Caspase-2 is required for skeletal muscle differentiation and myogenesis.

Caspase activation plays a crucial role in skeletal muscle differentiation. We previously found that caspase-2 activity increases during skeletal muscle cell differentiation; however, its direct effect on differentiation has not been fully investigated. Here, we found that caspase-2 activity transiently increased more than two-fold within 24h following induction of differentiation. Both pharmacological inhibition and shRNA-mediated knockdown of caspase-2 suppressed myogenic differentiation and dramatically impaired myotube formation. Furthermore, shRNA-mediated knockdown prevented induction of p21 and altered cell cycle profiles. Interestingly, caspase-3 activity was also dramatically reduced following caspase-2 inhibition or ablation. Moreover, caspase-2 and p21 were localized to the nucleus during early differentiation. Given the nuclear localization of caspase-2 and p21, as well as the impairment in p21 induction in caspase-2 knockdown cells, we propose that the role of caspase-2 is to regulate p21 induction at the onset of differentiation, which may regulate the myogenic program. Collectively, these results highlight a novel function for caspase-2 in myocyte differentiation and myogenesis.

Chronic corticosterone-induced impaired cognitive flexibility is not due to suppressed adult hippocampal neurogenesis.

Hippocampal neurogenesis has been implicated in the etiology of depression. Recent studies suggest new neurons add flexibility to hippocampal-dependent learning and memory. We hypothesized that suppressed hippocampal neurogenesis may contribute to impaired cognitive flexibility associated with depression. The chronic corticosterone (CORT)-induced animal model of depression was used. In Experiment 1, rats received either CORT (40mg/kg) or vehicle injections for 21days and were subjected to Water maze during the last six days of drug treatment. No group differences were found during the spatial learning phase; however, cognitive flexibility, measured by reversal training, was significantly impaired in the CORT-treated rats. The probe test revealed enhanced memory of the new platform location for the CORT-treated rats. Given the time newborn neurons require to mature, we presumed if impaired cognitive flexibility seen in Experiment 1 were due to suppressed neurogenesis, terminating CORT treatment 3days prior to behavioural testing should still induce the impairment. Therefore, Experiment 2 was similar to Experiment 1, except that CORT injections were terminated 3days prior to behavioural assessment. However, not only was spatial learning significantly enhanced in the CORT-treated rats, but there were also no group differences during reversal or probe tests. Bromodeoxyruidine, administered a day after the first drug treatments in both experiments, was quantified and revealed the number of new neurons were the same in both groups in both experiments. Results suggest cognitive flexibility is impaired in the CORT-induced animal model of depression; an effect that is reversible and independent of suppressed hippocampal neurogenesis.

Effect of acute and chronic autophagy deficiency on skeletal muscle apoptotic signaling, morphology, and function.

Autophagy is a catabolic process that targets and degrades cytoplasmic materials. In skeletal muscle, autophagy is required for the control of mass under catabolic conditions, but is also basally active in the maintenance of myofiber homeostasis. In this study, we found that some specific autophagic markers (LC3-I, LC3-II, SQSTM1) were basally lower in glycolytic muscle compared to oxidative muscle of autophagy competent mice. In contrast, basal autophagic flux was higher in glycolytic muscle. In addition, we used several skeletal muscle-specific Atg7 transgenic mouse models to investigate the effect of acute (iAtg7-/-) and chronic (cAtg7-/-) autophagy deficiency on skeletal muscle morphology, contractility, and apoptotic signaling. While acute autophagy ablation (iAtg7-/-) resulted in increased centralized nuclei in glycolytic muscle, it did not alter contractile properties or measures of apoptosis and proteolysis. In contrast, with chronic autophagy deficiency (cAtg7-/-) there was an increased proportion of centralized nuclei, as well as reduced force and altered twitch kinetics in glycolytic muscle. Glycolytic muscle of cAtg7-/- mice also displayed an increased level of the pro-apoptotic protein BAX, as well as calpain and proteasomal enzymatic activity. Collectively, our data demonstrate cumulative damage from chronic skeletal muscle-specific autophagy deficiency with associated apoptotic and proteasomal upregulation. These findings point towards the importance of investigating different muscle/fiber types when studying skeletal muscle autophagy, and the critical role of autophagy in the maintenance of myofiber function, integrity, and cellular health.

Oestrogen-dependent satellite cell activation and proliferation following a running exercise occurs via the PI3K signalling pathway and not IGF-1.

