Epitranscriptomic regulation in fasting hearts: implications for cardiac health

Figures & data

Table 1. The m⁶A and m⁶Am regulators.

Protein	Protein name	Function	Modification
METTL3	Methyltransferase-like 3	Writers	m^6A
METTL14	Methyltransferase-like 14		m^6A
WTAP	Willms‘ tumour 1-associating protein		m^6A
METTL5	Methyltransferase-like 5		m^6A
METTL16	Methyltransferase-like 16		m^6A
ZCCHC4	Zinc finger CCHC-type containing 4		m^6A
PCIF1	Phosphorylated CTD interacting factor 1		m^6Am
METTL4	Methyltransferase-like 4		m^6Am
FTO	Fat mass and obesity-associated protein	Erasers	m^6Am, m^6A
ALKBH5	AlkB family member 5		m^6A
YTHDF1	YTH domain-containing family protein 1	Readers	m^6A
YTHDF2	YTH domain-containing family protein 2		m^6A
YTHDF3	YTH domain-containing family protein 3		m^6A
YTHDC1	YTH domain-containing protein 1		m^6A
YTHDC2	YTH domain-containing protein 2		m^6A
eIF3a	Eukaryotic initiation factor 3a		m^6A
eIF3c	Eukaryotic initiation factor 3c		m^6A
eIF3g	Eukaryotic initiation factor 3g		m^6A
HNRNPA2B1	Heterogeneous nuclear ribonucleoprotein A2/B1		m^6A
HNRNPC	Heterogeneous nuclear ribonucleoprotein C		m^6A
HNRNPD	Heterogeneous nuclear ribonucleoprotein D		m^6A
RBMX (HNRNPG)	RNA-binding motif protein, X chromosome		m^6A
IGF2BP1	Insulin-like growth factor 2 mRNA binding protein 1		m^6A
IGF2BP2	Insulin-like growth factor 2 mRNA binding protein 2		m^6A
IGF2BP3	Insulin-like growth factor 2 mRNA binding protein 3		m^6A
FMR1	Fragile X messenger ribonucleoprotein 1		m^6A
PRRC2A	Proline rich-coil 2A		m^6A
G3BP1	G3BP stress granule assembly factor 1	m^6A-repelled proteins	m^6A
G3BP2	G3BP stress granule assembly factor 2		m^6A
ELAVL1 (HUR)	ELAV-like protein 1		m^6A
USP10	Ubiquitin specific peptidase 10		m^6A
CAPRIN1	Cell cycle associated protein 1		m^6A
RBM42	RNA binding motif protein 42		m^6A

The key regulators are in bold.

Table 2. Characteristics of the fasting model.

	Control rats	Fasting rats
BW change (%)	+3 ± 2.16	-17 ± 2.22*
HW/Tibia (%)	25.5 ± 1.50	22 ± 1.47***
Hematocrit (%)	40.3 ± 4.49	45.6 ± 2.97*
Glycemia (mmol/l)	6.2 ± 0.43	3.9 ± 0.77****
AWTd (mm)	1.96 ± 0.13	1.85 ± 0.15
AWTs (mm)	2.81 ± 0.09	2.66 ± 0.23
PWTd (mm)	1.83 ± 0.13	1.90 ± 0.19
PWTs (mm)	2.71 ± 0.14	2.67 ± 0.24
RWT (%)	49.88 ± 5.59	51.41 ± 3.67
LVDd (mm)	7.63 ± 0.42	7.23 ± 0.17*
LVDs (mm)	4.64 ± 0.27	4.72 ± 0.22
FS (%)	39.9 ± 1.8	35.1 ± 3.4*
HR (bpm)	350 ± 19.9	327 ± 22*
CI (ml/min/kg)	306 ± 62	276 ± 33
Pes (mmHg)	86.63 ± 5.69	89.38 ± 4.34
Ped (mmHg)	4.00 ± 1.38	4.28 ± 2.25
Pdev (mmHg)	82.63 ± 4.88	85.10 ± 4.47
+(dP/dt)max (mmHg/s)	7,008 ± 529	5,453 ± 417*
-(dP/dt)max (mmHg/s)	-7,080 ± 529	-6,592 ± 616

