Mitochondrial Calcium Retention Capacity Research Articles

Targeting Mitochondrial Dynamics during Lower-Limb Ischemia Reperfusion in Young and Old Mice: Effect of Mitochondrial Fission Inhibitor-1 (mDivi-1).

Peripheral arterial disease (PAD) strikes more than 200 million people worldwide and has a severe prognosis by potentially leading to limb amputation and/or death, particularly in older patients. Skeletal muscle mitochondrial dysfunctions and oxidative stress play major roles in this disease in relation with ischemia-reperfusion (IR) cycles. Mitochondrial dynamics through impairment of fission-fusion balance may contribute to skeletal muscle pathophysiology, but no data were reported in the setting of lower-limb IR despite the need for new therapeutic options. We, therefore, investigated the potential protective effect of mitochondrial division inhibitor-1 (mDivi-1; 50 mg/kg) in young (23 weeks) and old (83 weeks) mice submitted to two-hour ischemia followed by two-hour reperfusion on systemic lactate, muscle mitochondrial respiration and calcium retention capacity, and on transcripts specific for oxidative stress and mitochondrial dynamics. At the systemic levels, an IR-related increase in circulating lactate was still major despite mDivi-1 use (+305.9% p < 0.0001, and +269.4% p < 0.0001 in young and old mice, respectively). Further, IR-induced skeletal muscle mitochondrial dysfunctions (more severely impaired mitochondrial respiration in old mice (OXPHOS CI state, -68.2% p < 0.0001 and -84.9% p < 0.0001 in 23- and 83-week mice) and reduced calcium retention capacity (-46.1% p < 0.001 and -48.2% p = 0.09, respectively) were not corrected by mDivi-1 preconditioning, whatever the age. Further, mDivi-1 treatment did not oppose superoxide anion production (+71.4% p < 0.0001 and +37.5% p < 0.05, respectively). At the transcript level, markers of antioxidant enzymes (SOD 1, SOD 2, catalase, and GPx) and fission markers (Drp1, Fis) remained unchanged or tended to be decreased in the ischemic leg. Fusion markers such as mitofusin 1 or 2 decreased significantly after IR in both groups. In conclusion, aging enhanced the deleterious effects or IR on muscle mitochondrial respiration, and in this setting of lower-limb IR, mDivi-1 failed to protect the skeletal muscle both in young and old mice.

Effect of Fusidic Acid and Some Nitrogen-Containing Derivatives on Liposomal and Mitochondrial Membranes.

The paper assesses the membranotropic action of the natural antibiotic fusidic acid (FA) and its derivatives. It was found that a FA analogue with ethylenediamine moiety (derivative 2), in contrast to native FA and 3,11-dioxime analogue (derivative 1), is able to increase the mobility of the lipid bilayer in the zone of lipid headgroups, as well as to induce permeabilization of lecithin liposome membranes. A similar effect of derivative 2 is also observed in the case of rat liver mitochondrial membranes. We noted a decrease in the microviscosity of the mitochondrial membrane and nonspecific permeabilization of organelle membranes in the presence of this agent, which was accompanied by a decrease in mitochondrial Δψ and OXPHOS efficiency. This led to a reduction in mitochondrial calcium retention capacity. The derivatives also reduced the production of H2O2 by mitochondria. The paper considers the relationship between the structure of the tested compounds and the observed effects.

Disturbance of mitochondrial functions caused by N-acetylglutamate and N-acetylmethionine in brain of adolescent rats: Potential relevance in aminoacylase 1 deficiency

Aminoacylase 1 (ACY1) deficiency is a rare genetic disorder that affects the breakdown of short-chain aliphatic N-acetylated amino acids, leading to the accumulation of these amino acid derivatives in the urine of patients. Some of the affected individuals have presented with heterogeneous neurological symptoms such as psychomotor delay, seizures, and intellectual disability. Considering that the pathological mechanisms of brain damage in this disorder remain mostly unknown, here we investigated whether major metabolites accumulating in ACY1 deficiency, namely N-acetylglutamate (NAG) and N-acetylmethionine (NAM), could be toxic to the brain by examining their in vitro effects on important mitochondrial properties. We assessed the effects of NAG and NAM on membrane potential, swelling, reducing equivalents, and Ca2+ retention capacity in purified mitochondrial preparations obtained from the brain of adolescent rats. NAG and NAM decreased mitochondrial membrane potential, reducing equivalents, and calcium retention capacity, and induced swelling in Ca2+-loaded brain mitochondria supported by glutamate plus malate. Notably, these changes were completely prevented by the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP and by ruthenium red, implying the participation of MPT and Ca2+ in these effects. Our findings suggest that NAG- and NAM-induced disruption of mitochondrial functions involving MPT may represent relevant mechanisms of neuropathology in ACY1 deficiency.

