Mitochondrial Outer Membrane Protein Research Articles

Abstract 409: Investigating the Role of the Mitochondrial Calcium Uniporter (mcu) in Calcium Regulation of Cardiac Mitochondria Using Transmural Spectroscopy and an Integrating Sphere

Cardiac mitochondria uptake calcium through the mitochondrial calcium uniporter (MCU). To better understand the role of MCU and mitochondrial calcium in regulating heart physiology and pathophysiology, we developed a method to measure mitochondrial matrix calcium in beating, perfused hearts. Langendorff perfusing hearts are loaded with 4.5 uM rhod-2-AM at 30°C and then perfused at 37°C to washout uncleaved dye. We determined that rhod-2 localized primarily to the mitochondria under our loading conditions, as shown by loading in a heart expressing a GFP-tagged mitochondrial outer membrane protein and analyzing heart slices under super resolution microscopy. Further, addition of Mn 2+ , which quenches cytosolic rhod-2, has little effect on rhod-2 signal. We insert an optical catheter with both white light and 532nm laser into the left ventricle and interleaved the collection of spectra from both light sources. The perfused heart is center mounted in an integrating sphere for spectra collection. The light passes through the ventricle and reflects off the integrating sphere resulting in a near uniform sampling of the transmitted light. Spectral properties from both light sources are determined using a rapid scanning spectrophotometer. Myoglobin oxygenation, cytochrome redox state, and rhod-2 loading are determined by white light absorbance. Ca 2+ bound rhod-2 emission is determined by removing background tissue effects from laser emission spectra and normalizing to tissue absorbance. Using this method we are able to measure changes in Ca 2+ and cytochromes during treatments such as isoproterenol and ischemia-reperfusion. The use of an integrating sphere transmural spectroscopy provides us an unique method to study mitochondrial Ca 2+ signaling in perfused mouse heart loaded with Rhod-2.

Precision Neurology for Parkinson's Disease: Coupling Miro1-Based Diagnosis With Drug Discovery.

Parkinson's disease (PD) is a debilitating movement disorder, significantly afflicting the aging population. Efforts to develop an effective treatment have been challenged by the lack of understanding of the pathological mechanisms underlying neurodegeneration. We have shown that Miro1, an outer mitochondrial membrane protein, situates at the intersection of the complex genetic and functional network of PD. Removing Miro1 from the surface of damaged mitochondria is a prerequisite for mitochondrial clearance via mitophagy. Parkinson's proteins PINK1, Parkin, and LRRK2 are the molecular helpers to remove Miro1 from dysfunctional mitochondria destined for mitophagy. We have found a delay in clearing Miro1 and initiating mitophagy in postmortem brains and induced pluripotent stem cell-derived neurons from PD patients harboring mutations in LRRK2, PINK1, or Parkin, or from sporadic PD patients with no known mutations. In addition, we have shown that reducing Miro1 by both genetic and pharmacological approaches can correct this Miro1 phenotype and rescue Parkinson's-relevant phenotypes in human neurons and fly PD models. These results suggest that the Miro1 defect may be a common denominator for PD, and compounds that reduce Miro1 promise a new class of drugs to battle PD. We propose to couple this Miro1 phenotype with Miro1-based drug discovery in future therapeutic studies, which could significantly improve the success of clinical trials. © 2020 International Parkinson and Movement Disorder Society.

Disruption of endoplasmic reticulum-mitochondria tethering proteins in post-mortem Alzheimer's disease brain

