Aging-induced mitochondrial dysfunction: two distinct populations of mitochondria versus a combined population.

Mitochondrial function in aged hearts is impaired, and studies of isolated mitochondria are commonly used to assess their function. The two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), are affected by aging. However, the yield of these mitochondria, particularly SSM, is limited in the mouse heart because of the smaller heart size. To address this issue, the authors developed a method to isolate a mixed population (MIX) of SSM and IFM mitochondria from a single mouse heart. The aim of the study was to compare the mitochondrial function between SSM, IFM, and the MIX population from young and aged mouse hearts. The MIX population had a higher yield of total protein and citrate synthase activity from both young and aged hearts compared with the individual yields of SSM or IFM. Oxidative phosphorylation (OXPHOS) decreased in aged SSM and IFM compared with young SSM and IFM, as well as in the MIX population isolated from aged hearts compared with young hearts, when using complex I or IV substrates. Furthermore, aging barely affected the sensitivity to mitochondrial permeability transition pore (MPTP) opening in SSM, whereas the sensitivity was increased in IFM isolated from aged hearts and in the MIX population from aged hearts compared with the corresponding populations isolated from young hearts. These results suggest that mitochondrial dysfunction exists in aged hearts and the isolation of a MIX population of mitochondria from the mouse heart is a potential approach to studying mitochondrial function in the mouse heart.NEW & NOTEWORTHY We developed two methods to isolate mitochondria from a single mouse heart. We compared mitochondrial function in young and aged mice using mitochondria isolated with different methods. Both methods can be successfully used to isolate cardiac mitochondria from single mouse hearts. Our results provide the flexibility to isolate mitochondria from a single mouse heart based on the purpose of the study.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7 days) and consumed drinking water with or without 1 mM sodium nitrate. Compared with the contralateral control limb, 3 days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P < 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3 days decreased FSR in both SS (P = 0.009) and IMF (P = 0.031) mitochondria. Additionally, 3 days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7 days of immobilization. However, in contrast to 3 days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7 days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3 day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Ephrin-B1 regulates the adult diastolic function through a late postnatal maturation of cardiomyocyte surface crests.

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function.

Subcellular Specialization of Mitochondrial Form and Function in Skeletal Muscle Cells.

Across different cell types and within single cells, mitochondria are heterogeneous in form and function. In skeletal muscle cells, morphologically and functionally distinct subpopulations of mitochondria have been identified, but the mechanisms by which the subcellular specialization of mitochondria contributes to energy homeostasis in working muscles remains unclear. Here, we discuss the current data regarding mitochondrial heterogeneity in skeletal muscle cells and highlight potential new lines of inquiry that have emerged due to advancements in cellular imaging technologies.

High-fat diet affects skeletal muscle mitochondria comparable to pressure overload-induced heart failure.

In heart failure, high-fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.

Mitochondrial dysfunction plays a key role in the abrogation of cardioprotection by sodium hydrosulfide post-conditioning in diabetic cardiomyopathy rat heart.

Our previous study demonstrated that hydrogen sulfide post-conditioning (HPOC) renders cardioprotection against ischemia-reperfusion (I/R) injury in normal rat by preserving mitochondria. But its efficacy in ameliorating I/R in the diabetic heart with (DCM) or without cardiomyopathy (DM) is unclear and is the focus of the present study. Normal (N), diabetes mellitus (streptozotocin, 35 mg/kg; normal diet), and DCM (streptozotocin, 35 mg/kg; high-fat diet) rats were subjected to I/R (30 min global ischemia followed by 60 min reperfusion) in presence and absence of HPOC using ex vivo Langendorff perfusion system. At the end of heart perfusion, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) fractions from the tissue were isolated and measured for the ATP production, electron transport chain (ETC) enzyme activity, and membrane potential. The prominent I/R-associated injury in DCM rat was not subsequently attenuated by HPOC protocol unlike in the normal or diabetic rat heart (latter rat heart showed moderate protection) (HPOC recovery on infarct size: N 75% vs. DM 63% vs. DCM 48%). The baseline ATP content and subsequent ATP-producing capacity in DCM rat heart were low as compared with those in normal or DM rat heart, especially in SSM. HPOC protocol reversed the I/R-induced low mitochondrial ATP content and low ATP-producing capacity (both in non-energized and energized with glutamate/malate) significantly in normal and DM hearts, but not in DCM heart. Moreover in DCM, decreased activity of mitochondrial electron chain enzymes (complexes I, II, III, and IV) in SSM (26%, 88%, 57%, and 17%) and IFM (76%, 89%, 60%, and 13%) from sham control was maintained even after the conditioning of heart with hydrogen sulfide donor. Results altogether suggest that significantly higher levels of perturbing mitochondria in DCM rat heart underline the deteriorated cardiac recovery by HPOC.

