Highlights

•	Most CHD cases are attributed to nongenetic factors, whereas the mechanisms underlying nongenetic factor–induced CHDs are elusive. Maternal diabetes is one of the nongenetic factors inducing CHDs.
•	The study reveals an innovative epigenetic mechanism underlying maternal diabetes–induced CHDs.
•	Maternal diabetes-activated transcription factor 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, teriflunomide (an FDA-approved drug) and echinacoside (a naturally occurring compound), increase the expression level of Mfn1 and Mfn2, restore mitochondrial fusion, and prevent CHD formation. These 2 activators show potential value in preventing CHDs in diabetic pregnancy.

Summary

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.

Introduction

The United States has the highest infant mortality rate, which is the basic measure of public health, among developed countries.¹ Congenital heart defects (CHDs) are the most common cause of infant death.² Furthermore, CHDs are the most prevalent birth defects, occurring in approximately 4 to 10 per 1,000 live births.² Epidemiological studies in CHD prevention suggest a controversial effect of maternal folic acid supplementation,^3,4 which is the only effective intervention to prevent neural tube defects, another type of potentially fatal birth defect. However, a mechanism-based means of preventing CHDs is still lacking.

Human epidemiological studies have demonstrated that the major contributing factors to the occurrence of CHDs are nongenetic factors.^2,5 Among all nongenetic factors that cause CHDs, maternal diabetes is the major factor.^2,6 The rate of CHDs in infants born to mothers with diabetes is approximately 4 to 6 times higher than mothers without diabetes.^6-8 More than 60 million women of reproductive age worldwide have diabetes, and this number will likely double by 2030 due to the current global epidemic of obesity.⁹ Even under the best clinical care, women with diabetes are still 3 to 4 times more likely to have a child with CHDs than women without diabetes.¹⁰ Thus, given these incidences and the lack of means to prevent CHDs, its occurrence is an unmet clinical need, and therefore uncovering the cellular and molecular events underlying its development will aid in the identification of effective preventions.

Here, using a maternal diabetes mouse model of CHDs, we show that this pathology occurs due to activation of the transcription factor FoxO3a, which stimulates expression of miR-140 and miR-195 that, in turn, represses mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) expression, respectively. Treating this model with 2 activators of mitochondrial fusion, teriflunomide, a U.S. Food and Drug Administration (FDA)-approved drug, and echinacoside, a natural compound, we found prevention of CHDs. This amelioration correlated with re-expression of Mfn1 and Mfn2, improved mitochondrial dynamics and cell proliferation, and reduced apoptosis. Thus, pharmacological restoration of mitochondrial fusion may be an effective approach to reduce the risk of CHDs resulting from diabetic pregnancy.

Discussion

It is conventionally accepted that organ development is orchestrated from the cell nucleus and that the mitochondria simply follow along; however, a recent study demonstrated that mitochondria orchestrate developmental events of the mouse heart, and the disturbance of mitochondrial function contributes to CHD formation.¹⁶ Mitochondrial dynamics are governed by fusion and fission events essential for proper heart development.¹⁶ Mitochondria fuse via the function of Mfn1 and Mfn2. Deleting the Mfn1 and Mfn2 genes in early heart muscle cells results in severely underdeveloped hearts.¹⁶ Furthermore, mouse embryonic stem cells missing Mfn2 and Opa1 (optic atrophy protein 1), a mitochondrial fusion facilitator, do not develop into beating cardiomyocytes.¹⁶ Reduced mitochondrial fusion resulting from Mfn1 and Mfn2 deletion disrupts several signaling pathways implicated in CHDs.¹⁶ This evidence suggests that altered mitochondrial dynamics drive cardiac dysmorphogenesis.

