Beyond their role as a cellular powerhouse, mitochondria are emerging as integral players in molecular signaling and cell fate determination through reactive oxygen species (ROS). While ROS production has historically been portrayed as an unregulated process driving oxidative stress and disease pathology, contemporary studies reveal that ROS also facilitate normal physiology. Mitochondria are especially abundant in cardiac tissue; hence, mitochondrial dysregulation and ROS production are thought to contribute significantly to cardiac pathology. Moreover, there is growing appreciation that medical therapies designed to mediate mitochondrial ROS production can be important strategies to ameliorate cardiac disease. In this review, we highlight evidence from animal models that illustrates the strong connections between mitochondrial ROS and cardiac disease, discuss advancements in the development of mitochondria-targeted antioxidant therapies, and identify challenges faced in bringing such therapies into the clinic.

As development proceeds from the embryonic to fetal stages, cardiac energy demands increase substantially, and oxidative phosphorylation of ADP to ATP in mitochondria becomes vital. Relatively little, however, is known about the signaling mechanisms regulating the transition from anaerobic to aerobic metabolism that occurs during the embryonic period. The main objective of this study was to test the hypothesis that adrenergic hormones provide critical stimulation of energy metabolism during embryonic/fetal development. We examined ATP and ADP concentrations in mouse embryos lacking adrenergic hormones due to targeted disruption of the essential dopamine β-hydroxylase (Dbh) gene. Embryonic ATP concentrations decreased dramatically, whereas ADP concentrations rose such that the ATP/ADP ratio in the adrenergic-deficient group was nearly 50-fold less than that found in littermate controls by embryonic day 11.5. We also found that cardiac extracellular acidification and oxygen consumption rates were significantly decreased, and mitochondria were significantly larger and more branched in adrenergic-deficient hearts. Notably, however, the mitochondria were intact with well-formed cristae, and there was no significant difference observed in mitochondrial membrane potential. Maternal administration of the adrenergic receptor agonists isoproterenol or l-phenylephrine significantly ameliorated the decreases in ATP observed in Dbh-/- embryos, suggesting that α- and β-adrenergic receptors were effective modulators of ATP concentrations in mouse embryos in vivo. These data demonstrate that adrenergic hormones stimulate cardiac energy metabolism during a critical period of embryonic development.

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have approximately 10-fold less cardiac ATP compared to littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation or degradation were affected in adrenergic-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using liquid chromatography/mass spectrometry (LC/MS). Our results showed glucose-1-phosphate and glucose-6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentosephosphate pathways were significantly lower compared to controls. Further, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80% and 40%, respectively) lower in adrenergic-deficient hearts.Addition of pyruvate and to a lesser extent, ribose, led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.

The nuclear genome-encoded mitochondrial DNA (mtDNA) transcription factor A (TFAM) is indispensable for mitochondrial energy production in the developing and postnatal heart; a similar role for TFAM is inferred in adult heart. Here, we provide evidence that challenges this long-standing paradigm. Unexpectedly, conditionalTfam ablation in vivo in adult mouse cardiomyocytes resulted in a prolonged period of functional resilience characterized by preserved mtDNA content, mitochondrial function, and cardiac function, despite mitochondrial structural alterations and decreased transcript abundance. Remarkably, TFAM protein levels did not directly dictate mtDNA content in the adult heart, and mitochondrial translation was preserved with acute TFAM inactivation, suggesting maintenance of respiratory chain assembly/function. Long-term Tfam inactivation, however, downregulated the core mtDNA transcription and replication machinery, leading to mitochondrial dysfunction and cardiomyopathy. Collectively, in contrast to the developing heart, these data reveal a striking resilience of the differentiated adult heart to acute insults to mtDNA regulation.

