Distinct Mitochondrial Subpopulations Research Articles

Hyperbaric oxygen treatment reveals spatiotemporal OXPHOS plasticity in the porcine heart.

Cardiomyocytes meet their high ATP demand almost exclusively by oxidative phosphorylation (OXPHOS). Adequate oxygen supply is an essential prerequisite to keep OXPHOS operational. At least two spatially distinct mitochondrial subpopulations facilitate OXPHOS in cardiomyocytes, i.e. subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). Their intracellular localization below the sarcolemma or buried deep between the sarcomeres suggests different oxygen availability. Here, we studied SSM and IFM isolated from piglet hearts and found significantly lower activities of electron transport chain enzymes and F1FO-ATP synthase in IFM, indicative for compromised energy metabolism. To test the contribution of oxygen availability to this outcome, we ventilated piglets under hyperbaric hyperoxic (HBO) conditions for 240 min. HBO treatment raised OXPHOS enzyme activities in IFM to the level of SSM. Complexome profiling analysis revealed that a high proportion of the F1FO-ATP synthase in the IFM was in a disassembled state prior to the HBO treatment. Upon increased oxygen availability, the enzyme was found to be largely assembled, which may account for the observed increase in OXPHOS complex activities. Although HBO also induced transcription of genes involved in mitochondrial biogenesis, a full proteome analysis revealed only minimal alterations, meaning that HBO-mediated tissue remodeling is an unlikely cause for the observed differences in OXPHOS. We conclude that a previously unrecognized oxygen-regulated mechanism endows cardiac OXPHOS with spatiotemporal plasticity that may underlie the enormous metabolic and contractile adaptability of the heart.

FIsetin Preserves Interfibrillar Mitochondria to Protect Against Myocardial Ischemia-Reperfusion Injury.

According to our previous study, fisetin (3,3',4',7-tetrahydroxyflavone), a bioactive phytochemical (flavonol), reportedly showed cardioprotection against ischemia-reperfusion injury (IRI) by reducing oxidative stress and inhibiting glycogen synthase kinase 3β (GSK3β) [1]. GSK3β is said to exert a non-mitochondrial mediated cardioprotection; therefore, distinct mechanisms of GSK3β on the regulatory effect of mitochondria need to be addressed. The two distinct mitochondrial subpopulations in the heart, namely interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM), respond differently to disease states. The current study aimed to understand the effect of fisetin on the subpopulation-specific preservation of IFM and SSM while rendering cardioprotection against ischemia reperfusion (I/R). Rats were pre-treated with fisetin (20 mg/kg) intraperitoneally, and IRI was induced using Langendorff isolated heart perfusion technique. Hemodynamic parameters were recorded, and the cardiac injury was assessed using infarct size (IS), lactate dehydrogenase (LDH), and creatine kinase (CK) levels. Subpopulation-specific mitochondrial preservation was evaluated by electron transport chain (ETC), catalase, superoxide dismutase (SOD), and glutathione (GSH) activities. The bioavailability of fisetin in IFM and SSM was measured using the fluorescence method. The ability of fisetin to bind directly to the mitochondrial complex-1 and activating it through donating electrons to FMN was studied using molecular docking studies and further validated by in vitro rotenone sensitivity assay. Cardioprotective effects exhibited by fisetin were mainly mediated through IFM preservation. Mitochondrial bioavailability of fisetin is more in IFM than SSM in both ex vivo and in vitro conditions. Fisetin increased mitochondrial ATP production in I/R insult hearts by activating ETC complex 1. Inhibition of complex 1 prevents the ATP-producing capacity of fisetin. Our results provide evidence that fisetin plays a protective role in myocardial IRI, possibly by preserving the functional activities of IFM.

Assessing Mitochondrial Bioenergetics in Isolated Mitochondria from Mouse Heart Tissues Using Oroboros 2k-Oxygraph.

In this chapter, we describe detailed protocols for measuring high-resolution respirometry on mitochondria extracted from adult whole mouse heart using the Oroboros 2k-Oxygraph system. The method provides detailed procedures for the preparation of mitochondria and measurement of high-resolution respirometry in response to various respiration inhibitions. The method described in this chapter could discern the different respiration rate on mitochondria extracted from two spatially distinct mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM). These approaches can easily be translated to other cells and tissues.