Values are means ± SD; n = 8–10; *p < 0.01; ***p < 0.001, ****p < 0.0001. AWTd – end-diastolic anterior wall thickness; AWTs – end-systolic anterior wall thickness; bpm – beats per minute; BW – body weight; CI – cardiac index; FS – fractional shortening; HR – heart rate; HW – heart weight; LVDd – end-diastolic LV diameter; LVDs – end-systolic LV diameter; Ped – end-diastolic pressure; Pes – end-systolic pressure; Pdev – developed pressure; PWTd – end-diastolic posterior wall thickness; PWTs – end-systolic posterior wall thickness; RWT – relative wall thickness; +(dP/dt)max – peak rate of pressure development; -(dP/dt)max – peak rate of pressure decline.

Figure 1. Effect of fasting on plasma metabolites measured by a multiplatform LC-MS-based approach (A) PCA (principal component analysis) showing a clear separation between fasting and control rat plasma samples, suggesting distinct metabolomic profiles associated with the fasting state (B) volcano plot of all (677) metabolites indicating differential levels of metabolites in plasma samples of fasting and control rats (C) heat map of the 50 most significantly affected metabolites; n = 7.

Figure 2. Effect of 3-day fasting on gene expressions of m⁶A and m⁶Am regulators in the left ventricle assessed by RT-qPCR. Writers are displayed in blue, erasers in red, and readers in yellow. The average of the control values is set to 1. Values are means ± SD; n = 6–8; *p < 0.05; **p < 0.01 (t-test). Alkbh5 – AlkB family member 5; Fto – fat mass and obesity-associated; Mettl3 – methyltransferase-like 3; Mettl4 – methyltransferase-like 4; Pcif1 – phosphorylated CTD interacting factor 1; Ythdf1–3 – YTH domain-containing family protein 1–3; Ythdc1–2 – YTH domain-containing protein 1–2.

Figure 3. Effect of 3-day fasting on protein levels of m⁶A and m⁶Am regulators in the left ventricles assessed by Western blot (A). Writers are displayed in blue, erasers in red, and readers in yellow. The average of the control values is set to 1. Representative Western blot membranes (B). Protein loadings were 40 μg (YTHDF1, YTHDF3), 30 μg (YTHDC1), 20 μg (FTO, ALKBH5, YTHDC2), 15 μg (METTL3, YTHDF2), and 10 μg (METTL4, PCIF1). Values are means ± SD; n = 8; *p < 0.05; **p < 0.01; ***p < 0.001 (t-test). ALKBH5 – AlkB family member 5; C – control; F – fasting; FTO – fat mass and obesity-associated protein; METTL3 – methyltransferase-like 3; METTL4 – methyltransferase-like 4; PCIF1 – phosphorylated CTD interacting factor 1; YTHDF1–3 – YTH domain-containing family protein 1–3; YTHDC1–2 – YTH domain-containing protein 1–2.

Table 3. Targeted proteomic analysis – changes in peptide levels.