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions, many of which are perturbed in Alzheimer's disease. Moreover, damage to ER-mitochondria signaling is seen in cell and transgenic models of Alzheimer's disease. However, as yet there is little evidence that ER-mitochondria signaling is altered in human Alzheimer's disease brains. ER-mitochondria signaling is mediated by interactions between the integral ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 which act to recruit and “tether” regions of ER to the mitochondrial surface. The VAPB-PTPIP51 tethers are now known to regulate a number of ER-mitochondria signaling functions including delivery of Ca2+from ER stores to mitochondria, mitochondrial ATP production, autophagy and synaptic activity. Here we investigate the VAPB-PTPIP51 tethers in post-mortem control and Alzheimer's disease brains. Quantification of ER-mitochondria signaling proteins by immunoblotting revealed loss of VAPB and PTPIP51 in cortex but not cerebellum at end-stage Alzheimer's disease. Proximity ligation assays were used to quantify the VAPB-PTPIP51 interaction in temporal cortex pyramidal neurons and cerebellar Purkinje cell neurons in control, Braak stage III-IV (early/mid-dementia) and Braak stage VI (severe dementia) cases. Pyramidal neurons degenerate in Alzheimer's disease whereas Purkinje cells are less affected. These studies revealed that the VAPB-PTPIP51 tethers are disrupted in Braak stage III-IV pyramidal but not Purkinje cell neurons. Thus, we identify a new pathogenic event in post-mortem Alzheimer's disease brains. The implications of our findings for Alzheimer's disease mechanisms are discussed.

Deficiency of mitochondrial outer membrane protein 64 confers rice resistance to both piercing-sucking and chewing insects in rice.

The brown planthopper (BPH) and striped stem borer (SSB) are the most devastating insect pests in rice (Oryza sativa) producing areas. Screening for endogenous resistant genes is the most practical strategy for rice insect-resistance breeding. Forty-five mutants showing high resistance against BPH were identified in a rice T-DNA insertion population (11,000 putative homozygous lines) after 4 years of large-scale field BPH-resistance phenotype screening. Detailed analysis showed that deficiency of rice mitochondrial outer membrane protein 64 (OM64) gene resulted in increased resistance to BPH. Mitochondrial outer membrane protein 64 protein is located in the outer mitochondrial membrane by subcellular localization and its deficiency constitutively activated hydrogen peroxide (H2 O2 ) signaling, which stimulated antibiosis and tolerance to BPH. The om64 mutant also showed enhanced resistance to SSB, a chewing insect, which was due to promotion of Jasmonic acid biosynthesis and related responses. Importantly, om64 plants presented no significant changes in rice yield-related characters. This study confirmed OM64 as a negative regulator of rice herbivore resistance through regulating H2 O2 production. Mitochondrial outer membrane protein 64 is a potentially efficient candidate to improve BPH and SSB resistance through gene deletion. Why the om64 mutant was resistant to both piercing-sucking and chewing insects via a gene deficiency in mitochondria is discussed.

Mitochondrial carrier homolog 2 is necessary for AML survival

AbstractThrough a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.

RETREG1/FAM134B mediated autophagosomal degradation of AMFR/GP78 and OPA1 —a dual organellar turnover mechanism

ABSTRACT Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate “mitoplasts”. This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The “mitoplasts” generated are degraded by reticulo-mito-phagy – simultaneously affecting dual organelle turnover. Abbreviations: AMFR/GP78: autocrine motility factor receptor; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BFP: blue fluorescent protein; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CNBr: cyanogen bromide; ER: endoplasmic reticulum; ERAD: endoplasmic-reticulum-associated protein degradation; FL: fluorescence, GFP: green fluorescent protein; HA: hemagglutinin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN: mitofusin, MGRN1: mahogunin ring finger 1; NA: numerical aperature; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PRNP/PrP: prion protein; RER: rough endoplasmic reticulum; RETREG1/FAM134B: reticulophagy regulator 1; RFP: red fluorescent protein; RING: really interesting new gene; ROI: region of interest; RTN: reticulon; SEM: standard error of the mean; SER: smooth endoplasmic reticulum; SIM: structured illumination microscopy; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STOML2: stomatin like 2; TOMM20: translocase of outer mitochondrial membrane 20; UPR: unfolded protein response.

