Sobia Iqbal

Adaptive plasticity of autophagic proteins to denervation in aging skeletal muscle

Aging muscle exhibits a progressive decline in mass and strength, known as sarcopenia, and a decrease in the adaptive response to contractile activity. The molecular mechanisms mediating this reduced plasticity have yet to be elucidated. The purposes of this study were 1) to determine whether denervation-induced muscle disuse would increase the expression of autophagy genes and 2) to examine whether selective autophagy pathways (mitophagy) are altered in aged animals. Denervation reduced muscle mass in young and aged animals by 24 and 16%, respectively. Moreover, young animals showed a 50% decrease in mitochondrial content following denervation, an adaptation that was not matched by aged animals. Basal autophagy protein expression was higher in aged animals, whereas young animals exhibited a greater induction of autophagy proteins following denervation. Localization of LC3II, Parkin, and p62 was significantly increased in the mitochondrial fraction of young and aged animals following denervation. Moreover, the unfolded protein response marker CHOP and the mitochondrial dynamics protein Fis1 were increased by 17- and 2.5-fold, respectively, in aged animals. Lipofuscin granules within lysosomes were evident with aging and denervation. Thus reductions in the adaptive plasticity of aged muscle are associated with decreases in disuse-induced autophagy. These data indicate that the expression of autophagy proteins and their localization to mitochondria are not decreased in aged muscle; however, the induction of autophagy in response to disuse, along with downstream events such as lysosome function, is impaired. This may contribute to an accumulation of dysfunctional mitochondria in aged muscle.

Expression of mitochondrial fission and fusion regulatory proteins in skeletal muscle during chronic use and disuse.

Introduction: The mitochondrial network within cells is mediated by fission and fusion processes. Methods: We investigated the expression of the proteins responsible for these events during conditions of altered oxidative capacity. Results: With chronic contractile activity, the mitochondrial reticulum increased in size, along with concomitant increases in the fusion proteins Opa1 and Mfn2 (by 36% and 53%; P < 0.05). When we induced muscle disuse through denervation for 7 days, fragmented mitochondria were observed, along with significant decreases in the expression of Mfn2 and Opa1 (by 84% and 70%). To assess the effects of aging on mitochondrial morphology, young (5 month) and aged (35 month) Fisher 344 Brown Norway rats were used. Aged animals also possessed smaller mitochondria and displayed increased levels of fission proteins. Conclusions: Chronic muscle use increases the ratio of fusion:fission proteins, leading to reticular mitochondria, whereas muscle disuse and aging result in a decrease in this ratio, culminating in fragmented organelles. Muscle Nerve 48: 963–970, 2013

Oxidative stress-induced mitochondrial fragmentation and movement in skeletal muscle myoblasts

Mitochondria are dynamic organelles, capable of altering their morphology and function. However, the mechanisms governing these changes have not been fully elucidated, particularly in muscle cells. We demonstrated that oxidative stress with H2O2 resulted in a 41% increase in fragmentation of the mitochondrial reticulum in myoblasts within 3 h of exposure, an effect that was preceded by a reduction in membrane potential. Using live cell imaging, we monitored mitochondrial motility and found that oxidative stress resulted in a 30% reduction in the average velocity of mitochondria. This was accompanied by parallel reductions in both organelle fission and fusion. The attenuation in mitochondrial movement was abolished by the addition of N-acetylcysteine. To investigate whether H2O2-induced fragmentation was mediated by dynamin-related protein 1, we incubated cells with mDivi1, an inhibitor of dynamin-related protein 1 translocation to mitochondria. mDivi1 attenuated oxidative stress-induced mitochondrial fragmentation by 27%. Moreover, we demonstrated that exposure to H2O2 upregulated endoplasmic reticulum-unfolded protein response markers before the initiation of mitophagy signaling and the mitochondrial-unfolded protein response. These findings indicate that oxidative stress is a vital signaling mechanism in the regulation of mitochondrial morphology and motility.

Multiple signaling pathways regulate contractile activity-mediated PGC-1α gene expression and activity in skeletal muscle cells.

PGC‐1α is an important transcriptional coactivator that plays a key role in mediating mitochondrial biogenesis. Within seconds of the onset of contractile activity, a number of rapid cellular events occur that form part of the initial signaling processes involved in PGC‐1α gene regulation, such as elevations in cytoplasmic calcium, AMPK and p38 activation, and elevated ROS production. We observed that basal levels of PGC‐1α promoter activity were more sensitive to resting Ca2+ levels, compared to ROS, p38 or, AMPK signaling. Moreover, enhanced PGC‐1α transcription and post‐translational activity on DNA were a result of the activation of multiple signal transduction pathways during contractile activity of myotubes. AMPK, ROS, and Ca2+ appear to be necessary for the regulation of contractile activity‐induced PGC‐1α gene expression, governed partly through p38 MAPK and CaMKII activity. Whether these signaling pathways are arranged as a linear sequence of events, or as largely independent pathways during contractile activity, remains to be determined.