Changes in peptide levels of epitranscriptomic regulators
Protein	Protein Accession	Peptide	Change	P-value
Writers
METTL5	B0BNB3	LFDTVIMNPPFGTK	−50%	0.004
		YDLPALYNFHK	−41%	0.038
WTAP	D3ZPY0	TTSSEPVDQAEATSK	−13%	0.076 ×
Erasers
ALKBH5	D3ZKD3	YFFGEGYTYGAQLQK	67%	0.060 ×
Readers
eIF3a	Q1JU68	ALEVIKPAHILQEK	−37%	0.049
YTHDF2	E9PU11	LGSTEVASSVPK	−34%	0.003
		APGMNTIDQGMAALK	−30%	0.021
eIF3a	Q1JU68	LLDMDGIIVEK	−28%	0.003
YTHDF3	D3ZIY3	AITDGQAGFGNDTLSK	−27%	0.002
eIF3g	Q5RK09	GFAFISFHR	−26%	0.009
YTHDF3	D3ZIY3	HTTSIFDDFAHYEK	−26%	0.012
YTHDF1	Q4V8J6	HTTSIFDDFSHYEK	−25%	0.013
eIF3c	B5DFC8	LNEILQVR	−23%	0.011
RBMX	Q4V898	GGHMDDGGYSMNFTLSSSR	−16%	0.025
eIF3g	Q5RK09	LPGELEPVQAAQNK	−17%	0.086 ×
m^6A-repelled proteins
USP10	Q3KR59	QADFVQTPITGIFGGHIR	−29%	0.021
CAPRIN1	Q5M9G3	TVLELQYVLDK	−28%	0.005
G3BP2	Q6AY21	VDAKPEVQSQPPR	−26%	0.007
CAPRIN1	Q5M9G3	YQEVTNNLEFAK	−24%	0.039
G3BP1	D3ZYS7	DFFQSYGNVVELR	−23%	0.023
ELAVL1	B5DF91	VAGHSLGYGFVNYVTAK	−21%	0.033

Changes at the edge of significance are marked by ’×’. ALKBH5 – AlkB family member 5; CAPRIN1 – Cell cycle associated protein 1; eIF3a/c/g – Eukaryotic initiation factor 3a/c/g; ELAVL1 – ELAV-like protein 1; G3BP1 – G3BP stress granule assembly factor 1; G3BP2 – G3BP stress granule assembly factor 2; RBMX – RNA-binding motif protein, X chromosome; USP10 – Ubiquitin specific peptidase 10; WTAP – Willms’ tumour 1-associating protein; YTHDC1 – YTH domain-containing protein 1; YTHDF1–3 – YTH domain-containing family protein 1–3.

Figure 4. Levels of epitranscriptomic regulators in left ventricles of fasting rats assessed by RT-qPCR, Western blot, and proteomic analysis. Out of the two peptides measured for each protein in proteomic analyses, the peptide with more profound changes was depicted. ALKBH5 – AlkB family member 5; FTO – fat mass and obesity-associated protein; METTL3 – methyltransferase-like 3; METTL4 – methyltransferase-like 4; PCIF1 – phosphorylated CTD interacting factor 1; YTHDF1–3 – YTH domain-containing family protein 1–3; YTHDC1–2 – YTH domain-containing protein 1–2.

Figure 5. The difference between m⁶A/m methylation levels in total RNA from left ventricles of fasting rats. Values are means ± SD; n = 7–8; * p < 0.05 (t-test).

Figure 6. The m⁶A/m enrichment in specific mRnas isolated from left ventricles of fasting rats. The average of the control values is set to 1. Values are means ± SD; n = 3; ** p < 0.01 (t-test). Nfe2l2 – NFE2 like BZIP transcription factor 2; Sirt1 – sirtuin 1; Sirt3 – sirtuin 3; Prkaa2 – protein kinase AMP-activated catalytic subunit alpha 2; Rela – RELA proto-oncogene, NF-KB Subunit; Nox4 – NADPH oxidase 4; Hdac1 – histone deacetylase 1; Foxo3 – forkhead box O3; Hif1a – hypoxia inducible factor 1 subunit alpha.

Figure 7. Effect of ALKBH5 and FTO inhibition on hypoxic tolerance of AVCMs isolated from control and fasting rats. Values are means ± SD; n = 9; ** p < 0.01; *** p < 0.001 (one-way ANOVA); + p < 0.05 compared to normoxic untreated AVCMs; ++ p < 0.01 compared to normoxic untreated AVCMs. ALKBH5i – ALKBH5 inhibitor; AVCMs – adult rat left ventricular cardiomyocytes; FTOi – FTO inhibitor.