Heat-related illness remains a major consequence of global warming. While acute and prolonged heat exposure appear to result in different intercellular modifications (Gonzalez-Esquerra and Leeson, 2005), both can lead to clinical conditions. Clinical manifestation can vary from exacerbation of cardiovascular or gastrointestinal risk factors such as hypertension (Fonseca et al., 2015) or disruption of the intestinal barrier (Xu et al., 2015) to potentially life-threatening diseases, including kidney pathology (Glaser et al., 2016), coronary artery disease, or cardiac arrest (Kones, 2011). There were also a significant number of deaths reported due to heat waves in California (Knowlton et al., 2009), India (Azhar et al., 2014), and Pakistan (Ghumman and Horney, 2016) in the last decade. Heatstress can affect not only human health, but also agricultural animal welfare and bring associated economic losses. For example, the U.S. swine industry is conservatively estimated to lose $299 million annually (St-Pierre et al., 2003), mostly from decreased meat production (Pearce et al., 2014) and higher morbidity and mortality (St-Pierre et al., 2003). While the negative effects of heatstress are clearly established, little is known about molecular changes that underlie heat-induced injury. The lack of a mechanistic understanding of heatstress-mediated pathologies contributes to the unavailability of etiological treatments for heat illness leaving only rehydration and general cooling as standard procedures to treat heatstress.
126.96.36.199. The UPR in SkeletalMuscle and Exercise
Increasingly, evidence is continuing to mount to support a role of the UPR in skeletalmuscle in response to exercise, despite the low secretory output of this tissue. From a developmental perspective, induction of the UPR has been described to facilitate myoblast differentiation. UPR factors CHOP and BiP were upregulated during myogenesis, while ATF6 activation further proved to be critical in myotube formation (149). Conversely, in aging animal studies specific chaperones were decreased relative to young animals, while ER-stress and apoptotic markers were also elevated (37, 127, 148, 157). Together, these results indicate a potential link between ER function and UPR signaling in the maintenance of skeletalmuscle throughout the lifespan. As well, growing evidence indicates that the ER senses alterations in the cellular nutritional and metabolic states and appropriately activates the UPR to adjust, and balance metabolic activities (130). Deldique and colleagues found that excessive nutrient feeding, specifically of fats, resulted in heightened UPR signaling in skeletalmuscle (49). In the same study, they observed a crosstalk between ER stress and the mammalian target of rapamycin (mTOR) pathway, which they proposed was responsible for ER stress-induced downregulation of protein synthesis in C2C12 cells. Following this up, they found that the presence of ER stress prevented activation of the mTOR complex 1 (mTORC1), contributing to an increased anabolic resistance (48). Thus further providing a link between ER stress and other conditions of anabolic resistance such as aging, immobilization, and disuse.
2.3.3 Muscle metabolic plasticity with exercise
The numerous merits of regular physical activity on whole body metabolism and general health have been known for decades, making the field of exercise physiology busy exploring the how, and the why. Regular exercise has been reported to mediate widespread protective benefits ranging from glucose homeostasis during metabolic stress, to preservation of muscle mass during atrophic stimuli, and even improved mood and cognition with aging. Thus, exercise exerts health benefits for the body and the mind. Indeed, regular exercise can attenuate loss of muscle mass, health and function with disuse, ageing and cachexia. Exercise was also recently reported to reduce depression in stressed mice through a PGC-1α-mediated modulation of kynurenine metabolism (5). The molecular mechanisms underlying the merits of exercise have been under vigorous investigation, and although significant breakthroughs have been made, we are far from fully understanding the vast reaching consequences of exercise. Pioneering studies by Dr. John Holloszy and colleagues in the 1960s demonstrated that muscle oxidative capacity is induced with aerobic exercise training (90), and the implication of the transcriptional co-activator PGC- 1α in this process by the same group in the early 2000s indicated a true inflection point in exercise physiology (10). PGC-1α is strongly induced by an acute bout of exercise and is in itself sufficient to induce mitochondrial biogenesis in cells and tissues (10, 208, 251, 315). Although the need for PGC-1α in exercise-induced benefits is debated (208, 228, 294), its role in organelle biogenesis is a soundly supported conclusion (Fig. 5).