AIM: The purpose of this study was to determine whether 17ß-estradiol (E2) enhances the activation, proliferation and differentiation of muscle satellite cells (SC) following eccentric exercise either via insulin-like growth factor-1 (IGF-1) or through phosphatidylinositol 3-kinase (PI3K) signalling. METHODS: This study used 64, 9-week-old, ovariectomized Sprague-Dawley rats that were divided into eight treatments groups based on oestrogen status (0.25 mg oestrogen pellet or sham), exercise status (90 min run @ 17 m min(-1), -13.5° or unexercised) and PI3K signalling inhibition (0.7 mg wortmannin kg(-1) body weight or DMSO control). RESULTS: Significant increases in total SCs were found in both soleus and white gastrocnemius muscles (immunofluorescent co-localization of Pax7(+) nuclei) 72 h following eccentric exercise (P < 0.05). Oestrogen supplementation caused a further enhancement in total SCs in exercised rats (P < 0.05). In animals where the PI3K pathway was inhibited, regardless of oestrogen or exercise status, there was no significant enhancement of SC number in both the soleus or white gastrocnemius muscles. Interestingly, oestrogen supplementation lowered muscle levels of IGF-1 with this effect being most prominent in the soleus muscle. While IGF-1 was increased following exercise (P < 0.05), oestrogen supplementation abrogated this increase back to sedentary levels. CONCLUSION: These data suggest that the increase in SC population following exercise in oestrogen-supplemented females may be mediated via PI3K pathway signalling and not IGF-1.

Autophagy is altered in skeletal and cardiac muscle of spontaneously hypertensive rats.

AIM: Autophagy is a subcellular degradation mechanism important for muscle maintenance. Hypertension induces well-characterized pathological changes to the heart and is associated with impaired function and increased apoptotic signalling in skeletal muscle. We examined whether essential hypertension affects several autophagy markers in skeletal and cardiac muscle. METHODS: Immunoblotting and qRT-PCR were used to measure autophagy-related proteins/mRNA in multiple skeletal muscles as well as left ventricle (LV) of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). RESULTS: Skeletal muscles of hypertensive rats had decreased (P < 0.01) cross-sectional area of type I fibres (e.g. soleus WKY: 2952.9 ± 64.4 µm(2) vs. SHR: 2579.9 ± 85.8 µm(2)) and a fibre redistribution towards a 'fast' phenotype. Immunoblot analysis revealed that some SHR skeletal muscles displayed a decreased LC3II/I ratio (P < 0.05), but none showed differences in p62 protein. LC3 and LAMP2 mRNA levels were increased approx. 2-3-fold in all skeletal muscles (P < 0.05), while cathepsin activity, cathepsin L mRNA and Atg7 protein were increased 16-17% (P < 0.01), 2-3-fold (P < 0.05) and 29-49% (P < 0.01), respectively, in fast muscles of hypertensive animals. Finally, protein levels of BAG3, a marker of chaperone-assisted selective autophagy, were 18-25% lower (P < 0.05) in SHR skeletal muscles. In the LV of SHR, LC3I and p62 protein were elevated 34% (P < 0.05) and 47% (P < 0.01), respectively. Furthermore, p62 mRNA was 68% higher (P < 0.05), while LAMP2 mRNA was 45% lower (P < 0.05), in SHR cardiac muscle. There was no difference in Beclin1, Atg7, Bnip3 or BAG3 protein in the LV between strains. CONCLUSION: These results suggest that autophagy is altered in skeletal and cardiac muscle during hypertension.

In vivo corticosterone administration at levels occurring with intense exercise does not induce intestinal lymphocyte apoptosis in mice.

Intestinal lymphocyte apoptosis can occur following physiological and pathophysiological stress as well as exhaustive exercise. In this study we investigated whether corticosterone (CORT) administration at physiological concentrations observed following strenuous exercise induces intestinal lymphocyte apoptosis and cell loss in mice. CORT injection (14 mg/kg; i.p.) caused a four-fold increase in plasma CORT concentrations, but did not affect intestinal lymphocyte cell loss or alter baseline intestinal lymphocyte apoptosis, as measured by phosphatidylserine externalization, cell viability, mitochondrial membrane depolarization, caspase 3, Bcl-2 and cytosolic cytochrome c protein levels. These findings indicate that CORT at levels observed following strenuous exercise is not involved in intestinal lymphocyte apoptosis and cell loss.

Polyethylene glycol but not mifepristone prevents intestinal lymphocyte loss following treadmill exercise in mice.

