Mitochondrial Calcium Handling Research Articles

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid–protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease.

Mitochondrial calcium handling during ischemia-induced cell death in neurons

Mitochondria sense and shape cytosolic Ca 2+ signals by taking up and subsequently releasing Ca 2+ ions during physiological and pathological Ca 2+ elevations. Sustained elevations in the mitochondrial matrix Ca 2+ concentration are increasingly recognized as a defining feature of the intracellular cascade of lethal events that occur in neurons during cerebral ischemia. Here, we review the recently identified transport proteins that mediate the fluxes of Ca 2+ across mitochondria and discuss the implication of the permeability transition pore in decoding the abnormally sustained mitochondrial Ca 2+ elevations that occur during cerebral ischemia.

Exercise Training Decreases Arrhythmias But Is Not Associated With Improved Mitochondrial Calcium Retention Capacity

PURPOSE: To determine if 10 days of exercise training decreases cardiac arrhythmias in the isolated female rat heart, and to determine if mitochondrial adaptations are involved. We hypothesized that exercise would promote an anti-arrhythmic phenotype characterized by improved calcium tolerance, improved ROS scavenging by superoxide dismutase (SOD), and delayed mitochondrial permeability transition pore opening. METHODS: Female Sprague-Dawley rats were randomly assigned to either the sedentary handling control (Sed) or the exercise trained (Ex) group. Animals in the Ex group were placed on a motorized treadmill at speeds of 15/30/15 meters per minute for 15/30/15 minutes respectively. Animals in the Sed group were handled and placed on the treadmill each day. Exercise duration was 10 days. Twenty-four hours after the last session hearts were excised via thoracotomy and either instrumented for isolated heart experiments or used for mitochondrial isolations. Isolated hearts were exposed to global ischemia for 30 minutes, followed by 30 minutes of reperfusion. RESULTS: Ex led to a significant decrease in the severity of arrhythmias by: 1) reducing the arrhythmia score (5.7 ± 0.9 and 3.0 ± 0.6, for Sed and Ex group respectively, P < 0.05) and 2) decreasing the time spent in a ventricular arrhythmia (11 ± 3 and 2 ± 1 minutes, for Sed and Ex group respectively, P 0.05). However, during reperfusion coronary flow was significantly preserved in the Ex group (P < 0.05, RM ANOVA). Western blots for both SOD isoforms showed that neither SOD1 nor SOD2 protein levels were increased following Ex. In isolated mitochondria, the calcium retention capacity in Ex animals was significantly lower than Sed counterparts (433 ± 33 and 343 ± 10 nmoles of calcium, for Sed and Ex respectively, P < 0.05). CONCLUSIONS: Our results suggest that Ex causes intrinsic changes in the female heart that decrease the severity of arrhythmias without increases in protein expression of SOD1 or SOD2. Surprisingly, mitochondria of trained animals were more sensitive to calcium-induced opening of the mitochondrial permeability transition pore. Future experiments investigating the kinetics of calcium uptake and extrusion in isolated mitochondria will improve our understanding of how mitochondrial calcium handling influences the propensity for arrhythmias.

Nature and cause of mitochondrial dysfunction in Huntington’s disease: focusing on huntingtin and the striatum

Polyglutamine expansion mutation in huntingtin causes Huntington's disease (HD). How mutant huntingtin (mHtt) preferentially kills striatal neurons remains unknown. The link between mitochondrial dysfunction and HD pathogenesis stemmed from postmortem brain data and mitochondrial toxin models. Current evidence from genetic models, containing mHtt, supports mitochondrial dysfunction with yet uncertain nature and cause. Because mitochondria composition and function varies across tissues and cell-types, mitochondrial dysfunction in HD vulnerable striatal neurons may have distinctive features. This review focuses on mHtt and the striatum, integrating experimental evidence from patients, mice, primary cultures and striatal cell-lines. I address the nature (specific deficits) and cause (mechanisms linked to mHtt) of HD mitochondrial dysfunction, considering limitations of isolated vs. in situ mitochondria approaches, and the complications introduced by glia and glycolysis in brain and cell-culture studies. Current evidence relegates respiratory chain impairment to a late secondary event. Upstream events include defective mitochondrial calcium handling, ATP production and trafficking. Also, transcription abnormalities affecting mitochondria composition, reduced mitochondria trafficking to synapses, and direct interference with mitochondrial structures enriched in striatal neurons, are possible mechanisms by which mHtt amplifies striatal vulnerability. Insights from common neurodegenerative disorders with selective vulnerability and mitochondrial dysfunction (Alzheimer's and Parkinson's diseases) are also addressed.

