FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair

Abstract

Background: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration.

Methods: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes.

Results: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis.

Conclusions: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Keywords: FTO protein, mouse; N(6)-methyladenosine; RNA methylation; heart failure; myocardial ischemia.

Figures

Figure 1.. Increased m6A in RNA in failing human (both ischemic and non-ischemic), pig and mouse (post-myocardial infarction ischemic) hearts.

Quantification of m6A in total or polyA+ RNA in LV of A, human, n=6–11; from infarct/peri-infarct areain B, pig, n=3–6, C, mouse, n=5–11; from non-infarct area in D, pig, n=3 and E, mouse, n=3–6. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001, compared with non-failing or sham. n.s., non significant.

Figure 2.. Decreased FTO mRNA and protein expression in human and mouse failing hearts.

A,quantification of mRNA, n=3–6, B, representative immunoblots, C, densitometry quantification of protein, n=5–8 for m6A regulators in human non-failing and failing hearts. D, quantification of mRNA, n=3–7, E, representative immunoblots and F, densitometry quantification of protein, n=3–4 at different time points in mouse LV. G, qRT-PCR quantification of selected mRNA expressions in mouse LV, n=4–8. mRNA/protein data represented as F/MI normalized to NF/sham. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001, compared with non-failing or sham.

Figure 3.. Fto-mediated m6A demethylation regulates intracellular Ca2+ recycling and contractile dynamics in isolated adult rat primary cardiomyocytes.

Quantification of A, Fto mRNA, n=3–4, B, m6A in total RNA, n=3–5, C, cells with arrhythmic events, n=47–90 cells per group from 4–12 rats. D, Representative Ca2+ transients obtained from pacing-induced myocytes. Measurements of E, maximal Ca2+ amplitude, F, time to 50% decay, G, Tau, H, cell shortening, n=23–74 cells per group from 4–12 rats. Sarcomere and Ca2+ transients were recorded at 1Hz pacing stimulation frequency with MyoPacer Field Stimulator (IonOptix MA, USA). Abbreviations, Unt: untreated; siCtrl: siRNA control, siFto: siRNA-mediated Fto knockdown, adnull: adenovirus with empty CMV promoter, adFto: adenovirus with full length Fto. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001by one-way ANOVA.

Figure 4.. AAV9-mediated myocardial FTO gene transfer rescues cardiac function in mouse models of MI.

A, Design of aavFto study. B, representative immunoblots showing Fto protein expression at week 0 of the aavFto study. C, m6A quantification in total RNA at week 4 of aavFto study, n=4–7. Echocardiographic assessments of LV function showing D, ejection fraction (EF) and E, fractional shortening (FS) at 4w post-MI, n=10–13. F, representative M-Mode echocardiograms showing anterior and posterior LV wall motion at 4w post-MI in aavFto mice. Error bars represent SEM. G, Representative Masson trichrome staining images of histological cross-sections taken in bright-field mode processed from sham, aavgnp-MI or aavFto-MI. H,Scar Size was measured as percentage of total LV area after Masson trichrome staining in sham, aavgnp-MI and aavFto-MI at four weeks post-MI surgeries. Error bars represent SD. *P<0.05, ***P<0.001by one-way ANOVA.

Figure 5.. Adenovirus-mediated myocardial FTO gene transfer rescues cardiac function in mouse models of MI.

A, Design of adFto study. B, representative immunoblots showing Fto protein expression at week 4 of the adFto study. C, m6A quantification in total RNA at week 4 of adFto study, n=3–5. Echocardiographic assessments of LV function showing D, ejection fraction (EF) and E, fractional shortening (FS) at 4w post-MI, n=6–9. F, representative M-Mode echocardiograms showing anterior and posterior LV wall motion at 4w post-MI in adFto mice. Error bars represent SEM. G, Representative Masson trichrome staining images of histological cross-sections taken in bright-field mode processed from sham, adnull-MI or adFto-MI. H,Scar Size was measured as percentage of total LV area after Masson trichrome staining in sham, adnull-MI and adFto-MI at four weeks post-MI surgeries. Error bars represent SD. *P<0.05, ***P<0.001 by one-way ANOVA.

Figure 6.. Contractile transcripts hypermethylated in failing hearts are demethylated by Fto overexpression.

A, total MeRIP bound reads from mouse MeRIP-Seq. B, mouse heatmap showing total MeRIP reads normalized to total non-IP reads within each transcript relative to sham. For each condition, total MeRIP reads per transcript were calculated for reads that fully overlapped with a given feature, and were normalized to total non-IP reads per corresponding transcript. C, top 10 DAVID GO terms enriched (±2-fold + FDR<0.05) from mouse MeRIP-Seq. Differential peak analysis of m6A MeRIP-Seq datasets were carried out using a modification of exomePeak R/Bioconductor package to compare the ratio of the absolute number of MeRIP reads to non-IP reads at a given peak between two conditions. D, IGV plots of mouse MeRIP reads for selected transcripts, IGV numbers indicate scale for MeRIP reads for all conditions per transcript.

Figure 7.. Serca2a mRNA is hypermethylated in human failing hearts and demethylation by FTO overexpression induces Serca2a mRNA and protein expression.

A, human MeRIP-qPCR showing m6A enrichment in mRNA. B,FTO mRNA and C,SERCA2A mRNA, n=3 expressions in AC16 human myocytes. Fto mRNA expression in D, aavFto-MI and E, adFto mice 4w post-MI, n=3–5. F, Representative immunoblots showing Serca2a protein expression at 4w post-MI. Error bars represent SEM. *P<0.05, ***P<0.001. A-C, student t-test; D and E, one-way ANOVA.

Figure 8.. Proposed working model based on our hypothesis.

A, Healthy heart with physiological FTO and m6A levels, B, Failing heart with decreased FTO, increased m6A, increased contractile mRNA degradation and dysfunctional myofilament, C, Fto rescued failing heart with attenuated m6A and restored contractile protein expression and myofilament.