Mitochondria-Associated Degradation Pathway (MAD) Function beyond the Outer Membrane

SUMMARYThe mitochondria-associated degradation pathway (MAD) mediates ubiquitination and degradation of mitochondrial outer membrane (MOM) proteins by the proteasome. We find that the MAD, but not other quality-control pathways including macroautophagy, mitophagy, or mitochondrial chaperones and proteases, is critical for yeast cellular fitness under conditions of paraquat (PQ)-induced oxidative stress in mitochondria. Specifically, inhibition of the MAD increases PQ-induced defects in growth and mitochondrial quality and decreases chronological lifespan. We use mass spectrometry analysis to identify possible MAD substrates as mitochondrial proteins that exhibit increased ubiquitination in response to PQ treatment and inhibition of the MAD. We identify candidate substrates in the mitochondrial matrix and inner membrane and confirm that two matrix proteins are MAD substrates. Our studies reveal a broader function for the MAD in mitochondrial protein surveillance beyond the MOM and a major role for the MAD in cellular and mitochondrial fitness in response to chronic, low-level oxidative stress in mitochondria.

Cysteine Oxidations in Mitochondrial Membrane Proteins: The Case of VDAC Isoforms in Mammals.

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.

Exploring the FMN binding site in the mitochondrial outer membrane protein mitoNEET

MitoNEET is a mitochondrial outer membrane protein that hosts a redox active [2Fe-2S] cluster in the C-terminal cytosolic domain. Increasing evidence has shown that mitoNEET has an essential role in regulating energy metabolism in human cells. Previously, we reported that the [2Fe-2S] clusters in mitoNEET can be reduced by the reduced flavin mononucleotide (FMNH2) and oxidized by oxygen or ubiquinone-2, suggesting that mitoNEET may act as a novel redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone. Here, we explore the FMN binding site in mitoNEET by using FMN analogs and find that lumiflavin, like FMN, at nanomolar concentrations can mediate the redox transition of the mitoNEET [2Fe-2S] clusters in the presence of flavin reductase and NADH (100 μM) under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that both FMN and lumiflavin can dramatically change the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters and form a covalently bound complex with mitoNEET under blue light exposure, suggesting that FMN/lumiflavin has specific interactions with the [2Fe-2S] clusters in mitoNEET. In contrast, lumichrome, another FMN analog, fails to mediate the redox transition of the mitoNEET [2Fe-2S] clusters and has no effect on the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters under blue light exposure. Instead, lumichrome can effectively inhibit the FMNH2-mediated reduction of the mitoNEET [2Fe-2S] clusters, indicating that lumichrome may act as a potential inhibitor to block the electron transfer activity of mitoNEET.

Contact-ID, a tool for profiling organelle contact sites, reveals regulatory proteins of mitochondrial-associated membrane formation.

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.

The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria

BackgroundTransmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a recently identified cell adhesion molecule which is predominantly expressed by epithelial cells of the intestine and the kidney. Its expression is downregulated in both colon and renal cancer suggesting a tumor suppressive activity. The function of TMIGD1 at the cellular level is largely unclear. Published work suggests a protective role of TMIGD1 during oxidative stress in kidney epithelial cells, but the underlying molecular mechanisms are unknown.ResultsIn this study, we address the subcellular localization of TMIGD1 in renal epithelial cells and identify a cytoplasmic scaffold protein as interaction partner of TMIGD1. We find that TMIGD1 localizes to different compartments in renal epithelial cells and that this localization is regulated by cell confluency. Whereas it localizes to mitochondria in subconfluent cells it is localized at cell-cell contacts in confluent cells. We find that cell-cell contact localization is regulated by N-glycosylation and that both the extracellular and the cytoplasmic domain contribute to this localization. We identify Synaptojanin 2-binding protein (SYNJ2BP), a PDZ domain-containing cytoplasmic protein, which localizes to both mitochondria and the plasma membrane, as interaction partner of TMIGD1. The interaction of TMIGD1 and SYNJ2BP is mediated by the PDZ domain of SYNJ2BP and the C-terminal PDZ domain-binding motif of TMIGD1. We also find that SYNJ2BP can actively recruit TMIGD1 to mitochondria providing a potential mechanism for the localization of TMIGD1 at mitochondria.ConclusionsThis study describes TMIGD1 as an adhesion receptor that can localize to both mitochondria and cell-cell junctions in renal epithelial cells. It identifies SYNJ2BP as an interaction partner of TMIGD1 providing a potential mechanism underlying the localization of TMIGD1 at mitochondria. The study thus lays the basis for a better understanding of the molecular function of TMIGD1 during oxidative stress regulation.

