The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy

Abstract

Background: N6-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known.

Methods: To determine the role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function.

Results: We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis.

Conclusions: Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

Keywords: RNA processing, post-transcriptional; gene expression profiling; hypertrophy; mice, transgenic.

Figures

Figure 1.

m6A is a dynamic modification in cardiomyocytes. A, Distribution of m6A peaks throughout mRNAs. B, Percentage of m6A-methylated RNA in relation to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in unstimulated (Normal) and hypertrophic (Hyper.) neonatal rat cardiomyocytes (n=3 each). C, PANTHER analysis of enriched functional gene categories showing differential m6A peaks during hypertrophy. D and E, Visualization of representative m6A peaks under unstimulated or hypertrophic conditions from the indicated mRNAs using Integrative Genomics Viewer. *P≤0.05 versus normal. Intracell. indicates intracellular; IP, immunoprecipitation; Map3k6, mitogen-activated protein kinase kinase kinase 6; m6A, N6-Methyladenosine; PANTHER, Protein Analysis through Evolutionary Relationships; phosphor., phosphorylation; Ryr2, ryanodine receptor; and UTR, untranslated region.

Figure 2.

Generation and characterization of METTL3 overexpression models. A, Western blot from cardiomyocytes overexpressing β-galactosidase (Ad-Control) or METTL3 (Ad-Mettl3) using antibodies against the indicated proteins and GAPDH loading control. B, Densitometry quantification of the expression for the indicated proteins relative to GAPDH control. C, Quantification of cardiomyocyte cell area based on pixel size from cardiomyocytes overexpressing β-galactosidase control or METTL3 (n=3 each). D, Representative images of cardiomyocytes from the indicated treatments stained for α-actinin (green). Scale bar=20 µm. E, Schematic of cardiomyocyte-specific METTL3 gain-of-function mouse model. F, Western blot from cardiac extracts from control (Ctrl) or METTL3 transgenic (TG) line 1 and 2. GAPDH was used as loading control. G, percentage of m6A-methylated RNA relative to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in isolated cardiomyocytes from control mice and METTL3 TG line 1 mice (n=3 each). H, Gravimetric analysis of heart weight normalized to body weight (HW/BW) in the indicated genotypes at 3 months of age (n≥6 each). *P<0.05 versus control. A.U. indicates arbitrary units; m6A, N6-Methyladenosine; and METTL3, methyltransferase-like 3.

Figure 3.

METTL3 drives compensated hypertrophy in vivo. A, Heart weight to body weight ratios (HW/BW) in 8-month-old METTL3-TG animals or littermate controls (n≥7 each group). B and C, Wheat germ agglutinin (green)–stained cardiac cross-sections of 8-month-old METTL3-TG or littermate control mice with quantification of cardiomyocyte cross-sectional area using ImageJ software. Scale bar=50 µm (n≥100 cells/animal, n=6 mice each). D, Representative images of isolated cardiomyocytes from 8-month-old hearts from the indicated genotypes. Scale bar=50 µm. E, Length/width ratios of isolated cardiomyocytes from 8-month-old control and TG mice (n≥100 cells/animal; n=3 mice each). F, Representative images of Masson trichrome staining of cardiac cross-sections of 8-month-old METTL3-TG or littermate control mice. Scale bar=1 mm. G, Echocardiographic quantification of percentage fractional shortening (FS) for 8-month-old METTL3-TG animals or littermate controls (n=12 each). H, Echocardiographic quantification of percentage fractional shortening for Sham or TAC-operated METTL3-TG animals or littermate controls (n≥5 each). I, HW/BW in TAC-operated METTL3-TG animals or littermate controls (n≥5 each group). J and K, Wheat germ agglutinin (green)–stained cardiac cross-sections of TAC-operated METTL3-TG or littermate control mice with quantification of cardiomyocyte cross-sectional area using ImageJ software. Scale bar=100 µm (n≥100 cells/animal, n=5 mice each). *P≤0.05 versus Ctrl. Ctrl indicates control; METTL3, methyltransferase-like 3; TAC, transverse aortic constriction; and TG, transgenic.

Figure 4.