Mitochondrial Dysfunction in Parkinson's Disease

It is clear from a striking convergence of human tissue studies, neurotoxin models, and genetic models that mitochondrial dysregulation plays a central pathogenic role in Parkinson's disease (PD) and related neurodegenerative conditions. Impaired mitochondrial quality could result from both increased damage and decreased ability to repair or clear damaged mitochondria. In particular, common deficits in mitochondrial respiratory chain function, oxidative stress, morphology/dynamics, and calcium handling capacities have been described in multiple PD model systems employing complex I inhibitors, 6-hydroxydopamine and molecular manipulation of Parkinsonian genes including alpha-synuclein, PTEN-induced kinase 1, Parkin, DJ-1, and, to a lesser extent, leucine rich repeat kinase 2. The most recent and exciting work implicates alterations in the regulation of macroautophagy and likely of selective mitophagic clearance of damaged mitochondria, although additional studies are needed to resolve some issues in this area. Future studies emphasizing the normal mitoprotective function(s) of proteins associated with recessive loss-of-function causes of familial PD, as well as compensatory mechanisms operating in their absence, may offer particularly valuable insights into strategies to enhance mitochondrial health.

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.ResultsTo gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT) and G93A (SH-SY5YG93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation.ConclusionIn this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

Applications of Genetically Targeted and Optimized Calcium Sensors

We have recently developed a series of FRET-based genetically encoded sensors for calcium. These sensors (dubbed “cameleons”) are made from cyan and yellow fluorescent proteins combined with engineered calcium sensing elements. These optimized cameleon sensors have now been genetically targeted to specific locations within cells including: the ER, mitochondria, Golgi, nucleus, plasma membrane, and cytosol. In the present work we put these sensors to the test in both standard tissue culture cells and in primary hippocampal neurons. We have now used these sensors to define how ER and mitochondrial calcium handling is altered by mutations in the calcium regulatory protein presenilin. Presenilin is an integral membrane protein that localizes to the ER, secretory pathway, and plasma membrane. There is emerging evidence from a number of research groups that presenilin plays a critical role in modulating ER calcium signaling. Using a combination of genetically targeted and small molecule sensors we have identified how mutations in presenilin alter calcium homeostasis in the ER and calcium release through the IP3R. In another line of research aimed at testing the sensitivity and versatility of our sensors, we have identified localized calcium signals generated upon invasion of a host mammalian cell by bacteria. In this work we will discuss the strengths and weaknesses of genetically encoded calcium sensors and potential avenues for further improvement and optimization.

Computer-assisted live cell analysis of mitochondrial membrane potential, morphology and calcium handling

Mitochondria are crucial for many aspects of cellular homeostasis and a sufficiently negative membrane potential (Δ ψ) across the mitochondrial inner membrane (MIM) is required to sustain most mitochondrial functions including ATP generation, MIM fusion, and calcium uptake and release. Here, we present a microscopy approach for automated quantification of Δ ψ and mitochondrial position, shape and calcium handling in individual living cells. In the base protocol, cells are stained with tetramethyl rhodamine methyl ester (TMRM), a fluorescent cation that accumulates in the mitochondrial matrix according to Δ ψ, and visualized using video-microscopy. Next, the acquired images are processed to generate a mitochondria-specific binary image (mask) allowing simultaneous quantification of mitochondrial TMRM fluorescence intensity, shape and position. In a more advanced version of this protocol a mitochondria-targeted variant of green fluorescent protein (mitoAcGFP1) is expressed to allow mask making in TMRM-stained cells. The latter approach allows quantification of Δ ψ in cells with a substantially depolarized Δ ψ. For automated quantification of mitochondrial calcium handling in space and time mitoAcGFP1-expressing cells are stained with rhod-2, a fluorescent calcium indicator that accumulates in the mitochondrial matrix. In this paper, a detailed step-by-step description of the above approaches and its pitfalls is provided.

