ATP-generating Capacity Research Articles

The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.

Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.

Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor.

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 ns lifetime change from pH 5.5 to 9.0. Critically, we find that the sensor maintains a p Ka of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are robustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time-resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent sensor, we also demonstrate that mCherryTYG is spectrally compatible with the ATeam sensor and EGFP for simultaneous dual-color measurements of intracellular pH, ATP, and extracellular pH. In a proof-of-concept, we quantify acute respiration-dependent pH homeostasis that exhibits a stoichiometric relationship with the ATP-generating capacity of the carbon fuel choice in E. coli. Broadly speaking, our work presents a previously unemployed methodology that will greatly facilitate continuous pH quantification.

Mitochondrial Rich Proton Pumping Cells in the Kidney and Epididymis are Highly Glycolytic

The kidney collecting duct (CD) has a crucial role in regulating acid‐base balance. Alpha intercalated cells (ICs) of the CD maintain acid‐base homeostasis by acidifying the urine, via apical V‐ATPase H+ pumps. ICs are characterized in appearance by a very high density of mitochondria, but remarkably little is known about metabolism in these cells, due to a lack of suitable techniques to study this and the inability to generate a stable IC cell line. By using different innovative imaging techniques, we aimed to better understand how metabolism might be adapted in ICs to their specialized H+ pumping function.Using multiphoton microscopy of intact murine kidney slices, we have found that mitochondria in ICs have some highly unusual features compared with other tubular cells. IC mitochondria are considerably more mobile and dynamic, and display a high NADH signal but low uptake of voltage dependent dyes. Moreover, they are energized by a pH gradient that is functionally coupled to the proton pumping activity of the V‐ATPase. However, high resolution confocal microscopy and 3D electron microscopy (FIB‐SEM) in fixed mouse kidney tissue revealed that the mitochondria and V‐ATPase expressing vesicles were distant from each other, reducing the likelihood that the mitochondria are the only energy source for the V‐ATPase H+ pumps in ICs. Furthermore, we observed that the expression of respiratory chain complex V (ATP synthase) is lower in ICs than in surrounding cells, implying reduced oxidative capacity. Conversely, expression of enzymes in the glycolytic pathway, including lactate dehydrogenase, was markedly higher in ICs, consistent with high anaerobic ATP‐generating capacity. Interestingly, expression of glycolytic enzymes was also high in clear cells in the epididymis, suggesting that this is a common feature of proton pumping cells.As a surrogate of cellular ATP levels, we imaged cytosolic Ca2+ in kidney slices using a fluorescent reporter (GcaMP6s), and found that inhibition of mitochondrial ATP production with cyanide produced striking rises in Ca2+ in all tubular cell types except ICs. However, Ca2+ also increased in ICs when glycolysis was simultaneously inhibited. Finally, direct functional measurement of V‐ATPase function in microperfused isolated outermedullary collecting ducts (OMCDs) revealed that inhibition of glycolysis leads to a significant decrease in H+ pumping in ICs.In summary, we have provided several lines of evidence that mitochondrial rich cells in the kidney and epididymis are surprisingly glycolytic, and that glycolysis is functionally important for V‐ATPase activity. These findings provide new insights into coupling of transport and metabolism in proton pumping cells.Support or Funding InformationThis research is supported by The Swiss National Centre for Competence in Research Kidney Control of Homeostasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of the Mitochondrial DNA and Its Replicative Capacity in Induced Pluripotent Stem Cells.

The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.

Defects in mitochondrial localization and ATP synthesis in the mdx mouse model of Duchenne muscular dystrophy are not alleviated by PDE5 inhibition