Endurance exercise activates PGC-1␣ genes in skeletalmuscle of humans and activated PGC-1 ␣ is stimulating mito- chondrial biogenesis with two methods. 38 First, PGC-1 ␣’s activation becomes rapid in the early stage and after that, a long term increase follows based on the increased expres- sion of PGC-1 ␣. The phenomenon of an early activity increase was proven with various evidences and gene transcription and expression of ﬁrst mitochondrial protein is increasing in a similar speed or faster than PGC-1 ␣ expression caused by exer- cise stimuli. Second NRF-1 and NRF-2 will combine with their response promoters prior to PGC-1 ␣ expression. Third, most of PGC-1␣ will be found in the cytoplasm of skeletalmuscle during a rest period but it will move into the nucleus dur- ing exercise. To summarize, exercise rapidly activates PGC-1 ␣ prior to the increase of PGC-1 ␣ expression and thus increases mitochondrial biogenesis. 39
likely due to the poor resolution obtained when homogeniz- ing whole-muscle preparations and utilizing densitometry to parse out the MHC isoform contributions. As a more accurate and sensitive approach, we used single muscle fiber analyses for these studies. MHC analysis was performed on 197.8 ± 2.1 single fibers from each sample (n = 1978 total fibers) (Table 1). There were no differences in MHC I isoform composition between the heart failure and control group (33% ± 7% and 45% ± 5%, respectively). Additionally, there were no differences between the groups in MHC IIa compo- sition (33% ± 1% and 41% ± 3%, respectively). However, there were significantly more (P , 0.05) MHC isoforms coexpressing one or more pure MHC isoforms (hybrids) in the heart failure patients (30% ± 7%) compared with the control subjects (13% ± 2%). Additionally, a significant dif- ference (P , 0.05) was found with the MHC IIa/IIx hybrid isoforms between the two groups (heart failure: 24% ± 6%; control: 9% ± 2%). These results demonstrate a molecular shift in the muscle of heart failure patients to a highly fati- gable fiber type that may account for classical symptoms such as exercise intolerance.
effectively block autophagic degradation  (Figure 3). De- nervation resulted in increased accumulation of LC3B-II, p62, and Nix (Bnip3l) proteins in WT muscle. Colchicine treatment resulted in further accumulations of LC3B-II and p62 in denervated WT muscle, indicating a successful block in autophagic degradation with the drug (Figure 3A,B,C). In contrast, PGC-1α KO animals exhibited an attenuated LC3B-lipidation and expression of the mitophagy-specific receptor Nix both basally and in response to denervation (Figure 3A,B,D). No significant difference in p62 expression was observed basally between the genotypes, and p62 levels did not change in KO animals with either Den or Col treat- ment (Figure 3C). Importantly, both basal and denervation- induced p62 and LC3B-II flux were lower in KO animals, as evidenced by a smaller accumulation of LC3BII and p62 in KO animals treated with colchicine (Figure 3E,F). We further examined lysosomal abundance and autophagy flux in KO and WT animals using confocal microscopy (Fig- ure 3G). We did this by isolating single fibers from fixed EDL muscles and co-immunostaining them for LC3B and Lamp-2. KO animals had lower lysosomal abundance, as indicated by a diminished intensity and frequency of red fluorescence, and this was especially evident follow- ing denervation (Figure 3G and Additional file 1: Figure S2). We also noted an increase in the colocalization (yellow) of autophagosomes and lysosomes and their aggregation in the perinuclear region of denervated WT muscles (Figure 3G, Merge). This was not as evi- denced in the denervated KO muscle, as indicated by decreased yellow flourescence. Taken together, these re- sults indicate that the lack of PGC-1α results in lower lysosomal abundance and reduced autophagy flux in re- sponse to denervation.
8.1.2. Insulin resistance induced by endoplasmic reticulum stress in muscle cells: what is wrong?
UPR, inflammation and insulin resistance
In the context of obesity, extracellular and intracellular lipids appear to initiate the development of metainflammation, i.e., inflammation induced by metabolic factors. This abnormal inflammation, also called low-grade inflammation, must be distinguished from the normal response to pathogen infection. In type 2 diabetes, the development of insulin resistance is intimately linked to metainflammation (Hotamisligil, 2005), this causal relationship has been discovered for over a hundred years. Indeed, in 1876 Ebstein observed that sodium salicylate reduced glycosuria in diabetic subjects (Shoelson et al., 2006). However, the mechanisms by which metabolic signals are transduced into inflammatory response remain unclear and belong to the emerging field of immunometabolism. Recently, it has been proposed that ER could be an integration center, which senses metabolic stress and triggers an inflammatory signal (Hotamisligil, 2010, Hotamisligil, 2006). More precisely, UPR signaling is coupled with the NF-κB and MAPK inflammatory pathways (Zhang and Kaufman, 2008). In the next paragraphs, we will focus on the experiments that revealed this mechanism.