Estrogen regulates spatially distinct cardiac mitochondrial subpopulations

Increased susceptibility to permeability transition pore (mPTP) is a significant concern to decreased cardiac performance in postmenopausal females. The goal of this study was to assess the effects of estrogen deficiency on the two spatially distinct mitochondrial subpopulations from left ventricle: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on: morphology, membrane potential, oxidative phosphorylation, mPTP and reactive oxygen species production. Female rats (8weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with placebo (OVX), estrogen replacement (OVX+E2) and Sham for 60days. The yield for IFM was found higher in the OVX group and lower in the SSM. SSM internal complexity and size were higher in the OVX group, although membrane potential was not different. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate+malate as substrate, were higher in IFM and SSM from the OVX group. The respiratory control ratio (RCR - state3/state 4), was not different in both SSM and IFM with glutamate+malate. The ADP:O ratio was found lower in IFM and SSM from OVX compared to Sham. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was greater in IFM from OVX rats compared to Sham and the ADP/O ratio was decreased. The RCR was unchanged in both subpopulations. The IFM from OVX rats presented a lower Ca2+retention capacity compared to Sham, however, the SSM remained unchanged. Hydrogen peroxide formation was found increased in the IFM from OVX animals with glutamate+malate and rotenone+succinate as substrates. The SSM showed increased ROS production only with rotenone+succinate. Western blot analyzes showed decreased levels of PGC-1α and NRF-1 in the OVX group. Estrogen replacement was able to restore most of the alterations induced by ovariectomy. In conclusion, our data shows that estrogen deficiency has distinct effects on the two spatially distinct mitochondrial subpopulations in oxidative phosphorylation, morphology, calcium retention capacity and ROS production.

Sex differences in the regulation of spatially distinct cardiac mitochondrial subpopulations.

Spatially distinct mitochondrial subpopulation may mediate myocardial pathology through permeability transition pore opening (MPTP). The goal of this study was to assess sex differences on the two spatially distinct mitochondrial subpopulations: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on morphology, membrane potential, mitochondrial function, oxidative phosphorylation, and MPTP. Aged matched Wistar rats were used to study SSM and IFM. Mitochondrial size was larger in SSM than in IFM in both genders. However, SSM internal complexity, yield, and membrane potential were higher in male than in female. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate+malate as substrate, were higher in IFM and SSM in the male group compared to female. The respiratory control ratio (RCR-state3/state 4), was not different in both SSM and IFM with glutamate+malate. The ADP:O ratio was found higher in IFM and SSM from female compared to males. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was also greater in male IFM compared to female. The RCR increased in the SSM while IFM remained the same. State 4 was higher in male SSM while in the IFM remained the same. The IFM presented a higher Ca(2+) retention capacity compared with SSM, however, there was a greater sensitivity to Ca(2+)-induced MPTP in SSM and IFM in the male group compared to female. In conclusion, our data show that spatially distinct mitochondrial subpopulations have sex-based differences in oxidative phosphorylation, morphology, and calcium retention capacity.

Adaptations of skeletal muscle mitochondria to exercise training.

Mitochondrial volume density (Mito(VD)) is composed of two distinct mitochondrial subpopulations--intermyofibrillar mitochondria (Mito(IMF)) and subsarcolemmal mitochondria (Mito(SS)). With exercise training, Mito(VD) may increase by up to 40% and is, for the most part, related to an increase in Mito(IMF). Exercise-induced adaptations in mitochondrial function depend on the intensity of training and appear to be explained predominately by an increased expression of mitochondrial enzymes that facilitate aerobic metabolism. Although mitochondrial content often increases with training, it seems that mitochondrial adaptations are not needed to facilitate maximal oxygen uptake, whereas such adaptations are of greater importance for endurance capacity.

Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA in the Diabetic Heart.

