PGRMC2 is a pressure-volume regulator critical for myocardial responses to stress in mice

Figures

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  Fig. 1. PGRMC2 was expressed in both human and mouse models, and metabolic acidosis was observed in hypoxic KO mice. a IHC staining of PGRMC2 receptor in heart tissues of WT and KO mouse models. b Western Blot analyses of PGRMC2 and GAPDH in heart tissues of WT and KO mouse models (blue arrow heads). c Quantification of PGRMC2 expressions in heart tissues of WT and KO mouse models using two-tailed t-test. The protein expression of PGRMC2 was normalized with the GAPDH of the corresponding samples (n = 4 each; p = 0.0064). Blood gas analyses of pH (d; p = 0.0084 & 0.0006), pCO2 (e), BEecf (f; p = 0.0004 & 0.0005), and HCO3 (g; p = 0.0309, 0.0007 & <0.0001) were measured in WT and KO of normoxic or hypoxic mouse models. Blood hemoglobin (Hgb; h; p < 0.0001) and hematocrit (Hct; i; p < 0.0001) concentrations were also measured using i-STAT CG8+ cartridge. n = 6-12 for all data sets, n represents the number of mice. Statistical analysis of blood test is based on one-way ANOVA multiple comparisons, followed by Tukey’s post-hoc. Statistical significance between different groups is indicated in the graph with asterisk (*); data are presented as mean values ± SEM. Source data are provided in a…

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  Fig. 2. Prolonged J-wave in the ECG was accompanied by fibrotic myocardium, shorter time to exhaustion and lower SBP in hypoxic KO mice. a Electrocardiogram of WT and KO hypoxic mouse models. Red and blue lines indicated QT interval and J-wave, respectively; n = 6 for each group. b Comparison of gross heart anatomy between WT vs KO of normoxic/hypoxic mouse model. c, d Heart weight and heart-to-body weight ratio after hypoxia exposure in all four groups at 18 weeks-old; n = 8-11. e Masson’s trichrome staining of heart tissue in WT and KO of normoxic or hypoxic mouse model. f–k Right ventricle (RV) fibrosis (f; p < 0.0001), left ventricle (LV) fibrosis (g; p = 0.0002, <0.0001), RV thickness (h), LV thickness (i), RV inner circumference (j; p = 0.0011 & <0.0001), LV inner circumference (k; p = 0.0025, <0.0001) were measured. Exercise capacity (l; p = 0.0017, 0.0065) and sustained running speed, (m; p = 0.0015, 0.0015) were measured using Exer 3/6 treadmill. Systolic blood pressure (SBP; n; p = 0.035), mean arterial pressure (MAP; o) and heart rate (HR; p) were measured using tail cuff. n\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts}…

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  Fig. 3. Hypoxic KO mice had pulmonary hypertension and reduced RV systolic function. a Masson’s trichrome staining of pulmonary vascular remodeling and lesions of WT and KO of normoxic or hypoxic mouse models. Arrows indicated pathology associated with medial wall thickening, plexiform lesion, and concentric cellular laminar neointimal lesion. b Different views of cardiac magnetic resonance (CMR). c–e Statistical significance between WT and KO is indicated with black (*) and blue (*) asterisks for normoxic and hypoxic, respectively. Longitudinal comparison to the 10 weeks age of the corresponding groups is shown in black (#) and blue (#) hashtag in WT and KO, respectively. RV Ejection fraction (c; p = 0.0025, <0.0001 for the rest), RV stroke volume (d; p = 0.027, 0.0004, <0.0001), and RV cardiac output (e; p = 0.0007) were measured with CMR at 10-, 12-, and 14 weeks old WT and KO of normoxic or hypoxic mouse models. f, g Statistical significance between WT and KO normoxic is indicated with black asterisk (*); Statistical significance between WT and KO hypoxic is indicated with blue asterisk (*); longitudinal comparison to the 10 weeks age of the corresponding groups is shown in hashtag…

