David A. Hood

Coordination of metabolic plasticity in skeletal muscle

SUMMARY Skeletal muscle is a highly malleable tissue, capable of pronounced metabolic and morphological adaptations in response to contractile activity (i.e. exercise). Each bout of contractile activity results in a coordinated alteration in the expression of a variety of nuclear DNA and mitochondrial DNA (mtDNA) gene products, leading to phenotypic adaptations. This results in an increase in muscle mitochondrial volume and changes in organelle composition, referred to as mitochondrial biogenesis. The functional consequence of this biogenesis is an improved resistance to fatigue. Signals initiated by the exercise bout involve changes in intracellular Ca2+ as well as alterations in energy status (i.e. ATP/ADP ratio) and the consequent activation of downstream kinases such as AMP kinase and Ca2+-calmodulin-activated kinases. These kinases activate transcription factors that bind DNA to affect the transcription of genes, the most evident manifestation of which occurs during the post-exercise recovery period when energy metabolism is directed toward anabolism, rather than contractile activity. An important protein that is affected by exercise is the transcriptional coactivator PGC-1α, which cooperates with multiple transcription factors to induce the expression of nuclear genes encoding mitochondrial proteins. Once translated in the cytosol, these mitochondrially destined proteins are imported into the mitochondrial outer membrane, inner membrane or matrix space via specific import machinery transport components. Contractile activity affects the expression of the import machinery, as well as the kinetics of import, thus facilitating the entry of newly synthesized proteins into the expanding organelle. An important set of proteins that are imported are the mtDNA transcription factors, which influence the expression and replication of mtDNA. While mtDNA contributes only 13 proteins to the synthesis of the organelle, these proteins are vital for the proper assembly of multi-subunit complexes of the respiratory chain, when combined with nuclear-encoded protein subunits. The expansion of skeletal muscle mitochondria during organelle biogenesis involves the assembly of an interconnected network system (i.e. a mitochondrial reticulum). This expansion of membrane size is influenced by the balance between mitochondrial fusion and fission. Thus, mitochondrial biogenesis is an adaptive process that requires the coordination of multiple cellular events, including the transcription of two genomes, the synthesis of lipids and proteins and the stoichiometric assembly of multisubunit protein complexes into a functional respiratory chain. Impairments at any step can lead to defective electron transport, a subsequent failure of ATP production and an inability to maintain energy homeostasis.

Mitochondrial function and apoptotic susceptibility in aging skeletal muscle.

During aging, skeletal muscle undergoes sarcopenia, a condition characterized by a loss of muscle cell mass and alterations in contractile function. The origin of these decrements is unknown, but evidence suggests that they can be partly attributed to mitochondrial dysfunction. To characterize the nature of this dysfunction, we investigated skeletal muscle contractile properties, subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial biogenesis and function, as well as apoptotic susceptibility in young (6 months old) and senescent (36 months old) Fischer 344 Brown Norway rats. Muscle mass and maximal force production were significantly lower in the 36‐month group, which is indicative of a sarcopenic phenotype. Furthermore, contractile activity in situ revealed greater fatigability in the 36‐month compared to the 6‐month animals. This decrement could be partially accounted for by a 30% lower mitochondrial content in fast‐twitch muscle from 36‐month animals, as well as lower protein levels of the transcriptional coactivator peroxisome proliferator‐activated receptor γ coactivator‐1α. Enzyme activities and glutamate‐induced oxygen consumption rates in isolated SS and IMF mitochondria were similar between age groups. However, mitochondrial reactive oxygen species (ROS) production during state 3 respiration was ~1.7‐fold greater in mitochondria isolated from 36‐month compared to 6‐month animals, and was accompanied by a 1.8‐fold increase in the DNA repair enzyme 8‐oxoguanine glycosylase 1 in fast‐twitch muscle. Basal rates of release of cytochrome c and endonuclease G in SS mitochondria were 3.5‐ to 7‐fold higher from senescent animals. These data suggest that the age‐related sarcopenia and muscle fatigability are associated with enhanced ROS production, increased mitochondrial apoptotic susceptibility and reduced transcriptional drive for mitochondrial biogenesis.

