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

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.


Pgrmc1 KO mice on a HFD present various cardiac lipotoxic markers.
While the cardiac lipid accumulation and low ATP production were observed in HFD Pgrmc1 KO mice, the heart did not show hypertrophy, as shown in Fig. 5A. Conversely, the heart weight (HW) was decreased (p < 0.05, 86.5% vs. HFD WT) in HFD Pgrmc1 KO mice. However, the ratio of HW/body weight (BW) was not decreased in HFD Pgrmc1 KO mice. Consistently, HW/tibia length was similar between HFD WT and Pgrmc1 KO mice (Fig. 5A). In H&E staining, number of www.nature.com/scientificreports/ nuclei per area was increased (p < 0.05, 2.18-fold vs. HFD WT) in the hearts of HFD Pgrmc1 KO mice (Fig. 5B), suggesting the cardiac cell size is smaller. As indicators of ER stress, the ratio of pEIF2α/EIF2α protein and the expression of CHOP protein were increased (p < 0.05, 1.86-and 1.78-fold, respectively, vs. HFD WT) in the hearts of HFD Pgrmc1 KO mice (Fig. 5C). Furthermore, oxidative stress was increased in the hearts of HFD Pgrmc1 KO mice; the level of reduced glutathione (GSH) was decreased (p < 0.05, 85.9% vs. HFD WT), while the levels of oxidized glutathione (GSSG) and the GSSG/GSH ratio had increased (p < 0.05, 1.2-and 1.4-fold, respectively, vs. HFD WT), as shown in Fig. 5D. The mRNA expression of pro-inflammatory cytokine genes, including Tnf, Il-1β, and Il-6, was increased (p < 0.05, 1.37-, 25-, and 6.02-fold, respectively, vs. HFD WT) in the hearts of HFD Pgrmc1 KO mice (Fig. 5E). The level of plasma creatine phosphokinase (CPK) was increased (p < 0.05, 1.33-fold vs. HFD WT) in HFD Pgrmc1 KO mice (Fig. 5F). Abnormal stress led to the induction of cardiac fibrosis, as observed by Masson's trichrome staining. A significant increase (p < 0.05, 1.44-fold vs. HFD WT) in fibroblasts (blue staining) was observed in the hearts of HFD Pgrmc1 KO mice (Fig. 5G). Furthermore, as a cardiac failure marker, the expression of ANP was increased (p < 0.05, 2.44-fold vs. HFD WT) in the hearts of HFD Pgrmc1 KO mice (Fig. 5H). Based on these results, we concluded that the metabolic phenotype of Pgrmc1 KO mice leads to cardiac lipotoxicity under HFD conditions, but does not trigger cardiac hypertrophy.

Pgrmc1 increases mitochondrial respiration and fatty acid oxidation in H9c2 cells. H9c2 cells
were transfected with Pgrmc1 siRNA, and the protein expression of PGRMC1 was suppressed (p < 0.05, 67.8% vs. control siRNA (CON)) in the Pgrmc1 siRNA group (Fig. 6A). The number of mtDNA copies was decreased (p < 0.05, 54.1% vs. CON) in the Pgrmc1 siRNA group (Fig. 6B). Using the seahorse flux analyzer in H9c2 cells, we monitored the oxygen consumption rate (OCR), wherein other endocrine factors were not considered.
The level of maximal respiration was suppressed (p < 0.05, 72.2% vs. CON) in the Pgrmc1 siRNA group (Fig. 6C). To measure the rate of fatty acid oxidation, palmitate was conjugated with CD-BSA (charcoal dextranbovine serum albumin) and used for treatment. The level of maximal respiration was decreased (p < 0.05, 76.8% vs. CON) in the Pgrmc1 siRNA group (Fig. 6D). Conversely, the levels of glycolysis and glycolytic capacity, which were measured by the extracellular acidification rate (ECAR), were increased (p < 0.05, 1.97-and 1.76fold, respectively, vs. CON) in the Pgrmc1 siRNA group (Fig. 6E). In the palmitate-BSA-treated condition, the positive area for Oil-Red-O staining was increased (p < 0.05, 1.86-fold vs. CON) in the Pgrmc1 siRNA group (Fig. 6F). According to the in vitro results, we confirmed that Pgrmc1 is involved in mitochondrial metabolism, especially for fatty acid oxidation in cardiac cells.

