ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.

Mitochondrial Transfer to Host Cells from Ex Vivo Expanded Donor Hematopoietic Stem Cells.

Mitochondrial dysfunction is observed in various conditions, from metabolic syndromes to mitochondrial diseases. Moreover, mitochondrial DNA (mtDNA) transfer is an emerging mechanism that enables the restoration of mitochondrial function in damaged cells. Hence, developing a technology that facilitates the transfer of mtDNA can be a promising strategy for the treatment of these conditions. Here, we utilized an ex vivo culture of mouse hematopoietic stem cells (HSCs) and succeeded in expanding the HSCs efficiently. Upon transplantation, sufficient donor HSC engraftment was attained in-host. To assess the mitochondrial transfer via donor HSCs, we used mitochondrial-nuclear exchange (MNX) mice with nuclei from C57BL/6J and mitochondria from the C3H/HeN strain. Cells from MNX mice have C57BL/6J immunophenotype and C3H/HeN mtDNA, which is known to confer a higher stress resistance to mitochondria. Ex vivo expanded MNX HSCs were transplanted into irradiated C57BL/6J mice and the analyses were performed at six weeks post transplantation. We observed high engraftment of the donor cells in the bone marrow. We also found that HSCs from the MNX mice could transfer mtDNA to the host cells. This work highlights the utility of ex vivo expanded HSC to achieve the mitochondrial transfer from donor to host in the transplant setting.

Role of the ST6GAL1 sialyltransferase in regulating ovarian cancer cell metabolism.

The ST6GAL1 sialyltransferase, which adds α2-6-linked sialic acids to N-glycosylated proteins, is upregulated in many malignancies including ovarian cancer. Through its activity in sialylating select surface receptors, ST6GAL1 modulates intracellular signaling to regulate tumor cell phenotype. ST6GAL1 has previously been shown to act as a survival factor that protects cancer cells from cytotoxic stressors such as hypoxia. In the present study, we investigated a role for ST6GAL1 in tumor cell metabolism. ST6GAL1 was overexpressed (OE) in OV4 ovarian cancer cells, which have low endogenous ST6GAL1, or knocked-down (KD) in ID8 ovarian cancer cells, which have high endogenous ST6GAL1. OV4 and ID8 cells with modulated ST6GAL1 expression were grown under normoxic or hypoxic conditions, and metabolism was assessed using Seahorse technology. Results showed that cells with high ST6GAL1 expression maintained a higher rate of oxidative metabolism than control cells following treatment with the hypoxia mimetic, desferrioxamine (DFO). This enrichment was not due to an increase in mitochondrial number. Glycolytic metabolism was also increased in OV4 and ID8 cells with high ST6GAL1 expression, and these cells displayed greater activity of the glycolytic enzymes, hexokinase and phosphofructokinase. Metabolism maps were generated from the combined Seahorse data, which suggested that ST6GAL1 functions to enhance the overall metabolism of tumor cells. Finally, we determined that OV4 and ID8 cells with high ST6GAL1 expression were more invasive under conditions of hypoxia. Collectively, these results highlight the importance of sialylation in regulating the metabolic phenotype of ovarian cancer cells.

Mitochondrial DNA Variations Modulate Alveolar Epithelial Mitochondrial Function and Oxidative Stress in Newborn Mice Exposed to Hyperoxia.

Oxidative stress is an important contributor to bronchopulmonary dysplasia (BPD), a form of chronic lung disease that is the most common morbidity in very preterm infants. Mitochondrial functional differences due to inherited and acquired mutations influence the pathogenesis of disorders in which oxidative stress plays a critical role. We previously showed using mitochondrial-nuclear exchange (MNX) mice that mitochondrial DNA (mtDNA) variations modulate hyperoxia-induced lung injury severity in a model of BPD. In this study, we studied the effects of mtDNA variations on mitochondrial function including mitophagy in alveolar epithelial cells (AT2) from MNX mice. We also investigated oxidant and inflammatory stress as well as transcriptomic profiles in lung tissue in mice and expression of proteins such as PINK1, Parkin and SIRT3 in infants with BPD. Our results indicate that AT2 from mice with C57 mtDNA had decreased mitochondrial bioenergetic function and inner membrane potential, increased mitochondrial membrane permeability and were exposed to higher levels of oxidant stress during hyperoxia compared to AT2 from mice with C3H mtDNA. Lungs from hyperoxia-exposed mice with C57 mtDNA also had higher levels of pro-inflammatory cytokines compared to lungs from mice with C3H mtDNA. We also noted changes in KEGG pathways related to inflammation, PPAR and glutamatergic signaling, and mitophagy in mice with certain mito-nuclear combinations but not others. Mitophagy was decreased by hyperoxia in all mice strains, but to a greater degree in AT2 and neonatal mice lung fibroblasts from hyperoxia-exposed mice with C57 mtDNA compared to C3H mtDNA. Finally, mtDNA haplogroups vary with ethnicity, and Black infants with BPD had lower levels of PINK1, Parkin and SIRT3 expression in HUVEC at birth and tracheal aspirates at 28 days of life when compared to White infants with BPD. These results indicate that predisposition to neonatal lung injury may be modulated by variations in mtDNA and mito-nuclear interactions need to be investigated to discover novel pathogenic mechanisms for BPD.