Impaired mitochondrial fusion leads to mitochondrial dysfunction and subsequently alters cardiac morphogenesis. Mitochondrial dysfunction is an evident cellular defect in cardiomyocytes exposed to maternal diabetes.^17,24 Intrinsic abnormalities are present in cardiomyocytes derived from inducible pluripotent stem cells of patients with CHDs that lack an underlying genetic cause,²⁵ suggesting that cell development is a key factor in cardiac morphogenesis. These cellular organelle defects continue to persist after the establishment of CHDs and may contribute to sustainable cardiomyocyte dysfunction in patients with CHD. The present study illustrates for the first time that maternal diabetes increases 2 key miRNAs that impair mitochondrial fusion and enhance mitochondrial fragmentation in mouse embryonic cardiomyocytes in vitro and in vivo.

miRNAs are critically involved in virtually all aspects of cardiac development and disease.²⁶ miR-1 overexpression disrupts mouse embryonic heart development,²⁷ and miR-133a overexpression in cardiomyocytes leads to decreased cell proliferation and the formation of cardiac septation defects.²⁸ The present study demonstrates that the upregulation of miR-140 and miR-195 mediates the teratogenicity of maternal diabetes leading to CHDs. During development, miR-140 is predominantly expressed in embryonic chondrocytes.²⁹ miR-140 induces cardiomyocyte apoptosis via the intrinsic mitochondrial pathway.¹⁴ In contrast to miR-140, miR-195 is expressed early in the developing human heart.³⁰ miR-195 inhibits cell proliferation and induces apoptosis by repressing multiple prosurvival proteins.^31,32 Multiple lines of evidence suggest that miR-140 and miR-195 always work together. They both trigger mitochondrial dysfunction,^14,31 participate in stem cell aging,³¹ and are elevated in adult heart diseases.³³ In agreement with the coherence between these 2 miRNAs, we found that deleting the mir140 gene or the mir195 gene significantly ameliorated maternal diabetes–induced CHDs, and that overexpressing these 2 miRNAs in the heart mimicked maternal diabetes in inducing CHDs.

The transcription factor FoxO3a is activated by maternal diabetes.²³ FoxO3a upregulates miRNAs in cancer cells.³⁴ In the present study, FoxO3a transcriptionally induced miR-140 and miR-195 expression and thus inhibited mitochondrial fusion in embryonic cardiomyocytes. FoxO3a reduces the size of cardiomyocytes in rats.³⁵ FoxO3a is a cell death trigger that acts through the mitochondrial apoptosis pathway in conditions of heart failure and hypertrophy.^36,37 Our previous study indicated that the deletion of FoxO3a could inhibit maternal diabetes–induced apoptosis in cardiac progenitor cells in vivo.³⁸ Here, we showed that Foxo3a gene deletion ameliorates maternal diabetes–induced CHDs by suppressing mitochondrial fragmentation and dysfunction. Thus, we reveal the downstream effectors of FoxO3a, miR-140 and miR-195, in defective heart development.

Mitochondrial fusion, a prosurvival event, maintains mitochondrial homeostasis by removing dysfunctional mitochondria.^39,40 Cells lacking both Mfn1 and Mfn2 have completely fragmented mitochondria with no detectable mitochondrial fusion.⁴⁰ Mitochondrial fusion is important for the maintenance of mitochondrial morphology, cell growth, membrane potential, and respiration.³⁹ Reduced fusion could be a key factor contributing to diabetes- or miRNA-induced mitochondrial dysfunction. Maternal diabetes induces cellular dysfunction in cells required for cardiac septation leading to CHDs.^17,24 Enhanced mitochondrial fusion stimulates cell proliferation by promoting cell cycle progression.⁴¹ Cells with double knockout of Mfn1 and Mfn2 proliferate much slower than their corresponding wild-type counterparts.⁴² Here, we showed that reduced Mfn1 and Mfn2 expression cause cellular dysfunction and alterations in cardiac septation leading to CHDs under conditions of maternal diabetes exposure and miRNA overexpression.