Sarcopenia, the loss of muscle mass and function as we age, affects all individuals from approximately the 4 th decade of life and results in a poor quality of life. The mechanisms responsible for sarcopenia are unclear and it is likely that it is a multifactorial disease. However, it is hypothesised that an increase in systemic and/or muscle pro-inflammatory cytokine levels play a major role. Interferon induced protein 10 (IP10) is a chemokine that has been shown to be increased in serum as we age. Polyphenols are extracts from plants and have been shown to have anti-inflammatory effects with benefits in multiple diseases. Therefore, the main aim of this thesis was to establish an in vitro model to study the effect of increased levels of IP10 on muscle atrophy and inflammation and to examine whether resveratrol treatment was able to protect against IP10 induced effects on skeletalmuscle atrophy or inflammation.
19 is defined as increasing dysfunction in both tissue and organs as a whole and can be considered both as a chronological and biological process (Hekimi and Guarente, 2003, Tatar et al., 2003, Carter et al., 2007). A plethora of functional changes have been associated with this pathology including a reduction in tissue regenerative capacity, a decrease in endocrine response and increases in oxidative stress (Lightfoot et al., 2014, Carter et al., 2007). Not only does DR reduces mortality via the reduction of these age related changes it also reduces the prevalence of morbidity. Disease risk reductions during DR include, but are not limited to, the prevalence of type II diabetes, hypertension and obesity, as well as a decline in some cancers and cardio-vascular disease (Longo and Mattson, 2014, Lam et al., 2013, Carter et al., 2007). Therefore, regardless of the limited data to suggest that DR improves lifespan in humans, the implementation of DR reduces numerous physiological afflictions often associated with obesity and age and is therefore widely considered to improve healthspan. Skeletalmuscle is one such tissue that maybe particularly susceptible to age related changes and DR.
How caspase 3 activity translates to a beneficial stress adaptation in skeletalmuscle fibers is currently unknown. One probable mechanism may involve direct communica- tion between caspase 3 and other proteostatic control mechanisms within the muscle fiber (i.e., caspase signaling may serve to control both proteasome and autophagy- related signaling during muscle adaptation). To date, over 500 physiologic substrates have been identified for caspase 3/7, as such the ability of an effector caspase to target and integrate regulatory control over disparate proteolytic mechanisms is an entirely probable event. In Drosophila oogenesis, the effector caspase equivalent, death caspase-1 (DCP-1), has been shown to promote autophagy flux by cleaving and inhibiting the key autophagy suppressing protein SesB . Whereas autophagy has been generally associated with muscle atrophy/wasting, a number of studies have shown that resistance trained skeletalmuscle is associated with enhanced autophagic flux [62, 114]. The observations in Drosophila have established the existence of a caspase-directed autophagy signal, whether skeletalmuscle utilizes a similar beneficial regulatory cascade will require further investigation. Simultaneous caspase activa- tion and proteosome signaling in skeletalmuscle are understood to have generally negative outcomes, associ- ated with wasting and atrophy in a variety of disease set- tings [26, 71, 102]. Nevertheless, caspase 3 has been demonstrated to target and cleave subunits of the 19S proteasome (Rpt2 and Rpt6), leading to an obligatory increase in proteasome activity during myoblast differenti- ation . Mutation of the respective caspase 3 cleavage sites in Rpt2 and Rpt6 resulted in failure to up-regulate the 19S proteasome, with a profound block in the differ- entiation program. Clearly, the uncontrolled engagement of this signaling interaction would have dire consequences for myofiber viability, yet one can easily envision that a transient activation of these proteases may act to remodel the ultrastructure of the myofiber in response to physio- logic demands.
