Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified ...as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.
While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the "anti-aging" protein, ...α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.
Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Mitochondrial DNA (mtDNA) sequences that are predicted to form G4 are ...enriched on the heavy-strand and have been associated with formation of deletion breakpoints. Increasing evidence supports the ability of mtDNA to form G4 in cancer cells; however, the functional roles of G4 structures in regulating mitochondrial nucleic acid homeostasis in non-cancerous cells remain unclear. Here, we demonstrate by live cell imaging that the G4-ligand RHPS4 localizes primarily to mitochondria at low doses. We find that low doses of RHPS4 do not induce a nuclear DNA damage response but do cause an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. We also observe that RHPS4 interferes with mtDNA levels or synthesis both in cells and isolated mitochondria. Importantly, a mtDNA variant that increases G4 stability and anti-parallel G4-forming character shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-mediated. Taken together, our results indicate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells.
Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type ...2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN accelerates the onset of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.
Robust cellular bioenergetics is vital in the energy-demanding process of maintaining matrix homeostasis in the intervertebral disc. Age-related decline in disc cellular bioenergetics is hypothesised ...to contribute to the matrix homeostatic perturbation observed in intervertebral disc degeneration. The present study aimed to measure how ageing impacted disc cell mitochondria and bioenergetics. Age-related changes measured included matrix content and cellularity in disc tissue, as well as matrix synthesis, cell proliferation and senescence markers in cell cultures derived from annulus fibrosus (AF) and nucleus pulposus (NP) isolated from the discs of young (6-9 months) and older (36-50 months) New Zealand White rabbits. Cellular bioenergetic parameters were measured using a Seahorse XFe96 Analyzer, in addition to quantitating mitochondrial morphological changes and membrane potential. Ageing reduced mitochondrial number and membrane potential in both cell types. Also, it significantly reduced glycolytic capacity, mitochondrial reserve capacity, maximum aerobic capacity and non-glucose-dependent respiration in NP. Moreover, NP cells exhibited age-related decline in matrix synthesis and reduced cellularity in older tissues. Despite a lack of changes in mitochondrial respiration with age, AF cells showed an increase in glycolysis and altered matrix production. While previous studies report age-related matrix degenerative changes in disc cells, the present study revealed, for the first time, that ageing affected mitochondrial number and function, particularly in NP cells. Consequently, age-related bioenergetic changes may contribute to the functional alterations in aged NP cells that underlie disc degeneration.
First described as essential to the phagocytic activity of leukocytes, Nox2-derived ROS have emerged as mediators of a range of cellular and tissue responses across species from salubrious to ...deleterious consequences. Knowledge of their role in inflammation is limited, however. We postulated that TNFα-induced endothelial reactive oxygen species (ROS) generation and pro-inflammatory signaling would be ameliorated by targeting Nox2. Herein, we in silico-modelled two first-in-class Nox2 inhibitors developed in our laboratory, explored their cellular mechanism of action and tested their efficacy in in vitro and mouse in vivo models of inflammation. Our data show that these inhibitors (CPP11G and CPP11H) disrupted canonical Nox2 organizing factor, p47
, translocation to Nox2 in the plasma membrane; and abolished ROS production, markedly attenuated stress-responsive MAPK signaling and downstream AP-1 and NFκB nuclear translocation in human cells. Consequently, cell adhesion molecule expression and monocyte adherence were significantly inhibited by both inhibitors. In vivo, TNFα-induced ROS and inflammation were ameliorated by targeted Nox2 inhibition, which, in turn, improved hind-limb blood flow. These studies identify a proximal role for Nox2 in propagated inflammatory signaling and support therapeutic value of Nox2 inhibitors in inflammatory disease.
We tested the hypothesis that reflexes arising from working respiratory muscle can elicit increases in sympathetic vasoconstrictor
outflow to limb skeletal muscle, in seven healthy human subjects at ...rest.
We measured muscle sympathetic nerve activity (MSNA) with intraneural electrodes in the peroneal nerve while the subject inspired
(primarily with the diaphragm) against resistance, with mouth pressure ( P M ) equal to 60 % of maximal, a prolonged duty cycle ( T I / T Tot ) of 0.70, breathing frequency ( f b ) of 15 breaths min â1 and tidal volume ( V T ) equivalent to twice eupnoea. This protocol was known to reduce diaphragm blood flow and cause fatigue.
MSNA was unchanged during the first 1â2 min but then increased over time, to 77 ± 51 % (s.d.) greater than control at exhaustion
(mean time, 7 ± 3 min). Mean arterial blood pressure (+12 mmHg) and heart rate (+27 beats min â1 ) also increased.
When the V T , f b and T I / T Tot of these trials were mimicked with no added resistance, neither MSNA nor arterial blood pressure increased.
MSNA and arterial blood pressure also did not change in response to two types of increased central respiratory motor output
that did not produce fatigue: (a) high inspiratory flow rate and f b without added resistance; or (b) high inspiratory effort against resistance with P M of 95 % maximal, T I / T Tot of 0.35 and f b of 12 breaths min â1 . The heart rate increased by 5â16 beats min â1 during these trials.
