Adiponectin receptor 1 variants contribute to hypertrophic cardiomyopathy that can be reversed by rapamycin

Abstract

Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart muscle disease characterized by hypertrophy with preserved or increased ejection fraction in the absence of secondary causes. However, recent studies have demonstrated that a substantial proportion of individuals with HCM also have comorbid diabetes mellitus (~10%). Whether genetic variants may contribute a combined phenotype of HCM and diabetes mellitus is not known. Here, using next-generation sequencing methods, we identified novel and ultrarare variants in adiponectin receptor 1 (ADIPOR1) as risk factors for HCM. Biochemical studies showed that ADIPOR1 variants dysregulate glucose and lipid metabolism and cause cardiac hypertrophy through the p38/mammalian target of rapamycin and/or extracellular signal-regulated kinase pathways. A transgenic mouse model expressing an ADIPOR1 variant displayed cardiomyopathy that recapitulated the cellular findings, and these features were rescued by rapamycin. Our results provide the first evidence that ADIPOR1 variants can cause HCM and provide new insights into ADIPOR1 regulation.

Fig. 1. ADIPOR1 variants in patients with HCM.

(A) Pedigrees of HCM families with respective ADIPOR1 amino acid changes. Filled symbols represent affected individuals. Plus (+) and minus (−) signs indicate the presence and absence of the amino acid changes, respectively. (B) Alignment of ADIPOR1 protein sequences from various species with the amino acid changes altered in patients with HCM shown in highlights. (C) In silico analysis of the ADIPOR1 amino acid changes showing the predicted pathological nature. SIFT, Sorting Intolerant From Tolerant; PANTHER-PSEP, protein analysis through evolutionary relationships position-specific evolutionary preservation; PMUT, pathogenic mutation prediction; CADD, combined annotation dependent depletion.

Fig. 2. ADIPOR1 variants induce cardiomyocyte hypertrophy.

Representative images (A) and cell surface area measurements (B) in cardiomyocytes infected with Ad.βGal (vector control), Ad.AR1, Ad.F145I, and Ad.V146M and stained with phalloidin (actin), cardiac troponin T (cTnT), or Hoechst (nuclei) as indicated. Scale bars, 10 μm. Results presented as relative cell area compared to Ad.βGal (n = 20 cells in five different fields). (C) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of hypertrophic marker genes Anf, Bnp, and Myh7 in cardiomyocytes infected with Ad.βGal, Ad.AR1, Ad.F145I, and Ad.V146M, respectively. mRNA levels were normalized to 18S ribosomal RNA (rRNA) and presented as relative expression levels compared to the level in the Ad.βGal cardiomyocytes (n = 4 to 8). Values are shown as means ± SEM with each experiment performed in triplicate (n = 3). Significance was evaluated by Student’s t test or one-way analysis of variance (ANOVA) with post hoc Bonferroni’s test, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3. Individual ADIPOR1 variants modulate diverse signaling pathways in cardiomyocyte hypertrophy.

(A to D) Representative immunoblots with respective proteins from the total lysates of neonatal cardiomyocytes expressing Ad.βGal, Ad.AR1, Ad.V146M (A and B), or Ad.F145I (C and D), respectively. Expression levels were normalized to respective total proteins and presented as relative expression levels compared to the level in the Ad.βGal or Ad.AR1 cardiomyocytes, respectively. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin levels were used as a loading control. Values are shown as means ± SEM with each experiment performed in triplicate (n = 3). Significance was evaluated by Student’s t test or one-way ANOVA with post hoc Bonferroni’s test, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001. AU, arbitrary units.

Fig. 4. ADIPOR1 V146M interferes with glucose transport and insulin response in cardiomyocytes.

(A) Measurement of glucose uptake rates in adult rat cardiomyocytes expressing Ad.βGal, Ad.AR1, and Ad.V146M [APN treatment (±30 μg/ml) for 30 min]. The uptake rates were determined using [³H]–2-deoxyglucose (2DG) incorporation method with counts expressed in counts per minute and plotted as a percentage to the Ad.βGal. Data are displayed as the percentage difference in uptake relative to basal values (n = 3). (B) Quantitative RT-PCR analysis of glucose utilization–related genes (Slc2a4 and Pfkm) in adult cardiomyocytes expressing Ad.βGal, Ad.AR1, and Ad.V146M. mRNA levels were normalized to 18S rRNA and presented as relative expression levels compared to the level in the Ad.βGal cardiomyocytes (n = 4 to 8). (C and D) Representative immunoblots with respective proteins from the total lysates of adult cardiomyocytes expressing Ad.βGal, Ad.AR1, and Ad.V146M (±100 nM rapamycin treatment for 30 min or ±100 nM insulin treatment for 15 min), respectively. Expression levels of phospho-proteins were normalized to respective total proteins and presented as relative expression levels and compared accordingly. GAPDH levels were used as loading control. (E and F) Quantitative RT-PCR analysis of glucose utilization–related genes (Slc2a4 and Pfkm) in adult rat cardiomyocytes expressing Ad.βGal, Ad.AR1, and Ad.V146M (±100 nM rapamycin treatment for 30 min). mRNA levels were normalized to 18S rRNA and presented as relative expression levels compared to the level in the untreated Ad.βGal cardiomyocytes (n = 3). All the values are shown as means ± SEM with each experiment performed in duplicate. Significance was evaluated by Student’s t test or one-way ANOVA with post hoc Bonferroni’s test, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5. ADIPOR1 mutant (Cre-V146M) mice activate p38/mTOR pathways and modulate metabolism-associated gene expressions.