Mitochondrial haplotype modulates genome expression and mitochondrial structure/function in cardiomyocytes following volume overload.

Mitochondrial DNA (mtDNA) haplotype regulates mitochondrial structure/function and reactive oxygen species in aortocaval fistula (ACF) in mice. Here, we unravel the mitochondrial haplotype effects on cardiomyocyte mitochondrial ultrastructure and transcriptome response to ACF in vivo. Phenotypic responses and quantitative transmission electron microscopy (TEM) and RNA sequence at 3 days were determined after sham surgery or ACF in vivo in cardiomyocytes from wild-type (WT) C57BL/6J (C57n:C57mt) and C3H/HeN (C3Hn:C3Hmt) and mitochondrial nuclear exchange mice (C57n:C3Hmt or C3Hn:C57mt). Quantitative TEM of cardiomyocyte mitochondria C3HWT hearts have more electron-dense compact mitochondrial cristae compared with C57WT. In response to ACF, mitochondrial area and cristae integrity are normal in C3HWT; however, there is mitochondrial swelling, cristae lysis, and disorganization in both C57WT and MNX hearts. Tissue analysis shows that C3HWT hearts have increased autophagy, antioxidant, and glucose fatty acid oxidation-related genes compared with C57WT. Comparative transcriptomic analysis of cardiomyocytes from ACF was dependent upon mtDNA haplotype. C57mtDNA haplotype was associated with increased inflammatory/protein synthesis pathways and downregulation of bioenergetic pathways, whereas C3HmtDNA showed upregulation of autophagy genes. In conclusion, ACF in vivo shows a protective response of C3Hmt haplotype that is in large part driven by mitochondrial nuclear genome interaction.NEW & NOTEWORTHY The results of this study support the effects of mtDNA haplotype on nuclear gene expression in cardiomyocytes. Currently, there is no acceptable therapy for volume overload due to mitral regurgitation. The findings of this study could suggest that mtDNA haplotype activates different pathways after ACF warrants further investigations on human population of heart disease from different ancestry backgrounds.

Scientific opportunities in resilience research for cardiovascular health and wellness. Report from a National Heart, Lung, and Blood Institute workshop.

Exposure of biological systems to acute or chronic insults triggers a host of molecular and physiological responses to either tolerate, adapt, or fully restore homeostasis; these responses constitute the hallmarks of resilience. Given the many facets, dimensions, and discipline-specific focus, gaining a shared understanding of "resilience" has been identified as a priority for supporting advances in cardiovascular health. This report is based on the working definition: "Resilience is the ability of living systems to successfully maintain or return to homeostasis in response to physical, molecular, individual, social, societal, or environmental stressors or challenges," developed after considering many factors contributing to cardiovascular resilience through deliberations of multidisciplinary experts convened by the National Heart, Lung, and Blood Institute during a workshop entitled: "Enhancing Resilience for Cardiovascular Health and Wellness." Some of the main emerging themes that support the possibility of enhancing resilience for cardiovascular health include optimal energy management and substrate diversity, a robust immune system that safeguards tissue homeostasis, and social and community support. The report also highlights existing research challenges, along with immediate and long-term opportunities for resilience research. Certain immediate opportunities identified are based on leveraging existing high-dimensional data from longitudinal clinical studies to identify vascular resilience measures, create a 'resilience index,' and adopt a life-course approach. Long-term opportunities include developing quantitative cell/organ/system/community models to identify resilience factors and mechanisms at these various levels, designing experimental and clinical interventions that specifically assess resilience, adopting global sharing of resilience-related data, and cross-domain training of next-generation researchers in this field.