Testosterone deficiency impairs cardiac interfibrillar mitochondrial function and myocardial contractility while inducing oxidative stress.

Clinical studies have shown that low levels of endogenous testosterone are associated with cardiovascular diseases. Considering the intimate connection between oxidative metabolism and myocardial contractility, we determined the effects of testosterone deficiency on the two spatially distinct subpopulations of cardiac mitochondria, subsarcolemmal (SSM) and interfibrillar (IFM). We assessed cardiac function and cardiac mitochondria structure of SSM and IFM after 12 weeks of testosterone deficiency in male Wistar rats. Results show that low testosterone reduced myocardial contractility. Orchidectomy increased total left ventricular mitochondrial protein in the SSM, but not in IFM. The membrane potential, size and internal complexity in the IFM after orchidectomy were higher compared to the SHAM group. However, the rate of oxidative phosphorylation with all substrates in the IFM after orchidectomy was lower compared to the SHAM group. Testosterone replacement restored these changes. In the testosterone-deficient SSM group, oxidative phosphorylation was decreased with palmitoyl-L-carnitine as substrate; however, the mitochondrial calcium retention capacity in IFM was increased. There was no difference in swelling of the mitochondria in either group. These changes in IFM were followed by a reduction in phosphorylated form of AMP-activated protein kinase (p-AMPK-α), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) translocation to mitochondria and decreased mitochondrial transcription factor A (TFAM). Testosterone deficiency increased NADPH oxidase (NOX), angiotensin converting enzyme (ACE) protein expression and reduced mitochondrial antioxidant proteins such as manganese superoxide dismutase (Mn-SOD) and catalase in the IFM. Treatment with apocynin (1.5 mM in drinking water) normalized myocardial contractility and interfibrillar mitochondrial function in the testosterone depleted animals. In conclusion, our findings demonstrate that testosterone deficiency leads to reduced myocardial contractility and impaired cardiac interfibrillar mitochondrial function. Our data suggest the involvement of reactive oxygen species, with a possibility of NOX as an enzymatic source.

Erythropoietin-mediated cardioprotection in hearts subjected to ischemia reperfusion.

Several studies provide evidence that erythropoietin (EPO) could play an important role in the recovery of the heart subjected to ischemia-reperfusion. In this regard, it has been suggested that EPO could be involved in protein kinase B (Akt) activation as a cell survival protein. The aim of the present study was to investigate the effects of EPO on the Akt/glycogen synthase kinase 3 beta (GSK-3β) pathway in the presence or absence of wortmannin (W, Akt inhibitor) and its relationship with mitochondrial morphology and function preservation in ischemic-reperfused rat hearts. EPO improved the functional recovery of the heart subjected to ischemia-reperfusion, reduced the release of CK and the infarct size, and promoted preservation of the mitochondrial structure. Moreover, it reduced tissue lactate content and preserved glycogen in order to prevent ischemia. The results showed greater Akt activation, accompanied by preservation of swelling and mitochondrial calcium retention capacity, as well as an increase in ATP synthesis capacity. These results were accompanied by an inhibition of GSK-3β, suggesting regulation of Akt on the opening of the mitochondrial permeability transition pore. All these beneficial effects exerted by acute treatment with EPO were prevented by W. The present study provided novel evidence that EPO not only enhances intrinsic activation of Akt during myocardial ischemia-reperfusion but also promotes GSK-3β inhibition, contributing to mitochondrial structure and function preservation.