Cardiomyocytes are rich in mitochondria which are situated in spatially distinct subcellular regions, including those under the plasma membrane, subsarcolemmal mitochondria, and those between the myofibrils, interfibrillar mitochondria. We previously observed subpopulation-specific differences in mitochondrial proteomes following diabetic insult. The objective of this study was to determine whether mitochondrial genome-encoded proteins are regulated by microRNAs inside the mitochondrion and whether subcellular spatial location or diabetes mellitus influences the dynamics. Using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a pool of mitochondrial microRNAs, termed mitomiRs, that are redistributed in spatially distinct mitochondrial subpopulations in an inverse manner following diabetic insult. Redistributed mitomiRs displayed distinct interactions with the mitochondrial genome requiring specific stoichiometric associations with RNA-induced silencing complex constituents argonaute-2 (Ago2) and fragile X mental retardation-related protein 1 (FXR1) for translational regulation. In the presence of Ago2 and FXR1, redistribution of mitomiR-378 to the interfibrillar mitochondria following diabetic insult led to downregulation of mitochondrially encoded F0 component ATP6. Next generation sequencing analyses identified specific transcriptome and mitomiR sequences associated with ATP6 regulation. Overexpression of mitomiR-378 in HL-1 cells resulted in its accumulation in the mitochondrion and downregulation of functional ATP6 protein, whereas antagomir blockade restored functional ATP6 protein and cardiac pump function. We propose mitomiRs can translationally regulate mitochondrially encoded proteins in spatially distinct mitochondrial subpopulations during diabetes mellitus. The results reveal the requirement of RNA-induced silencing complex constituents in the mitochondrion for functional mitomiR translational regulation and provide a connecting link between diabetic insult and ATP synthase function.

Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies.

Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial subpopulations have revealed differences in morphological features including shape, absolute size, and internal cristae arrangement. These findings have been complemented by functional studies indicating differences in biochemical parameters and, potentially, functional roles for the ATP generated, based upon subcellular location. Consequently, mitochondrial subpopulations appear to be influenced differently during cardiac pathologies including ischemia/reperfusion, heart failure, aging, exercise, and diabetes mellitus. These influences may be the result of specific structural and functional disparities between mitochondrial subpopulations such that the stress elicited by a given cardiac insult differentially impacts subcellular locales and the mitochondria contained within. The goal of this review is to highlight some of the inherent structural and functional differences that exist between spatially distinct cardiac mitochondrial subpopulations as well as provide an overview of the differential impact of various cardiac pathologies on spatially distinct mitochondrial subpopulations. As an outcome, we will instill a basis for incorporating subcellular spatial location when evaluating the impact of cardiac pathologies on the mitochondrion. Incorporation of subcellular spatial location may offer the greatest potential for delineating the influence of cardiac pathology on this critical organelle.

Of mice and men (and muscle mitochondria)

Of mice and men (and muscle mitochondria)

Characterization of functionally distinct mitochondrial subpopulations

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca(2+) concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca(2+)-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30% of the mitochondria showed changes in response to Ca(2+), which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca(2+) over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca(2+)-induced swelling.

Rat Cardiac Mitochondrial Sub-populations Show Distinct Features of Oxidative Phosphorylation during Ischemia, Reperfusion and Ischemic Preconditioning

Background: Inter-fibrillar (IFM) and sub-sarcolemmal (SSM) mitochondria are two distinct mitochondrial sub-populations and are expected to behave differently during pathological conditions. This study was undertaken to compare functional oxidative phosphorylation (OXPHOS) in IFM and SSM during ischemia, reperfusion and ischemic preconditioning. Methods: Langendorff perfused Wistar rat hearts were subjected to 35minutes ischemia, 60minutes reperfusion and ischemic preconditioning (IPC) procedure (3cycles of 2-minutes ischemia followed by 3-minutes reperfusion).Subsequently IFM and SSM were isolated, and mitochondrial electron transport chain (ETC) enzyme activities and respiration were measured immediately. Results: Functional enzyme activities of ETC in IFM and SSM showed prominent differences especially in the proximal part of ETC enzymes during ischemia and reperfusion. SSM favor FADH₂while IFM prefer NADH as the main reducing equivalent for electron transport during ischemia and reperfusion. IPC preserved ETC enzyme activities in both IFM and SSM rendering cardio protection. Similarly IPC preserve ADP stimulated respiration with glutamate and malate as substrate in both sub populations, but not in IFM, with succinate as substrate. Apparently, the preconditioning imparts enhanced protection more to SSM than IFM during ischemia and reperfusion and especially to the proximal part of the ETC. Conclusion: We propose that mitochondrial dysfunction, one of the major targets of myocardial ischemia reperfusion injury needs to be evaluated by the synergic effect of both IFM and SSM.

