Small Molecule Activators of Mitochondrial Fusion Prevent Congenital Heart Defects Induced by Maternal Diabetes

Abstract

Most congenital heart defect (CHD) cases are attributed to nongenetic factors; however, the mechanisms underlying nongenetic factor-induced CHDs are elusive. Maternal diabetes is one of the nongenetic factors, and this study aimed to determine whether impaired mitochondrial fusion contributes to maternal diabetes-induced CHDs and if mitochondrial fusion activators, teriflunomide and echinacoside, could reduce CHD incidence in diabetic pregnancy. We demonstrated maternal diabetes-activated FoxO3a increases miR-140 and miR-195, which in turn represses Mfn1 and Mfn2, leading to mitochondrial fusion defects and CHDs. Two mitochondrial fusion activators are effective in preventing CHDs in diabetic pregnancy.

Keywords: congenital heart defect; maternal diabetes; microRNA; mitochondrial fusion; mitofusin 1; mitofusin 2.

Graphical abstract

Figure 1

Mitochondrial Fusion Activators Ameliorate Maternal Diabetes–Induced CHDs (A and J) Primary cardiomyocytes transfected with mitochondrial matrix-targeted photoactive green fluorescent protein (mito-PAGFP) and percentage of mitochondria (>2 μm) per area in cells (n = 5). (B) Protein levels in embryonic day 12.5 (E12.5) hearts (n = 3). (C) mito-PAGFP in primary cardiomyocytes. Red circles: photoactivated regions. (D) Mito-PAGFP intensity (n = 3). (E) Time to first fusion event (n = 5). (F and I) Messenger RNA levels in E12.5 hearts (n = 3). (G) Heart vessels (upper) and sections (lower). (H and K) Numbers of embryos. Data are presented as mean ± SD in A, B, D, E, F, I, and J. Student’s t-test in A and B. One-way analysis of variance with Tukey’s post hoc test in D, E, F, I, and J. Chi-square test in H and K. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. AO = aorta; CHD = congenital heart defect; ECH = echinacoside; HLHS = hypoplastic left heart syndrome; LA/RA = left/right atrium; LV/RV = left/right ventricle; PA = pulmonary artery; PTA = persistent truncus arteriosus; TERI = teriflunomide.

Figure 2

Mitochondrial Fusion Activators Reduce CHDs Induced by miR-195/140 Transgenic Overexpression MicroRNA (miRNA) levels in primary cardiomyocytes (n = 3) (A) and in embryonic day 12.5 (E12.5) hearts (B) (n = 3). (C and H) mRNA levels in E12.5 hearts (n = 3). (D) Time-lapse images of mitochondrial matrix-targeted photoactive green fluorescent protein (mito-PAGFP) in primary cardiomyocytes. (E) mito-PAGFP fluorescence intensity (n = 3). (F) Time to first mitochondrial fusion event (n = 5). (G and I) Numbers of E17.5 embryos. Data are presented as mean ± SD in A, B, C, E, F, and H. One-way analysis of variance with Tukey’s post hoc test in A, C, E, F, and H. Student’s t-test in (B). Chi-square test in G and I. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. dTg = miR-195 and miR-140 double transgenic; WT = wild-type; other abbreviations as in Figure 1.

Figure 3

FoxO3a Upregulates miR-140/195 and Represses Mitochondrial Fusion (A) FoxO3a immunostaining in primary cardiomyocytes and quantification of fluorescence intensity in nuclei (n = 3). FoxO3a binding sites on the Mir195 (B) and Mir140 (C) promoters and promoter-driven luciferase reporter activity in H9C2 cells (n = 3). (D) MicroRNA (miRNA) levels in embryonic day 12.5 (E12.5) hearts (n = 3). (E) E17.5 heart vessels (upper) and sections (lower). (F) Numbers of E17.5 embryos. (G) Representative images of primary cardiomyocytes. (H) Percentage of mitochondria (>2 μm) per area in cells (n = 5). (I and J) Flow cytometry analysis and ratios of fluorescence intensity (red/green) in primary cardiomyocytes (n = 3). (K) Protein levels (n = 3). Data are presented as mean ± SD in A, B, C, D, H, J, and K. Student’s t-test in A. One-way analysis of variance with Tukey’s post hoc test in B, C, D, H, J, and K. Chi-square test in F. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. DM = diabetes mellitus; ND = nondiabetic; TSS = transcription start site; VSD = ventricular septum defect; other abbreviations as in Figures 1 and 2.