The novel cyclophilin inhibitor C105SR reduces hepatic ischaemia–reperfusion injury via mitoprotection

Mitochondrial permeability transition pore (mPTP) opening is critical for mediating cell death during hepatic ischaemia-reperfusion injury (IRI). Blocking mPTP opening by inhibiting cyclophilin D (CypD) is a promising pharmacological approach for the treatment of IRI. Here, we show that diastereoisomers of a new class of small-molecule cyclophilin inhibitors (SMCypIs) have properties that make them attractive candidates for the development of therapeutic agents against liver IRI. Derivatives of the parent SMCypI were synthesised and evaluated for their ability to inhibit CypD peptidyl-prolyl cis-trans isomerase (PPIase) activity and for their mitoprotective properties, evaluated by measuring mitochondrial swelling and calcium retention capacity in liver mitochondria. The ability of the selected compounds to inhibit mPTP opening was evaluated in cells subjected to hypoxia/reoxygenation using a calcein/cobalt assay. Their ability to inhibit cell death was evaluated in cells subjected to hypoxia/reoxygenation by measuring lactate dehydrogenase (LDH) release, propidium iodide staining, and cell viability. The compound performing best invitro was selected for invivo efficacy evaluation in a mouse model of hepatic IRI. The two compounds that showed the strongest inhibition of CypD PPIase activity and mPTP opening, C105 and C110, were selected. Their SR diastereoisomers carried the activity of the racemic mixture and exhibited mitoprotective properties superior to those of the known macrocyclic cyclophilin inhibitors cyclosporin A and alisporivir. C105SR was more potent than C110SR in inhibiting mPTP opening and prevented cell death in a model of hypoxia/reoxygenation. Finally, C105SR substantially protected against hepatic IRI invivo by reducing hepatocyte necrosis and apoptosis. We identified a novel cyclophilin inhibitor with strong mitoprotective properties both invitro and invivo that represents a promising candidate for cellular protection in hepatic IRI. Hepatic ischaemia-reperfusion injury (IRI) is one of the main causes of morbidity and mortality during or after liver surgery. However, no effective therapies are available to prevent or treat this devastating syndrome. An attractive strategy to prevent hepatic IRI aims at reducing cell death by targeting mitochondrial permeability transition pore opening, a phenomenon regulated by cyclophilin D. Here, we identified a new small-molecule cyclophilin inhibitor, and demonstrated the enhanced mitoprotective and hepatoprotective properties of one of its diastereoisomers both invitro and invivo, making it an attractive lead compound for subsequent clinical development.

Heat Shock Protein 70 Is Involved in the Efficiency of Preconditioning with Cyclosporine A in Renal Ischemia Reperfusion Injury by Modulating Mitochondrial Functions.

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Uncovering the relationship between optic atrophy-1 protein and mitochondria function

Mitochondria, known as the powerhouse of the cell, also participate in the regulation of ion homeostasis, redox status, cell growth and differentiation, lipid metabolism, and cell death. The inner mitochondrial membrane (IMM) plays a crucial role in the regulation of mitochondrial metabolism and function. The dynamin-related GTPase optic atrophy-1 (OPA1) localized in the IMM is responsible for mitochondria fusion. In addition, OPA1 has been shown to play an important role in maintaining the structural integrity and functional activity of mitochondrial cristae. We have recently shown that mitochondrial swelling stimulates enzymatic cleavage of OPA1 by OMA1, a zinc metallopeptidase, which converts long-OPA1 (L-OPA1) to short-OPA1 (S-OPA1). Cleavage of L-OPA1 was suggested to diminish mitochondrial fusion and bioenergetics leading to cell death. The role of OPA1 cleavage and OMA1 activation under diverse swelling conditions has not yet been elucidated. In this study, we investigated the role of OPA1 cleavage under various mitochondrial swelling conditions in the presence and absence of Myls22 (an OPA1 inhibitor), and TPEN (a zinc-chelator and OMA1 inhibitor). Cardiac mitochondria were isolated from Sprague Dawley rats and their function was assessed under Ca2+-induced mitochondrial swelling with oxoglutarate and malate (OM) with/without ADP. Our dose-dependent experiments revealed that Myls22 and TPEN exert the maximum effect at 50 and 5 μM, respectively. Next, we found that the maximum respiration rate (state 3) of mitochondria for complex I is diminished by both inhibitors. However, neither Myls22 nor TPEN prevented alterations in mitochondrial respiration rates, swelling, membrane potential, and calcium retention capacity induced by Ca2+. Finally, we evaluated the protein expression of L-OPA1 via western blot and found more OPA1 cleavage under state 3 compared to state 2 respiration rate suggesting the regulatory role of S-OPA1 in mitochondrial respiration. Thus, our data demonstrate that although mitochondrial swelling stimulates L-OPA cleavage associated with mitochondrial dysfunction, inhibition of OPA1 cleavage/OMA1 activity by Mysl22/TPEN is ineffective in ameliorating the effects of Ca2+-induced mitochondria swelling. This study was supported by the National Science Foundation (Grant 2006477). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Melatonin and Exercise Counteract Sarcopenic Obesity through Preservation of Satellite Cell Function.

