Cardiac myocyte KLF5 regulates body weight via alteration of cardiac FGF21

Abstract

Cardiac metabolism affects systemic energetic balance. Previously, we showed that Krüppel-like factor (KLF)-5 regulates cardiomyocyte PPARα and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (αMHC-KLF5^-/-) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT). Alterations in cardiac expression of the mediator complex subunit 13 (Med13) modulates obesity. αMHC-KLF5^-/- mice had reduced cardiac Med13 expression likely because KLF5 upregulates Med13 expression in cardiomyocytes. We then investigated potential mechanisms that mediate cross-talk between cardiomyocytes and WAT. High fat diet-fed αMHC-KLF5^-/- mice had increased levels of cardiac and plasma FGF21, while food intake, activity, plasma leptin, and natriuretic peptides expression were unchanged. Consistent with studies reporting that FGF21 signaling in WAT decreases sumoylation-driven PPARγ inactivation, αMHC-KLF5^-/- mice had less SUMO-PPARγ in WAT. Increased diet-induced obesity found in αMHC-KLF5^-/- mice was absent in αMHC-[KLF5^-/-;FGF21^-/-] double knockout mice, as well as in αMHC-FGF21^-/- mice that we generated. Thus, cardiomyocyte-derived FGF21 is a component of pro-adipogenic crosstalk between heart and WAT.

Keywords: FGF21; Heart; High fat diet; Krüppel-like factor; Obesity.

Figure 1:. Cardiomyocyte-specific KLF5 deletion accelerates diet-induced obesity –

A: Representative pictures of control floxed or αMHC-KLF5^−/− mice after 6 weeks of HFD. B-C: Cumulative body weight gain (g) (B), body weight gain (%) (C) of control floxed (n = 6) and αMHC-KLF5^−/−(n = 13) mice after 6 weeks HFD (*p<0.05, **p<0.01, ***p<0.001 vs floxed with 2way ANOVA (B) or Student’s T-Test (C)). Data are presented as mean ± SEM. (See also Figure S1A–D) HFD: high fat diet, KLF: Krüppel-like factor.

Figure 2:. Cardiomyocyte-specific KLF5 deletion promotes adipose tissue expansion and hepatic lipid accumulation –

A: posterior subcutaneous WAT weight of control floxed (n = 4) and αMHC-KLF5^−/− (n = 7) mice after 6 weeks HFD. B: mRNA levels of lipid metabolism markers Pparg1, Pparg2, Lpl, Cd36, Dgat2 and Glut4 in WAT isolated from control floxed or αMHC-KLF5^−/− mice after 6 weeks of HFD (n=3; **p<0.01, ***p<0.001 with Student’s T-Test). C-D: Representative pictures of H&E staining of WAT and BAT (C), and Oil Red O staining of liver (D) of control floxed and αMHC-KLF5^−/− mice after 6 weeks HFD. Data are presented as mean ± SEM. BAT: brown adipose tissue, Dgat: diglyceride acyltransferase, Glut: glucose transporter, HFD: high fat diet, KLF: Krüppel-like factor, Lpl: lipoprotein lipase, WAT: white adipose tissue.

Figure 3:. Cardiomyocyte-specific KLF5 deletion does not alter significantly respiratory exchange ratio, caloric intake, activity, and energy expenditure at the early stage of HFD –

Respiratory exchange ratio (A), feeding timecourse (B), activity time course (C), and energy expenditure (D) from metabolic cage analysis of control floxed (n = 6) and αMHC-KLF5^−/− (n = 5) mice on HFD for 3 weeks. Data are presented as mean ± SEM. HFD: high fat diet, KLF: Krüppel-like factor.

Figure 4:. KLF5 is a miR-208-independent, direct positive regulator of cardiac MED13 –

A-B: Cardiac Med13 mRNA levels (n=4-6; ***p<0.001 with Student’s T-Test) (A) and miR-208a levels (n=3-4) (B) in male and female control floxed and αMHC-KLF5^−/− mice. C: Klf5 and Med13 mRNA levels in HL-1 cells treated with Ad-GFP or Ad-KLF5 (n=4, *p<0.05; **p<0.01 vs ad-GFP with Student’s T-Test). D: Predicted KLF-binding sites by in silico promoter analysis on aligned mouse and human Med13 promoters (highlighted in yellow). E-F: Enrichment of −730/−713 bp region (E) or −142/−125 bp region (F) (highlighted in yellow) of mouse Med13 promoter with KLF5 of chromatin samples from HL-1 cells treated with Ad-GFP or Ad-KLF5 (n=3; ***p<0.001 vs Ad-GFP with Student’s T-Test). Data are presented as mean ± SEM. Ad: adenovirus, KLF: Krüppel-like factor, Med13: mediator complex subunit 13.

