Bioenergetics Consequences of Mitochondrial Transplantation in Cardiomyocytes.

Background Mitochondrial transplantation has been recently explored for treatment of very ill cardiac patients. However, little is known about the intracellular consequences of mitochondrial transplantation. This study aims to assess the bioenergetics consequences of mitochondrial transplantation into normal cardiomyocytes in the short and long term. Methods and Results We first established the feasibility of autologous, non-autologous, and interspecies mitochondrial transplantation. Then we quantitated the bioenergetics consequences of non-autologous mitochondrial transplantation into cardiomyocytes up to 28 days using a Seahorse Extracellular Flux Analyzer. Compared with the control, we observed a statistically significant improvement in basal respiration and ATP production 2-day post-transplantation, accompanied by an increase in maximal respiration and spare respiratory capacity, although not statistically significantly. However, these initial improvements were short-lived and the bioenergetics advantages return to the baseline level in subsequent time points. Conclusions This study, for the first time, shows that transplantation of non-autologous mitochondria from healthy skeletal muscle cells into normal cardiomyocytes leads to short-term improvement of bioenergetics indicating "supercharged" state. However, over time these improved effects disappear, which suggests transplantation of mitochondria may have a potential application in settings where there is an acute stress

Impaired cardiac and skeletal muscle bioenergetics in children, adolescents, and young adults with Barth syndrome

Barth syndrome (BTHS) is an X‐linked condition characterized by altered cardiolipin metabolism and cardioskeletal myopathy. We sought to compare cardiac and skeletal muscle bioenergetics in children, adolescents, and young adults with BTHS and unaffected controls and examine their relationships with cardiac function and exercise capacity. Children/adolescents and young adults with BTHS (n = 20) and children/adolescent and young adult control participants (n = 23, total n = 43) underwent (31)P magnetic resonance spectroscopy ((31)P‐MRS) of the lower extremity (calf) and heart for estimation of skeletal muscle and cardiac bioenergetics. Peak exercise testing (VO (2peak)) and resting echocardiography were also performed on all participants. Cardiac PCr/ATP ratio was significantly lower in children/adolescents (BTHS: 1.5 ± 0.2 vs. Control: 2.0 ± 0.3, P < 0.01) and adults (BTHS: 1.9 ± 0.2 vs. Control: 2.3 ± 0.2, P < 0.01) with BTHS compared to Control groups. Adults (BTHS: 76.4 ± 31.6 vs. Control: 35.0 ± 7.4 sec, P < 0.01) and children/adolescents (BTHS: 71.5 ± 21.3 vs. Control: 31.4 ± 7.4 sec, P < 0.01) with BTHS had significantly longer calf PCr recovery (τ PCr) postexercise compared to controls. Maximal calf ATP production through oxidative phosphorylation (Qmax‐lin) was significantly lower in children/adolescents (BTHS: 0.5 ± 0.1 vs. Control: 1.1 ± 0.3 mmol/L per sec, P < 0.01) and adults (BTHS: 0.5 ± 0.2 vs. Control: 1.0 ± 0.2 mmol/L sec, P < 0.01) with BTHS compared to controls. Blunted cardiac and skeletal muscle bioenergetics were associated with lower VO(2peak) but not resting cardiac function. Cardiac and skeletal muscle bioenergetics are impaired and appear to contribute to exercise intolerance in BTHS

Premutation in the Fragile X Mental Retardation 1 (FMR1) Gene Affects Maternal Zn-milk and Perinatal Brain Bioenergetics and Scaffolding.