The calpains are a family of Ca 2+ -dependent cysteine proteases – skeletalmuscle fibres contain both the ubi- quitous isoforms μ-calpain, m-calpain, and calpain-10, as well as the muscle-specific form, calpain-3 . Those calpains activated within a physiologically relevant [Ca 2+ ] range are calpain-3 and μ-calpain [13,14]. Calpain 3 plays a role in remodelling and maintaining normal sarcomeric structures, whereas μ-calpain is associated with dismant- ling sarcomeric structures; and a balance in their activities is important for skeletalmuscle integrity (see review ). Intracellular Ca 2+ concentrations above resting cytosolic levels cause autolysis of μ-Calpain and Calpain-3, thus in- creasing their proteolytic activity [13,16]. Over-activation of calpains due to Ca 2+ overload has been implicated in many pathological conditions including, Parkinson’s dis- ease and muscular dystrophy [17,18]. As there is substan- tial evidence suggesting dysregulation of intracellular Ca 2+ homeostasis in T2D (for review see ), it is possible that calpains are being over-activated by excessive intracellular Ca 2+ accumulation in this disease. Thus far, calpain activity has not been investigated for a role in T2D- associated atrophy.
In this study it was demonstrated how perimysium organisation changes after deformation applications. In compression in the fibre direction (Comp-F), the collagen fibrils in longitudinal direction are wavy (w2) and bear no load, but since the collagen fibrils are straight in the transverse plane (w1) for both chicken and porcine tissues they are assumed to take part in load bearing in this condition (see Figure 5.8i-iii). In compression in the cross-fibre direction (Comp-XF) there is both wavy and straight perimysium in the transverse plane (w1). Takaza et al. (2014), through polarised light images, showed that 30% compressive deformation in the cross-fibre direction caused the collagen fibrils to be pushed together and showed an increase in waviness, which agrees with the findings of this study; that in Comp-XF, where the perimysium is aligned with the deformation direction, the collagen fibrils become wavy. However, the portion of perimysium which is aligned perpendicular to the applied deformation (straight fibrils) is greater than the proportion that is wavy, indicating that perimysium mostly reorients to bear the load. In their study, the colour changes of perimysium strands within the tissue were an indicator of the perimysium reorganization. However, in the current study, the waviness of perimysium collagen fibres were clearly visible in confocal images and it was shown how the perimysium responds under different deformation conditions. Takaza et al. (2014) also showed when the tissue is compressed perpendicular to the muscle fibres, the perimysium showed a preferred orientation to become perpendicular to the load direction and this preferred alignment is accompanied with more stretch in collagen fibrils. On the other hand, the collagen fibrils in the longitudinal direction under Comp-XF become straight, taking part in passive load bearing (see Figure 5.8i-iii). The compressive stress-stretch response in skeletalmuscle reported in Chapter 3 showed generally that the tissue (either chicken or porcine) is stiffer in Comp-XF. It can be concluded that the stiffer response in Comp-XF than Comp-F (regardless of small difference between them) is because in Comp-XF collagen is involved in load bearing in two perpendicular planes (the less wavy collagen seen in the longitudinal direction induced by the deformation (Figure 5.7(c), w2)) as well as the stretched collagen in the transverse plane caused by direct deformation (Figure 5.6i(c), w1)). However in Comp- F only induced straightness in the transverse plane is responsible for passive load bearing.
Over the past century, understanding the mechanisms underlying muscle fatigue and weakness has been the focus of much investigation. However, the dominant theory in the field, that lactic acidosis causes muscle fatigue, is unlikely to tell the whole story. Recently, dysregulation of sarcoplasmic reticulum (SR) Ca 2+ release has been associated with impaired muscle function induced by a wide range of stressors, from dystrophy to heart failure to muscle fatigue. Here, we address current understandings of the altered regulation of SR Ca 2+ release during chronic stress, focusing on the role of the SR Ca 2+ release channel known as the type 1 ryanodine receptor.