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  Fig. 4. Hypoxic KO mice had a reduced LV systolic function, abdominal ascites, pulmonary and hepatic congestion, and elevated heart failure biomarkers. a–d Statistical significance between WT and KO is indicated with black (*) and blue (*) asterisks for normoxic and hypoxic, respectively. Longitudinal comparison to the 10 weeks age of the corresponding groups is shown in black (#) and blue (#) hashtag in WT and KO, respectively. LV ejection fraction (a; p = 0.014, 0.045, 0.0048, 0.0078, 0.0078, <0.0001 for asterisk; p = 0.0272, 0.0250, 0.0138, 0.0084 for hashtag), stroke volume (b; p = 0.0290, 0.0084, 0.0053), cardiac output (c; p = 0.0071), and thickness (d), were measured with CMR at 10-, 12-, and 14 weeks old of WT and KO of normoxic or hypoxic mouse models. e Abdominal transverse and longitudinal planes of the WT and KO were taken from T2 Weighted TurboRARE MRI imaging, which was used to examine free abdominal fluid (red arrows). f, g Masson’s trichrome staining of lung (f) and liver (g) tissues from female mice to indicate the alveolar wall thickness and hyperemic liver. Male mice can be found in Supplementary Fig. 11c, d, h– j. Atrial natriuretic peptide (ANP; h; p < 0.0001), brain…

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  Fig. 5. LV systolic pressure and function from the ex-vivo working heart system were abnormal in KO mouse model heart. PV loop was studied in 14 weeks old WT and KO of normoxic or hypoxic mouse models (a). LVV left ventricle volume, LVP left ventricle pressure. Left ventricle pressure maximum (LVPmax), end systolic pressure (ESP), and end diastolic pressure (EDP) parameters were analyzed (b). Ejection fraction, stroke volume, and cardiac output were analyzed to indicate LV functions (c). Adrenalin (4 mg/L) and diltiazem (0.08 mg/L) were used as a stress test. n = 6 for all data sets; n represents the number of mice. Statistical analysis is based on one-way ANOVA multiple comparisons, followed by Tukey’s post-hoc. Statistical significance between control and treatment is indicated with asterisk (*); comparison between WT and KO is shown in hashtag (#). *p = 0.021; ****p < 0.0001; #p = 0.025; ####p < 0.0001; data are presented as mean values ± SEM. Source data are provided in a Source Data file.

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  Fig. 6. KO mouse cardiomyocytes were not responsive to estradiol, progesterone, and hydrocortisone. a Representatives of 340 nm/380 nm excitation ratio using Fura-2AM test in WT, KO, and rescued cardiomyocytes were shown with treatment of estradiol (15 ng/mL), progesterone (40 ng/mL), and hydrocortisone (10 μg/mL) after baseline calcium level was recorded for 2–3 min. Ionomycin (5 μM) was used as a positive control. b–d Cytosolic calcium was quantified and compared between WT and KO cardiomyocytes in normoxic (b), hypoxic (c), and after rescued (d). n = 3–9 represents the number of cardiomyocytes (or independent experiments) from a total of 3 mice in each group. Statistical analysis is based on two-tailed t-test. Statistical significance between different groups is indicated with asterisk (*); *p = 0.025; **p = 0.0053; ***p = 0.0001; ****p < 0.0001; data are presented as mean values ± SEM. Source data are provided in a Source Data file.

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  Fig. 7. Hypoxic KO mouse heart tissue has higher nucleus HIF-1α translocation and expressions of VEGF, TGF-β1R, p-PKC, and p-AKT. a, b IHC staining (a) and quantification (b; p = 0.0029 or <0.0001) of HIF-1α in heart tissue. c. Western blot analyses of mouse heart tissue for VEGF, TGF-β1, TGF-β1 receptor (TGF-β1R), p-PKC, and p-AKT. d–h Expression levels of VEGF (d; p = 0.032, 0.0040, 0.0006), TGF-β1 (e), TGF-β1R (f; p = 0.0054, 0.0002), p-AKT (g; p = 0.018, 0.0025, 0.0059), and p-PKC (h; p = 0.013, 0.023, 0.031) were quantified. All protein expressions were normalized with the GAPDH of the corresponding samples. i. Graphical summary of PGRMC2 role in the heart during hypoxia. n = 6–9 for all data sets, n represents the number of mice. Statistical analysis is based on one-way ANOVA multiple comparisons, followed by Tukey’s post-hoc. Statistical significance between different groups is indicated with asterisk (*); data are presented as mean values ± SEM. Source data are provided in a Source Data file.