Mesenchymal stem cell bioenergetics and apoptosis are associated with risk for bronchopulmonary dysplasia in extremely low birth weight infants.

Oxidant stress contributes significantly to the pathogenesis of bronchopulmonary dysplasia (BPD) in extremely low birth weight (ELBW) infants. Mitochondrial function regulates oxidant stress responses as well as pluripotency and regenerative ability of mesenchymal stem cells (MSCs) which are critical mediators of lung development. This study was conducted to test whether differences in endogenous MSC mitochondrial bioenergetics, proliferation and survival are associated with BPD risk in ELBW infants. Umbilical cord-derived MSCs of ELBW infants who later died or developed moderate/severe BPD had lower oxygen consumption and aconitase activity but higher extracellular acidification-indicative of mitochondrial dysfunction and increased oxidant stress-when compared to MSCs from infants who survived with no/mild BPD. Hyperoxia-exposed MSCs from infants who died or developed moderate/severe BPD also had lower PINK1 expression but higher TOM20 expression and numbers of mitochondria/cell, indicating that these cells had decreased mitophagy. Finally, these MSCs were also noted to proliferate at lower rates but undergo more apoptosis in cell cultures when compared to MSCs from infants who survived with no/mild BPD. These results indicate that mitochondrial bioenergetic dysfunction and mitophagy deficit induced by oxidant stress may lead to depletion of the endogenous MSC pool and subsequent disruption of lung development in ELBW infants at increased risk for BPD.

Mito-Mendelian interactions alter in vivo glucose metabolism and insulin sensitivity in healthy mice.

The regulation of euglycemia is essential for human health with both chronic hypoglycemia and hyperglycemia having detrimental effects. It is well documented that the incidence of type 2 diabetes increases with age and exhibits racial disparity. Interestingly, mitochondrial DNA (mtDNA) damage also accumulates with age and its sequence varies with geographic maternal origins (maternal race). From these two observations, we hypothesized that mtDNA background may contribute to glucose metabolism and insulin sensitivity. Pronuclear transfer was used to generate mitochondrial-nuclear eXchange (MNX) mice to directly test this hypothesis, by assessing physiologic parameters of glucose metabolism in nuclear isogenic C57BL/6J mice harboring either a C57BL/6J (C57n:C57mt wild type-control) or C3H/HeN mtDNA (C57n:C3Hmt-MNX). All mice were fed normal chow diets. MNX mice were significantly leaner, had lower leptin levels, and were more insulin sensitive, with lower modified Homeostatic Model Assessment of Insulin Resistance (mHOMA-IR) values and enhanced insulin action when compared with their control counterparts. Further interrogation of muscle insulin signaling revealed higher phosphorylated Akt/total Akt ratios in MNX animals relative to control, consistent with greater insulin sensitivity. Overall, these results are consistent with the hypothesis that different mtDNA combinations on the same nuclear DNA (nDNA) background can significantly impact glucose metabolism and insulin sensitivity in healthy mice.NEW & NOTEWORTHY Different mitochondrial DNAs on the same nuclear genetic background can significantly impact body composition, glucose metabolism, and insulin sensitivity in healthy mice.

Hedgehog Signaling Regulates Metabolism and Polarization of Mammary Tumor-Associated Macrophages.

Elevated infiltration of immunosuppressive alternatively polarized (M2) macrophages is associated with poor prognosis in patients with cancer. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages. Here we identify a novel role for oncogenic Hedgehog (Hh) signaling in programming signature metabolic circuitries that regulate alternative polarization of tumor-associated macrophages. Two immunocompetent orthotopic mouse models of mammary tumors were used to test the effect of inhibiting Hh signaling on tumor-associated macrophages. Treatment with the pharmacologic Hh inhibitor vismodegib induced a significant shift in the profile of tumor-infiltrating macrophages. Mass spectrometry-based metabolomic analysis showed Hh inhibition induced significant alterations in metabolic processes, including metabolic sensing, mitochondrial adaptations, and lipid metabolism. In particular, inhibition of Hh in M2 macrophages reduced flux through the UDP-GlcNAc biosynthesis pathway. Consequently, O-GlcNAc-modification of STAT6 decreased, mitigating the immune-suppressive program of M2 macrophages, and the metabolically demanding M2 macrophages shifted their metabolism and bioenergetics from fatty acid oxidation to glycolysis. M2 macrophages enriched from vismodegib-treated mammary tumors showed characteristically decreased O-GlcNAcylation and altered mitochondrial dynamics. These Hh-inhibited macrophages are reminiscent of inflammatory (M1) macrophages, phenotypically characterized by fragmented mitochondria. This is the first report highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. These data describe a novel immunometabolic function of Hh signaling that can be clinically exploited. SIGNIFICANCE: These findings illustrate that Hh activity regulates a metabolic and bioenergetic regulatory program in tumor-associated macrophages that promotes their immune-suppressive polarization.