Interactions between ROS and AMP kinase activity in the regulation of PGC-1α transcription in skeletal muscle cells

Reactive oxygen species (ROS) play an important role in cellular function via the activation of signaling cascades. ROS have been shown to affect mitochondrial biogenesis, morphology, and function. Their beneficial effects are likely mediated via the upregulation of transcriptional regulators such as peroxisome proliferator-activated receptor-gamma coactivator-1 protein-alpha (PGC-1alpha). However, the ROS signals that regulate PGC-1alpha transcription in skeletal muscle are not understood. Here we examined the effect of H2O2 on the regulation of PGC-1alpha expression, and its relationship to AMPK activation. We demonstrate that 24 h of exogenous H2O2 treatment increased PGC-1alpha promoter activity and mRNA expression. Both effects were blocked with the addition of N-acetylcysteine, a ROS scavenger. These effects were mediated, in part, via upstream stimulatory factor-1/Ebox DNA binding and involved 1) interactions with downstream sequences and 2) the activation of AMPK. Elevated ROS led to the activation of AMPK, likely via a decline in ATP levels. The activation of AMPK using 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside increased PGC-1alpha promoter activity and mRNA levels but reduced ROS production. Thus the net effect of AMPK activation on PGC-1alpha expression was a result of increased transcriptional activation, counterbalanced by reduced ROS production. The effects of H2O2 on PGC-1alpha expression differed depending on the level of ROS within the cell. Low levels of ROS result in reduced PGC-1alpha mRNA in the absence of an effect on PGC-1alpha promoter activation. In contrast, elevated levels of H2O2 induce PGC-1alpha transcription indirectly, via AMPK activation. These data identify unique interactions between ROS and AMPK activation on the expression of PGC-1alpha in muscle cells.

Mechanisms of exercise-induced mitochondrial biogenesis in skeletal muscle.

Acute exercise initiates rapid cellular signals, leading to the subsequent activation of proteins that increase gene transcription. The result is a higher level of mRNA expression, often observed during the recovery period following exercise. These molecules are translated into precursor proteins for import into preexisting mitochondria. Once inside the organelle, the protein is processed to its mature form and either activates mitochondrial DNA gene expression, serves as a single subunit enzyme, or is incorporated into multi-subunit complexes of the respiratory chain devoted to electron transport and substrate oxidation. The result of this exercise-induced sequence of events is the expansion of the mitochondrial network within muscle cells and the capacity for aerobic ATP provision. An understanding of the molecular processes involved in this complex pathway of organelle synthesis is important for therapeutic purposes, and is a primary research undertaking in laboratories involved in the study of mitochondrial biogenesis. This pathway in muscle becomes impaired with chronic inactivity and aging, which leads to a reduced muscle aerobic capacity and an increased tendency for mitochondrially mediated apoptosis, a situation that can contribute to muscle atrophy. The resumption, or adoption, of an active lifestyle can ameliorate this metabolic dysfunction, improve endurance, and help maintain muscle mass.

Regulation of mitochondrial biogenesis in muscle by endurance exercise.

Behavioural and hereditary conditions are known to decrease mitochondrial volume and function within skeletal muscle. This reduces endurance performance, and is manifest both at high- and low-intensity levels of exertion. A programme of regular endurance exercise, undertaken over a number of weeks, produces significant adaptations within skeletal muscle such that noticeable improvements in oxidative capacity are evident, and the related decline in endurance performance can be attenuated. Notwithstanding the important implications that this has for the highly trained endurance athlete, an improvement in mitochondrial volume and function through regular physical activity also endows the previously sedentary and/or aging population with an improved quality of life, and a greater functional independence. An understanding of the molecular and cellular mechanisms that govern the increases in mitochondrial volume with repeated bouts of exercise can provide insights into possible therapeutic interventions to care for those with mitochondrially-based diseases, and those unable to withstand regular physical activity. This review focuses on the recent developments in the molecular aspects of mitochondrial biogenesis in chronically exercising muscle. Specifically, we discuss the initial signalling events triggered by muscle contraction, the activation of transcription factors involved in both nuclear and mitochondrial DNA transcription, as well as the post-translational import mechanisms required for mitochondrial biogenesis. We consider the importance and relevance of chronic physical activity in the induction of mitochondrial biogenesis, with particular emphasis on how an endurance training programme could positively affect the age-related decline in mitochondrial content and delay the progression of age- and physical inactivity-related diseases.

AMP-Activated Protein Kinase-Regulated Activation of the PGC-1α Promoter in Skeletal Muscle Cells

The mechanisms by which PGC-1α gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1α using AICAR, an activator of AMPK, that is known to increase PGC-1α expression. A 2.2 kb fragment of the human PGC-1α promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-κB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1α promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at −495 within the PGC-1α promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1α promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1α promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1α promoter activity. The USF-1-mediated increase in PGC-1α promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1α gene expression. This could represent a potential therapeutic target to control PGC-1α expression in skeletal muscle.