Sarcopenic obesity (SO) is characterized by atrophic skeletal muscle impairment (sarcopenia) and obesity, which is associated with adverse outcomes of morbidity and mortality in elderly people. We investigated the effects of melatonin and exercise training on SO in 32-week-old senescence-accelerated mouse-prone-8 (SAMP8) mice fed a normal diet or a high-fat diet for 16 weeks. Melatonin, exercise, or melatonin and exercise for 8 weeks displayed reductions in the SO-induced impairment of skeletal muscle function and atrophy. Specifically, a decrease in mitochondrial calcium retention capacity in skeletal muscles observed in the HFD-con group was attenuated in melatonin and/or exercise intervention groups. More importantly, HFD-con mice displayed a lower number of Pax7+ satellite cells (SCs) and higher expression of p16ink than P8ND mice, which were attenuated by melatonin and/or exercise interventions. The cellular senescence in SC-derived primary myoblasts from HFD-con mice was significantly attenuated in myoblasts from the melatonin and/or exercise groups, which was reproduced in a senescence model of H2O2-treated C2C12 myoblasts. Our results suggest that melatonin and exercise training attenuate SO-induced skeletal muscle dysfunction, at least in part, through preserving the SC pool by inhibiting cellular senescence and attenuating mitochondrial dysfunction.

Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime.

Duchenne muscular dystrophy (DMD) is associated with distinct mitochondrial stress responses. Here, we aimed to determine whether the prospective mitochondrial-enhancing compound Olesoxime, prevents early-stage mitochondrial stress in limb and respiratory muscle from D2.mdx mice using a proof-of-concept short-term regimen spanning 10-28 days of age. As mitochondrial-cytoplasmic energy transfer occurs via ATP- or phosphocreatine-dependent phosphate shuttling, we assessed bioenergetics with or without creatine in vitro. We observed that disruptions in Complex I-supported respiration and mH2O2 emission in D2.mdx quadriceps and diaphragm were amplified by creatine demonstrating mitochondrial creatine insensitivity manifests ubiquitously and early in this model. Olesoxime selectively rescued or maintained creatine sensitivity in both muscles, independent of the abundance of respiration-related mitochondrial proteins or mitochondrial creatine kinase cysteine oxidation in quadriceps. Mitochondrial calcium retention capacity and glutathione were altered in a muscle-specific manner in D2.mdx but were generally unchanged by Olesoxime. Treatment reduced serum creatine kinase (muscle damage) and preserved cage hang-time, microCT-based volumes of lean compartments including whole body, hindlimb and bone, recovery of diaphragm force after fatigue, and cross-sectional area of diaphragm type IIX fiber, but reduced type I fibers in quadriceps. Grip strength, voluntary wheel-running and fibrosis were unaltered by Olesoxime. In summary, locomotor and respiratory muscle mitochondrial creatine sensitivities are lost during early stages in D2.mdx mice but are preserved by short-term treatment with Olesoxime in association with specific indices of muscle quality suggesting early myopathy in this model is at least partially attributed to mitochondrial stress.

Effects of Subnormothermic Regulated Hepatic Reperfusion on Mitochondrial and Transcriptomic Profiles in a Porcine Model.

We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

Calcium Overload and Mitochondrial Metabolism.

Mitochondria calcium is a double-edged sword. While low levels of calcium are essential to maintain optimal rates of ATP production, extreme levels of calcium overcoming the mitochondrial calcium retention capacity leads to loss of mitochondrial function. In moderate amounts, however, ATP synthesis rates are inhibited in a calcium-titratable manner. While the consequences of extreme calcium overload are well-known, the effects on mitochondrial function in the moderately loaded range remain enigmatic. These observations are associated with changes in the mitochondria ultrastructure and cristae network. The present mini review/perspective follows up on previous studies using well-established cryo-electron microscopy and poses an explanation for the observable depressed ATP synthesis rates in mitochondria during calcium-overloaded states. The results presented herein suggest that the inhibition of oxidative phosphorylation is not caused by a direct decoupling of energy metabolism via the opening of a calcium-sensitive, proteinaceous pore but rather a separate but related calcium-dependent phenomenon. Such inhibition during calcium-overloaded states points towards mitochondrial ultrastructural modifications, enzyme activity changes, or an interplay between both events.

