterone receptor membrane component 1 (Pgrmc1) suppressed de novo lipogenesis in a previous study, it was questioned whether cardiac Pgrmc1 protects against lipotoxicity. Hence, we focused on the role of cardiac Pgrmc1 in basal (Resting), glucose-dominant (Refed) and lipid-dominant high-fat diet (HFD) conditions. Pgrmc1 KO mice showed high FFA levels and low glucose levels compared to wild-type (WT) mice. Pgrmc1 KO mice presented low number of mitochondrial DNA copies in heart, and it was concomitantly observed with low expression of TCA cycle genes and oxidative phosphorylation genes. Pgrmc1 absence in heart presented low fatty acid oxidation activity in all conditions, but the production of acetyl-CoA and ATP was in pronounced suppression only in HFD condition. Furthermore, HFD Pgrmc1 KO mice resulted in high cardiac fatty acyl-CoA levels and TG level. Accordingly, HFD Pgrmc1 KO mice were prone to cardiac lipotoxicity, featuring high levels in markers of inflammation, endoplasmic reticulum stress, oxidative stress, fibrosis, and heart failure. In vitro study, it was also confirmed that Pgrmc1 enhances rates of mitochondrial respiration and fatty acid oxidation. This study is clinically important because mitochondrial defects in Pgrmc1 KO mice hearts represent the late phase of cardiac failure.

Figure 1

Pgrmc1 KO mice show low cardiac mitochondrial metabolism in resting state. (A) Schematic diagram which presents experimental schedule for resting state. (B) Levels of blood glucose (mg/dL) and plasma FFA (µM) in resting WT and Pgrmc1 KO mice. (C) Expression of mitochondrial DNA (mtDNA) in hearts of resting WT and Pgrmc1 KO mice. Nuclear DNA was used for an internal control. mRNA expression of TCA cycle and OXPHOS in hearts of resting WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (D) mRNA expression of contractility markers in hearts of resting WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (E) Fatty acid oxidation activity in hearts of resting WT and Pgrmc1 KO mice. mRNA expression of fatty acid oxidation genes in hearts of resting WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (F) Western blot analysis and quantification of glycolysis and glucose oxidation genes in hearts of resting WT and Pgrmc1 KO mice. β-actin was used for an internal control. (G) Cardiac FFA level (µM) and fatty acyl-CoA levels (pmol/mg) in resting WT and Pgrmc1 KO mice. (H) Levels of glucose metabolites (unit) in resting WT and Pgrmc1 KO mice. (I) Levels of coenzyme A (CoA) and acetyl-CoA in resting WT and Pgrmc1 KO mice. (J) Levels of TCA cycle intermediates in resting WT and Pgrmc1 KO mice. (K) Ratio of ATP per ADP in resting WT and Pgrmc1 KO mice. Values represent means ± SD. *p < 0.05. Student’s t test was performed. Total numbers of mice used for experiment were 5 (control WT), 4 (control Pgrmc1 KO).

Figure 2

Pgrmc1 KO mice show high levels of cardiac FFA and paltimoyl-CoA, but low levels of cardiac glucose metabolites and TCA cycle intermediates in refed state. (A) Schematic diagram which presents experimental schedule for refed state. (B) Levels of blood glucose (mg/dl) and plasma FFA (µM) in refed WT and Pgrmc1 KO mice. (C) Expression of mitochondrial DNA (mtDNA) in hearts of refed WT and Pgrmc1 KO mice. Nuclear DNA was used for an internal control. mRNA expression of TCA cycle and OXPHOS in hearts of refed WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (D) mRNA expression of contractility markers in hearts of refed WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (E) Fatty acid oxidation activity in hearts of refed WT and Pgrmc1 KO mice. mRNA expression of fatty acid oxidation genes in hearts of refed WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (F) Western blot analysis and quantification of glycolysis and glucose oxidation genes in hearts of refed WT and Pgrmc1 KO mice. β-Actin was used for an internal control. (G) Cardiac FFA level (µM) and fatty acyl-CoA levels (pmol/mg) in refed WT and Pgrmc1 KO mice. (H) Levels of glucose metabolites (unit) in refed WT and Pgrmc1 KO mice. (I) Levels of coenzyme A (CoA) and acetyl-CoA in refed WT and Pgrmc1 KO mice. (J) Levels of TCA cycle intermediates in refed WT and Pgrmc1 KO mice. (K) Ratio of ATP per ADP in refed WT and Pgrmc1 KO mice. Values represent means ± SD. *p < 0.05. Student’s t test was performed. Total numbers of mice used for experiment were 8 (refed WT), and 6 (refed Pgrmc1 KO).