Plasticity of Skeletal Muscle Mitochondria in Response to Contractile Activity

Regularly performed exercise in the form of endurance training produces a well‐established adaptation in skeletal muscle termed mitochondrial biogenesis. The physiological benefit of this is an enhanced performance of muscle when subject to endurance exercise. This is not only of great advantage for athletic endeavours, but it also clearly improves the quality of life of previously sedentary individuals and those involved in injury rehabilitation. Here we review the molecular basis for mitochondrial biogenesis in muscle, from the initial signals arising in contracting muscle, to the transcription factors involved in mitochondrial and nuclear DNA transcription, as well as the post‐translational import mechanisms required for the synthesis of the organelle. We discuss specific protein components associated with reactive oxygen species production, and suggest some questions which remain unanswered with respect to the role of exercise‐induced mitochondrial biogenesis in ageing, apoptosis and disease.

Role of p53 in mitochondrial biogenesis and apoptosis in skeletal muscle

p53 is a tumor suppressor protein that also plays a role in regulating aerobic metabolism. Since skeletal muscle is a major source of whole body aerobic respiration, it is important to delineate the effects of p53 on muscle metabolism. In p53 knockout (KO) mice, we observed diminished mitochondrial content in mixed muscle and lowered peroxisome proliferator-activated receptor-gamma (PPARgamma) coactivator (PGC)-1alpha protein levels in gastrocnemius muscle. In intermyofibrillar (IMF) mitochondria, lack of p53 was associated with reduced respiration and elevated reactive oxygen species production. Permeability transition pore kinetics remained unchanged; however, IMF mitochondrial cytochrome c release was reduced and DNA fragmentation was lowered, illustrating a resistance to mitochondrially driven apoptosis in muscle of KO mice. p53-null animals displayed similar muscle strength but greater fatigability and less locomotory endurance than wild-type (WT) animals. Surprisingly, the adaptive responses in mitochondrial content to running were similar in WT and KO mice. Thus p53 may be important, but not necessary, for exercise-induced mitochondrial biogenesis. In WT animals, acute muscle contractions induced the phosphorylation of p53 in concert with increased activation of upstream kinases AMP-activated protein kinase and p38, indicating a pathway through which p53 may initiate mitochondrial biogenesis in response to contractile activity. These data illustrate a novel role for p53 in maintaining mitochondrial biogenesis, apoptosis, and performance in skeletal muscle.

The role of PGC-1α on mitochondrial function and apoptotic susceptibility in muscle

Mitochondria are critical for cellular bioenergetics, and they mediate apoptosis within cells. We used whole body peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) knockout (KO) animals to investigate its role on organelle function, apoptotic signaling, and cytochrome-c oxidase activity, an indicator of mitochondrial content, in muscle and other tissues (brain, liver, and pancreas). Lack of PGC-1alpha reduced mitochondrial content in all muscles (17-44%; P < 0.05) but had no effect in brain, liver, and pancreas. However, the tissue expression of proteins involved in mitochondrial DNA maintenance [transcription factor A (Tfam)], import (Tim23), and remodeling [mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1)] did not parallel the decrease in mitochondrial content in PGC-1alpha KO animals. These proteins remained unchanged or were upregulated (P < 0.05) in the highly oxidative heart, indicating a change in mitochondrial composition. A change in muscle organelle composition was also evident from the alterations in subsarcolemmal and intermyofibrillar mitochondrial respiration, which was impaired in the absence of PGC-1alpha. However, endurance-trained KO animals did not exhibit reduced mitochondrial respiration. Mitochondrial reactive oxygen species (ROS) production was not affected by the lack of PGC-1alpha, but subsarcolemmal mitochondria from PGC-1alpha KO animals released a greater amount of cytochrome c than in WT animals following exogenous ROS treatment. Our results indicate that the lack of PGC-1alpha results in 1) a muscle type-specific suppression of mitochondrial content that depends on basal oxidative capacity, 2) an alteration in mitochondrial composition, 3) impaired mitochondrial respiratory function that can be improved by training, and 4) a greater basal protein release from subsarcolemmal mitochondria, indicating an enhanced mitochondrial apoptotic susceptibility.

Transcriptional and post-transcriptional regulation of mitochondrial biogenesis in skeletal muscle: Effects of exercise and aging

Acute contractile activity of skeletal muscle initiates the activation of signaling kinases. This promotes the phosphorylation of transcription factors, leading to enhanced DNA binding and transcriptional activation and/or repression. The mRNA products of nuclear genes encoding mitochondrial proteins are translated in the cytosol and imported into pre-existing mitochondria. When contractile activity is repeated, the recapitulation of these cellular events progressively leads to an expansion of the mitochondrial reticulum within muscle. This has physiologically relevant health benefit, including enhanced lipid metabolism and reduced muscle fatigability. In aging skeletal muscle, the response to contractile activity appears to be attenuated, suggesting that a greater contractile stimulus is required to attain a similar phenotype adaptation. This review summarizes our current understanding of the effects of exercise on the gene expression pathway leading to organelle biogenesis in muscle.