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.

Ischemia time impacts on respiratory chain functions and Ca2+-handling of cardiac subsarcolemmal mitochondria subjected to ischemia reperfusion injury.

BACKGROUND: Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in heart surgery. We determined the effect of increasing ischemia time on the respiratory chain (RC) function, the inner membrane polarization and Ca2+ homeostasis of rat cardiac subsarcolemmal mitochondria (SSM). METHODS: Wistar rat hearts were divided into 4 groups of stop-flow induced warm global IR using a pressure-controlled Langendorff system: 0, 15, 30 and 40 min of ischemia with 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records (dP/dt, dPmax) with an intraventricular balloon. Following reperfusion, SSM were isolated and analyzed regarding electron transport chain (ETC) coupling by polarography (Clark-Type electrode), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+-induced swelling and Ca2+-uptake/release (Calcium Green-5 N® fluorescence). RESULTS: LV contractility and systolic pressure during reperfusion were impaired by increasing ischemic times. Ischemia reduced ETC oxygen consumption in IR40/30 compared to IR0/30 at complex I-V (8.1 ± 1.2 vs. 18.2 ± 2.0 nmol/min) and II-IV/V (16.4 ± 2.6/14.8 ± 2.3 vs. 2.3 ± 0.6 nmol/min) in state 3 respiration (p < 0.01). Relative membrane potential revealed a distinct hyperpolarization in IR30/30 and IR40/30 (171.5 ± 17.4% and 170.9 ± 13.5%) compared to IR0/30 (p < 0.01), wearing off swiftly after CCCP-induced uncoupling. Excess mitochondrial permeability transition pore (mPTP)-gated Ca2+-induced swelling was recorded in all groups and was most pronounced in IR40/30. Pyruvate addition for mPTP blocking strongly reduced SSM swelling in IR40/30 (relative AUC, ± pyruvate; IR0/30: 1.00 vs. 0.61, IR15/30: 1.68 vs. 1.00, IR30/30: 1.42 vs. 0.75, IR40/30: 1.97 vs. 0.85; p < 0.01). Ca2+-uptake remained unaffected by previous IR. Though Ca2+-release was delayed for ≥30 min of ischemia (p < 0.01), Ca2+ retention was highest in IR15/30 (RFU; IR0/30: 6.3 ± 3.6, IR 15/30 42.9 ± 5.0, IR30/30 15.9 ± 3.8, IR40/30 11.5 ± 6.6; p ≤ 0.01 for IR15/30 against all other groups). CONCLUSIONS: Ischemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Ischemia time should be considered an important factor influencing IR experimental data on subsarcolemmal mitochondria. Periods of warm global ischemia should be minimized during cardiac surgery to avoid excessive damage to SSMs.

Endurance training restores spatially distinct cardiac mitochondrial function and myocardial contractility in ovariectomized rats.