Figure 3

Pgrmc1 KO mice increases cardiac fatty acyl-CoA levels, but suppresses production of acetyl-CoA and ATP. (A) Schematic diagram which presents experimental schedule for HFD state. (B) Levels of blood glucose (mg/dl) and plasma FFA (µM) in HFD WT and Pgrmc1 KO mice. (C) Expression of mitochondrial DNA (mtDNA) in hearts of HFD WT and Pgrmc1 KO mice. Nuclear DNA was used for an internal control. mRNA expression of TCA cycle and OXPHOS in hearts of HFD WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (D) mRNA expression of contractility markers in hearts of HFD WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (E) Fatty acid oxidation activity in hearts of HFD WT and Pgrmc1 KO mice. mRNA expression of fatty acid oxidation genes in hearts of HFD WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (F) Western blot analysis and quantification of glycolysis and glucose oxidation genes in hearts of HFD WT and Pgrmc1 KO mice. β-actin was used for an internal control. (G) Cardiac FFA level (µM) and fatty acyl-CoA levels (pmol/mg) in HFD WT and Pgrmc1 KO mice. (H) Levels of glucose metabolites (unit) in HFD WT and Pgrmc1 KO mice. (I) Levels of coenzyme A (CoA) and acetyl-CoA in HFD WT and Pgrmc1 KO mice. (J) Levels of TCA cycle intermediates in HFD WT and Pgrmc1 KO mice. (K) Ratio of ATP per ADP in HFD WT and Pgrmc1 KO mice. Values represent means ± SD. *p < 0.05. Student’s t test was performed. Total numbers of mice used for experiment were 10 (HFD WT) and 7 (HFD Pgrmc1 KO).

Figure 4

HFD Pgrmc1 KO mice present high cardiac lipid accumulation. (A) mRNA expression of fatty acid elongation and desaturation genes in hearts of HFD WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (B) mRNA expression of fatty acid esterification and desaturation genes in hearts of HFD WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (C) Western blot analysis and quantification of SPT1 in hearts of HFD WT and Pgrmc1 KO mice. β-Actin was used for an internal control. (D) Oil-Red-O staining and quantification in hearts of HFD WT and Pgrmc1 KO mice (scale bar 25 µm) Positive area was measured by Image J program. Red area was set as positive. (E) Relative TG levels in HFD WT and Pgrmc1 KO mice. Values represent means ± SD. *p < 0.05. Student’s t test was performed. Total numbers of mice used for experiment were 10 (HFD WT) and 7 (HFD Pgrmc1 KO).

Figure 5

Pgrmc1 KO mice shows lipotoxicity in HFD condition. (A) Gross image of hearts of HFD WT and Pgrmc1 KO mice. Heart weight (HW), HW per body weight (BW) and HW per tibia length were measured. (B) H&E staining of hearts of HFD WT and Pgrmc1 KO mice (scale bar 50 µm). Nucleus per area was measured by Image J program. (C) Western blot analysis and quantification of ER stress genes in hearts of HFD WT and Pgrmc1 KO mice. β-Actin was used for an internal control. (D) Cardiac levels of reduced (GSH) and oxidized (GSSG) glutathione (µM), and ratio of GSSG:GSH in HFD WT and Pgrmc1 KO mice. (E) mRNA expression of pro-inflammatory genes in hearts of HFD WT and Pgrmc1 KO mice. Rplp0 was used for an internal control. (F) Plasma CPK (U/I) level of HFD WT and Pgrmc1 KO mice. (G) Masson trichrome staining of hearts of HFD WT and Pgrmc1 KO mice (scale bar 30 µm). Positive area was measured by Image J program. Blue fibroblasts were set as positive. (H) Western blot analysis and quantification of ANP in hearts of HFD WT and Pgrmc1 KO mice. β-Actin was used for an internal control. Values represent means ± SD. *p < 0.05. Student’s t test was performed. Total numbers of mice used for experiment were 10 (HFD WT) and 7 (HFD Pgrmc1 KO).

Figure 6

Pgrmc1 increases mitochondrial respiration and fatty acid oxidation in H9c2 cells. (A) Western blot analysis and quantification of PGRMC1 in H9c2 cells. β-Actin was used for an internal control. (B) Copies of mitochondrial DNA (mtDNA) in H9c2 cells. Nuclear DNA (nDNA) was used for an internal control. (C) Mitochondrial respiration measured by using flux analyzer in H9c2 cells. Values were normalized to baseline. (D) Glycolysis rate measured by using flux analyzer in H9c2 cells. Values were normalized to baseline. (E) Fatty acid oxidation rate measured by mitochondrial stress test using flux analyzer in palmitate-BSA treated condition in H9c2 cells. Values were normalized to baseline. (F) Oil-Red-O staining in palmitate-BSA treated condition in H9c2 cells (scale bar 100 µm). Positive area was measured by Image J program. Red area was set as positive. Values represent means ± SD. *p < 0.05. Student’s t test was performed. All experiments were repeated at least 3.