The role of mitochondria in apoptosis

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca2+ signaling. Massive accumulation of Ca2+ in the mitochondria leads to apoptosis. The Ca2+ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.

Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons

In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells.

Acute Tobacco Smoke Exposure Promotes Mitochondrial Permeability Transition in Rat Heart

Chronic exposure to tobacco smoke is known to impair mitochondrial function. However, the effect of acute tobacco smoke exposure (ATSE) in vivo, as might occur in social settings, on mitochondrial function and calcium handling of cardiac cells has not been examined. It was hypothesized that ATSE might adversely modify mitochondrial function as reflected in mitochondrial energetics, membrane potential, and calcium transport. Mitochondria were isolated from the hearts of adult rats either exposed to 6 h of environmental tobacco smoke (∼60 mg/mm3 tobacco smoke particles) or sham exposure. To model a calcium stress similar to ischemia/reperfusion, mitochondria were exposed to a Ca2+ bolus with measurement of membrane potential, energetics, Ca2+uptake and release, and redox state. ATSE mitochondria were characterized by significantly higher ADP-stimulated ATP production and a more reduced redox state (NADH ratio) under basal conditions without observed changes in resting Ψm. Exposure of ATSE mitochondria to Ca2+stress resulted in significantly more rapid depolarization of Ψm. The initial rate of Ca2+uptake was not altered in ATSE mitochondria, but CsA-sensitive Ca2+ release was significantly increased. ATSE does not significantly alter resting mitochondrial function. However, ATSE modifies the response of cardiac mitochondria to calcium stress, resulting in a more rapid depolarization and subsequent release of Ca2+ via the mitochondrial permeability transition (MPT). This research was supported by a Philip Morris External Research program grant. We thank Mike Goldsmith and Dale Uyeminami at the Center for Health and Environment for invaluable assistance in the smoke exposure experiments.

Cross-talk between the sarcoplasmic reticulum and the mitochondrial calcium handling systems may play an important role in the regulation of contraction in anococcygeus smooth muscle

Mitochondrial Ca 2+ and its relation with the contraction induced by phenylephrine was investigated. In normal Ca 2+, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP) and oligomycin produced contraction similar to that promoted by phenylephrine. Phenylephrine-induced contraction was reduced by FCCP+oligomycin. In Ca 2+-free, FCCP+oligomycin did not induce contraction. Response to FCCP+oligomycin was reduced upon Ca 2+ repletion and this response was lower than that to phenylephrine. Ca 2+ concentration was increased by FCCP+oligomycin. Since a profuse net of sarcoplasmic reticulum encloses mitochondria, a cross-talk between the two organelles may play an important role in the phenylephrine-induced contraction in presence of Ca 2+ encountered in both sarcoplasmic reticulum and extracellular medium of anococcygeus cells.

Expression Profiling of a Hypercontraction-induced Myopathy in Drosophila Suggests a Compensatory Cytoskeletal Remodeling Response

Mutations that alter muscle contraction lead to a large array of diseases, including muscular dystrophies and cardiomyopathies. Although the molecular lesions underlying many hereditary muscle diseases are known, the downstream pathways that contribute to disease pathogenesis and compensatory muscle remodeling are poorly defined. We have recently identified and characterized mutations in Myosin Heavy Chain (Mhc) that lead to hypercontraction and subsequent degeneration of flight muscles in Drosophila. To characterize the genomic response to hypercontraction-induced myopathy, we performed expression analysis using Affymetrix high density oligonucleotide microarrays in Drosophila Mhc hypercontraction alleles. The altered transcriptional profile of dystrophic Mhc muscles suggests an actin-dependent remodeling of the muscle cytoskeleton. Specifically, a subset of the highly up-regulated transcripts is involved in actin regulation and structural support for the contractile machinery. In addition, we identified previously uncharacterized proteins with putative actin-interaction domains that are up-regulated in Mhc mutants and differentially expressed in muscles. Several of the up-regulated proteins, including the dystrophin-related protein, MSP-300, and the homolog of the neuronal activity-regulated protein, ARC, localize to specific subcellular muscle structures that may provide key structural sites for cytoskeletal remodeling in dystrophic muscles. Defining the genome-wide transcriptional response to muscle hypercontraction in Drosophila has revealed candidate loci that may participate in the pathogenesis of muscular dystrophy and in compensatory muscle repair pathways through modulation of the actin cytoskeleton.