UNLABELLED: Circulating lymphocyte numbers decrease following intense physical activity, possibly due to exercise-induced apoptosis. Increased reactive oxygen species (ROS) and glucocorticoids (GC) following exercise contribute to lymphocyte apoptosis. Intestinal lymphocyte (IL) numbers also decrease following exercise. AIM: The purpose of this study was to determine the contribution of GC to exercise-induced IL loss. METHODS: Female C57BL/6 mice (n = 178) were randomized to five drug conditions: (1) single injection of the glucocorticoid receptor antagonist mifepristone (MIF) solubilized in polyethylene glycol (PEG); (2) three injections of MIF (repeated MIF) PEG; (3) single injection of PEG (PEG); (4) three injections of PEG (repeated PEG); or (5) repeated injections of saline (SAL). Within each drug group mice were further randomized to exercise conditions: (1) control condition (non-exercised); (2) treadmill running with sacrifice immediately following the exercise; or (3) treadmill running with sacrifice 24 h after completion of the exercise. RESULTS: There was a significant exercise effect, across all T lymphocyte subsets, in SAL (P < 0.01), PEG (P < 0.01) and MIF (P < 0.01) treated mice but not in mice given repeated PEG or repeated MIF exposure. The exercise effect was due to reduced IL numbers 24 h post-exercise compared with non-exercised controls. CONCLUSION: These results suggest that GC are not directly responsible for IL cell loss following exercise. Repeated exposure to PEG may confer protection in the gastrointestinal tract from exercise-induced lymphocyte depletion. Because PEG inhibits ROS generation in experimental cell injury, the mechanisms for IL loss after exercise may involve oxidative stress.

N-Acetyl-L-cysteine prevents exercise-induced intestinal lymphocyte apoptosis by maintaining intracellular glutathione levels and reducing mitochondrial membrane depolarization.

Intense exercise leads to post-exercise lymphocytopenia and immunosuppression, possibly by triggering lymphocyte apoptosis. To test the role of oxidative stress on exercise-induced lymphocyte apoptosis, we administered the antioxidant N-acetyl--cysteine (NAC) and measured apoptosis in intestinal lymphocytes (IL) from exhaustively exercised animals. Eighty-seven female C57BL/6 mice were randomly assigned to receive NAC (1 g/kg) or saline 30 min prior to treadmill exercise for 90 min at 2degrees slope (30 min at 22 m min(-1), 30 min at 25 m min(-1), and 30 min at 28 m min(-1)) and sacrificed immediately (Imm) or 24 hours (24 h) after cessation of exercise. Control mice (nonexercised) were exposed to treadmill noise and vibration without running. Exercise increased IL phosphatidylserine externalization (p<0.001), mitochondrial membrane depolarization (p<0.05), and decreased intracellular glutathione concentrations (p<0.05) immediately following exercise in saline relative to nonexercised mice. At 24 h post-exercise, saline injected mice had fewer total (p<0.001) and CD3+ (p<0.005) IL compared to nonexercised animals. NAC injection in mice maintained intracellular glutathione levels, prevented phosphatidylserine externalization, mitochondrial membrane depolarization, and loss of IL immediately and 24 h after exercise. These data suggest that lymphocyte apoptosis precedes post-exercise lymphocytopenia and may be due to oxidative stress.

Adrenalectomy in mice does not prevent loss of intestinal lymphocytes after exercise.

Exhaustive exercise is associated with an increase in circulating glucocorticoids (GCs), lymphocyte apoptosis, and a reduction in intestinal lymphocyte number. The present study examined the role of GCs on the numerical changes seen in intestinal lymphocytes after exercise. Female C57BL/6 mice were bilaterally adrenalectomized (ADX; n = 18) or given sham surgery (Sham; n = 18) and assigned to one of three exercise conditions: treadmill running (28 m/min, 90 min, 2 degrees slope) and killed immediately or after 24 h recovery, or not exercised and killed immediately after 90-min exposure to the treadmill environment. Lymphocytes were isolated from the intestines with CD45(+) cells collected by positive selection using magnetic bead separation columns, and lymphocyte subpopulations were analyzed by flow cytometry for CD45(+), CD3alphabeta(+), CD3gammadelta(+), CD8beta(+), CD8alpha(+), CD4(+), and NK(+) phenotypic markers. ADX mice had significantly more intestinal CD45(+) leukocytes (P < 0.05) and CD3alphabeta(+) (P < 0.05), CD3gammadelta(+) (P < 0.01), CD8alpha(+) (P < 0.001), and NK(+) (P < 0.05) intestinal lymphocytes than Sham mice. There was a significant effect of exercise condition on total intestinal CD45(+) leukocytes (P < 0.01) and CD3alphabeta(+) (P < 0.05), CD8alpha(+) (P < 0.001), and CD4(+) (P < 0.05) intestinal lymphocytes, with fewer cells at 24 h postexercise compared with the other treatment conditions. There were no surgical x exercise interaction effects on the CD3 and CD8 phenotype numbers. Plasma corticosterone was virtually nil in ADX mice regardless of exercise condition but was significantly elevated in Sham mice immediately postexercise (P < 0.001). The data indicate that ADX does not prevent the loss of lymphocytes from the intestinal mucosa 24 h after strenuous exercise and GCs are not directly causal in the leukopenia of exercise.