Mitochondrial membrane protein mitofusin 2 as a potential therapeutic target for treating free fatty acid–induced hepatic inflammation in dairy cows during early lactation

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and β-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1β, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase β activity, and the abundance of NLRP3, caspase-1, IL-1β, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation.

TRNA: Splicing and Intron Turnover

In most eukaryotic genomes a subset of tRNA genes contains introns. Introns in tRNAs genes are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by a conserved heterotetrameric protein enzyme, tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast was surprisingly discovered to occur on the mitochondrial surface (Yoshihisa et al. 2003). We recently reported that the SEN complex also locates to the mitochondrial surface in fission yeast (Wan & Hopper 2018) and, therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for at least 500 million years. The mitochondrial outer membrane protein, Tom70, is required for efficient SEN subunit location to mitochondria in both budding and fission yeast (Wan & Hopper 2018). The freed introns are rarely detected in cells and until recently it was unknown how are they are turned over. We identified at least 5 separate, species‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate, possibly indicating their functions in stress response (Peltier, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNAs (Bao, unpublished), also indicative of their possible roles in gene regulation. Our initial experiments support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA (Nostramo, unpublished).Support or Funding InformationNIH GMS 122884

Fluctuating Weak Magnetic Fields Induce Marked Mitophagy

Previous epidemiological studies have revealed that occupational exposure to extremely low frequency weak magnetic fields (ELF‐WMF) is associated with the development of neurodegenerative diseases including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). The molecular mechanism of these detrimental effects, however, remain mostly unelucidated.We examined the effects of extremely low frequency fluctuation of weak magnetic fields (ELF‐WMF) on cultured mammalian cells. While applying ELF‐WMF, we examined the temporal profiles of expression levels of VDAC1, a mitochondrial outer membrane protein, and seven electron transport complex (ETC) proteins of NDUFS1 (ETC I), NDUFB8 (ETC I), SDHB (ETC II), UQCRFS1 (ETC III), UQCRC2 (ETC III), MTCOI (ETC IV), and ATP5A (ETC V). ELF‐WMF downregulated the amounts of these proteins at 3 h and recovered them at 12 h. Quantification of the amount of mitochondria with MitoTracker Green showed that the mitochondrial mass was decreased to 70% at 3 h and recovered at 12 h. In addition, mitophagy‐related proteins of PINK1 and LC3‐II were increased by ELF‐WMF at 1.5–2.5 h. Quantification of ETC enzymatic activities in protein homogenates revealed that ELF‐WMF exclusively suppressed the activity of ETC II in 10 min.We report that ELF‐WMF much weaker than geomagnetic fields suppresses the enzymatic activity of ETC II, and provokes mitophagy. Mitophagy is a cellular quality‐assurance system to eliminate damaged mitochondria. Mitophagy induced by ELF‐WMF may be causally associated with the development and progression of AD and ALS. Contrarily, ELF‐WMF finely tuned to preferentially eliminate damaged mitochondria may be able to ameliorate Parkinson’s disease, in which compromised mitophagy is a cause of the disease.Support or Funding InformationThis research was supported by grants‐in‐aids from MEXT, MHLW, and AMED of Japan.