A needle in a haystack:focus on “Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart”

diabetes mellitus is increasingly common throughout the world, due to both an increase in incidence and to decreased mortality of those with diabetes. Unfortunately, diabetes more than doubles the risk of cardiovascular disease in adults, and it even causes its own peculiar form of “diabetic”

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.

Mitochondrial Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase 4 (mPHGPx) Provides Cardioprotection From Type 1 Diabetes Mellitus Insult

Evidence suggests that the inner mitochondria membrane (IMM) is a target for the deleterious effects of type 1 diabetes mellitus (DM). Maintenance of IMM integrity represents a specific locus for testing directed prophylactic intervention. We previously reported that type 1 DM imparts differential effects on spatially distinct mitochondrial subpopulations, subsarcolemmal (SSM) and interfibrillar (IFM). The goal of this study was to determine whether overexpression of a mitochondrially‐targeted antioxidant enzyme, mPHGPx, could elicit protection to mitochondria during type 1 DM. MPHGPx transgenic mice and littermate controls were made diabetic through low dose injections of streptozotocin. Five weeks post hyperglycemia onset, ejection fraction and fractional shortening were decreased in the diabetic heart (*P&lt;0.05), and were reversed with mPHGPx overexpression (*P&lt;0.05). MPHGPx overexpression in the diabetic heart was associated with increased electron transport chain complex activities in IFM as compared to diabetic controls (*P&lt;0.05), with no differences on SSM. Hydrogen peroxide and lipid peroxidation were attenuated in the mPHGPx diabetic IFM as compared to diabetic controls (*P&lt;0.05), with no changes in the SSM. These results indicate that mPHGPx overexpression provides cardioprotection to the diabetic heart, which is associated with mitochondrial subpopulation‐specific protection.

Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of subcellular spatial location

Cardiovascular complications, such as diabetic cardiomyopathy, account for the majority of deaths associated with diabetes mellitus. Mitochondria are particularly susceptible to the damaging effects of diabetes mellitus and have been implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac mitochondria consist of two spatially distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The goal of this study was to determine whether subcellular spatial location is associated with apoptotic propensity of cardiac mitochondrial subpopulations during diabetic insult. Swiss Webster mice were subjected to intraperitoneal injection of streptozotocin or citrate saline vehicle. Ten weeks following injection, diabetic hearts displayed increased caspase-3 and caspase-9 activities, indicating enhanced apoptotic signaling (P < 0.05, for both). Mitochondrial size (forward scatter) and internal complexity (side scatter) were decreased in diabetic IFM (P < 0.05, for both) but not in diabetic SSM. Mitochondrial membrane potential (Delta(Psim)) was lower in diabetic IFM (P < 0.01) but not in diabetic SSM. Mitochondrial permeability transition pore (mPTP) opening was increased in diabetic compared with control IFM (P < 0.05), whereas no differences were observed in diabetic compared with control SSM. Examination of mPTP constituents revealed increases in cyclophilin D in diabetic IFM. Furthermore, diabetic IFM possessed lower cytochrome c and BcL-2 levels and increased Bax levels (P < 0.05, for all 3). No significant changes in these proteins were observed in diabetic SSM compared with control. These results indicate that diabetes mellitus is associated with an enhanced apoptotic propensity in IFM, suggesting a differential apoptotic susceptibility of distinct mitochondrial subpopulations based upon subcellular location.

Reply to “Letter to the editor: ‘Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?’”