Fragile X premutation alleles have 55-200 CGG repeats in the 5' UTR of the FMR1 gene. Altered zinc (Zn) homeostasis has been reported in fibroblasts from &gt;60 years old premutation carriers, in which Zn supplementation significantly restored Zn-dependent mitochondrial protein import/processing and function. Given that mitochondria play a critical role in synaptic transmission, brain function, and cognition, we tested FMRP protein expression, brain bioenergetics, and expression of the Zn-dependent synaptic scaffolding protein SH3 and multiple ankyrin repeat domains 3 (Shank3) in a knock-in (KI) premutation mouse model with 180 CGG repeats. Mitochondrial outcomes correlated with FMRP protein expression (but not FMR1 gene expression) in KI mice and human fibroblasts from carriers of the pre- and full-mutation. Significant deficits in brain bioenergetics, Zn levels, and Shank3 protein expression were observed in the Zn-rich regions KI hippocampus and cerebellum at PND21, with some of these effects lasting into adulthood (PND210). A strong genotype × age interaction was observed for most of the outcomes tested in hippocampus and cerebellum, whereas in cortex, age played a major role. Given that the most significant effects were observed at the end of the lactation period, we hypothesized that KI milk might have a role at compounding the deleterious effects on the FMR1 genetic background. A higher gene expression of ZnT4 and ZnT6, Zn transporters abundant in brain and lactating mammary glands, was observed in the latter tissue of KI dams. A cross-fostering experiment allowed improving cortex bioenergetics in KI pups nursing on WT milk. Conversely, WT pups nursing on KI milk showed deficits in hippocampus and cerebellum bioenergetics. A highly significant milk type × genotype interaction was observed for all three-brain regions, being cortex the most influenced. Finally, lower milk-Zn levels were recorded in milk from lactating women carrying the premutation as well as other Zn-related outcomes (Zn-dependent alkaline phosphatase activity and lactose biosynthesis-whose limiting step is the Zn-dependent β-1,4-galactosyltransferase). In premutation carriers, altered Zn homeostasis, brain bioenergetics and Shank3 levels could be compounded by Zn-deficient milk, increasing the risk of developing emotional and neurological/cognitive problems and/or FXTAS later in life

Extra-matrix Mg\u3csup\u3e2+\u3c/sup\u3e Limits Ca\u3csup\u3e2+\u3c/sup\u3e Uptake and Modulates Ca\u3csup\u3e2+\u3c/sup\u3e Uptake-independent Respiration and Redox State in Cardiac Isolated Mitochondria

Cardiac mitochondrial matrix (m) free Ca2+ ([Ca2+]m) increases primarily by Ca2+ uptake through the Ca2+ uniporter (CU). Ca2+ uptake via the CU is attenuated by extra-matrix (e) Mg2+ ([Mg2+]e). How [Ca2+]m is dynamically modulated by interacting physiological levels of [Ca2+]e and [Mg2+]e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg2+]e modulates Ca2+ uptake via the CU, it also alters bioenergetics in a matrix Ca2+–induced and matrix Ca2+–independent manner. To test this, we measured changes in [Ca2+]e, [Ca2+]m, [Mg2+]e and [Mg2+]m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl2 (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl2 (0–2 mM). In parallel, we assessed effects of added CaCl2 and MgCl2 on NADH, membrane potential (ΔΨm), and respiration. We found that \u3e0.125 mM MgCl2 significantly attenuated CU-mediated Ca2+ uptake and [Ca2+]m. Incremental [Mg2+]e did not reduce initial Ca2+uptake but attenuated the subsequent slower Ca2+ uptake, so that [Ca2+]m remained unaltered over time. Adding CaCl2 without MgCl2 to attain a [Ca2+]m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca2+]m did not additionally enhance NADH oxidation or respiration. Adding MgCl2 did not increase [Mg2+]m but it altered bioenergetics by its direct effect to decrease Ca2+ uptake. However, at a given [Ca2+]m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg2+]e. Thus, [Mg2+]e without a change in [Mg2+]m can modulate bioenergetics independently of CU-mediated Ca2+ transport

Cyclin B1/CDK1-regulated mitochondrial bioenergetics in cell cycle progression and tumor resistance.

A mammalian cell houses two genomes located separately in the nucleus and mitochondria. During evolution, communications and adaptations between these two genomes occur extensively to achieve and sustain homeostasis for cellular functions and regeneration. Mitochondria provide the major cellular energy and contribute to gene regulation in the nucleus, whereas more than 98% of mitochondrial proteins are encoded by the nuclear genome. Such two-way signaling traffic presents an orchestrated dynamic between energy metabolism and consumption in cells. Recent reports have elucidated the way how mitochondrial bioenergetics synchronizes with the energy consumption for cell cycle progression mediated by cyclin B1/CDK1 as the communicator. This review is to recapitulate cyclin B1/CDK1 mediated mitochondrial activities in cell cycle progression and stress response as well as its potential link to reprogram energy metabolism in tumor adaptive resistance. Cyclin B1/CDK1-mediated mitochondrial bioenergetics is applied as an example to show how mitochondria could timely sense the cellular fuel demand and then coordinate ATP output. Such nucleus-mitochondria oscillation may play key roles in the flexible bioenergetics required for tumor cell survival and compromising the efficacy of anti-cancer therapy. Further deciphering the cyclin B1/CDK1-controlled mitochondrial metabolism may invent effect targets to treat resistant cancers