(A) Representative immunoblots with respective proteins from the total lysates of mouse ventricular tissues isolated from Cre or Cre-V146M (n = 2). GAPDH levels were used as loading control. (B) Quantitative RT-PCR analyses of lipid oxidation genes (Slc27a1, Acox1, Cpt1a, Ppargc1a, and Ppara) in Cre or Cre-V146M mice (n = 4). (C to E) Quantitative RT-PCR analyses of glucose utilization genes (Slc2a1, Slc2a4, and Pfkm) in ventricular cardiac tissues from Cre or Cre-V146M groups (n = 4) as indicated. Mice were fasted overnight and received a single intraperitoneal injection of insulin (10 mU/g), and hearts were harvested 10 min later. mRNA levels were normalized to 18S rRNA and presented as relative expression levels compared to the level in the Cre mouse heart tissues, respectively. Values are shown as means ± SEM with each experiment performed in duplicate. Significance was evaluated by Student’s t test. *P < 0.05 and **P < 0.01. (F) Representative immunoblots with respective proteins in human heart tissue biopsies from healthy individuals (C1 and C2) or ADIPOR1 V146M–mutated patient. GAPDH levels were used as loading control.

Fig. 6. Rapamycin reverses cardiac failure in ADIPOR1 Cre-V146M mice.

(A) Representative M-mode echocardiography and (B) percentage FS in vehicle or rapamycin-treated Cre and Cre-V146M mice (Cre-Veh, Cre.V146M-Veh, or Cre.V146M-Rapa), respectively (n = 12). n.s., not significant. (C) Representative immunoblots with indicated proteins from the total lysates of the respective mouse heart tissues (Cre-Veh, Cre.V146M-Veh, or Cre.V146M-Rapa) (n = 3). Expression levels were normalized to respective total proteins and presented as relative expression levels compared to the level in Cre-Veh or Cre.V146M-Veh. β-Actin levels were used as loading control. (D) Representative cardiomyocyte cross-sectional area (in square micrometers) measurements (n = 5 and 20 to 30 cells per mouse) in Cre-Veh, Cre-Rapa, Cre.V146M-Veh, and Cre.V146M-Rapa mouse ventricular sections, respectively. (E) Quantitative RT-PCR analysis of hypertrophic-related genes (Anf and Myh7) in heart tissues from Cre-Veh, Cre.V146M-Veh, and Cre.V146M-Rapa. mRNA levels were normalized to 18S rRNA and presented as relative expression levels compared to the levels in the Cre-Veh or Cre.V146M-Veh. Individual experiment was performed in duplicate (n = 9 to 12), respectively. All the values are shown as means ± SEM. Significance was evaluated by Student’s t test or one-way ANOVA with post hoc Bonferroni’s test, respectively. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 7. Rapamycin reduces cardiac fibrosis and restores mTOR pathway in ADIPOR1 Cre-V146M mice.

(A) Representative micrographs of Masson’s trichrome–stained LV sections and quantification of fibrosis (collagen area) in the indicated mice, respectively (n = 5, four to five fields per mouse). (B) Representative immunoblots with respective proteins from the total lysates of mouse ventricular tissues isolated from Cre-Veh, Cre.V146M-Veh, or Cre.V146M-Rapa (n = 3). Expression levels were normalized to respective total proteins and presented as relative expression levels compared to the level in Cre-Veh or Cre.V146M-Veh, respectively. β-Actin levels were used as loading control. All the values are shown as means ± SEM. Significance was evaluated by Student’s t test or one-way ANOVA with post hoc Bonferroni’s test, respectively. **P < 0.01 and ***P < 0.001. (C) Proposed mechanisms of ADIPOR1 mutants leading to hypertrophy and insulin resistance, or hypertrophy alone, respectively, events that ultimately cause cardiomyopathy.