Small molecule drugs are the pillars of traditional medicine. Teriflunomide, a small molecule compound, is approved by the FDA for use in the treatment of multiple sclerosis; however, studies also showed that teriflunomide could activate mitochondrial fusion. One study indicated that teriflunomide upregulates mitofusins and also induces mitochondrial elongation by depletion of the cellular pyrimidine pool secondary to the inhibition of dihydroorotate dehydrogenase.²⁰ Another study indicated that teriflunomide increases Mfn2 transcriptional activity and mitofusin mRNA levels in Hela cells.⁴³ Echinacoside, another small molecule compound, is currently being investigated for anti-apoptotic and neuroprotective effects.^44,45 Similarly, this compound also can function as a mitochondrial fusion activator. A study found that echinacoside selectively binds to the previously uncharacterized casein kinase 2 (CK2) α′ subunit (CK2α′) as a direct cellular target and allosterically regulates CK2α′ conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex, and then the CK2α′/BTF3 complex facilitates β-catenin nuclear translocation to activate T-cell factor/lymphoid enhancer factor transcription factors and stimulates transcription of the mitochondrial fusion gene Mfn2.²¹ These findings are consistent with our current study. We demonstrated that teriflunomide and echinacoside, acting as mitochondrial fusion activators, increase the expression levels of Mfn1 and Mfn2 and improve mitochondrial fusion in cardiomyocytes under diabetic conditions, and in turn, prevent CHD formation in diabetic pregnancy. We did not observe any side effect of the 2 compounds in pregnant mice or embryos at the adopted dosage (15 mg/kg); however, we found teriflunomide often caused abortion at a higher dosage (30 mg/kg) in our preliminary study.

The STZ-induced type 1 diabetic embryopathy mouse model, which can mimic hyperglycemia in human maternal diabetes, is widely accepted in the field of maternal diabetes–induced birth defects.^11,46,47 STZ used to induce diabetes is not a complicating factor because STZ is cleared from the bloodstream rapidly (serum half-life is 5 minutes with no drug measurable by 2 hours),⁴⁸ and pregnancy is not established until 1 to 2 weeks after STZ injection.⁴⁹ Insulin treatment of STZ-induced diabetic embryopathy effectively reduces hyperglycemia and embryonic malformations,⁴⁹ indicating that hyperglycemia is the primary cause of teratogenicity and that pregestational STZ injections do not cause any toxicity to the developing embryo. In the current study, this mouse model produced 20% to 40% CHDs, including ventricular septum defect, persistent truncus arteriosus, transposition of the great arteries, and hypoplastic left heart syndrome, in embryos exposed to diabetes, whereas embryos from nondiabetic controls had zero CHDs. Among all the CHD cases, ventricular septum defect cases were the most common, which is almost identical to that in humans.^6,50 In addition, we observed very few neural tube defect cases at E17.5 when embryos were harvested for CHD rate determination, as well as the infrequent cases of kidney defects and eye defects. We used 2 miRNA modified mouse models in this study, miR-140 global knockout and miR-195 conditional knockout mice, and did not observe any cardiac or noncardiac defects because of the deletion of each miRNA.

Unlike neural tube defects, which can be reduced by folate supplementation, prevention methods for CHDs are lacking. The present study reveals a mechanism-based method for the prevention of CHDs induced by nongenetic factors that are primarily causal factors in humans. Treatment of the diabetic pregnant dams with small molecule activators of mitochondrial fusion restored Mfn1 and Mfn2 expression at the transcriptional level and subsequently rescued mitochondrial fusion in cardiomyocytes. We also uncovered the molecular pathway that leads to the inhibition of mitochondrial fusion in the CHD models, thus providing new targets for the design of prevention approaches.

Conclusions

We conclude that reduced mitochondrial fusion is a key event in the formation of CHDs induced by either miR-195/miR-140 Tg expression or exposure to a maternal diabetic milieu. Maternal diabetes–activated FoxO3a increases miR-140 and miR-195, which in turn represses Mfn1 and Mfn2, leading to mitochondrial fusion defects and CHDs. Maternal treatment with either of teriflunomide and echinacoside restores Mfn1 and Mfn2 expression and mitochondrial fusion in cardiomyocytes of embryonic hearts exposed to diabetes, implicating that activating mitochondrial fusion could be a potent means to prevent CHDs induced by maternal nongenetic factors.