Recombinant Mitochondrial Transcription Factor A with N-terminal Mitochondrial Transduction Domain Increases Respiration and Mitochondrial Gene Expression in G11778A Leber&#x27;s Hereditary Optic Neuropathy Cybrid Cells

Diseases involving mitochondrial defects usually manifest themselves in high-energy, post-mitotic tissues such as brain, retina, skeletal and cardiac muscle and frequently cause deficiencies in mitochondrial bioenergetics. We have developed a scalable procedure to produce recombinant human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its &#x22;mitochondrial transduction domain&#x22; (MTD,=PTD+MLS). _In vitro_ studies in a classic mitochondrial disease cell model demonstrated that Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment. MTD-TFAM treatment of these cell lines reversibly increased oxygen consumption (respiration) rates 3-fold, levels of respiratory proteins and mitochondrial gene expression. _In vivo_ results demonstrated that respiration increased to lesser degrees in mitochondria from tissues of mice injected with MTD-TFAM. MTD-TFAM can alter mitochondrial bioenergetics and holds promise for treatment of mitochondrial diseases involving deficiencies of energy production

Enhanced charge-independent Mitochondrial Free Ca\u3csup\u3e2+\u3c/sup\u3e and Attenuated ADP-induced NADH Oxidation by Isoflurane: Implications for Cardioprotection

Modulation of mitochondrial free Ca2 + ([Ca2 +]m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca2 +]m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca2 +]m and mitochondrial redox state (NADH), membrane potential (ΔΨm), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl2 (≈ 200 nM free Ca2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca2 +]m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨm and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨm, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨm, and matrix contraction with PM substrates. These findings suggest that isoflurane\u27s effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca2 + uptake by inhibiting the Na+/Ca2 + exchanger (NCE), independent of changes in ΔΨm and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca2 +]m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level

Isoflurane Modulates Cardiac Mitochondrial Bioenergetics by Selectively Attenuating Respiratory Complexes

Mitochondrial dysfunction contributes to cardiac ischemia–reperfusion (IR) injury but volatile anesthetics (VA) may alter mitochondrial function to trigger cardioprotection. We hypothesized that the VA isoflurane (ISO) mediates cardioprotection in part by altering the function of several respiratory and transport proteins involved in oxidative phosphorylation (OxPhos). To test this we used fluorescence spectrophotometry to measure the effects of ISO (0, 0.5, 1, 2 mM) on the time-course of interlinked mitochondrial bioenergetic variables during states 2, 3 and 4 respiration in the presence of either complex I substrate K+-pyruvate/malate (PM) or complex II substrate K+-succinate (SUC) at physiological levels of extra-matrix free Ca2 + (~ 200 nM) and Na+ (10 mM). To mimic ISO effects on mitochondrial functions and to clearly delineate the possible ISO targets, the observed actions of ISO were interpreted by comparing effects of ISO to those elicited by low concentrations of inhibitors that act at each respiratory complex, e.g. rotenone (ROT) at complex I or antimycin A (AA) at complex III. Our conclusions are based primarily on the similar responses of ISO and titrated concentrations of ETC. inhibitors during state 3. We found that with the substrate PM, ISO and ROT similarly decreased the magnitude of state 3 NADH oxidation and increased the duration of state 3 NADH oxidation, ΔΨm depolarization, and respiration in a concentration-dependent manner, whereas with substrate SUC, ISO and ROT decreased the duration of state 3 NADH oxidation, ΔΨm depolarization and respiration. Unlike AA, ISO reduced the magnitude of state 3 NADH oxidation with PM or SUC as substrate. With substrate SUC, after complete block of complex I with ROT, ISO and AA similarly increased the duration of state 3 ΔΨm depolarization and respiration. This study provides a mechanistic understanding in how ISO alters mitochondrial function in a way that may lead to cardioprotection

Tyrosine Nitration of Voltage-dependent Anion Channels in Cardiac Ischemia-reperfusion: Reduction by Peroxynitrite Scavenging

Excess superoxide (O2−) and nitric oxide (NO) forms peroxynitrite (ONOO−) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO−. Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O2−/ONOO− during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15 kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC–MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35 kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO−. We also found that ONOO− directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO− formed oligomers. Resveratrol (RES), a scavenger of O2−/ONOO−, reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O2− and ONOO− levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO− during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function