University of Alabama at Birmingham Nathan Shock Center: comparative energetics of aging.

The UAB Nathan Shock Center focuses on comparative energetics and aging. Energetics, as defined for this purpose, encompasses the causes, mechanisms, and consequences of the acquisition, storage, and use of metabolizable energy. Comparative energetics is the study of metabolic processes at multiple scales and across multiple species as it relates to health and aging. The link between energetics and aging is increasingly understood in terms of dysregulated mitochondrial function, altered metabolic signaling, and aberrant nutrient responsiveness with increasing age. The center offers world-class expertise in comprehensive, integrated energetic assessment and analysis from the level of the organelle to the organism and across species from the size of worms to rats as well as state-of-the-art data analytics. The range of services offered by our three research cores, (1) The Organismal Energetics Core, (2) Mitometabolism Core, and (3) Data Analytics Core, is described herein.

An evolutionary, or "Mitocentric" perspective on cellular function and disease.

The incidence of common, metabolic diseases (e.g. obesity, cardiovascular disease, diabetes) with complex genetic etiology has been steadily increasing nationally and globally. While identification of a genetic model that explains susceptibility and risk for these diseases has been pursued over several decades, no clear paradigm has yet been found to disentangle the genetic basis of polygenic/complex disease development. Since the evolution of the eukaryotic cell involved a symbiotic interaction between the antecedents of the mitochondrion and nucleus (which itself is a genetic hybrid), we suggest that this history provides a rational basis for investigating whether genetic interaction and co-evolution of these genomes still exists. We propose that both mitochondrial and Mendelian, or "mito-Mendelian" genetics play a significant role in cell function, and thus disease risk. This paradigm contemplates the natural variation and co-evolution of both mitochondrial and nuclear DNA backgrounds on multiple mitochondrial functions that are discussed herein, including energy production, cell signaling and immune response, which collectively can influence disease development. At the nexus of these processes is the economy of mitochondrial metabolism, programmed by both mitochondrial and nuclear genomes.

Unlocking the Secrets of Mitochondria in the Cardiovascular System: Path to a Cure in Heart Failure­A Report from the 2018 National Heart, Lung, and Blood Institute Workshop

Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches.

Mitochondrial - nuclear genetic interaction modulates whole body metabolism, adiposity and gene expression in vivo.

We hypothesized that changes in the mitochondrial DNA (mtDNA) would significantly influence whole body metabolism, adiposity and gene expression in response to diet. Because it is not feasible to directly test these predictions in humans we used Mitochondrial-Nuclear eXchange mice, which have reciprocally exchanged nuclear and mitochondrial genomes between different Mus musculus strains. Results demonstrate that nuclear-mitochondrial genetic background combination significantly alters metabolic efficiency and body composition. Comparative RNA sequencing analysis in adipose tissues also showed a clear influence of the mtDNA on regulating nuclear gene expression on the same nuclear background (up to a 10-fold change in the number of differentially expressed genes), revealing that neither Mendelian nor mitochondrial genetics unilaterally control gene expression. Additional analyses indicate that nuclear-mitochondrial genome combination modulates gene expression in a manner heretofore not described. These findings provide a new framework for understanding complex genetic disease susceptibility.

Vascular Endothelial Mitochondrial Function Predicts Death or Pulmonary Outcomes in Preterm Infants.

