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ImportanceThe pathophysiology of exercise intolerance in patients with heart failure with preserved ejection fraction (HFpEF) remains incompletely understood. Multiple lines of evidence suggest that abnormal skeletal muscle metabolism is a key contributor, but the mechanisms underlying metabolic dysfunction remain unresolved.ObjectiveTo evaluate the associations of skeletal muscle mitochondrial function using respirometric analysis of biopsied muscle fiber bundles from patients with HFpEF with exercise performance.Design, Setting, and ParticipantsIn this cross-sectional study, muscle fiber bundles prepared from fresh vastus lateralis biopsies were analyzed by high-resolution respirometry to provide detailed analyses of mitochondrial oxidative phosphorylation, including maximal capacity and the individual contributions of complex I–linked and complex II-linked respiration. These bioenergetic data were compared between patients with stable chronic HFpEF older than 60 years and age-matched healthy control (HC) participants and analyzed for intergroup differences and associations with exercise performance. All participants were treated at a university referral center, were clinically stable, and were not undergoing regular exercise or diet programs. Data were collected from March 2016 to December 2017, and data were analyzed from November 2020 to May 2021.Main Outcomes and MeasuresSkeletal muscle mitochondrial function, including maximal capacity and respiration linked to complex I and complex II. Exercise performance was assessed by peak exercise oxygen consumption, 6-minute walk distance, and the Short Physical Performance Battery.ResultsOf 72 included patients, 50 (69%) were women, and the mean (SD) age was 69.6 (6.1) years. Skeletal muscle mitochondrial function measures were all markedly lower in skeletal muscle fibers obtained from patients with HFpEF compared with HCs, even when adjusting for age, sex, and body mass index. Maximal capacity was strongly and significantly correlated with peak exercise oxygen consumption (R = 0.69; P R = 0.70; P R = 0.46; P Conclusions and RelevanceIn this study, patients with HFpEF had marked abnormalities in skeletal muscle mitochondrial function. Severely reduced maximal capacity and complex I–linked and complex II–linked respiration were associated with exercise intolerance and represent promising therapeutic targets.

Ubiquinol cytochrome c reductase hinge protein (UQCRH) is required for the electron transfer between cytochrome c1 and c of the mitochondrial cytochrome bc1 Complex (CIII). A two-exon deletion in the human UQCRH gene has recently been identified as the cause for a rare familial mitochondrial disorder. Deletion of the corresponding gene in the mouse (Uqcrh-KO) resulted in striking biochemical and clinical similarities including impairment of CIII, failure to thrive, elevated blood glucose levels, and early death. Here, we set out to test how global ablation of the murine Uqcrh affects cardiac morphology and contractility, and bioenergetics. Hearts from Uqcrh-KO mutant mice appeared macroscopically considerably smaller compared to wildtype littermate controls despite similar geometries as confirmed by transthoracic echocardiography (TTE). Relating TTE-assessed heart to body mass revealed the development of subtle cardiac enlargement, but histopathological analysis showed no excess collagen deposition. Nonetheless, Uqcrh-KO hearts developed pronounced contractile dysfunction. To assess mitochondrial functions, we used the high-resolution respirometer NextGen-O2k allowing measurement of mitochondrial respiratory capacity through the electron transfer system (ETS) simultaneously with the redox state of ETS-reactive coenzyme Q (Q), or production of reactive oxygen species (ROS). Compared to wildtype littermate controls, we found decreased mitochondrial respiratory capacity and more reduced Q in Uqcrh-KO, indicative for an impaired ETS. Yet, mitochondrial ROS production was not generally increased. Taken together, our data suggest that Uqcrh-KO leads to cardiac contractile dysfunction at 9 weeks of age, which is associated with impaired bioenergetics but not with mitochondrial ROS production. Graphical abstract Global ablation of the Uqcrh gene results in functional impairment of CIII associated with metabolic dysfunction and postnatal developmental arrest immediately after weaning from the mother. Uqcrh-KO mice show dramatically elevated blood glucose levels and decreased ability of isolated cardiac mitochondria to consume oxygen (O2). Impaired development (failure to thrive) after weaning manifests as a deficiency in the gain of body mass and growth of internal organ including the heart. The relative heart mass seemingly increases when organ mass calculated from transthoracic echocardiography (TTE) is normalized to body mass. Notably, the heart shows no signs of collagen deposition, yet does develop a contractile dysfunction reflected by a decrease in ejection fraction and fractional shortening.

Two-dimensional cell cultures are established models in research for studying and perturbing cell-type specific functions. However, many limitations apply to the cell growth in a monolayer using standard cell culture media. Although they have been used for decades, their formulations do not mimic the composition of the human cell environment. In this study, we analyzed the impact of a newly formulated human plasma-like media (HPLM) on cell proliferation, mitochondrial bioenergetics, and alterations of drug efficacies using three distinct cancer cell lines. Using high-resolution respirometry, we observed that cells grown in HPLM displayed significantly altered mitochondrial bioenergetic profiles, particularly related to mitochondrial density and mild uncoupling of respiration. Furthermore, in contrast to standard media, the growth of cells in HPLM unveiled mitochondrial dysfunction upon exposure to the FDA-approved kinase inhibitor sunitinib. This seemingly context-dependent side effect of this drug highlights that the selection of the cell culture medium influences the assessment of cancer drug sensitivities. Thus, we suggest to prioritize media with a more physiological composition for analyzing bioenergetic profiles and to take it into account for assigning drug efficacies in the cell culture model of choice.

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.

Two-dimensional cell cultures are established models in research for studying and perturbing cell-type specific functions. The simplicity of these models facilitates the reproducible manipulation of cultured cells. Limitations, however, relate to cell growth in a monolayer using standard cell culture media. Although such media are in use for decades, their formulations do not mimic the composition of the originating human cell environment in physiological or pathological conditions. In this study, we selected three frequently-used cancer cell lines SW620 (colon cancer), MCF7 (breast cancer), and A375 (melanoma) to determine the impact of a newly formulated human plasma-like medium (HPLM) on cell proliferation and mitochondrial function. Mitochondrial bioenergetic profiles were analyzed by high-resolution respirometry, revealing metabolic reprogramming in cells cultured in standard and HPLM media. Notably, the culturing of differentially proliferating cells in HPLM affected key mechanism of cellular respiration. All three cancer cell lines displayed an amending bioenergetic profile, particularly related to mitochondrial density and mild uncoupling of respiration. Importantly, only cells cultured in HPLM displayed mitochondrial dysfunction upon exposure with the clinically prescribed cancer drug and kinase inhibitor sunitinib. These findings highlight that cell culture media redirect mitochondrial function and affect cancer drug sensitivities. To advance the translational potential of cell culture models we propose to prioritize media with a human plasma-like composition for analyzing bioenergetic profiles and for determining target-oriented drug efficacies.

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extentin organelloin real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U-13C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system inferred that NAD+regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U-13C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.