Phenotypes of maternal vascular malperfusion placental pathology and adverse pregnancy outcomes: A retrospective cohort study.

OBJECTIVE: To identify which components of maternal vascular malperfusion (MVM) pathology are associated with adverse pregnancy outcomes and to investigate the morphological phenotypes of MVM placental pathology and their relationship with distinct clinical presentations of pre-eclampsia and/or fetal growth restriction (FGR). DESIGN: Retrospective cohort study. SETTING: Tertiary care hospital in Toronto, Canada. POPULATION: Pregnant individuals with low circulating maternal placental growth factor (PlGF) levels (<100 pg/mL) and placental pathology analysis between March 2017 and December 2019. METHODS: Association between each pathological finding and the outcomes of interest were calculated using the chi-square test. Cluster analysis and logistic regression was used to identify phenotypic clusters, and their association with adverse pregnancy outcomes. Cluster analysis was performed using the K-modes unsupervised clustering algorithm. MAIN OUTCOME MEASURES: Preterm delivery <34+0 weeks of gestation, early onset pre-eclampsia with delivery <34+0 weeks of gestation, birthweight <10th percentile (small for gestational age, SGA) and stillbirth. RESULTS: The diagnostic features of MVM most strongly associated with delivery <34+0 weeks of gestation were: infarction, accelerated villous maturation, distal villous hypoplasia and decidual vasculopathy. Two dominant phenotypic clusters of MVM pathology were identified. The largest cluster (n = 104) was characterised by both reduced placental mass and hypoxic ischaemic injury (infarction and accelerated villous maturation), and was associated with combined pre-eclampsia and SGA. The second dominant cluster (n = 59) was characterised by infarction and accelerated villous maturation alone, and was associated with pre-eclampsia and average birthweight for gestational age. CONCLUSIONS: Patients with placental MVM disease are at high risk of pre-eclampsia and FGR, and distinct pathological findings correlate with different clinical phenotypes, suggestive of distinct subtypes of MVM disease.

Pan-tissue mitochondrial phenotyping reveals lower OXPHOS expression and function across cancer types.

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type.

Medium-chain fatty acid oxidation is independent of l-carnitine in liver and kidney but not in heart and skeletal muscle.

Medium-chain fatty acid (MCFA) consumption confers a wide range of health benefits that are highly distinct from long-chain fatty acids (LCFAs). A major difference between the metabolism of LCFAs compared with MCFAs is that mitochondrial LCFA oxidation depends on the carnitine shuttle, whereas MCFA mitochondrial oxidation is not. Although MCFAs are said to range from 6 to 14 carbons long based on physicochemical properties in vitro, the biological cut-off length of acyl chains that can bypass the carnitine shuttle in different mammalian tissues is unknown. To define the range of acyl chain length that can be oxidized in the mitochondria independent of carnitine, we determined the oxidative metabolism of free fatty acids (FFAs) from 6 to 18 carbons long in the liver, kidney, heart, and skeletal muscle. The liver oxidized FFAs 6 to 14 carbons long, whereas the kidney oxidized FFAs from 6 to 10 carbons in length. Heart and skeletal muscle were unable to oxidize FFAs of any chain length. These data show that while the liver and kidney can oxidize MCFAs in the free form, the heart and skeletal muscle require carnitine for the oxidative metabolism of MCFAs. Together these data demonstrate that MCFA oxidation independent of carnitine is tissue-specific.NEW & NOTEWORTHY This work demonstrates that the traditional concept of mitochondrial medium-chain fatty acid oxidation as unregulated and independent of carnitine applies only to liver metabolism, and to kidney to a lesser extent, but not the heart or skeletal muscle. Thus, the benefits of dietary medium-chain fatty acids are set by liver metabolic activity and peripheral tissues are unlikely to receive direct benefits from medium-chain fatty acid metabolism, but rather metabolic byproducts of liver's medium-chain oxidative metabolism.

Weight loss increases skeletal muscle mitochondrial energy efficiency in obese mice.