We previously demonstrated that the loss of female hormones induces cardiac and mitochondrial dysfunction in the female heart. Here, we show the impact of endurance training for twelve weeks, a nonpharmacological therapy against cardiovascular disease caused by ovariectomy and its contribution to cardiac contractility, mitochondrial quality control, bioenergetics and oxidative damage. We found that ovariectomy induced cardiac hypertrophy and dysfunction by decreasing SERCA2 and increasing phospholamban protein expression. Endurance training restored myocardial contractility, SERCA2 levels, increased calcium transient in ovariectomized rats but did not change phospholamban protein expression or cardiac hypertrophy. Additionally, ovariectomy decreased the amount of intermyofibrillar mitochondria and induced mitochondrial fragmentation that were accompanied by decreased levels of mitofusin 1, PGC-1α, NRF-1, total AMPK-α and mitochondrial Tfam. Endurance training prevented all these features except for mitofusin 1. Ovariectomy reduced O2 consumption, elevated O2.- release and increased Ca2+-induced mitochondrial permeability transition pore opening in both mitochondrial subpopulations. Ovariectomy also increased NOX-4 protein expression in the heart, reduced mitochondrial Mn-SOD, catalase protein expression and increased protein carbonylation in both mitochondrial subpopulations, which were prevented by endurance training. Taken together, our findings show that endurance training prevented cardiac contractile dysfunction and mitochondrial quality control in ovariectomized rats.

Structural evidence for a new elaborate 3D-organization of the cardiomyocyte lateral membrane in adult mammalian cardiac tissues.

AIMS: This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. METHODS AND RESULTS: Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. CONCLUSION: Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.

Effect of Sodium Thiosulfate Postconditioning on Ischemia-Reperfusion Injury Induced Mitochondrial Dysfunction in Rat Heart.

The recent research on the therapeutic applications of sodium thiosulfate (STS) has gained importance in the treatment of cardiovascular diseases. Progressively through the present work, we have demonstrated that postconditioning of isolated rat heart subjected to ischemia-reperfusion injury using STS had preserved the mitochondrial structure, function, and number. Heart comprising of two mitochondrial subpopulations interfibrillar (IFM-involved in contractile function) and subsarcolemmal (SSM-involved in metabolic function), STS postconditioning imparted a state of hypometabolism to SSM, thereby reducing the metabolic demand of the reperfused heart. The IFM, on the other hand, provided the energy required to maintain contraction. Moreover, the hypometabolic state induced in SSM can lower the free radical release in addition to STS innate ability to act as an antioxidant and radical scavenger, all of which collectively provided cardioprotection. Therefore, drugs targeting IFM specifically or those reducing the energy demand for SSM can be suitable targets for myocardial ischemia-reperfusion injury.

MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload.

Heart failure remains a major public-health problem with an increase in the number of patients worsening from this disease. Despite current medical therapy, the condition still has a poor prognosis. Heart failure is complex but mitochondrial dysfunction seems to be an important target to improve cardiac function directly. Our goal was to analyze the effects of MitoQ (100 µM in drinking water) on the development and progression of heart failure induced by pressure overload after 14 weeks. The main findings are that pressure overload-induced heart failure in rats decreased cardiac function in vivo that was not altered by MitoQ. However, we observed a reduction in right ventricular hypertrophy and lung congestion in heart failure animals treated with MitoQ. Heart failure also decreased total mitochondrial protein content, mitochondrial membrane potential in the intermyofibrillar mitochondria. MitoQ restored membrane potential in IFM but did not restore mitochondrial protein content. These alterations are associated with the impairment of basal and stimulated mitochondrial respiration in IFM and SSM induced by heart failure. Moreover, MitoQ restored mitochondrial respiration in heart failure induced by pressure overload. We also detected higher levels of hydrogen peroxide production in heart failure and MitoQ restored the increase in ROS production. MitoQ was also able to improve mitochondrial calcium retention capacity, mainly in the SSM whereas in the IFM we observed a small alteration. In summary, MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload, by decreasing hydrogen peroxide formation, improving mitochondrial respiration and improving mPTP opening.

Unique morphological characteristics of mitochondrial subtypes in the heart: the effect of ischemia and ischemic preconditioning.