Mitochondria are recognized as signaling organelles because, under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased post-translational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel crosstalk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

Background Excessive alcohol consumption during pregnancy is associated with high risk of congenital heart defects, but it is unclear how alcohol specifically affects heart development during the acute aftermath of a maternal binge drinking episode. We hypothesize that administration of a single maternal binge dose of alcohol to pregnant mice at embryonic day 9.5 (E9.5) causes perturbations in the expression patterns of specific genes in the developing heart in the acute period (1–3 days) following the binge episode. To test this hypothesis and identify strong candidate ethanol‐sensitive target genes of interest, we adapted a mouse binge alcohol model that is associated with a high incidence of congenital heart defects as described below. Methods/Results Pregnant mice were administered a single dose of alcohol (2.5 g/kg in saline) or control (saline alone) via oral gavage. To evaluate the impact of maternal binge alcohol on cardiac gene expression profiles, we isolated embryonic hearts from both groups (n = 5/group) at 24, 48, and 72 h post‐gavage for transcriptomic analyses. RNA was extracted and evaluated using quantitative RNA‐sequencing (RNA‐Seq) methods. To identify a cohort of binge‐altered cardiac genes, we set the threshold for change at >2.0‐fold difference with adjusted p < 0.05 versus control.  RNA‐Seq analysis of cardiac gene expression revealed that of the 17 genes that were altered within the first 48 h post‐binge, with the largest category consisting of transcription factors (Alx1, Alx4, HoxB7, HoxD8, and Runx2), followed by signaling molecules (Adamts18, Dkk2, Rtl1, and Wnt7a). Furthermore, multiple comparative and pathway analyses suggested that several of the candidate genes identified through differential RNA‐Seq analysis may interact through certain common pathways. To investigate this further, we performed gene‐specific qPCR analyses for three representative candidate targets: Runx2, Wnt7a, and Mlxipl. Notably, only Wnt7a showed significantly (p < 0.05) decreased expression in response to maternal binge alcohol in the qPCR assays. Conclusions These findings identify Wnt7a and a short list of potential other candidate genes and pathways for further study, which could provide mechanistic insights into how maternal binge alcohol consumption produces congenital cardiac malformations.

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Cardiac energy demands increase during embryonic development, requiring activation of oxidative phosphorylation to convert ADP to ATP in mitochondria. We have recently shown that adrenergic hormones are required to maintain sufficient cardiac energy metabolism during embryonic development, but the specific mechanism(s) underlying this regulation are not known. Mouse embryos lacking the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to targeted loss of the dopamine β-hydroxylase ( Dbh ) gene, have markedly (>50-fold) decreased steady-state ATP/ADP ratios. Rates of ATP synthesis and hydrolysis did not differ between adrenergic-deficient and competent embryos suggesting the enzymatic machinery required for ATP production/consumption is functional. We hypothesized that adrenergic-deficient embryonic hearts are metabolically starved of nutrients leading to energy depletion. To identify changes in metabolism in adrenergic-deficient hearts, we performed LC-MS metabolomics, which showed decreases in all nucleotide triphosphates (NTPs) and NAD(H) confirming energy depletion. Additionally, products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G-6-PDH), and pyruvate dehydrogenase (PDH) were significantly diminished compared to controls, suggesting impaired activity. Enzymatic activities of GAPDH, G-6-PDH, PDH, and glycogen phosphorylase (GP); a well-known enzyme under adrenergic regulation, were measured from the rate of NAD(P)H production. GAPDH, G-6-PDH, and GP activities were significantly decreased (~80%, 40%, and 70% reduction, respectively) compared to controls. Interestingly, GAPDH, G-6-PDH, and GP protein levels, examined by western blot, did not differ from adrenergic-competent controls, thereby suggesting that adrenergic hormones regulate posttranslational activity of these enzymes. These results indicate that mitochondria are metabolically starved due to impairments in glycogenolysis, glycolysis, and pentose phosphate pathways. These findings reveal new mechanistic insights into global adrenergic regulation of major metabolic pathways during embryonic heart development.