Extracellular pH Modifies Mitochondrial Control of Capacitative Calcium Entry in Jurkat Cells

It was found that a collapse of the mitochondrial calcium buffering caused by the protonophoric uncoupler CCCP, antimycin A plus oligomycin, or the inhibitor of the mitochondrial Ca2+/Na+ exchanger led to a strong inhibition of thapsigargin-induced capacitative Ca2+ entry (CCE) into Jurkat cells suspended in a medium at pH 7.2. The effect of these inhibitors was markedly less significant at higher extracellular pH. Moreover, dysfunction of the mitochondrial calcium handling greatly decreased CCE sensitivity to extracellular Ca2+ when the pH of extracellular solution was 7.2 (apparent Kd toward extracellular Ca2+ rose from 2.3 +/- 0.6 mm in control cells to 11.0 +/- 1.7 mM in CCCP-treated cells) as compared with pH 7.8 (apparent Kd toward extracellular Ca2+ increased from 1.3 +/- 0.4 mM in control cells to 2.4 +/- 0.4 mM in uncoupler-treated cells). Changes in intracellular pH triggered by methylamine did not influence Ca2+ influx. This suggests that, in Jurkat cells, store-operated calcium channels sense extracellular pH change as a parameter that modifies their sensitivity to intracellular Ca2+. In contrast, in human osteosarcoma cells, changes in extracellular pH as well as mitochondrial uncoupling did not exert any inhibitory effects on CCE.

P3-307 Identification of a domain in the amyloid precursor protein (APP) required for the LRP-mediated endocytosis of APP: protease complexes

<dm:abstracts xmlns:dm="http://www.elsevier.com/xml/dm/dtd"><ce:abstract xmlns:ce="http://www.elsevier.com/xml/common/dtd" view="all" class="author" id="aep-abstract-id1"><ce:section-title>Publisher Summary</ce:section-title><ce:abstract-sec view="all" id="aep-abstract-sec-id1"><ce:simple-para id="fsabs015" view="all">This chapter discusses the role of excitotoxicity in the molecular pathogenesis of Huntington's disease (HD). HD is a progressive neurological disorder characterized by involuntary movements, emotional disturbances, and dementia. The underlying genetic lesion is an expansion of a CAG trinucleotide repeat in the <ce:italic>HD</ce:italic> gene that results in an expanded polyglutamine (polyQ) stretch at the N-terminus of the huntingtin protein (htt). The cardinal neuropathological feature of HD is a selective loss in the striatum of medium-sized spiny neurons. Recent evidence strongly implicates aberrant glutamate signaling, disrupted neuronal calcium handling, and the accompanying excitotoxicity in the pathogenesis of HD. The involvement of excitotoxicity in the pathogenesis of HD was first suggested by rodent studies in which intrastriatal injections of kainic acid (KA) or quinolinic acid (QA, an NMDA receptor agonist) produced lesions that mimicked many of the neurochemical and histopathological features of HD, and was associated with HD-like behavioral deficits. A number of human and animal studies have since identified defects in NMDA and mGluR5 signaling, as well as mitochondrial calcium handling in HD patients and animal models of HD. Together, these studies give rise to a coherent, multifactorial model of mutant huntingtin-mediated alteration of glutamate receptor activity, and calcium signaling as a primary contributor to neuronal degeneration in HD.</ce:simple-para></ce:abstract-sec></ce:abstract></dm:abstracts>

Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood?*1

Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood?*1

Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood?