Discussion
Obesity is characterized by the gain of body weight and fat mass. When fat mass accumulates around abdominal organs, visceral obesity occurs, leading to susceptibility to cardiovascular conditions 14 . Regarding physiological aspects of the heart, cardiac TG accumulation is a common characteristic of most animal models of obesity, as the heart promotes fatty acid uptake even in a lipotoxic state 15 . Therefore, fatty acid disposal in the heart is crucial to maintain intracardiac lipid balance, but fatty acid oxidation is limited under condition of failing heart. Compensatory glucose utilization is activated, but ATP production cannot be fully replenished. Consequently, the lack of fatty acid oxidation leads to impairment of cardiac contractility 16 . In a HFD mouse model, Pgrmc1 KO hearts presented lipid accumulation and suppressed ATP production. This was due to impaired fatty acid oxidation and mitochondrial dysfunction of Pgrmc1 KO hearts. Cardiac mitochondrial function is crucial for energy metabolism because the heart relies on oxidative phosphorylation for energy production 17 . Cardiac cells possess large numbers of mitochondria 18 , and ATP produced by mitochondrial oxidative phosphorylation is used for cardiac contractile function, as it is hydrolyzed to ADP 19 .  In a pathological murine model, a high-fat, high-sucrose (HFHS) diet causes cardiac mitochondrial dysfunction and decreased cardiac ATP production 20 . From a biochemical perspective, ATP is generated in the electron transport system by NADH and FADH 2 , which are produced in the TCA cycle. Fatty acid and glucose oxidation both produce acetyl-CoA, which drives the TCA cycle, and the acetyl-CoA/CoA ratio indicates acetyl-CoA production 21 . Fatty acid oxidation is the primary pathway for ATP production in the heart, while glucose oxidation is the more efficient pathway 19 . Fatty acid oxidation inhibits glucose oxidation, thereby balancing cardiac metabolism 19,22 . While Pgrmc1 KO hearts lack fatty acid oxidation activity, the acetyl-CoA production was maintained in a resting and refed state. This result can be interpreted in two ways; (1) decrease of fatty acid oxidation was not enough to trigger a physiological response. (2) acetyl-CoA from glucose source compensated the level. Especially in the refed state, where glucose is enriched, the increased expression of glycolysis and glucose oxidation and low level of glucose metabolites in Pgrmc1 KO hearts were observed, suggesting compensatory recovery of acetyl-CoA from glucose. Conversely, in fat-rich condition (HFD), Pgrmc1 KO hearts presented compensatory response from glucose according to the induction of glucose metabolic pathway and low glucose metabolite levels, but acetyl-CoA production was suppressed. Furthermore, TCA cycle intermediates levels were low, especially for citrate/isocitrate, fumarate, and malate. According to the low capacity of ATP production in HFD Pgrmc1 KO hearts, these comparative results suggest that lack of fatty acid oxidation activity in fat-enriched conditions leads Pgrmc1 KO hearts to pronounced impairment of ATP production. While Pgrmc1 KO hearts www.nature.com/scientificreports/ could spend the cellular fatty acid in resting and refed conditions, they failed to fully use the cellular fatty acid in long-term HFD conditions. Low fatty acid usage can result in excessive residual fatty acyl-CoA and induce lipid accumulation in the heart. Palmitoyl-CoA is the first form of fatty acyl-CoA synthesized from acetyl-CoA, which is converted to stearoyl-CoA by elongation. Scd1 desaturates both palmitoyl-CoA and stearoyl-CoA to form oleoyl-CoA. Afterward, desaturated fatty acyl-CoA is esterified through catalysis by a series of enzymes, including Gpam, Agpat1, Mogat1, and Dgat1, to form triglycerides 8 . Meanwhile, ceramide also would be synthesized in the condition of lipid overload by conjugation of serine and palmitoyl CoA. The first and rate-limiting step of ceramide synthesis is controlled by SPT1 23 . When the heart of HFD Pgrmc1 KO mice has high levels of fatty acyl-CoA due to limited fatty acid oxidation activity, they could be successfully esterified to triglycerides or used for ceramide synthesis considering the expression of genes. Accordingly, lipid accumulation was observed in the heart of HFD Pgrmc1 KO mice.
Lipotoxicity triggered by lipid is controversial as it differs by lipid species, including TG, DAG, and ceramide 24 . Nonetheless, as the heart is not a conventional lipid-storing organ, lipid overload in the heart is still unusual and can cause cardiac abnormalities 25 . Lipid-induced heart failure accompanies hypertrophy, fibrosis, inflammation, and mitochondrial dysfunction, which lead to contractile dysfunction 26 . HFD Pgrmc1 KO hearts was not linked with cardiac hypertrophy according to similar HW/BW and HW/tibial length to those of HFD WT mice. Instead, HW and BW of HFD Pgrmc1 KO mice were both low. As weight gain during the HFD period was similar between WT and Pgrmc1 KO mice (Fig. S1), Pgrmc1 KO mice are phenotypically lighter than WT mice. Conversely, HFD Pgrmc1 KO hearts induced various heart failure markers. Increased pEIF2α and CHOP expression, high GSSG and low GSH levels, and increased inflammatory gene expression in Pgrmc1 KO heart are indicative of heart failur [27][28][29][30] . Plasma CPK was also increased, which serves as a cardiac damage marker 31 . Finally, fibrosis 27 and cardiac failure markers 32 suggest the cardiac failure of Pgrmc1 KO mice.
Our hypothesis that was based on the in vivo study was confirmed in an in vitro study. Pgrmc1 knockdown resulted in low mtDNA copy number and suppression of mitochondrial respiration in H9c2 cells. Furthermore, consistent to in vivo study, induction of glycolysis was observed in the Pgrmc1 knockdown group of H9c2 cells. Finally, suppression of fatty acid oxidation resulted in remarkable lipid accumulation in Pgrmc1 knockdown H9c2 cells.
Collectively, this study highlights the role of Pgrmc1 as a metabolic modulator of cardiac health. Since suppression of fatty acid oxidation without decreasing mitochondrial biogenesis is observed in the early stages of heart failure 33 , and characteristic mitochondrial dysfunction is seen in the late stages of heart failure 34 , individuals with low cardiac Pgrmc1 expression might be prone to mitochondrial impairment and progression to heart failure. According to our study, induction of Pgrmc1 expression improves the fundamental cardiac energy capacity and reduces cardiac lipid accumulation by increasing fatty acid oxidation and mitochondrial respiration. This is important because a fundamental increase in ATP production capacity is suggested for the targeted treatment of cardiomyopathy 35 .