Weight loss from an overweight state is associated with a disproportionate decrease in whole-body energy expenditure that may contribute to the heightened risk for weight regain. Evidence suggests that this energetic mismatch originates from lean tissue. Although this phenomenon is well documented, the mechanisms have remained elusive. We hypothesized that increased mitochondrial energy efficiency in skeletal muscle is associated with reduced expenditure under weight loss. Wildtype (WT) male C57BL6/N mice were fed with high fat diet for 10 weeks, followed by a subset of mice that were maintained on the obesogenic diet (OB) or switched to standard chow to promote weight loss (WL) for additional 6 weeks. Mitochondrial energy efficiency was evaluated using high-resolution respirometry and fluorometry. Mass spectrometric analyses were employed to describe the mitochondrial proteome and lipidome. Weight loss promoted ~50% increase in the efficiency of oxidative phosphorylation (ATP produced per O2 consumed, or P/O) in skeletal muscle. However, weight loss did not appear to induce significant changes in mitochondrial proteome, nor any changes in respiratory supercomplex formation. Instead, it accelerated the remodeling of mitochondrial cardiolipin (CL) acyl-chains to increase tetralinoleoyl CL (TLCL) content, a species of lipids thought to be functionally critical for the respiratory enzymes. We further show that lowering TLCL by deleting the CL transacylase tafazzin was sufficient to reduce skeletal muscle P/O and protect mice from diet-induced weight gain. These findings implicate skeletal muscle mitochondrial efficiency as a novel mechanism by which weight loss reduces energy expenditure in obesity.

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.

Phenotype-Directed Management of Hypertension in Pregnancy.

Hypertensive disorders of pregnancy are among the most serious conditions that pregnancy care providers face; however, little attention has been paid to the concept of tailoring clinical care to reduce associated adverse maternal and perinatal outcomes based on the underlying disease pathogenesis. This narrative review discusses the integration of phenotype-based clinical strategies in the management of high-risk pregnant patients that are currently not common clinical practice: real-time placental growth factor testing at Mount Sinai Hospital, Toronto and noninvasive hemodynamic monitoring to guide antihypertensive therapy at the University of Washington Medical Center, Seattle. Future work should focus on promoting more widespread integration of these novel strategies into obstetric care to improve outcomes of pregnancies at high risk of adverse maternal-fetal outcomes from these complications of pregnancy.

Circulating maternal placental growth factor responses to low-molecular-weight heparin in pregnant patients at risk of placental dysfunction.

BACKGROUND: Patients at high risk of severe preeclampsia and fetal growth restriction have low circulating levels of placental growth factor and features of maternal vascular malperfusion placental pathology at delivery. Multimodal screening and commencement of aspirin prophylaxis at 11 to 13 weeks' gestation markedly reduces the risk of preterm delivery with preeclampsia. However, the additional role of low-molecular-weight heparin and mechanisms of action remain uncertain. Because low-molecular-weight heparin augments the production and release of placental growth factor in vitro by both placental villi and vascular endothelium, it may be effective to suppress the risk of severe preeclampsia in a niche group of high-risk patients with low circulating placental growth factor in the early second trimester. OBJECTIVE: This study aimed to define a gestational age-specific reference range for placental growth factor and to test the hypothesis that prophylactic low-molecular-weight heparin administered in the early second trimester may restore deficient circulating placental growth factor levels and thereby prolong pregnancy. STUDY DESIGN: Centile curves for circulating placental growth factor levels from 12 to 36 weeks' gestation were derived using quantile regression of combined data from a published cohort of 4207 unselected nulliparous patients in Cambridge, United Kingdom, at 4 sampling time points (12, 20, 28, and 36 weeks' gestation) and the White majority (n=531) of a healthy nulliparous cohort in Toronto, Canada, at 16 weeks' gestation using the same test platform. Within a specialty high-risk clinic in Toronto, a niche group of 7 patients with a circulating placental growth factor at the <10th centile in the early second trimester received daily prophylactic low-molecular-weight heparin (enoxaparin; 40 mg subcutaneously) and were followed up until delivery (group 1). Their baseline characteristics, delivery details, and placental pathologies were compared with 5 similar patients who did not receive low-molecular-weight heparin during the observation period (group 2) and further with 21 patients who delivered with severe preeclampsia (group 3) in the same institution. RESULTS: A gestational age-specific reference range for placental growth factor levels at weekly intervals between 12 and 36 weeks was established for White women with singleton pregnancies. Within group 1, 5 of 7 patients demonstrated a sustained increase in circulating placental growth factor levels, whereas placental growth factor levels did not increase in group 2 or group 3 patients who did not receive low-molecular-weight heparin. Group 1 patients receiving low-molecular-weight heparin therapy exhibited a later gestation at delivery, relative to groups 2 and 3 (36 weeks [33-37] vs 23 weeks [22-26] and 28 weeks [27-31], respectively), and consequently had higher birthweights (1.93 kg [1.1-2.7] vs 0.32 kg [0.19-0.39] and 0.73 kg [0.52-1.03], respectively). The incidence of stillbirth was lowest in group 1 (14% [1 of 7]), relative to groups 2 and 3 (80% [4 of 5] and 29% [6 of 21], respectively). Maternal vascular malperfusion was the most common placental pathology found in association with abnormal uterine artery Doppler. CONCLUSION: In patients at high risk of a serious adverse pregnancy outcome owing to placental disease, the addition of low-molecular-weight heparin to aspirin prophylaxis in the early second trimester may restore deficient circulating placental growth factor to mediate an improved perinatal outcome. These data support the implementation of a multicenter pilot randomized control trial where patients are recruited primarily based on the assessment of placental function in the early second trimester.

Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia.

Currently there is great interest in targeting mitochondrial oxidative phosphorylation (OXPHOS) in cancer. However, notwithstanding the targeting of mutant dehydrogenases, nearly all hopeful 'mito-therapeutics' cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. Using acute myeloid leukemia (AML) as a model, herein, we leveraged an in-house diagnostic biochemical workflow to identify 'actionable' bioenergetic vulnerabilities intrinsic to cancerous mitochondria. Consistent with prior reports, AML growth and proliferation was associated with a hyper-metabolic phenotype which included increases in basal and maximal respiration. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic OXPHOS limitations. Remarkably, by performing experiments across a physiological span of ATP free energy, we provide direct evidence that leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of oxidative ATP synthesis proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Together, these findings argue against ATP being the primary output of leukemic mitochondria and provide proof-of-principle that restoring, rather than disrupting, OXPHOS may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

PlGF (Placental Growth Factor) Testing in Clinical Practice: Evidence From a Canadian Tertiary Maternity Referral Center.

[Figure: see text].

Second Trimester Placental Growth Factor Levels and Placental Histopathology in Low-Risk Nulliparous Pregnancies.

OBJECTIVE: Placental growth factor (PlGF) levels are lower at delivery in pregnancies with preeclampsia or fetuses small for gestational age (SGA). These obstetrical complications are typically mediated by placental dysfunction, most commonly related to the specific placental phenotype termed placental maternal vascular malperfusion (MVM). The objective of this study was to determine the relationship between PlGF levels in the second trimester and the development of placental diseases that underlie adverse perinatal outcomes. METHODS: We performed a secondary analysis of the prospective Placental Health Study in unselected healthy nulliparous women (n = 773). Maternal demographic data, Doppler ultrasound measurements, and plasma PlGF levels at 15 to 18 weeks gestation were analyzed for association with pregnancy outcomes and placental pathology following delivery. RESULTS: Low PlGF levels in the second trimester (<10th percentile; <72 pg/mL) was associated with preterm delivery (<37 weeks; 26% vs. 6%, P < 0.001; unadjusted odds ratio (OR) 5.75, 95% CI 3.2-10.5), reduced mean birth weight (2998 vs. 3320 g, P < 0.001), SGA deliveries (25% vs. 11%, P = 0.001; OR 2.6, 95% CI 1.5-4.6), and preeclampsia (7% vs. 2%, P = 0.02; OR 4.3, 95% CI 1.5-12.8) relative to normal PlGF levels (≥10th percentile; ≥72 pg/mL). Low PlGF was associated with lower mean placental weight (447 vs. 471 g, P = 0.01), aberrant cord insertion (25% vs. 12%, P = 0.001) and a pathologic diagnosis of MVM (18% vs. 11%, P = 0.04; OR 1.9, 95% CI 1.01-3.55) but not with other placental pathologies. CONCLUSION: MVM placental pathology and related adverse perinatal outcomes are associated with low PlGF in the early second trimester for healthy nulliparous women.

Aglycemic growth enhances carbohydrate metabolism and induces sensitivity to menadione in cultured tumor-derived cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent form of liver malignancy and carries poor prognoses due to late presentation of symptoms. Treatment of late-stage HCC relies heavily on chemotherapeutics, many of which target cellular energy metabolism. A key platform for testing candidate chemotherapeutic compounds is the intrahepatic orthotopic xenograft (IOX) model in rodents. Translational efficacy from the IOX model to clinical use is limited (in part) by variation in the metabolic phenotypes of the tumor-derived cells that can be induced by selective adaptation to subculture conditions. METHODS: In this study, a detailed multilevel systems approach combining microscopy, respirometry, potentiometry, and extracellular flux analysis (EFA) was utilized to examine metabolic adaptations that occur under aglycemic growth media conditions in HCC-derived (HEPG2) cells. We hypothesized that aglycemic growth would result in adaptive "aerobic poise" characterized by enhanced capacity for oxidative phosphorylation over a range of physiological energetic demand states. RESULTS: Aglycemic growth did not invoke adaptive changes in mitochondrial content, network complexity, or intrinsic functional capacity/efficiency. In intact cells, aglycemic growth markedly enhanced fermentative glycolytic substrate-level phosphorylation during glucose refeeding and enhanced responsiveness of both fermentation and oxidative phosphorylation to stimulated energy demand. Additionally, aglycemic growth induced sensitivity of HEPG2 cells to the provitamin menadione at a 25-fold lower dose compared to control cells. CONCLUSIONS: These findings indicate that growth media conditions have substantial effects on the energy metabolism of subcultured tumor-derived cells, which may have significant implications for chemotherapeutic sensitivity during incorporation in IOX testing panels. Additionally, the metabolic phenotyping approach used in this study provides a practical workflow that can be incorporated with IOX screening practices to aid in deciphering the metabolic underpinnings of chemotherapeutic drug sensitivity.

Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs.

Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Isolated mitochondria are a powerful model for separating global cellular changes from intrinsic mitochondrial alterations. However, common laboratory practices for isolating mitochondria (e.g., differential centrifugation) routinely results in organelle preparations with variable mitochondrial purity. To overcome this issue, we developed a mass spectrometry-based method that quantitatively evaluates sample-specific percent mitochondrial enrichment. Sample-specific mitochondrial enrichment was then used to correct various biochemical readouts of mitochondrial function to a 'fixed' amount of mitochondrial protein, thus allowing for intrinsic mitochondrial bioenergetics, relative to the underlying proteome, to be assessed across multiple mouse tissues (e.g., heart, brown adipose, kidney, liver). Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues. This method can easily be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome.

Hemodynamic Complications in Pregnancy: Preeclampsia and Beyond.

Normal pregnancy is a complex and dynamic process that requires significant adaptation from the maternal system. Failure of this adaptive process in pregnancy contributes to many pregnancy related disorders, including the hypertensive disorders of pregnancy. This article discusses placental development and how abnormalities in the process of vascular remodeling contribute to the multisystem maternal and fetal disease that is preeclampsia and fetal growth restriction. We review some of the consequences of this condition on the mother and fetus, aspects of the clinical management of preeclampsia and how it can influence both mother and infant in the postnatal period and beyond.

Low molecular weight heparin promotes transcription and release of placental growth factor from endothelial cells.

Circulating levels of placental growth factor (PlGF) are significantly reduced in women who develop preeclampsia. Low molecular weight heparin (LMWH) has been shown to acutely elevate circulating PlGF levels in pregnant women at increased risk of preeclampsia. The objective of the current investigation was to determine the mechanisms by which LMWH mediates the extracellular release of PlGF from endothelial cells. Cultured human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) were exposed to LMWH; PlGF transcription, translation, mobilization, and secretion were then assessed. LMWH significantly increased the release of PlGF from both HAECs and HUVECs. LMWH treatment promoted a significant increase of PlGF-1 mRNA expression in HAECs, accompanied by the intracellular transport and release of PlGF into the conditioned media. LMWH-mediated release of PlGF from HAECs was not directly mediated by extracellular mobilization, synthesis, or stability of PlGF mRNA/protein. LMWH exposure promotes the release of PlGF from endothelial cells through the upregulation of PlGF-1 mRNA expression. Stimulation of circulating PlGF levels by LMWH may be an important mechanism by which LMWH could reduce the risk of preeclampsia or minimize disease severity.NEW & NOTEWORTHY There are few therapeutic options available for the prevention of preeclampsia, a serious hypertensive disorder of pregnancy. Women who subsequently develop preeclampsia exhibit significantly reduced circulating levels of the proangiogenic placental growth factor protein. Low molecular weight heparin (LMWH) has previously been investigated as a preventative therapy against the development of preeclampsia; however, its mechanism of action is not known. The current study determined that LMWH promotes the transcription and release of placental growth factor protein from endothelial cells, providing a mechanistic basis by which LMWH could reduce the risk of preeclampsia or minimize disease severity.

Subcellular proteomics combined with bioenergetic phenotyping reveals protein biomarkers of respiratory insufficiency in the setting of proofreading-deficient mitochondrial polymerase.