Restoration of the full anabolic effect of FS by IGF-I in STZ animals, despite the persistence of hyperglycemia, indicates that IGF-I may substitute for the anabolic properties but not for the hypoglycemic effect of insulin. A Similar observation was already made for bone growth. Indeed, IGF-I treatment re- stored the longitudinal bone growth of STZ-treated rats without normalization of glycemia (55). The fact that FS retains its hypertrophic action in IGF-I-treated STZ animals despite the presence of hyperglycemia suggests that the failure of FS to stimulate muscle hypertrophy in STZ animals is not due to glucose toxicity. However, the observation that IGF-I restores growth in STZ-diabetic animals without restoring normogly- cemia cannot be interpreted as the result of the IGF-I binding to the IGF-IR. Indeed, our work did not investigate which, IGF-IR or insulin receptor (INS-R), is required for FS to exert its anabolic action. Although the ligands insulin and IGF-I can substitute one for the other, this phenomenon does not seem to apply to their receptors. Indeed, deletion of the INS-R (48), like the IGF-IR (43), is associated with decreased muscle growth, suggesting that both receptors are required for proper physiological muscle growth. Moreover, expression of the dominant-negative form of IGF-IR, or MKR, which inhibits the FS-inducedmuscle hypertrophy (34), impairs both insulin and IGF-I signaling in muscle due to hybrid receptor formation (22). Therefore, since the two receptors are mandatory for proper physiological muscle growth, we cannot specify which of them, INS-R or IGF-IR, is involved in the FS-inducedmuscle hypertrophy. Ultimately, our study establishes the need of insulin or IGF-I for the full anabolic effect of FS toward skeletalmuscle. When insulin or IGF-I alone is missing, FS retains its full anabolic effect. But, when both are deficient, as
We have recently demonstrated that angiotensin II infusion in the rat produced a marked reduction in body weight accompa- nied by depression of circulating and skeletalmuscle IGF-1 (5). These findings suggested that downregulation of IGF-1 signaling in skeletalmuscle could mediate the wasting effect of angiotensin II. Recent studies using in vitro models of muscle atrophy have indicated that IGF-1 acts through Akt and Foxo to suppress atro- gin-1/muscle ring finger–1 (atrogin-1/MuRF-1) transcription (6). Atrogin-1 and MuRF-1 are ubiquitin ligases whose expression is elevated in various muscle atrophy models (7). In vivo studies have indicated that apoptosis is also involved in muscle wasting (8, 9). Furthermore, we have recently shown that activation of caspase-3 leading to actin cleavage contributes to proteolysis in catabolic conditions such as uremia or diabetes and leaves a char- acteristic 14-kDa actin fragment in muscle (10). In view of the potent anabolic and antiapoptotic effects of IGF-1 (11, 12), we hypothesized that downregulation of IGF-1 signaling in response to angiotensin II would lead to a coordinated activation of both caspase-3–mediated apoptosis and activation of the ubiquitin- proteasome (Ub-P’some) pathway, resulting in loss of skeletalmuscle. We used skeletalmuscle–specific IGF-1–transgenic mice to
Cancer cachexia is a syndrome characterized by loss of skeletalmuscle mass, inflammation, anorexia and anemia, contributing to patient fatigue and reduced quality of life. In addition to nutritional approaches, exercise training (EX) has been proposed as a suitable tool to manage cachexia. In the present work the effect of mild exercise training, coupled to erythropoietin (EPO) administration to prevent anemia, has been tested in tumor-bearing mice. In the C26 hosts, acute exercise does not prevent and even worsens muscle wasting. Such pattern is prevented by EPO co-administration or by the adoption of a chronic exercise protocol. EX and EPO co-treatment spares oxidative myofibers from atrophy and counteracts the oxidative to glycolytic shift, inducing PGC-1α. LLC hosts are responsive to exercise and their treatment with the EX-EPO combination prevents the loss of muscle strength and the onset of mitochondrial ultrastructural alterations, while increases muscle oxidative capacity and intracellular ATP content, likely depending on PGC-1α induction and mitophagy promotion. Consistently, muscle-specific PGC-1α overexpression prevents LLC-inducedmuscle atrophy and Atrogin-1 hyperexpression. Overall, the present data suggest that low intensisty exercise can be an effective tool to be included in combined therapeutic approaches against cancer cachexia, provided that anemia is coincidently treated in order to enhance the beneficial action of exercise.