Corrigendum for Skyschally et al., volume 315, 2018, p. H159–H172

CorrigendumCorrigendum for Skyschally et al., volume 315, 2018, p. H159–H172Published Online:24 May 2022https://doi.org/10.1152/ajpheart.00152.2018_COROriginal articleMoreSectionsPDF (179 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Skyschally A, Kleinbongard P, Lieder H, Gedik N, Stoian L, Amanakis G, Elbers E, Heusch G. Humoral transfer and intramyocardial signal transduction of protection by remote ischemic perconditioning in pigs, rats, and mice. Am J Physiol Heart Circ Physiol 315: H159–H172, 2018. First published March 23, 2018. doi:10.1152/ajpheart.00152.2018.In the published article, there was an error in Fig. 5, A and B. Due to an internal calibration error and an improper focus of the fluorescence detector over the well plates containing the samples, the reported absolute numbers for mitochondrial ATP and ROS formation were incorrect by an order of magnitude. The authors were unable to reconstruct the true absolute numbers and have requested the removal of these data. The authors apologize for this error and state that it does not change the scientific conclusions of the article in any way. The corrected Fig. 5 with revised legend appears below.Figure 5.Mitochondrial calcium retention capacity in isolated rat hearts with infusion of pig plasma. A and B: original data removed. C and D: data without (C) and with (D) cyclosporin A. TC, time control; I/R, ischemia-reperfusion; pPLA, plasma taken from pigs after placebo (PLA); pRPER, plasma taken from pigs after remote ischemic perconditioning (RPER); aliquots of pPLA and pRPER samples (individual open symbols with the same shape within pPLA or pRPER represent plasma samples from the same pig, respectively) were retested in rat hearts subjected to reperfusion injury salvage kinase (RISK; +RISK-BL) or survivor activating factor enhancement (SAFE; +SAFE-BL) blockade, respectively. Closed symbols: means ± SD; *P < 0.05 vs. TC, #P < 0.05 vs. pRPER and I/R (one-way ANOVA; Fisher’s LSD post hoc tests). LSD, least significant difference.Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated articlesHumoral transfer and intramyocardial signal transduction of protection by remote ischemic perconditioning in pigs, rats, and mice 09 Jul 2018American Journal of Physiology-Heart and Circulatory Physiology More from this issue > Volume 322Issue 6June 2022Pages H1086-H1086 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajpheart.00152.2018_CORPubMed35608821History Published online 24 May 2022 Published in print 1 June 2022 Metrics Downloaded 103 times

Mitochondrial calcium retention capacity correlates with markers of muscle mass, strength, power and functional capacities in young and old adults

RationaleThe aging‐related loss of muscle mass and function is a major risk factor for falls, fractures, frailty, loss of independence, institutionalization and even death. Although the underlying cellular and molecular mechanisms remain partly understood, impaired mitochondrial permeability transition pore (mPTP) function has been proposed as a potential mechanism. Indeed, altered mitochondrial calcium retention capacity (mCRC), a marker of mPTP function, has been documented with aging in both rodent and human skeletal muscles and mPTP opening was shown to cause muscle atrophy. However, studies on the impact of aging on mPTP function in humans remain scarce, and whether impaired mPTP function correlates with markers of muscle mass, performance and functional capacities remain unknown.ObjectiveThe present study aimed at investigating whether mCRC is associated with markers of muscle mass, muscle function and functional capacities in humans.MethodsTwelve young (29.4 ± 4.4y) and fourteen old (80 ± 7.4y) healthy men were enrolled in the present study. Leg lean mass (leg LM) was assessed using DEXA. Maximal isometric knee extension strength (iKES) and power were measured using a strain gauge system attached to a chair and the Nottingham Leg Extensor Power rig, respectively. To assess functional capacities, the performance at the 6‐min walk test and the step test (maximum number of steps in 20s) were evaluated. To assess mPTP function, mCRC was measured in permeabilized myofibers prepared from muscle biopsies obtained in the vastus lateralis.ResultsAs compared to young adults, old individuals displayed significantly lower leg LM (p&lt;0.01), iKES (p&lt;0.01), and power (p&lt;0.01). The performance at the 6‐min walk test (p&lt;0.01) and the step test (p&lt;0.01) were also lower in old individuals. Old individuals also displayed a significantly lower mCRC (p&lt;0.01). mCRC was significantly correlated with leg LM (r=0.34; p=0.045), iKES (r=0.35; p=0.04) and muscle power (r=0.41; p=0.02). In addition, mCRC was significantly correlated with the performance at the 6‐min walking test (r=0.42;p=0.01) and step test (r=0.46; p&lt;0.01).ConclusionsThese results indicate that mPTP function is altered with aging in humans and highlight positive associations between mPTP, muscle mass, strength, power, and functional capacities. These findings therefore strengthen the emerging view that altered mPTP function contributes to the aging‐related loss of muscle mass and function in humans.