Downregulation of Fis1 in Endothelial Cells Does Not Impact Normal Endothelial Cell Mitochondrial Bioenergetics, Metabolism, or Mitochondrial Protein Content

Our prior data suggest that knockdown of Fis1 protein improves vascular endothelial health by reversing endothelial dysfunction associated with dysglycemic conditions and reducing endothelial inflammation and permeability of the endothelium. Fis1 is an outer mitochondrial membrane protein that triggers mitochondrial fission when bound by dynamin‐related protein‐1. Fis1 is important to mitochondrial fission process specifically under pathophysiological conditions making Fis1 an attractive therapeutic target. We hypothesize that reducing Fis1 expression will not alter other mitochondrial proteins and mitochondrial bioenergetics under different glycemic conditions. We measured mitochondrial proteins, and mitochondrial bioenergetics in wild type immortalized human microvascular endothelial cells (HMEC‐1) and HMEC‐1 cells that were transfected with Fis1 siRNA (20 nM) or scrambled siRNA (20 nM). Upon transfection, cells were exposed to different glycemic conditions: normal (5mM), high (30mM for six hours), and low (2mM for two hours) glucose levels. Mitochondrial proteins expression in Fis1 siRNA HMEC‐1 cell lysates and scrambled control cell lysates were assessed under different glycemic conditions by high throughput immunodetection WES system. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in transfected cells under different glycemic conditions were measured by Seahorse mitochondrial and glycolytic stress tests. There were no significant differences in mitochondrial bioenergetics (OCR and ECAR; n=5, p>0.05), and expression of other mitochondrial proteins (MFF, MFN1, MFN2, Drp1, OPA1, Cyt‐c, TOMM20, MFF, pAMPKa, POLG, P62, NDUFB8, GABARAP) between siFis1 cells and scrambled controls in any of the glycemic conditions (n=4, p>0.05). There is significant increase in expression of other mitochondrial proteins (MFF, MFN1, Cyt‐c, TOMM20, MFF, pAMPKa, POLG, P62, NDUFB8, GABARAP) under low glucose conditions (n=4, p<0.05) compared to normal and high glucose conditions. Reducing the expression of Fis1 did not alter other mitochondrial proteins and mitochondrial bioenergetics that are critical for normal physiological processes. Therapeutic potential of Fis1 knockdown is enhanced by a lack of impact on endothelial cell metabolism and other mitochondrial protein expression.Support or Funding InformationThis project was supported by the National Institutes of Health, through Grant Number R01‐HL128240 and HL125409.

Unbiased Proteomics Supports a Key Role for Mitochondria in Skeletal Muscle of Highly Functioning Octogenarians

IntroductionSkeletal muscle mass and function decline with aging, and more precipitously after the age 75 y. To help provide insights into biologically relevant mechanisms for preserving muscle mass and function in advanced age, in this study we performed proteomics on skeletal muscle of world class octogenarian track and field athletes in comparison to healthy octogenarian non‐athletes.MethodsMuscle cross‐sectional area by MRI and a vastus lateralis muscle biopsy were performed in 15 octogenarian world class track and field athletes (8 of whom were world record holders in their discipline at the time of testing) and 14 non‐athlete age‐ and sex‐matched non‐athlete controls. From these subjects, a portion of muscle from a subset of 12 master athletes (MA mean age 81.19 ± 5.1 y) and 12 non‐athlete controls (NA mean age 80.94 ± 4.5 y) was used for liquid‐chromatography mass spectrometry to generate quantitative tandem mass tag proteomics data. In addition, we measured mtDNA copy number, COX/SDH histochemistry to identify respiratory compromised fibers, and Western blot of mitochondrial inner and outer membrane proteins.ResultsMuscle cross‐sectional area was higher in MA. Tandem mass spectrometry identified over 6000 proteins, and significant differences in abundance were found between NA controls and MA for more than 800 proteins. A pathway analysis revealed that pathways involved in mitochondria (e.g., TCA cycle, respiratory electron transport, cristae formation, sirtuins) were higher in MA, while proteins in pathways involved in the spliceosome and nuclear pore were downregulated in MA. Finally, 8 mtDNA‐encoded proteins that were included in the analysis were elevated in MA versus NA. These proteomics data are consistent with phenotypic data showing MA have higher mtDNA copy number, fewer respiratory chain compromised muscle fibers, and an increased ratio of ETC subunits (inner mitochondrial membrane proteins) relative to VDAC (outer mitochondrial membrane protein), suggesting an increase in cristae formation.ConclusionOur data underscore that mitochondrial pathways are key to maintaining a high level of physical function in advanced age. With the current study design we cannot determine the degree to which these differences are attributable to the physical activity habits of MA, but it is likely to play a role.Support or Funding InformationCIHR (MOP 125986 to RTH)NIA Intramural Research program