protein, which is considered a speci ﬁ c quantitative compo- nent of MAM, was signi ﬁ cantly decreased, whereas swim- ming increased its expression in the skeletalmuscle of diabetic mice. These ﬁ ndings suggested that swimming can somewhat attenuate MAM impairment induced by dia- betes. Notably, MAM disruption is an essential subcellular alteration associated with muscle insulin resistance in mice and humans, indicating that reduced ER-mitochondria cou- pling could be a common change in several insulin sensitive tissues that play key roles in altered glucose homeostasis in the context of obesity and T2DM. 52 We also identi ﬁ ed a positive correlation between levels of Sigma-1R protein and MMP, suggesting that mitochondrial function is posi- tively associated with the quantity of MAM in the patho- genesis of diabetes.
since the P trial received no carbohydrate into the recov- ery period, it is quite possible that the greater fat oxida- tion during the later stages of exercise continued into recovery in the P trial and subsequently attenuated the UCP3 mRNA expression. This is supported by evidence that elevated circulating fatty acids are associated with the upregulation of skeletalmuscle expression of UCP3 [14,41-43]. We do not have evidence of circulating free fatty acids (FFA) in the current study, but it is well established that fasted exercise in the absence of carbo- hydrate delivery elevates FFA compared to carbohydrate trials . Although fat oxidation appears to coincide with UCP3 expression, the metabolic role of this protein in skeletalmuscle remains unclear as it suggests a loss of exercise efficiency by uncoupling the proton gradient created in the electron transport chain from ATP syn- thesis. However, besides fat oxidation, UCP3 has been implicated as being important in the control of thermo- genesis and the regulation of oxidative stress . The long term implications of the attenuation of UCP3 ex- pression following exercise with carbohydrate supple- mentation in this study and others has yet to be determined [14,43]. It is intriguing to think that lower UCP3 mRNA may play a role in previous evidence of the carbohydrate attenuating effect on fat oxidation with exercise training [44,46]. These studies demonstrated that low carbohydrate availability (fat adapted) resulted in greater rates of fat oxidation even when glycogen levels were restored with a day on a high carbohydrate diet. Our study and others have shown that UCP3 is the gene most consistently attenuated with the consumption of exogenous carbohydrate. How UCP3 expression is af- fected during longer periods of low carbohydrate
Assessments of both groups included questionnaires, physical exams for disease status, fasting blood collec- tion, intravenous glucose tolerance tests for insulin sen- sitivity, 7 days of accelerometer-measured physical activity, computed tomography (CT) imaging of abdo- men and thigh, and vastus lateralis muscle biopsies . Disability (health assessment questionnaire-disability index (HAQ-DI) and co-morbidities (co-morbidity index) were assessed by previously published question- naires [6, 7]. Disease activity assessed by the disease ac- tivity score in 28 joints (DAS-28) was determined from a patient-completed visual analog scale, physician- determined numbers of tender and swollen joints, and erythrocyte sedimentation rate . Plasma concentra- tions of inflammatory markers and cytokines were determined by immunoassay  and nuclear magnetic resonance (NMR) spectroscopy (GlycA) . Insulin sensitivity was determined using Bergman’s minimal model  and concentrations of glucose and insulin (glucose: Beckman-CoulterDXC600; insulin: electroche- miluminscent assay from Meso Scale Discovery) at each of 29 time points during the intravenous glucose toler- ance test.
Oxidative stress and mitochondrial dysfunction are associated with the aging process. However, the role of nuclear factor erythroid 2 -related factor 2 (Nrf2) in skeletalmuscle during aging remains to be clarified. In the current study, we assessed whether the lack of Nrf2, which is known as a master regu- lator of redox homeostasis, promotes age-related mitochondrial dysfunction and muscle atrophy in skeletalmuscle. Here, we demonstrated that mitochon- drial 4-hydroxynonenal and protein carbonyls, markers of oxidative stress, were robustly elevated in aged Nrf2 knockout (KO) mice because of the decreased expression of Nrf2-target antioxidant genes. Mitochondrial respira- tion declined with aging; however, there was no difference between Nrf2 KO and age-matched WT mice. Similarly, cytochrome c oxidase activity was lower in aged WT and Nrf2 KO mice compared with young WT mice. The expres- sion of Mfn1 and Mfn2 mRNA was lower in aged Nrf2 KO muscle. Mito- chondrial reactive oxygen species production per oxygen consumed was elevated in aged Nrf2 KO mice. There was no effect of Nrf2 KO on muscle mass normalized to body weight. These results suggest that Nrf2 deficiency exacerbates age-related mitochondrial oxidative stress but does not affect the decline of respiratory function in skeletalmuscle.