Comparative analysis of mitochondrial CRC in permeabilized cells and isolated cell mitochondria

Mitochondria undergo permeability transition (PT) resulting in the opening of the non‐selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass &lt;1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+‐sensitive fluorescence probe Calcium Green‐5N. Results demonstrated the interference of dye‐associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin‐permeabilized cells was higher than the CRC of digitonin‐permeabilized cells. In addition, the mitochondrial CRC of saponin‐permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin‐permeabilized cells has more advantages than isolated mitochondria.

Mitofilin downregulation/Loss favors the initiation and development of dilated cardiomyopathy that progresses to acute heart failure and cardiac arrest

RationalHeart failure (HF) is a clinical syndrome characterized by a constellation of symptoms that are caused by a structural and/or functional cardiac abnormality, ultimately resulting in reduced cardiac output and/or elevated intracardiac pressures. Despite the fact that many cardiovascular diseases end in HF, the condition too often fails to attract the awareness and emphasis it deserves. Worryingly, the mechanisms leading to HF are still not clearly defined. Therefore, research to decipher mechanisms of cardiac decompensation in HF with reduced ejection fraction (HFrEF) and to identify new treatments is urgently needed.ObjectiveThe objective of this study is to determine the role of the mitochondrial inner membrane protein (Mitofilin/Mic60), which plays a crucial role in maintaining mitochondrial cristae morphology, downregulation and loss in the onset and progression of HF and in increasing the prevalence of sudden death in the mouse.Material and methodsWe developed a Mitofilin‐floxed mouse model and crossed this mouse with a transgenic mouse that expresses Tamoxifen inducible Myh6‐Cre recombinase to generate a cardiomyocyte‐specific Mitofilin KO (cMitofilin‐/‐) mouse. Kaplan Meier Curve was used to determine the rate of mice survival, mitochondrial integrity was measured by analyzing cristae morphology and ROS production was assessed using amplex red method. The calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore opening was measured using calcium green dye. Cardiac function was measured by echocardiography and Mic60 mRNA assessed by qRT‐PCR.ResultsWe found an dramatic increase in sudden cardiac death in cMitofilin‐/‐ mice within 5 days post Tamoxifen treatment. Mitofilinfl+/fl++Cre(+) mice treated TAM (cMitofilin‐/‐) displayed significantly increase in cardiac size and reduction of cardiac function indicative of acute dilated cardiomyopathy (DCM) compared to vehicle. cMitofilin‐/‐mice showed mitochondrial cristae disruption and dysfunction including reduced mitochondrial CRC and increased ROS production compared to WT. We observed that full‐body Mitofilin‐/‐ mice do not survive after multiple crossing of full‐body heterozygote Mitofilin+/‐ mice, suggesting that a single allele expression of Mitofilin is sufficient to rescue the DCM phenotype. To determine the role of Mitofilin downregulation in DCM, we compared Mitofilin levels in WT mice treated with Doxorubicin (DOX) versus vehicle. DOX is a highly effective anticancer drug that causes acute ventricular dysfunction, and may induce late‐onset cardiomyopathy and HF. We found a dramatic reduction of Mitofilin in DOX‐treated animals that exhibited a higher death rate versus controls; this finding was associated with an increase in mitochondrial oxidative stress and dysfunction. Furthermore, mice treated with DOX displayed a decrease in Mitofilin mRNA, suggesting reduced Mitofilin expression during HF is at least in part mediated by Mitofilin transcriptional dysregulation. Interestingly, Mitofilin overexpression in H9c2 cardiomyocytes prevented DOX‐induced increase in cell survival.Conclusion. Together, these results indicate that Mitofilin downregulation/loss induces the onset and progression of DCM that is associated with HF and cardiac arrest. Our results provide a better understanding of mitochondrial‐dependent mechanism of DCM and represent a novel potential therapeutic target against DCM and HF.