RATIONALE: Three subsets of mitochondria have been described in adult cardiomyocytes - intermyofibrillar (IMF), subsarcolemmal (SSM), and perinuclear (PN). They have been shown to differ in physiology, but whether they also vary in morphological characteristics is unknown. Ischemic preconditioning (IPC) is known to prevent mitochondrial dysfunction induced by acute myocardial ischemia/reperfusion injury (IRI), but whether IPC can also modulate mitochondrial morphology is not known. AIMS: Morphological characteristics of three different subsets of adult cardiac mitochondria along with the effect of ischemia and IPC on mitochondrial morphology will be investigated. METHODS: Mouse hearts were subjected to the following treatments (N=6 for each group): stabilization only, IPC (3x5 min cycles of global ischemia and reperfusion), ischemia only (20 min global ischemia); and IPC and ischemia. Hearts were then processed for electron microscopy and mitochondrial morphology was assessed subsequently. RESULTS: In adult cardiomyocytes, IMF mitochondria were found to be more elongated and less spherical than PN and SSM mitochondria. PN mitochondria were smaller in size when compared to the other two subsets. SSM mitochondria had similar area to IMF mitochondria but their sphericity measures were similar to PN mitochondria. Ischemia was shown to increase the sphericity parameters of all 3 subsets of mitochondria; reduce the length of IMF mitochondria, and increase the size of PN mitochondria. IPC had no effect on mitochondrial morphology either at baseline or after ischemia. CONCLUSION: The three subsets of mitochondria in the adult heart are morphologically different. IPC does not appear to modulate mitochondrial morphology in adult cardiomyocytes.

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.

Hydrogen sulfide preconditioning shows differential protection towards interfibrillar and subsarcolemmal mitochondria from isolated rat heart subjected to revascularization injury.

BACKGROUND: Hydrogen sulfide (H2S) is well known to protect the heart from ischemia reperfusion injury by specifically modulating the mitochondrial adenosine-triphosphate-linked potassium channel, thereby preserving mitochondrial function. The present study is designed to investigate the H2S preconditioning effect specifically on the mitochondrial subpopulation, namely, interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria. METHODS: Isolated heart perfusion model with the method of Langendorff was used to induce reperfusion injury in rat hearts. The animals were randomly divided into five groups: normal, ischemia (ISC), reperfusion (IR), preconditioning (IPC), and H2S preconditioning (HIPC). All the groups, except normal and ischemia, were subjected to 30-min global ischemia followed by 60-min reperfusion with Krebs-Henseleit buffer. RESULTS: Our study results show that H2S at a dose of 20 µM significantly (P<.05) reduced the infarct size (59%) and the creatine kinase and lactate dehydrogenase activity in cardiac tissue. DNA fragmentation as observed in ischemia reperfusion control was absent in case of H2S-preconditioned heart. On comparing the classical protection provided by IPC with H2S, a significant recovery was seen in the IFM fraction in case of HIPC, whereas the SSM could not recover as evidenced by better mitochondrial respiration rate and electron chain enzyme activities. Studies on isolated mitochondrial subpopulation from normal, IR, and IPC hearts exposed to H2S in vitro support the above observation. CONCLUSION: The present study concluded that IFM shows major contribution towards H2S-mediated cardioprotection, whereas classical IPC recovered both subpopulations from IR injury.

Hydrogen sulfide modulates sub-cellular susceptibility to oxidative stress induced by myocardial ischemic reperfusion injury.

In this study, we compared the impact of H2S pre (HIPC) and post-conditioning (HPOC) on oxidative stress, the prime reason for myocardial ischemia reperfusion injury (I/R), in different compartments of the myocardium, such as the mitochondria beside its subpopulations (interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria) and microsomal fractions in I/R injured rat heart. The results demonstrated that compared to I/R rat heart, HIPC and HPOC treated hearts shows reduced myocardial injury, enhanced antioxidant enzyme activities and reduced the level of TBARS in different cellular compartments. The extent of recovery (measured by TBARS and GSH levels) in subcellular fractions, were in the following descending order: microsome > SSM > IFM in both HIPC and HPOC. In summary, oxidative stress mediated mitochondrial dysfunction, one of the primary causes for I/R injury, was partly recovered by HIPC and HPOC treatment, with significant improvement in SSM fraction compared to the IFM.

Hydrogen sulfide post-conditioning preserves interfibrillar mitochondria of rat heart during ischemia reperfusion injury.