METTL3 inhibition prevents the development of cardiomyocyte hypertrophy. A, qPCR analysis for METTL3 expression in response to Ctrl or METTL3 siRNA-mediated knockdown (n=3 each). B, Representative images of cardiomyocytes treated with control siRNA (si-Ctrl) or siRNA targeting METTL3 (si-M3) stained for α-actinin (green). Scale bar=20 µm. C, Quantification of cardiomyocyte cell area (n≥50 cells/well, n=3 independent experiments/treatment). *P<0.05 versus baseline; #P<0.05 versus si-Ctrl hypertrophy. Ctrl indicates control; METTL3, methyltransferase-like 3; siRNA, small interfering RNA; and qPCR, quantitative polymerase chain reaction.

Figure 5.

Generation and characterization of METTL3 cardiac-restricted knockout mice. A, Schematic of METTL3 loss-of-function mouse model. B, Western blot for METTL3 from cardiac extracts from control (Ctrl) or cardiomyocyte-specific METTL3 knockout mice (cKO). GAPDH and Ponceau staining were used as loading control. C, percentage of m6A-methylated RNA relative to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in isolated cardiomyocytes from control and METTL3-cKO mice (n=3 each). D, Representative Masson trichrome–stained cardiac cross-sections from 3-month-old METTL3-cKO mice and controls. Scale bar=1 mm. E, Quantification of heart weight to body weight ratio (HW/BW) for 3-month-old METTL3-cKO or control mice (n=5 each). F, Echocardiographic quantification of percentage fractional shortening (FS) for 3-month-old METTL3-cKO or control mice (n=12 each). G and H, Wheat germ agglutinin (green)–stained cardiac cross-sections of 3-month-old METTL3-cKO or control mice with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥4 mice each). *P≤0.05 versus Ctrl. m6A indicates N6-Methyladenosine; and METTL3, methyltransferase-like 3.

Figure 6.

METTL3 loss-of-function causes maladaptive cardiomyocyte remodeling and cardiac dysfunction with aging. A and B, Wheat germ agglutinin–stained cardiac cross-sections of 8-month-old METTL3-cKO or control mice with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥5 mice each). C, Quantification of heart weight to body weight ratio (HW/BW) for 8-month-old METTL3-cKO or control mice (n≥13 each). D and F, Representative bright field image of isolated cardiomyocytes from the indicated genotypes at 8 months of age (D) and measurements of length (E) and length/width ratios (F) (n≥100 cells/animal; n=3 animals each). Scale bar=50 µm. G, Representative Masson trichrome–stained cardiac cross-sections from 8-month-old METTL3-cKO mice and control mice. Scale bar=1 mm. H through J, Representative images of short-axis (M-mode) and long-axis (B-mode) echocardiographic analysis (H), and echocardiographic quantification of left ventricular chamber end-diastolic dimensions (LVEDd) (I) and percentage fractional shortening (FS) (J) in 8-month-old METTL3-cKO and control mice (n=18 each). *P≤0.05 versus Ctrl. cKO indicates cardiomyocyte-specific METTL3 knockout mice; Ctrl, control; and METTL3, methyltransferase-like 3.

Figure 7.

METTL3 loss-of-function causes maladaptive cardiomyocyte remodeling and cardiac dysfunction poststress. A, Echocardiographic quantification of percentage fractional shortening (FS) in 3-month-old METTL3-cKO and control mice subjected to sham or TAC surgery for the indicated times (n=8 each). B and C, Wheat germ agglutinin–stained cardiac cross-sections of METTL3-cKO or control mice subjected to 6 weeks of TAC with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n=7 mice each). D, Echocardiographic quantification of percentage FS in 3-month-old METTL3-cKO and control mice subjected to vehicle (Veh) or angiotensin/phenylephrine infusion (Ang/PE) for 4 weeks (n=5 each). E and F, Wheat germ agglutinin–stained cardiac cross-sections of METTL3-cKO or control mice subjected to 4 weeks of vehicle orAng/PE with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥3 mice each). *P≤0.05 versus Ctrl. cKO indicates cardiomyocyte-specific METTL3 knockout mice; Ctrl, control; METTL3, methyltransferase-like 3; and TAC, transverse aortic constriction.