Given the crucial roles for mitochondria in ATP energy supply, Ca(2+) handling and cell death, mitochondrial dysfunction has long been suspected to be an important pathogenic feature in Duchenne muscular dystrophy (DMD). Despite this foresight, mitochondrial function in dystrophin-deficient muscles has remained poorly defined and unknown in vivo. Here, we used the mdx mouse model of DMD and non-invasive spectroscopy to determine the impact of dystrophin-deficiency on skeletal muscle mitochondrial localization and oxidative phosphorylation function in vivo. Mdx mitochondria exhibited significant uncoupling of oxidative phosphorylation (reduced P/O) and a reduction in maximal ATP synthesis capacity that together decreased intramuscular ATP levels. Uncoupling was not driven by increased UCP3 or ANT1 expression. Dystrophin was required to maintain subsarcolemmal mitochondria (SSM) pool density, implicating it in the spatial control of mitochondrial localization. Given that nitric oxide-cGMP pathways regulate mitochondria and that sildenafil-mediated phosphodiesterase 5 inhibition ameliorates dystrophic pathology, we tested whether sildenafil's benefits result from decreased mitochondrial dysfunction in mdx mice. Unexpectedly, sildenafil treatment did not affect mitochondrial content or oxidative phosphorylation defects in mdx mice. Rather, PDE5 inhibition decreased resting levels of ATP, phosphocreatine and myoglobin, suggesting that sildenafil improves dystrophic pathology through other mechanisms. Overall, these data indicate that dystrophin-deficiency disrupts SSM localization, promotes mitochondrial inefficiency and restricts maximal mitochondrial ATP-generating capacity. Together these defects decrease intramuscular ATP and the ability of mdx muscle mitochondria to meet ATP demand. These findings further understanding of how mitochondrial bioenergetic dysfunction contributes to disease pathogenesis in dystrophin-deficient skeletal muscle in vivo.

Mitochondrial viability in mouse and human postmortem brain

Neuronal function in the brain requires energy in the form of ATP, and mitochondria are canonically associated with ATP production in neurons. The electrochemical gradient, which underlies the mitochondrial transmembrane potential (DeltaPsi(mem)), is harnessed for ATP generation. Here we show that DeltaPsi(mem) and ATP-production can be engaged in mitochondria isolated from human brains up to 8.5 h postmortem. Also, a time course of postmortem intervals from 0 to 24 h using mitochondria isolated from mouse cortex reveals that DeltaPsi(mem) in mitochondria can be reconstituted beyond 10 h postmortem. It was found that complex I of the mitochondrial electron transport chain was affected adversely with increasing postmortem intervals. Mitochondria isolated from postmortem mouse brains maintain the ability to produce ATP, but rates of production decreased with longer postmortem intervals. Furthermore, we show that postmortem brain mitochondria retain their DeltaPsi(mem) and ATP-production capacities following cryopreservation. Our finding that DeltaPsi(mem) and ATP-generating capacity can be reinitiated in brain mitochondria hours after death indicates that human postmortem brains can be an abundant source of viable mitochondria to study metabolic processes in health and disease. It is also possible to archive these mitochondria for future studies.

Modulation of Cell Motility by Spatial Repositioning of Enzymatic ATP/ADP Exchange Capacity

ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities. Although already much is known about the role of ATP in fundamental processes of living systems, data about its compartmentalization are rather scarce, and we still have only very limited understanding of whether patterns in the distribution of intracellular ATP concentration ("ATP inhomogeneity") do exist and have a regulatory role. Here we report on the analysis of coupling of local ATP supply to regulation of actomyosin behavior, a widespread and dynamic process with conspicuous high ATP dependence, which is central to cell shape changes and cell motility. As an experimental model, we use embryonic fibroblasts from knock-out mice without major ATP-ADP exchange enzymes, in which we (re)introduce the ATP/ADP exchange enzyme adenylate kinase-1 (AK1) and deliberately manipulate its spatial positioning by coupling to different artificial location tags. By transfection-complementation of AK1 variants and comparison with yellow fluorescent protein controls, we found that motility and spreading were enhanced in cells with AK1 with a focal contact guidance tag. Intermediary enhancement was observed in cells with membrane-targeted or cytosolic AK1. Use of a heterodimer-inducing approach for transient translocation of AK1 to focal contacts under conditions of constant global AK1 activity in the cell corroborated these results. Based on our findings with these model systems, we propose that local ATP supply in the cell periphery and "on site" fuelling of the actomyosin machinery, when maintained via enzymes involved in phosphoryl transfer, are codetermining factors in the control of cell motility.