Lymphocyte distribution in mouse submandibular lymph nodes in response to acute treadmill exercise.

The submandibular lymph nodes (LN), part of the nasal-associated lymphoid tissue (NALT), are involved in local immune responses in the eye, upper respiratory tract (URT), and oral mucosa. Although athletes have been reported to be at increased risk for URT and ocular infections, little is known about the impact of exercise on LN included in the NALT. The purpose of this study was to examine the impact of intense acute exercise on submandibular lymphocyte distribution. Female C57BL/6 mice were randomly assigned to a nonexercised control condition or a single session of treadmill exercise (32 m.min-1, 8 degrees grade for 90 min) and sacrificed immediately, 2, and 24 h after exercise. Running resulted in a significant increase in plasma corticosterone immediately following exercise compared with other times (p < 0.001). Percentages and total numbers of CD3+ and CD4+CD8- T lymphocytes in submandibular LN were significantly lower 24 h after exercise compared with controls. The percentage of pan-NK and CD19+ B cells increased immediately and 24 h after exercise, respectively, but the total numbers were not affected. The results suggest that decreased percentages and absolute numbers of T cells in submandibular LN following a single session of intense exercise may be partially mediated by increased corticosterone concentrations and may have consequences for ocular health among athletes.

Treadmill exercise in mice increases intestinal lymphocyte loss via apoptosis.

UNLABELLED: Strenuous exercise is associated with a transient decline in circulating lymphocytes, possibly through increased apoptosis. Intestinal lymphocytes are important effector cells of intestinal immune function but have not been studied in relation to exercise. AIM: The purpose of the present study was to examine the effect of exercise on intestinal lymphocyte phenotypes and apoptosis. METHODS: Female C57BL/6 mice (n = 112) were randomized to: (1) treadmill running (90 min, 32 m min-1, 8 degrees grade) and killed immediately after exercise, (2) treadmill running and killed 2 h after exercise, (3) treadmill running and killed 24 h after exercise or (4) a non-exercised control condition with exposure to treadmill noise and vibration without running. RESULTS: Flow cytometry indicated that the total intestinal CD3+T (P < 0.01), CD4+T (P < 0.005), CD8+T (P < 0.05), pan-NK (P < 0.005) and CD19+B (P < 0.05) lymphocytes were significantly lower 24 h after exercise compared with non-exercised controls. Significantly more CD3+T (P < 0.05) and CD8+T (P < 0.05) intestinal lymphocytes stained positive for annexin V, a marker of apoptosis, at 24 h after exercise compared with intestinal lymphocytes from non-exercised controls. Plasma corticosterone and 8-isoprostane concentrations were also significantly higher immediately after exercise compared with other exercise conditions. CONCLUSION: Acute strenuous exercise increases intestinal T (CD3+ and CD8+) lymphocyte loss and apoptosis. The extent to which the exercise-induced apoptosis in intestinal lymphocytes is mediated by increased glucocorticoid concentrations in the gastrointestinal tract will require further studies.

Physical activity and colon cancer. A systematic review of potential mechanisms.

Colorectal cancer is a multifactorial disease, with complex interactions between genetic and environmental factors. Physical activity has been shown to have a significant inverse relationship with colon cancer risk. Studies in experimental animals suggest that physical activity is protective in chemical carcinogenesis in the colon. Various mechanisms for this protective effect have been extensively cited throughout the literature, although few have been empirically tested. The purpose of this review was to review the published evidence on physical activity and the hypothesized mechanisms. A systematic review of the literature was conducted using the Medline database from 1970-2002, using the terms "colorectal cancer, colon cancer, neoplasm, carcinoma, exercise, fitness, and physical activity". This yielded 330 articles of which 23 review articles were searched for proposed mechanisms of this association. Following identification of possible mechanisms, additional searches were conducted to identify empirical studies in the exercise literature examining these mechanisms. These mechanisms include changes in gastrointestinal transit time, altered immune function and prostaglandin levels as well as changes in insulin levels, insulin-like growth factors, bile acid secretion, serum cholesterol and gastrointestinal and pancreatic hormone profiles. There is currently little empirical data to support any of the hypothesized biological mechanisms for the protective effect of exercise on colon cancer. Moreover, it is likely that no one mechanism is responsible for the risk reduction observed in epidemiological and animal studies and, therefore, the observed benefits of physical activity in colon cancer may be a combination of these and other factors. A greater understanding of the biological mechanisms involved will be an important step in developing exercise prescriptions targeted to reduce the burden of colon cancer across different populations.