Cardiac mitochondrial dysfunction is considered to be the main manifestation in the pathology of ischemia reperfusion injury, and by restoring its functional activity, hydrogen sulfide (H2S), a novel endogenous gaseotransmitter renders cardioprotection. Given that interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria are the two main types in the heart, the present study investigates the specific H2S-mediated action on IFM and SSM during ischemic reperfusion in the Langendorff rat heart model. Rats were randomly divided into five groups, namely normal, ischemic control, reperfusion control (I/R), ischemic post-conditioning (POC), and H2S post-conditioning (POC_H2S). In reperfusion control, cardiac contractility decreased, and lactate dehydrogenase, creatine kinase, and infracted size increased compared to both normal and ischemic group. In hearts post-conditioned with H2S and the classical method improved cardiac mechanical function and decreased cardiac markers in the perfusate and infarct size significantly. Both POC and POC_H2S exerts its cardioprotective effect of preserving the IFM, as evident by significant improvement in electron transport chain enzyme activities and mitochondrial respiration. The in vitro action of H2S on IFM and SSM from normal and I/R rat heart supports H2S and mediates cardioprotection via IFM preservation. Our study indicates that IFM play an important role in POC_H2S mediated cardioprotection from reperfusion injury.

Mechanical ventilation triggers abnormal mitochondrial dynamics and morphology in the diaphragm.

The diaphragm is a unique skeletal muscle designed to be rhythmically active throughout life, such that its sustained inactivation by the medical intervention of mechanical ventilation (MV) represents an unanticipated physiological state in evolutionary terms. Within a short period after initiating MV, the diaphragm develops muscle atrophy, damage, and diminished strength, and many of these features appear to arise from mitochondrial dysfunction. Notably, in response to metabolic perturbations, mitochondria fuse, divide, and interact with neighboring organelles to remodel their shape and functional properties-a process collectively known as mitochondrial dynamics. Using a quantitative electron microscopy approach, here we show that diaphragm contractile inactivity induced by 6 h of MV in mice leads to fragmentation of intermyofibrillar (IMF) but not subsarcolemmal (SS) mitochondria. Furthermore, physical interactions between adjacent organellar membranes were less abundant in IMF mitochondria during MV. The profusion proteins Mfn2 and OPA1 were unchanged, whereas abundance and activation status of the profission protein Drp1 were increased in the diaphragm following MV. Overall, our results suggest that mitochondrial morphological abnormalities characterized by excessive fission-fragmentation represent early events during MV, which could potentially contribute to the rapid onset of mitochondrial dysfunction, maladaptive signaling, and associated contractile dysfunction of the diaphragm.

Biology of Muscle Atrophy and of its Recovery by FES in Aging and Mobility Impairments: Roots and By-Products.

There is something in our genome that dictates life expectancy and there is nothing that can be done to avoid this; indeed, there is not yet any record of a person who has cheated death. Our physical prowess can vacillate substantially in our lifetime according to our activity levels and nutritional status and we may fight aging, but we will inevitably lose. We have presented strong evidence that the atrophy which accompanies aging is to some extent caused by loss of innervation. We compared muscle biopsies of sedentary seniors to those of life long active seniors, and show that these groups indeed have a different distribution of muscle fiber diameter and fiber type. The senior sportsmen have many more slow fiber-type groupings than the sedentary people which provides strong evidence of denervation-reinnervation events in muscle fibers. It appears that activity maintains the motoneurons and the muscle fibers. Premature or accelerated aging of muscle may occur as the result of many chronic diseases. One extreme case is provided by irreversible damage of the Conus and Cauda Equina, a spinal cord injury (SCI) sequela in which the human leg muscles may be completely and permanently disconnected from the nervous system with the almost complete disappearance of muscle fibers within 3-5 years from SCI. In cases of this extreme example of muscle degeneration, we have used 2D Muscle Color CT to gather data supporting the idea that electrical stimulation of denervated muscles can retain and even regain muscle. We show here that, if people are compliant, atrophy can be reversed. A further example of activity-related muscle adaptation is provided by the fact that mitochondrial distribution and density are significantly changed by functional electrical stimulation in horse muscle biopsies relative to those not receiving treatment. All together, the data indicate that FES is a good way to modify behaviors of muscle fibers by increasing the contraction load per day. Indeed, it should be possible to defer the muscle decline that occurs in aging people and in those who have become unable to participate in physical activities. Thus, FES should be considered for use in rehabilitation centers, nursing facilities and in critical care units when patients are completely inactive even for short periods of time.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.