Role of p53 within the regulatory network controlling muscle mitochondrial biogenesis.

The tumor suppressor protein p53 is recognized to contribute significantly to the regulation of mitochondrial content. Mice without p53 have reduced endurance capacity and muscle performance. However, the function of p53 in muscle remains to be fully established. Understanding how p53 coordinates mitochondrial homeostasis will facilitate a better comprehension of how exercise could constitute as a therapy for cancer treatment.

Effect of p53 on mitochondrial morphology, import, and assembly in skeletal muscle

The purpose of this study was to investigate whether p53 regulates mitochondrial function via changes in mitochondrial protein import, complex IV (COX) assembly, or the expression of key proteins involved in mitochondrial dynamics and degradation. Mitochondria from p53 KO mice displayed ultra-structural alterations and were more punctate in appearance. This was accompanied by protein-specific alterations in fission, fusion, and mitophagy-related proteins. However, matrix-destined protein import into subsarcolemmal or intermyofibrillar mitochondria was unaffected in the absence of p53, despite mitochondrial subfraction-specific reductions in Tom20, Tim23, mtHsp70, and mtHsp60 in the knockout (KO) mitochondria. Complex IV activity in isolated mitochondria was also unchanged in KO mice, but two-dimensional blue native-PAGE revealed a reduction in the assembly of complex IV within the IMF fractions from KO mice in tandem with lower levels of the assembly protein Surf1. This observed defect in complex IV assembly may facilitate the previously documented impairment in mitochondrial function in p53 KO mice. We suspect that these morphological and functional impairments in mitochondria drive a decreased reliance on mitochondrial respiration as a means of energy production in skeletal muscle in the absence of p53.

The role of mitochondrial fusion and fission in skeletal muscle function and dysfunction.

Classic textbook depictions of mitochondria portray these organelles to be static bean-shaped structures. However the mitochondrial population is quite heterogeneous, and can form small individual organelles or extended reticula throughout muscle. This morphological plasticity is controlled by fission and opposing fusion events. Skeletal muscle mitochondrial morphology has been demonstrated to be altered under various disease conditions, including diabetes, denervation, as well as during development, aging, and exercise. This implies that mitochondrial fission and fusion machinery components are involved in regulating the architecture of the organelle during various states of muscle use and disuse. Furthermore, disruptions in either of these opposing processes have been demonstrated to result in diseases, suggesting that proper maintenance of mitochondrial morphology is critical for proper cell function.

Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle

The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

Effect of Age on the Processing and Import of Matrix-Destined Mitochondrial Proteins in Skeletal Muscle

Deregulation of muscle mitochondrial biogenesis may explain the altered mitochondrial properties associated with aging. Maintenance of the mitochondrial network requires the continuous incorporation of nascent proteins into their subcompartments via the protein import pathway. We examined whether this pathway was impaired in muscle of aged animals, focusing on the subsarcolemmal and intermyofibrillar mitochondrial populations. Our results indicate that the import of proteins into the mitochondrial matrix was unaltered with age. Interestingly, import assays supplemented with the cytosolic fraction illustrated an attenuation of protein import, and this effect was similar between age groups. We observed a 2.5-fold increase in protein degradation in the presence of the cytosolic fraction obtained from aged animals. Thus, the reduction of mitochondrial content and/or function observed with aging may not rely on altered activity of the import pathway but rather on the availability of preproteins that are susceptible to elevated rates of degradation by cytosolic factors.

Cytoskeletal regulation of mitochondrial movements in myoblasts.

Mitochondria are distributed in the cell to match the energy demands, and it is their interaction with the cytoskeleton that controls their movement and displacement. Our purpose was to determine which cytoskeletal components are primarily responsible for mitochondrial movement in muscle cells. Live‐cell imaging was used to visualize mitochondrial dynamics in myoblasts. Destabilization of microtubules (MT) reduced the total path length and average speed traveled by mitochondria by 64–74%, whereas actin disruption only reduced these variables by 37‐40%. Downregulation of the microtubule motor proteins, Kif5B and dynein, by siRNA resulted in decreases in the average speed of mitochondrial movements, by 30 to 40%. We observed a reduction in the average speed of mitochondrial movements (by 22 to 48%) under high calcium conditions. This attenuation in the presence of calcium was negated in cells pre‐treated with siRNA targeted to the microtubule motor protein adaptor, Milton, suggesting that Milton is involved in mediating mitochondrial arrest in the presence of high calcium within muscle cells. Thus, we have demonstrated that, in myoblasts, mitochondria primarily move along microtubules tracks with the aid of the motor proteins Kif5B and dynein, in a manner which is inhibited by calcium. These observations will eventually help us understand organelle movements in more complex muscle systems, such as mature myotubes subjected to elevated calcium levels and contractile activity.