The mitochondrial mutator mouse is a well-established model of premature aging. In addition to accelerated aging, these mice develop hypertrophic cardiomyopathy at ~13 months of age, presumably due to overt mitochondrial dysfunction. Despite evidence of bioenergetic disruption within heart mitochondria, there is little information about the underlying changes to the mitochondrial proteome that either directly underly or predict respiratory insufficiency in mutator mice. Herein, nLC-MS/MS was used to interrogate the mitochondria-enriched proteome of heart and skeletal muscle of aged mutator mice. The mitochondrial proteome from heart tissue was then correlated with respiratory conductance data to identify protein biomarkers of respiratory insufficiency. The majority of downregulated proteins in mutator mitochondria were subunits of respiratory complexes I and IV, including both nuclear and mitochondrial-encoded proteins. Interestingly, the mitochondrial-encoded complex V subunits, were unchanged or upregulated in mutator mitochondria, suggesting a robustness to mtDNA mutation. Finally, the proteins most strongly correlated with respiratory conductance were PPM1K, NDUFB11, and C15orf61. These results suggest that mitochondrial mutator mice undergo a specific loss of mitochondrial complexes I and IV that limit their respiratory function independent of an upregulation of complex V. Additionally, the role of PPM1K in responding to mitochondrial stress warrants further exploration.

One Week of L-Citrulline Supplementation Improves Performance in Trained Cyclists.

Stanelle, ST, McLaughlin, KL, and Crouse, SF. One week of L-citrulline supplementation improves performance in trained cyclists. J Strength Cond Res 34(3): 647-652, 2020-L-citrulline (CIT) is a nonessential amino acid that is touted as an ergogenic aid for athletic performance because of its purported ability to stimulate nitric oxide production. Although previous research has demonstrated that CIT supplementation over a period of days improves physiological factors such as V[Combining Dot Above]O2 kinetics, no studies to date have explored whether there is a direct benefit to endurance performance. This study used a randomized, double-blind, crossover design to test whether chronic supplementation with pure CIT improves cycling performance over a maltodextrin placebo (PLAC). Nine trained male cyclists (24 ± 3 years; 181 ± 7 cm; 76 ± 13 kg; 4.18 ± 0.51 L·min V[Combining Dot Above]O2max) completed two 7-day supplementation periods (6 g·d of CIT or PLAC) separated by a 7-day washout. Subjects consumed the final 6-g dose 2 hours before the cycling performance evaluation, which consisted of a 40-km time trial (TT) followed by a supramaximal sprint repeat task (SRT). Paired t-tests and repeated-measures analysis of variance (α = 0.05) were used to analyze TT and SRT data, respectively. CIT supplementation produced an improvement in TT time of 5.2% that trended toward significance (p = 0.08). Furthermore, CIT promoted a significant increase in average heart rate, average rating of perceived exertion, and average power throughout the TT (p < 0.05). However, supplementation with CIT did not prevent fatigue during the SRT. Overall, this study is the first to demonstrate that CIT supplementation may provide a modest improvement to endurance cycling performance in trained athletes.

Intracardiac administration of ephrinA1-Fc preserves mitochondrial bioenergetics during acute ischemia/reperfusion injury.

AIMS: Intracardiac injection of recombinant EphrinA1-Fc immediately following coronary artery ligation in mice reduces infarct size in both reperfused and non-reperfused myocardium, but the cellular alterations behind this phenomenon remain unknown. MAIN METHODS: Herein, 10 wk-old B6129SF2/J male mice were exposed to acute ischemia/reperfusion (30minI/24hrsR) injury immediately followed by intracardiac injection of either EphrinA1-Fc or IgG-Fc. After 24 h of reperfusion, sections of the infarct margin in the left ventricle were imaged via transmission electron microscopy, and mitochondrial function was assessed in both permeabilized fibers and isolated mitochondria, to examine mitochondrial structure, function, and energetics in the early stages of repair. KEY FINDINGS: At a structural level, EphrinA1-Fc administration prevented the I/R-induced loss of sarcomere alignment and mitochondrial organization along the Z disks, as well as disorganization of the cristae and loss of inter-mitochondrial junctions. With respect to bioenergetics, loss of respiratory function induced by I/R was prevented by EphrinA1-Fc. Preservation of cardiac bioenergetics was not due to changes in mitochondrial JH2O2 emitting potential, membrane potential, ADP affinity, efficiency of ATP production, or activity of the main dehydrogenase enzymes, suggesting that EphrinA1-Fc indirectly maintains respiratory function via preservation of the mitochondrial network. Moreover, these protective effects were lost in isolated mitochondria, further emphasizing the importance of the intact cardiomyocyte ultrastructure in mitochondrial energetics. SIGNIFICANCE: Collectively, these data suggest that intracardiac injection of EphrinA1-Fc protects cardiac function by preserving cardiomyocyte structure and mitochondrial bioenergetics, thus emerging as a potential therapeutic strategy in I/R injury.

Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform.

Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.