LETTERS TO THE EDITORReply to “Letter to the editor: ‘Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?’”Erinne R. Dabkowski and John M. HollanderErinne R. Dabkowski and John M. HollanderPublished Online:01 Aug 2009https://doi.org/10.1152/ajpheart.00539.2009MoreSectionsPDF (29 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat reply: We would like to address the letter by Schwarzer and Doenst (4), regarding our recent study (2) investigating the differential influence of streptozotocin-induced diabetes mellitus on interfibrillar and subsarcolemmal mitochondria in mice. This study identified a multitude of differential responses associated with the diabetic phenotype, which included cardiac contractile abnormalities, mitochondrial morphological changes, increases in reactive oxygen species, enhanced oxidative stress, changes in a specific cardiolipin species, and impaired “resting” mitochondrial respiratory rates (1).We are in agreement with Drs. Schwarzer and Doenst (4) that the inclusion of state 3 respiration analyses would have strengthened our study and enabled a more conclusive interpretation of the data on state 4 respiration. This is not to say that an examination of state 4 respiration independent of state 3 respiration is entirely without value. In fact, in a similar model of diabetes mellitus (streptozotocin), it was shown that decreases in state 3 respiration can occur concomitantly with decreases in state 4 respiration (3).We would also like to address the second point made by Schwarzer and Doenst (4), regarding the inclusion of two different methodologies for assessing mitochondrial function. In this regard, it is important to point out that at no time do we make comparisons between different electron transport chain complexes (i.e., complex I vs. complex III, etc.). Rather, we only make comparisons of electron transport chain function between treatment groups (i.e., control vs. diabetic, subsarcolemmal mitochondria vs. interfibrillar mitochondria, etc.) in which the same methodology is being employed. Thus the utilization of different methods for assessment should not confound the interpretation of the findings for comparisons made solely between treatment groups.In conclusion, we hope that our response will help to clarify the interpretation of the data presented regarding the mitochondrial functional parameters presented in Dabkowski et al. (2).REFERENCES1 Chance B, Williams GR. The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17: 65–134, 1956.PubMed | Google Scholar2 Dabkowski ER, Williamson CL, Bukowski VC, Chapman RS, Leonard SS, Peer CJ, Callery PS, Hollander JM. Diabetic cardiomyopathy-associated dysfunction in spatially distinct mitochondrial subpopulations. Am J Physiol Heart Circ Physiol 296: H359–H369, 2009.Link | ISI | Google Scholar3 King KL, Young ME, Kerner J, Huang H, O'Shea KM, Alexson SE, Hoppel CL, Stanley WC. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart. J Lipid Res 48: 1511–1517, 2007.Crossref | PubMed | ISI | Google Scholar4 Schwarzer M, Doenst T. Letter to the editor: “Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?” Am J Physiol Heart Circ Physiol. doi:10.1152/ajpheart.00434.2009.Link | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: J. M. Hollander, West Virginia Univ. School of Medicine, Div. of Exercise Physiology, Center for Interdisciplinary Research in Cardiovascular Sciences, 1 Medical Center Dr., Morgantown, WV 26506 (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 297Issue 2August 2009Pages H890-H890 Copyright & PermissionsCopyright © 2009 the American Physiological Societyhttps://doi.org/10.1152/ajpheart.00539.2009History Published online 1 August 2009 Published in print 1 August 2009 Metrics

Skeletal Muscle Mitochondrial Subpopulation Response in a Type 2 Diabetic Mouse Model

Dysfunctional mitochondria are central to the pathogenesis of type 2 diabetes mellitus. In skeletal muscle, two spatially distinct mitochondrial subpopulations exist. The interfibrillar mitochondria (IFM) which are situated between myofibrils and the subsarcolemmal mitochondria (SSM) which reside directly below the sarcolemma. The objective of this study was to determine how type 2 diabetes mellitus differentially affects distinct mitochondrial subpopulations in skeletal muscle. Db/db mice and littermate controls were sacrificed at 20 weeks of age and mitochondrial subpopulations were isolated from the gastrocnemius muscle. State 3 respiration in db/db mice was significantly lower in both IFM and SSM populations compared to respective controls leading to a decrease in RCR ratios (P&lt;0.05). Electron transport chain complex I and IV activities were significantly decreased in the diabetic IFM with no change in the SSM (P&lt;0.05 for both). Superoxide production was increased in the diabetic IFM and SSM, although to a greater extent in the IFM. These findings indicate that both mitochondrial subpopulations in mouse gastrocnemius muscle are affected during type 2 diabetes mellitus, however, the IFM exhibit a greater dysfunctional profile.