Protective Effect of Diltiazem on Hepatic Ischemia-Reperfusion Injury in Rats by Improving Liver Tissue Blood Flow

Background Cytosolic calcium ions are known to play an important role in ischemia-reperfusion (IR) injury. However, the protective effect of calcium channel blockers remains controversial in liver IR injury. Moreover, calcium channel blockers improve hepatic IR injury not due to blocking an increase in hepatic calcium concentration. Therefore, we hypothesized that calcium antagonists protected a liver from IR injury by a vasodilatory action rather than by the inhibition of an increase in Ca 2+ within parenchymal cells. This study evaluated the effects of diltiazem, a calcium channel blocker, on liver energy metabolism and blood flow after IR injury. Methods Twenty-seven rats underwent hepatic ischemia for 30 minutes followed by 60 minutes of reperfusion. The animals were allocated into group C (without drug); group D5 (diltiazem, 5 μg/kg per min); or group D10 (diltiazem, 10 μg/kg per min). Diltiazem was infused before laparotomy and then throughout the experiment. Results After 60 minutes of reperfusion, liver tissue blood flow and ATP concentrations were significantly higher in group D10 than the other animals (both, P < .05). Changes in ATP values strongly correlated those observed in blood flow (R = 0.80, P < .001). Conclusion Diltiazem improved ATP-generating capacity during reperfusion by improving liver tissue blood flow. An improvement in hepatic tissue perfusion may be a therapeutic strategy for liver IR injury.

Skeletal Muscle Aging in F344BN F1-Hybrid Rats: II. Improved Contractile Economy in Senescence Helps Compensate for Reduced ATP-Generating Capacity

We used a pump-perfused rat hind-limb preparation to compare young adult (YA: 8-9- month-old), late middle-aged (LMA: 28-29-month-old), and senescent (SEN: 36-month-old) rats at similar rates of convective O(2) delivery during a 4-minute contraction bout. We hypothesized that not only would VO(2) and lactate production be reduced, but also that contractile economy would be altered with aging. Peak tension was lower in LMA (42%) and SEN (71%) versus YA. VO(2) and lactate efflux was progressively lower with increasing age. Estimated adenosine triphosphate per N of force was increased in LMA (35%) and reduced in SEN (31%) versus YA. Myosin heavy chain (MHC) analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a lower MHC type IIb and higher MHC type IIa/IIx in SEN versus YA. Therefore, whereas contractile economy is impaired in LMA, it is improved in SEN, and this latter effect may be due in part to reduced type IIb MHC.

Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation.

In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP-generating capacity of the cells is not limiting.

Mechanism of myocardial protective action of dilazep during ischaemia and reperfusion

The aim of this study was to investigate if dilazep is able to reduce with a direct protective action on the myocardium the deleterious effects caused by ischaemia and reperfusion. For this purpose we used an isolated rabbit heart preparation. The hearts were either perfused aerobically or made totally ischaemic for 60 min (by abolishing coronary flow) or made ischaemic for 60 min and then reperfused for 30 min. Ischaemic and reperfusion damage was measured in terms of alteration in mechanical function, lactate and CPK release, mitochondrial function and tissue content of Adenosine Triphosphate (ATP), Creatine Phosphate (CP) and calcium. Dilazep (10 −5 M) was administered in the perfusate either 20 minutes before ischaemia or only during post-ischaemic reperfusion. Ischaemia induced a decline of the endogenous stores of ATP and CP, followed by an alteration of calcium homeostasis with increase of diastolic pressure, mitochondria calcium overload and impairment of the oxidative phosphorylating capacities. On reperfusion, tissue and mitochondrial calcium increase the capacity of the mitochondria to use O 2 for state III respiration was further impaired and the ATP-generating capacity reduced. Diastolic pressure increased and there was only a small recovery of active tension generation associated with massive CPK release. Administration of dilazep before ischaemia induced a negative inotropic effect which, in turn, resulted in a slowing of the rate of CP and ATP depletion during ischaemia. This protected the hearts against the ischemic, and reperfusion-induced decline in the ATP-generating and O 2-utilizing capacities of the mitochondria. In addition, there was a less marked increase in tissue and mitochondrial Ca ++, CPK and lactate release were reduced and the recovery of developed pressure on reperfusion was significantly increased. Administration of dilazep during reperfusion failed to modify the exacerbation of ischaemic damage caused by the readmission of coronary flow. These data suggest that dilazep benefits the ischaemic myocardium via an ATP sparing action.