Figure 5:. αMHC-KLF5^−/− mice on HFD have neither higher leptin and adiponectin nor lower natriuretic peptide levels –

A-B: Leptin (A) and adiponectin (B) levels in plasma obtained from control floxed and αMHC-KLF5^−/− mice after 6 weeks of HFD (n=3-5). C: Cardiac Anp and Bnp mRNA expression from control floxed and αMHC-KLF5^−/− mice after 6 weeks HFD diet (n=3-5, ***p<0.001 with Student’s T-Test). D: BNP levels in plasma from control floxed and αMHC-KLF5^−/− mice after 6 weeks in HFD (n=3-9). Data are presented as mean ± SEM. ANP; atrial natriuretic peptide, BNP: brain natriuretic peptide, HFD: high fat diet, KLF: Krüppel-like factor. (See also Figure S3)

Figure 6:. αMHC-KLF5^−/− mice on HFD have increased FGF21 signaling –

A-D: Cardiac Fgf21 mRNA expression (n=4-5, ***p<0.001 with 2way ANOVA) (A), and plasma FGF21 levels (n=8, *p<0.05 with Student’s T-Test; 95% CI 69.7-138.9 pg/ml in control floxed and 118.7-233.5 pg/ml in αMHC-KLF5^−/− mice) (B) in control floxed and αMHC-KLF5^−/− mice after 6 weeks in HFD. C: Fgf21r and Klotb mRNA levels in WAT obtained from control floxed and αMHC-KLF5^−/− mice after 6 weeks in HFD (n=3, **p<0.01, ***p<0.001 vs floxed with Student’s T-Test). D: Representative western blot image and quantitative analysis of sumo-PPARγ and PPARγ protein levels obtained with immunoprecipitation from WAT isolated from control floxed and αMHC-KLF5^−/− mice after 6 weeks in HFD (n=3-4, *p<0.05 with Student’s T-Test). The lanes were run on the same gel but were noncontiguous. Data are presented as mean ± SEM. HFD: high fat diet, KLF: Krüppel-like factor.

Figure 7:. Cardiomyocyte FGF21 is associated with increased weight gain of HFD-fed αMHC-KLF5^−/− mice –

A: Cardiac Fgf21 mRNA levels in control floxed and αMHC-[KLF5^−/−;FGF21^−/−] mice (n= 8, *p<0.05 vs floxed with Student’s T-Test). B-D: Cumulative body weight gain (B), body weight gain (C), and posterior subcutaneous WAT weight (D) of control floxed (n = 21) and αMHC-[KLF5^−/−;FGF21^−/−] (B and C: n = 9, D: n = 5) mice after 6 weeks on HFD. E: Plasma FGF21 levels in control floxed (n = 34; 95% CI 102.4-225.2 pg/ml), and αMHC-[KLF5^−/−;FGF21^−/−] (n = 8; 95% CI 1.0-115.4 pg/ml) mice after 6 weeks HFD (*p<0.05 vs floxed with Student’s T-Test). F: mRNA levels of lipid metabolism markers Pparg1, Pparg2, Lpl, Cd36, Dgat2 and Glut4 in WAT obtained from control floxed (n = 25) and aMHC-[KLF5^−/−;FGF21^−/−] (n = 6) mice. Data are presented as mean ± SEM. cmDKO is αMHC-[KLF5^−/−;FGF21^−/−]. Dgat: diglyceride acyltransferase, Glut: glucose transporter, HFD: high fat diet, KLF: Krüppel-like factor, Lpl: lipoprotein lipase, WAT: white adipose tissue. (See also Figure S1E–G, and Figure S3)

Figure 8:. Cardiomyocyte FGF21 ablation negates the proadipogenic effect of KLF5 deletion –

A: Representative pictures of H&E staining of WAT and BAT, and Oil Red O staining of liver of control floxed and aMHC-[KLF5^−/−;FGF21^−/−] mice after 6 weeks HFD. B: Quantification of WAT adipocyte size (n = 4) of control floxed and aMHC-[KLF5^−/−;FGF21^−/−] mice after 6 weeks HFD. C: Western Blotting analysis for sumo-PPARγ and PPARγ protein levels obtained with immunoprecipitation from WAT isolated from control floxed and aMHC-[KLF5^−/−;FGF21^−/−] mice after 6 weeks in HFD (n=3-6). D: Cardiac Med13 mRNA levels in HFD-fed control floxed and aMHC-[KLF5^−/−;FGF21^−/−] mice (n=4, *p<0.05 vs floxed with Student’s T-Test). Data are presented as mean ± SEM. cmDKO is aMHC-[KLF5^−/−;FGF21^−/−]. BAT: brown adipose tissue, HFD: high fat diet, KLF: Krüppel-like factor, WAT: white adipose tissue.