Knockout of VDAC1 in H9c2 Cells Promotes tBHP‐induced Cell Apoptosis Through Decreased Mitochondrial HK II Binding and Enhanced Glycolytic Stress

VDAC1, the most abundant mitochondrial outer membrane protein, transports ions and metabolites between the cytoplasm and mitochondria. It is also involved in cellular energy production and apoptosis, but the role of VDAC1 in cell death remains controversial. Silencing or upregulation of VDAC1 may stimulate or inhibit cell apoptosis, growth and survival depending on cell types and stimuli. Using VDAC1 knockout (Vdac1−/−) embryonic stem cells (ES) and mouse embryonic fibroblast (MEF), it was revealed that VDAC1 was dispensable for both mitochondrial membrane permeability transition pore (mPTP) opening and Bax and Bid‐driven cell death. The effect of Vdac1−/− on oxidative stress‐induced cell death in a cardiac cell line has not been reported. We hypothesized that although VDAC1 is dispensable for mPTP and is not involved in mPTP‐mediated cell death, VDAC1 is an essential regulator of oxidative stress‐induced cell apoptosis in cardiac cells. To this end, we knocked out VDAC1 in the cardiac fibroblast cell line, H9c2 (Vdac1−/− H9c2) by the CRISPR‐cas9 technique and examined whether VDAC1 plays a role in cell death via oxidative stress induced by tert‐butylhydroperoxide (tBHP), an organic peroxide. We found that under physiologic conditions, Vdac1−/− did not affect H9c2 cell proliferation, and mitochondrial respiration. However, tBHP treatment enhanced the extracellular acidification rate (ECAR) and proton production rate (PPR) produced from glycolysis and promoted cell apoptosis in Vdac1−/− H9c2 cells compared to the wildtype. Vdac1−/− H9c2 cells also showed markedly reduced binding of mitochondria hexokinase II (HK II), a key glycolytic enzyme. Rescue of VDAC1 in Vdac1−/− H9c2 cells reinstated mitochondria HK II binding and concomitantly decreased tBHP‐induced cell death. Mitochondrial respiration remained the same after tBHP treatment in Vdac1−/− and wild type H9c2 cells. Our results suggest that Vdac1−/− in H9c2 cells enhances tBHP‐induced oxidative stress‐mediated cell apoptosis by reducing mitochondria HK II binding and by increasing ECAR and PPR produced from glycolytic stress.Support or Funding InformationNational Institutes of Health (R01 HL‐131673‐01A1), Veterans Administration (BX‐002539‐01), United States.