Scavenging mitochondrial hydrogen peroxide by peroxiredoxin 3 overexpression attenuates contractile dysfunction and muscle atrophy in a murine model of accelerated sarcopenia.

Age‐related muscle atrophy and weakness, or sarcopenia, are significant contributors to compromised health and quality of life in the elderly. While the mechanisms driving this pathology are not fully defined, reactive oxygen species, neuromuscular junction (NMJ) disruption, and loss of innervation are important risk factors. The goal of this study is to determine the impact of mitochondrial hydrogen peroxide on neurogenic atrophy and contractile dysfunction. Mice with muscle‐specific overexpression of the mitochondrial H2O2 scavenger peroxiredoxin3 (mPRDX3) were crossed to Sod1KO mice, an established mouse model of sarcopenia, to determine whether reduced mitochondrial H2O2 can prevent or delay the redox‐dependent sarcopenia. Basal rates of H2O2 generation were elevated in isolated muscle mitochondria from Sod1KO, but normalized by mPRDX3 overexpression. The mPRDX3 overexpression prevented the declines in maximum mitochondrial oxygen consumption rate and calcium retention capacity in Sod1KO. Muscle atrophy in Sod1KO was mitigated by ~20% by mPRDX3 overexpression, which was associated with an increase in myofiber cross‐sectional area. With direct muscle stimulation, maximum isometric specific force was reduced by ~20% in Sod1KO mice, and mPRDX3 overexpression preserved specific force at wild‐type levels. The force deficit with nerve stimulation was exacerbated in Sod1KO compared to direct muscle stimulation, suggesting NMJ disruption in Sod1KO. Notably, this defect was not resolved by overexpression of mPRDX3. Our findings demonstrate that muscle‐specific PRDX3 overexpression reduces mitochondrial H2O2 generation, improves mitochondrial function, and mitigates loss of muscle quantity and quality, despite persisting NMJ impairment in a murine model of redox‐dependent sarcopenia.

Beta-Amyloid Instigates Dysfunction of Mitochondria in Cardiac Cells.

Alzheimer’s disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ1-40 and Aβ1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ1-40 and Aβ1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ1-42 had more adverse effects than Aβ1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ1-40 and Aβ1-42 significantly increased Ca2+ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells.

Simultaneous Acquisition of Mitochondrial Calcium Retention Capacity and Swelling to Measure Permeability Transition Sensitivity.

The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant inducers of mitochondrial swelling is matrix calcium (Ca2+). Respiring mitochondria will intake available Ca2+ into their matrix until a threshold is reached which triggers the opening of the mitochondrial permeability transition pore (MPTP). Upon opening of the pore, mitochondrial membrane potential dissipates and the mitochondria begin to swell, rendering them dysfunctional. The total amount of Ca2+ taken up by a mitochondrion prior to the engagement of the MPTP is referred to as mitochondrial Ca2+ retention capacity (CRC). The CRC/swelling assay is a useful tool for observing the dose-dependent event of mitochondrial dysfunction in real-time. In this technique, isolated mitochondria are treated with specific boluses of Ca2+ until they reach CRC and undergo swelling. A fluorometer is utilized to detect an increase in transmitted light passing through the sample as the mitochondria lose cristae density, and simultaneously measures calcium uptake by way of a Ca2+-specific membrane impermeable fluorescent dye. Here we provide a detailed protocol describing the mitochondrial CRC/swelling assay and we discuss how varying amounts of mitochondria and Ca2+ added to the system affect the dose-dependency of the assay. We also report how to validate the assay by using MPTP and calcium uptake inhibitors and troubleshooting common mistakes that occur with this approach.

Age-Dependent Changes in the Function of Mitochondrial Membrane Permeability Transition Pore in Rat Liver Mitochondria

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.