Diabetic cardiomyopathy-associated dysfunction in spatially distinct mitochondrial subpopulations

Diabetic cardiomyopathy is the leading cause of heart failure among diabetic patients, and mitochondrial dysfunction has been implicated as an underlying cause in the pathogenesis. Cardiac mitochondria consist of two spatially, functionally, and morphologically distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). SSM are situated beneath the plasma membrane, whereas IFM are embedded between myofibrils. The goal of this study was to determine whether spatially distinct cardiac mitochondrial subpopulations respond differently to a diabetic phenotype. Swiss-Webster mice were subjected to intraperitoneal injections of streptozotocin or citrate saline vehicle. Five weeks after injections, diabetic hearts displayed decreased rates of contraction, relaxation, and left ventricular developed pressures (P < 0.05 for all three). Both mitochondrial size (forward scatter, P < 0.01) and complexity (side scatter, P < 0.01) were decreased in diabetic IFM but not diabetic SSM. Electron transport chain complex II respiration was decreased in diabetic SSM (P < 0.05) and diabetic IFM (P < 0.01), with the decrease being greater in IFM. Furthermore, IFM complex I respiration and complex III activity were decreased with diabetes (P < 0.01) but were unchanged in SSM. Superoxide production was increased only in diabetic IFM (P < 0.01). Oxidative damage to proteins and lipids, indexed through nitrotyrosine residues and lipid peroxidation, were higher in diabetic IFM (P < 0.05 and P < 0.01, respectively). The mitochondria-specific phospholipid cardiolipin was decreased in diabetic IFM (P < 0.01) but not SSM. These results indicate that diabetes mellitus imposes a greater stress on the IFM subpopulation, which is associated, in part, with increased superoxide generation and oxidative damage, resulting in morphological and functional abnormalities that may contribute to the pathogenesis of diabetic cardiomyopathy.

Quantitative proteomic analysis of distinct mitochondrial subpopulations in diabetic myocardium

Reactive oxygen species resulting from diabetes mellitus contribute to damage and loss of mitochondrial proteins. Two spatially distinct mitochondria populations exist in the cardiac myocyte, interfibrillar mitochondria (IFM), which situate between the myofibrils, and subsarcolemmal mitochondria (SSM), which exist beneath the sarcolemma. The goal of this study was to determine the impact of diabetic insult on proteomic profiles of distinct mitochondrial populations. Swiss Webster mice were made diabetic by streptozotocin injection and sacrificed following diabetes onset. Using 2D‐differential gel electrophoresis and iTRAQTM technology, we determined that proteomic profiles of IFM were affected to a greater extent than SSM, as a result of diabetes. Specific alterations included decreases in ATP synthesizing proteins (ATP synthase, P&lt;0.05) and electron transport chain proteins (cytochrome C oxidase, P&lt;0.05; flavoprotein ubiquinone oxidoreductase, P&lt;0.001). Further, aconitase, a protein prone to oxidation, was decreased in diabetic IFM (P&lt;0.002), while adenine nucleotide translocase, a protein associated with enhanced permeability transition pore opening, was elevated in diabetic IFM (P&lt;0.05). Our results suggest that the IFM are at greater risk for protein loss as a result of diabetes mellitus, which may contribute to the pathogenesis of diabetic cardiomyopathy.(Supported by AHA 0665237B)

Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: Possible physiological role?

Heterogeneity of mitochondria has been reported for a number of various cell types. Distinct mitochondrial subpopulations may be present in the cell and may be differently involved in physiological and pathological processes. However, the origin and physiological roles of mitochondrial heterogeneity are still unknown. In mice skeletal muscle, a much higher oxidized state of subsarcolemmal mitochondria as compared with intermyofibrillar mitochondria has been demonstrated. Using confocal imaging technique, we present similar phenomenon for rat soleus and gastrocnemius muscles, where higher oxidative state of mitochondrial flavoproteins correlates also with elevated mitochondrial calcium. Moreover, subsarcolemmal mitochondria demonstrate distinct arrangement and organization. In HL-1 cardiomyocytes, long thread mitochondria and small grain mitochondria are observed irrespective of a particular cellular region, showing also heterogeneous membrane potential and ROS production. Possible physiological roles of intracellular mitochondrial heterogeneity and specializations are discussed.