Miro1 as a novel regulator of hypertrophy in neonatal rat cardiomyocytes

Cardiac hypertrophy is an adaptive response to manage an excessive cardiac workload and maintain normal cardiac function. However, sustained hypertrophy leads to cardiomyopathy, cardiac failure, and death. Adrenergic receptors play a key role in regulating cardiac function under normal and pathological conditions. Mitochondria are responsible for 90% of ATP production in cardiomyocytes. Mitochondrial function is dynamically regulated by fusion and fission processes. Changes in mitochondrial dynamics and metabolism are central issues in cardiac hypertrophy. Stimulating cardiomyocytes with adrenergic agonists generates hypertrophy and increases mitochondrial fission, which in turn is associated with decreased ATP synthesis. Miro1 is a mitochondrial outer membrane protein involved in mitochondrial dynamics and transport in neurons. The objective of this work was to evaluate whether Miro1 regulates cardiomyocyte hypertrophy through changes in mitochondrial dynamics. In neonatal rat ventricular myocytes, we showed that phenylephrine induced cardiomyocyte hypertrophy and increased Miro1 mRNA and protein levels. Moreover, alpha-adrenergic stimulation provoked a mitochondrial fission pattern in the cardiomyocytes. Miro1 knockdown prevented both the cardiomyocyte hypertrophy and mitochondrial fission pattern. Our results suggest that Miro1 participates in phenylephrine-induced cardiomyocyte hypertrophy through mitochondrial fission.

Effects of NIX-mediated mitophagy on ox-LDL-induced macrophage pyroptosis in atherosclerosis.

Pyroptosis is a form of cell death that is uniquely dependent on caspase-1. Pyroptosis involved in oxidized low-density lipoprotein (ox-LDL)-induced human macrophage death through the promotion of caspase-1 activation is important for the formation of unstable plaques in atherosclerosis. The mitochondrial outer membrane protein NIX directly interacts with microtubule-associated protein 1 light chain 3 (LC3). Although we previously showed that NIX-mediated mitochondrial autophagy is involved in the clearance of damaged mitochondria, how NIX contributes to ox-LDL-induced macrophage pyroptosis remains unknown. Here, immunoperoxidase staining Nix expression decreased in human atherosclerosis. When we silenced NIX expression in murine macrophage cell, active caspase-1, and mature interleukin-1β expression levels were increased and LC3 was reduced. In addition, LDH release and acridine orange and ethidium bromide staining indicated that damage to macrophage cell membranes induced by ox-LDL was substantially worse. Moreover, intracellular reactive oxygen species and NLRP3 inflammasome levels increased. Taken together, these results demonstrated that NIX inhibits ox-LDL-induced macrophage pyroptosis via autophagy in atherosclerosis.

N-degron-mediated degradation and regulation of mitochondrial PINK1 kinase.

Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by a gradual loss of a specific group of dopaminergic neurons in the substantia nigra. Importantly, current treatments only address the symptoms of PD, yet not the underlying molecular causes. Concomitantly, the function of genes that cause inherited forms of PD point to mitochondrial dysfunction as a major contributor in the etiology of PD. An inherent challenge that mitochondria face is the continuous exposure to diverse stresses including high levels of reactive oxygen species and protein misfolding, which increase their likelihood of dysregulation. In response, eukaryotic cells have evolved sophisticated quality control mechanisms to identify, repair and/or eliminate abnormal dysfunctional mitochondria. One such mechanism is mitophagy, a process which involves PTEN-induced putative kinase 1 (PINK1), a mitochondrial Ser/Thr kinase and Parkin, an E3 ubiquitin ligase, each encoded by genes responsible for early-onset autosomal recessive familial PD. Over 100 loss-of-function mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been reported to cause autosomal recessive early-onset PD. PINK1 acts upstream of Parkin and is essential for the mitochondrial localization and activation of Parkin. Upon mitochondrial damage, PINK1 builds up on the outer mitochondrial membrane (OMM) and mediates the activation of Parkin. Activated Parkin then ubiquitinates numerous OMM proteins, eliciting mitochondrial autophagy (mitophagy). As a result, damaged mitochondrial components can be selectively eliminated. Thus, PINK1 acts a sensor of damage via fine-tuning of its levels on mitochondria, where it activates Parkin to orchestrate the clearance of unhealthy mitochondria. Previous work has unveiled that the Arg-N-end rule degradation pathway (Arg-N-degron pathway) mediates the degradation of PINK1, and thus fine-tune PINK1-dependent mitochondrial quality control pathway. Herein, we briefly discuss the interconnection between N-end rule degradation pathways and mitophagy in the context of N-degron mediated degradation of mitochondrial kinase PINK1 and highlight some of the future prospects.