RATIONALE: Vascular endothelial mitochondrial dysfunction contributes to the pathogenesis of several oxidant stress-associated disorders. Oxidant stress is a major contributor to the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity that often leads to sequelae in adult survivors. OBJECTIVES: This study was conducted to identify whether differences in mitochondrial bioenergetic function and oxidant generation in human umbilical vein endothelial cells (HUVECs) obtained from extremely preterm infants were associated with risk for BPD or death before 36 weeks postmenstrual age. METHODS: HUVEC oxygen consumption and superoxide and hydrogen peroxide generation were measured in 69 infants. MEASUREMENTS AND MAIN RESULTS: Compared with HUVECs from infants who survived without BPD, HUVECs obtained from infants who developed BPD or died had a lower maximal oxygen consumption rate (mean ± SEM, 107 ± 8 vs. 235 ± 22 pmol/min/30,000 cells; P < 0.001), produced more superoxide after exposure to hyperoxia (mean ± SEM, 89,807 ± 16,616 vs. 162,706 ± 25,321 MitoSOX Red fluorescence units; P < 0.05), and released more hydrogen peroxide into the supernatant after hyperoxia exposure (mean ± SEM, 1,879 ± 278 vs. 842 ± 119 resorufin arbitrary fluorescence units; P < 0.001). CONCLUSIONS: Our results indicating that endothelial cells of premature infants who later develop BPD or die have impaired mitochondrial bioenergetic capacity and produce more oxidants at birth suggest that the vascular endothelial mitochondrial dysfunction seen at birth in these infants persists through their postnatal life and contributes to adverse pulmonary outcomes and increased early mortality.

Mitochondrial DNA mutations and cardiovascular disease.

PURPOSE OF REVIEW: Cardiovascular disease (CVD) is responsible for more morbidity and mortality worldwide than any other ailment. Strategies for reducing CVD prevalence must involve identification of individuals at high risk for these diseases, and the prevention of its initial development. Such preventive efforts are currently limited by an incomplete understanding of the genetic determinants of CVD risk. In this review, evidence for the involvement of inherited mitochondrial mutations in development of CVD is examined. RECENT FINDINGS: Several forms of CVD have been documented in the presence of pathogenic mitochondrial DNA (mtDNA) mutations, both in isolation and as part of larger syndromes. Other 'natural' mtDNA polymorphisms not overtly tied to any pathology have also been associated with alterations in mitochondrial function and individual risk for CVD, but until very recently these studies have been merely correlative. Fortunately, novel animal models are now allowing investigators to define a causal relationship between inherited 'natural' mtDNA polymorphisms, and cardiovascular function and pathology. SUMMARY: Cardiovascular involvement is highly prevalent among patients with pathogenic mtDNA mutations. The relationship between CVD susceptibility and 'natural' mtDNA polymorphisms requires further investigation, but will be aided in the near future by several novel experimental models.

Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass.

Mitophagy occurs during ischemia/reperfusion (I/R) and limits oxidative stress and injury. Mitochondrial turnover was assessed in patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). Paired biopsies of right atrial appendage before initiation and after weaning from CPB were processed for protein analysis, mitochondrial DNA/nuclear DNA ratio (mtDNA:nucDNA ratio), mtDNA damage, mRNA, and polysome profiling. Mitophagy in the post-CPB samples was evidenced by decreased levels of mitophagy adapters NDP52 and optineurin in whole tissue lysate, decreased Opa1 long form, and translocation of Parkin to the mitochondrial fraction. PCR analysis of mtDNA comparing amplification of short vs. long segments of mtDNA revealed increased damage following cardiac surgery. Surprisingly, a marked increase in several mitochondria-specific protein markers and mtDNA:nucDNA ratio was observed, consistent with increased mitochondrial biogenesis. mRNA analysis suggested that mitochondrial biogenesis was traniscription independent and likely driven by increased translation of existing mRNAs. These findings demonstrate in humans that both mitophagy and mitochondrial biogenesis occur during cardiac surgery involving CPB. We suggest that mitophagy is balanced by mitochondrial biogenesis during I/R stress experienced during surgery. Mitigating mtDNA damage and elucidating mechanisms regulating mitochondrial turnover will lead to interventions to improve outcome after I/R in the setting of heart disease.

Generation of Mitochondrial-nuclear eXchange Mice via Pronuclear Transfer.

The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated embryo of a different strain of the same species (e.g., C57BL/6 and C3H/HeN, Mus musculus), generating a re-constructed zygote harboring nuclear and mitochondrial genomes from different strains. Two-cell embryos are transferred to the ostia of oviducts in CD-1 pseudopregnant mice and developed to term. Nuclear genotype and mtDNA haplotype are verified in offspring, and females selected as founders for desired MNX colonies. By utilizing MNX models, many new avenues for the in vivo study for mitochondrial and nuclear genetics, or mito-Mendelian genetics, are now possible.