Protection of the myocardium against postischemic reperfusion damage: The combined effect of hypothermia and nifedipine

This study tests the hypothesis that the efficacy of hypothermia as a cardiacprotective agent can be increased by adding the slow channel--blocking agent, nifedipine. Isolated, electrically paced rabbit hearts were made ischemic for 60 to 180 minutes at 37 degrees, 34 degrees, 28 degrees, 25 degrees, 15 degrees, 12 degrees, and 5 degrees C and then reperfused at 37 degrees C. Recovery was assessed in terms of recovery of mechanical function, tissue stores of adenosine triphosphate (ATP), oxidative phosphorylative and ATP-generating capacity of the mitochondria, and mitochondrial Ca2+ content. Half of the hearts received 50 micrograms/L nifedipine. The results show that the protective effects of hypothermia and nifedipine are additive.

Protective effect of pretreatment with verapamil, nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium

To establish whether the prophylactic use of verapamil, nifedipine or propranolol protects heart muscle against the deleterious effects of global ischemia and reperfusion, rabbits were injected subcutaneously twice daily with 2.0 mg/kg of one of these drugs for 4 to 5 days. The hearts were then isolated, paced and either perfused aerobically, made totally ischemic for 90 minutes or made ischemic for 90 minutes and then reperfused. At the end of this time some of the hearts were assayed for adenosine triphosphate (ATP), creatine phosphate (CP) and calcium (Ca ++). Other hearts were homogenized, the mitochondria harvested and monitored for oxidative phosphorylating and ATP-generating capacity and Ca ++ content. The effect of Ca ++ on the ATP-generatlng capacity of cardiac mitochondria was also determined. Hearts that were made ischemic gained Ca ++. The endogenous stores of ATP and CP were depleted; the mitochondria accumulated Ca ++ and the oxidative phosphorylating activity (respiratory control index and oxygen quotient) was impaired. During reperfusion, tissue and mitochondrial Ca ++ was substantially increased, the capacity of the mitochondria to use O 2 for state III respiration was further impaired and the ATP-generating capacity reduced. Resting tension increased and there was only a small recovery of active tension generation. Hearts from the rabbits treated with propranolol, verapamil or nifedipine were protected against ischemia, and ischemia with reperfusion-induced decline in the ATP-generating and O 2-utillzing capacity of the mitochondria. There was also a less marked increase in tissue and mitochondrial Ca ++ and the systolic tension-generating capacity of the hearts was better maintained.

Effect of growth conditions on the involvement of cytochrome c in electron transport, proton translocation and ATP synthesis in the facultative methylotroph Pseudomonas AM1

The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O(2) have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. -->H(+)/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the -->H(+)/O ratio increased to about 6. Cell yields with respect to succinate and O(2) were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH(4) (+)-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the ;needs' of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a(3). In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a(3) occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a(3), hence becoming involved in proton translocation and ATP synthesis during NADH and succinate oxidation. An alternative possibility is that in wild-type bacteria the cytochrome c is always involved in electron transport, but that its involvement in measurable proton translocation only occurs in carbon-limited conditions.

Oxidative phosphorylation in mitochondria isolated from small intestinal epithelium of thiamphenicol-treated rats and control rats

Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-P i exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled.

The effects of acclimation temperature on oxidative phosphorylation by turtle-heart mitochondria

1. 1. After prolonged periods of cold exposure turtle-heart mitochondria exhibit a decreased rate of respiration in the presence of glutamate and/gb-hydroxybutyrate-malate; respiration in the presence of succinate is not affected. 2. 2. Cold acclimation influences the ATP-generating capacity of turde-heart mitochondria as evidenced by a decreased sensitivity of phosphorylation to F − inhibition. In addition, P/O ratios estimated at 10° C. were increased in heart mitochondria from the cold-acclimated animals. 3. 3. It is to be concluded that the turtle, Chrystmys picta, does regulate its subcellular functions when subjected to cold temperatures. This is accomplished by way of an increased efficiency in energy metabolism.