Figure 4

miR-195 Deletion Restores Mitochondrial Fusion (A) Images of embryonic day 17.5 (E17.5) heart vessels (upper) and sections (lower). (B) Numbers of E17.5 embryos. TUNEL-positive cells in E9.5 hearts (C) and the quantification (D) (n = 3). p-H3-positive cells in E9.5 hearts (E) and the quantification (F) (n = 3). (G) Mitochondrial matrix-targeted photoactive green fluorescent protein (mito-PAGFP) intensity in primary cardiomyocytes (n = 3). (H) Time to first mitochondrial fusion event (n = 9). (I) Representative images of primary cardiomyocytes and percentage of mitochondria (>2 μm) per area in cells (n = 9). (J) Flow cytometry analysis and ratios of fluorescence intensity (red/green) in primary cardiomyocytes (n = 3). Data are presented as mean ± SD in D, F, G, H, I, and J. Chi-square test in (B). One-way analysis of variance with Tukey’s post hoc test in D, F, G, H, I, and J. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. OFT = outflow tract; other abbreviations as in Figure 1, Figure 2, Figure 3.

Figure 5

Inhibition of miR-195 Improves Mitochondrial Fusion by Upregulating Mfn2 (A) Representative images of primary cardiomyocytes. (B) Percentage of mitochondria (>2 μm) per area in cells (n = 9 or 5). (C) miR-195 binding site on Mfn2 mRNA. (D) Mfn2 mRNA level bound to biotin-labeled miR-195 (n = 3). (E) miR-195 and Mfn2 mRNA in RNA co-immunoprecipitation (IP) in embryonic day 12.5 (E12.5) hearts (n = 3). (F) Mfn2 mRNA in primary cardiomyocytes (n = 3). (G) Mfn2 mRNA and protein levels (n = 3). Data are presented as mean ± SD in B, D, E, F, and G. One-way analysis of variance with Tukey’s post hoc test in B, F, and G. Student’s t-test in D and E. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. IgG = immunoglobulin G; IP = immunoprecipitation; UTR = untranslated region; other abbreviations as in Figure 1, Figure 2, Figure 3.

Figure 6

miR-140 Deficiency Restores Mfn1 Expression and Mitochondrial Fusion (A) The miR-140 binding site on Mfn1 mRNA. (B) Mfn1 mRNA bond to biotin-labeled miR-140 (n = 3). (C) miR-140 and Mfn1 mRNA levels in RNA immunoprecipitation (n = 3). (D) Mfn1 mRNA and protein levels (n = 3). (E) Representative images of primary cardiomyocytes. (F) Percentage of mitochondria (>2 μm) per area in cells (n = 4). (G) Mitochondrial matrix-targeted photoactivable green fluorescent protein intensity in primary cardiomyocytes (n = 3). (H) Time to first mitochondrial fusion event (n = 9). (I) Images of embryonic day 17.5 heart vessels (upper) and sections (lower). Data are presented as mean ± SD in B, C, D, F, G, and H. Student’s t-test in B and C. One-way analysis of variance with Tukey’s post hoc test in D, F, G, and H. Chi-square test in I. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5.

Figure 7

Restoring Mfn1 Expression Abrogates the Teratogenicity of Maternal Diabetes (A) Representative images of primary cardiomyocytes and percentage of mitochondria (>2 μm) per area in cells (n = 4). (B) Time-lapse images of mitochondrial matrix-targeted photoactivable green fluorescent protein (mito-PAGFP) in primary cardiomyocytes. (C) mito-PAGFP intensity in primary cardiomyocytes (n = 3). (D) Time to first mitochondrial fusion event (n = 5). (E) Embryonic day 17.5 (E17.5) heart vessels (upper) and sections (lower). (F and I) Numbers of E17.5 embryos. (G) Cleaved caspase-3 levels (n = 3). (H) TUNEL-positive cells in E9.5 hearts and the quantification (n = 3). Data are presented as mean ± SD in A, C, D, G, and H. One-way analysis of variance with Tukey’s post hoc test in A, C, D, G, and H. Chi-square test in F and I. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. TGA = transposition of the great arteries; other abbreviations as in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5.