Over the past few years, extensive progress has been made in elucidating the role of calcium in the signaling of apoptosis. This has led to the characterization of calcium's role in the induction of apoptosis and in the regulation of effector proteases. In this review, we attempt to summarize the current knowledge regarding a segment of these studies, the interaction between the endoplasmic reticulum (ER) and mitochondria. This interface has been shown to play a crucial role in transferring agonist induced Ca 2+ signals to mitochondria during physiological processes. Recent evidence, however, extended the role of this Ca 2+ transfer to apoptotic pathways, showing that modulation of mitochondrial Ca 2+ uptake from the ER side has a prominent role in modulating cellular fate.

Interplay between mitochondria and cellular calcium signalling.

Mitochondria are increasingly ascribed central roles in vital cell signalling cascades. These organelles are now recognised as initiators and transducers of a range of cell signals, including those central to activation and amplification of apoptotic cell death. Moreover, as the main source of cellular ATP, mitochondria must be responsive to fluctuating energy demands of the cell. As local and global fluctuations in calcium concentration are ubiquitous in eukaryotic cells and are the common factor in a dizzying array of intra- and inter-cellular signalling cascades, the relationships between mitochondrial function and calcium transients is currently a subject of intense scrutiny. It is clear that mitochondria not only act as local calcium buffers, thus shaping spatiotemporal aspects of cytosolic calcium signals, but that they also respond to calcium uptake by upregulating the tricarboxylic acid cycle, thus reacting metabolically to local signalling. In this chapter we review current knowledge of mechanisms of mitochondrial calcium uptake and release and discuss the consequences of mitochondrial calcium handling for cell function, particularly in conjunction with mitochondrial oxidative stress.

In vitro effects of polyglutamine tracts on Ca 2+-dependent depolarization of rat and human mitochondria: relevance to Huntington’s disease

The mechanisms by which neurons die in CAG triplet repeat (polyglutamine) disorders, such as Huntington’s disease, are uncertain; however, mitochondrial dysfunction and disordered calcium homeostasis have been implicated. We previously demonstrated abnormal mitochondrial calcium handling in Huntington’s disease cell lines and transgenic mice. To examine whether these abnormalities might arise in part from direct effects of the expanded polyglutamine tract contained in mutant huntingtin, we have exposed normal rat liver and human lymphoblast mitochondria to glutathione S-transferase fusion proteins containing polyglutamine tracts of 0, 19, or 62 residues. Similar to bovine serum albumin, each of the protein constructs nonspecifically inhibited succinate-supported respiration, independent of polyglutamine tract length. There was a small but significant reduction of mitochondrial membrane potential (state 4) only in the presence of the pathological-length polyglutamine tract. With successive addition of small Ca 2+ aliquots, mitochondria exposed to pathological-length polyglutamine tracts (∼5 μM) depolarized much earlier and to a greater extent than those exposed to the other protein constructs. These results suggest that the mitochondrial calcium handling defects seen in Huntington’s disease cell lines and transgenic mice may be due, in part, to direct, deleterious effects of mutant huntingtin on mitochondria.

Time course of skeletal muscle repair and gene expression following acute hind limb ischemia in mice.

DNA microarrays were used to measure the time course of gene expression during skeletal muscle damage and regeneration in mice following femoral artery ligation (FAL). We found 1,289 known sequences were differentially expressed between the FAL and control groups. Gene expression peaked on day 3, and the functional cluster "inflammation" contained the greatest number of genes. Muscle function was depressed for 3 days postligation, but returned to normal by day 7. Decreased muscle function was accompanied by reduced expression of genes involved in mitochondrial energy production, muscle contraction, and calcium handling. The induction of MyoD on day 1 denoted the beginning of muscle regeneration and was followed by the reemergence of the embryonic forms of muscle contractile proteins, which peaked at day 7. Transcriptional analysis indicated that the ischemic skeletal muscle may transition through a functional adaptation stage with recovery of contractile force prior to full regeneration. Several members of the insulin-like growth factor axis were coordinately induced in a time frame consistent with their playing a role in the regenerative process.