Methods
Animals. C57BL/6J mice were housed in a pathogen-free facility at Chungnam National University under a standard 12 h light:12 h dark cycle and fed standard chow or high-fat diet, with water provided ad libitum. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Republic of Korea), and in compliance with ARRIVE guidelines. Animal experiment was approved by the Institutional Animal Care and Use Committee of Chungnam National University (IACUC; approval no. CNU-01145). As reported previously 9 , Pgrmc1 KO mice and wild-type littermates were used in this study. Refed conditions were induced by 6 h of feeding after 18 h of fasting. HFD conditions were induced by injecting streptozotocin (30 mg/kg) and feeding a high-fat diet for 8 weeks according to previous studies 36 . The high-fat diet (#D12492, Research Diets, Inc., New Brunswick, NJ) was composed of carbohydrate (20% kcal), protein (20% kcal), and fat (60% kcal).

Measurements of metabolites (HPLC-MS/MS). Standard metabolites and internal standards were
purchased from Sigma-Aldrich and CDN isotopes. All solvents including water were purchased from J. T. Baker. Tissue was homogenized using TissueLyzer (Qiagen) after adding 400 μL chloroform/methanol (2/1, v/v) and 100 μL internal standard solution containing 10 μM 13C5-glutamine for metabolites related to energy metabolism and 5 μM malonyl-13C3 CoA for fatty acyl CoAs. Metabolites were extracted from aqueous phase by liquid-liquid extraction 37 . The aqueous phase was dried using vacuum centrifuge, and the sample was reconstituted with 50 μL of H 2 O/acetonitrile (50/50 v/v) prior to LC-MS/MS analysis. Metabolites were analyzed with LC-MS/MS equipped with 1290 HPLC (Agilent), Qtrap 5500 (ABSciex), and reverse phase columns (Synergi fusion RP 50 × 2 mm for metabolites related to energy metabolism and Zorbax Extend-C18 3.5 μm, 2.1 × 12.5 mm for fatty acyl-CoA). Multiple reaction monitoring (MRM) was used, and the extracted ion chromatogram (EIC) corresponding to the specific transition for each metabolite was used for quantitation. Area under the curve of each EIC was normalized to that of EIC of internal standard, and used for quantitation.
Measurement of cardiac TG. Lipid extraction from heart was done with Folch method. Briefly, tissue was homogenized with beads and 0.9% NaCl solution. Homogenates were mixed with chloroform and methanol and incubated. Chloroform and distilled water were added and homogenates were centrifuged, and lower phase was collected. Steps after homogenization were 3 times repeated. Samples were dried and dissolved in chloroform and 2-propanol. TG level was analyzed by TG measurement solution (AM157S-K, Asan-Set).

Measurement of cardiac FFA. Commercial kit (BM-FFA100) for FFA measurement was obtained from
Biomax. Samples were processed according to manufacturer's protocol, and the level of cardiac FFA was measured.
RNA isolation, reverse transcription, and qRT-PCR. Heart was homogenized with TRIzol Reagent (Thermo Fisher Scientific, MA, USA) and the homogenate was mixed with chloroform (C2432, Sigma). After centrifugation, supernatant was mixed with isopropanol (1.09634.1011, Merck) and incubated for overnight on −20 °C. RNA pellet was attained by centrifugation, and the pellet was washed once with 70% ethanol and dissolved in DEPC (E174, Amresco)-treated water. cDNA was acquired by using a Reverse Transcriptase Kit (SG-cDNAS100, Smartgene, United Kingdom). mRNA expression was evaluated by real-time PCR using cDNA and specific primers (shown in Table 1). Excel Taq  Masson's trichrome staining. Sections were cut from paraffin blocks and processed using a commercial kit (MST-100 T, BIOGNOST). The fibrotic area was quantified by analysis with Image J program.
Statistical analysis. Data are reported as mean ± SD. Student's t test was performed using Graph Pad Software (GraphPad Inc., San Diego, CA, USA).