Thus, in the absence of any effect of increased central respiratory motor output per se on limb MSNA, we attributed the time-dependent increase in MSNA during high resistance, prolonged duty cycle breathing to
a reflex arising from a diaphragm that was accumulating metabolic end products in the face of high force output plus compromised
blood flow.
1 John Rankin Laboratory of Pulmonary Medicine, Department
Preventive Medicine, University of Wisconsin, Madison, Wisconsin 53705;
and 2 Department of Kinesiology, Kansas State University,
...Manhattan, Kansas 66506
The
normal respiratory muscle effort at maximal exercise requires a
significant fraction of cardiac output and causes leg blood flow to
fall. We questioned whether the high levels of respiratory muscle work
experienced in heavy exercise would affect performance. Seven male
cyclists maximal O 2 consumption
( O 2 ) 63 ± 5 ml · kg 1 · min 1 each
completed 11 randomized trials on a cycle ergometer at a workload
requiring 90% maximal O 2 . Respiratory
muscle work was either decreased (unloading), increased (loading), or
unchanged (control). Time to exhaustion was increased with unloading in 76% of the trials by an average of 1.3 ± 0.4 min or 14 ± 5% and decreased with loading in 83% of the trials by an average of
1.0 ± 0.6 min or 15 ± 3% compared with control
( P < 0.05). Respiratory muscle unloading during
exercise reduced O 2 , caused
hyperventilation, and reduced the rate of change in perceptions of
respiratory and limb discomfort throughout the duration of exercise.
These findings demonstrate that the work of breathing normally incurred
during sustained, heavy-intensity exercise (90%
O 2 ) has a significant influence on
exercise performance. We speculate that this effect of the normal
respiratory muscle load on performance in trained male cyclists is due
to the associated reduction in leg blood flow, which enhances both the
onset of leg fatigue and the intensity with which both leg and
respiratory muscle efforts are perceived.
blood flow distribution; dyspnea; work of breathing
The purpose was to examine, for a subset of a large random survey of men and women, the age-related changes in the parameters of aerobic function, maximal oxygen consumption (VO2max), and ventilatory ...threshold (T(VE)).
A "ramp-like" treadmill protocol was designed to measure VO2max and T(VE) on a total of 298 subjects (152 men and 146 women), aged 55-86 yr.
Data for VO2max (and HRmax) and T(VE) by 5-yr age groups provide "normative" results. Age-related declines in VO2max and T(VE) were fit by a linear model; however, age explained at most 37% of the variance across ages 55-86 yr. In this restricted age range, the rate of decline in VO2max, in both men (-0.034 L x min(-1) x yr(-1)) and women (-0.019 L x min(-1) x yr(-1)), was similar to that of previous reports for linear regression with age. Men, but not women, showed a decrease in body mass across age. Thus, the decline in VO2max expressed relative to body mass was similar in men (0.31 mL x kg(-1) x min(-1) x yr(-1)) and women (0.25); however, across this older age the decline is slower than noted for younger groups. The minimum level of aerobic power compatible with an independent life at age 85 yr was approximately 18 mL x kg(-1) x min(-1) in men and 15 mL x kg(-1) x min(-1) in women. Regression analysis showed HRmax across this age span is not well predicted by age. T(VE) across age declined at about one-half the rate of the VO2max, and in older age was approximately 85% of the VO2max.
The study provides "normative" cardiorespiratory function data of a random sample of independently living men and women aged 55-86 yr.
We measured muscle sympathetic nerve activity (MSNA, peroneal microneurography) in 5 healthy humans under conditions of matched tidal volume, breathing frequency, and end-tidal CO2, but varying ...respiratory motor output as follows(1) passive positive pressure mechanical ventilation, (2) voluntary hyperventilation, (3) assisted mechanical ventilation that required the subject to generate −2.5 cm H2O to trigger each positive pressure breath, and (4) added inspiratory resistance. Spectral analyses showed marked respiratory periodicities in MSNA; however, the amplitude of the peak power was not changed with changing inspiratory effort. Time domain analyses showed that maximum MSNA always occurred at end expiration (25% to 30% of total activity) and minimum activity at end inspiration (2% to 3% of total activity), and the amplitude of the variation was not different among conditions despite marked changes in respiratory motor output. Furthermore, qualitative changes in intrathoracic pressure were without influence on the respiratory modulation of MSNA. In all conditions, within-breath changes in MSNA were inversely related to small changes in diastolic pressure (1 to 3 mm Hg), suggesting that respiratory rhythmicity in MSNA was secondary to loading/unloading of carotid sinus baroreceptors. Furthermore, at any given diastolic pressure, within-breath MSNA varied inversely with lung volume, demonstrating an additional influence of lung inflation feedback on sympathetic discharge. Our data provide evidence against a significant effect of respiratory motor output on the within-breath modulation of MSNA and suggest that feedback from baroreceptors and pulmonary stretch receptors are the dominant determinants of the respiratory modulation of MSNA in the intact human.
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