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Background: During aging, skeletal muscle mass constantly diminishes and myogenic potential declines. At the cellular level, a decline in mitochondrial function is a hallmark of the aging process and the deficiency of the mitochondrial network contributes to a progressive reduction in muscle mass. Autophagic clearance of mitochondria through the process of mitophagy is required to remove impaired or damaged mitochondria, while mitophagy is a key regulator of muscle maintenance. Dysfunctional degradation of mitochondria is increasingly associated with aging (mitophaging), while mechanical stimuli have been shown to ameliorate the aging-induced impaired muscle mass and function; however, less is known about the potential effects of mechanical loading on mitophaging. The aim of the present study was to investigate the effect of mechanical stretching on mitophagy in aged myoblasts, in vitro.

Methods: Cell senescence was replicated using a multiple cell division model of C2C12 myoblasts. The control and aged cells were cultured on elastic membranes and underwent passive stretching using a mechanical loading protocol of 15% elongation for 12 h at a frequency of 1 Hz. Cell signaling and gene expression responses of mitophagy-associated and myogenic regulatory factors (MRFs) were assessed through immunoblotting and qRT-PCR of the cell lysates derived from stretched and non-stretched control and aged myoblasts.

Results: Mitophagy factor AMP-activated protein kinase (AMPK), mitochondrial biogenesis stimulator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), and mitophagy/mitochondrial biogenesis factor Parkin were downregulated in control stretched myoblasts compared to non-stretched cells, while the specific mechanical loading protocol used also reduced the phosphorylation of unc-51-like autophagy-activating kinase 1 (p-ULK1) (p < 0.05), as well as the expression of myogenic factor 5 (Myf5) and myogenic factor 4 (myogenin) (p < 0.001). Interestingly, this mechanical loading resulted in increased PGC-1a and Parkin expression (p < 0.05) and induced the previously undetected BCL2 interacting protein 3-like (BNIP3L/NIX) and AMPK expression and p-ULK1 activation in the aged myoblasts. In addition, mechanical stretching differentially affected the expression of MRFs in aged cells, upregulating the early differentiation factor, Myf5 (p < 0.01), while downregulating the late differentiation factor myogenin (p < 0.001).

Conclusions: These findings suggest the beneficial effects of mechanical loading on the impaired mitophagy and early differentiation in aged myoblasts, as indicated by the mitophagy initiation and the promotion of mitochondrial biogenesis in these cells. The mechanical loading-induced downregulation of mitophagy and myogenesis in the control myoblasts might indicate their loading-specific differential responses compared to the aged cells.

Background: Regular exercise training provides significant health benefits among cancer survivors and is associated with lower breast cancer mortality and reduced risk of recurrence. Both exercise-induced factors secreted into circulation (exerkines) and bioactive molecules contained in skeletal muscle secretome have been proposed to affect the tumor microenvironment and mediate some of the anti-carcinogenic effects of exercise. This study utilized exercise-conditioned human serum obtained from breast cancer patients during chemotherapy and skeletal myotubes’ secretome after mechanical loading to investigate their effects on breast cancer cells in vitro.

Methods: Breast cancer patients participated in a 12-week exercise training program during their chemotherapy, and blood serum was collected immediately before and after an exercise session in the 2nd and 12th weeks of training. Skeletal myoblasts were differentiated into myotubes and subjected to mechanical stretching to collect their secretome (stretch medium (SM)). Hormone-sensitive Michigan Cancer Foundation-7 (MCF-7) and triple-negative M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cells were treated with either human serum or with the skeletal myotubes’ secretome to examine their metabolic activity, migration, cytotoxicity levels and apoptosis regulation.

Results: The exercise-conditioned serum obtained from breast cancer patients who were subjected to the 12-week training during chemotherapy resulted in reduced metabolic activity (p < 0.001) and increased lactate dehydrogenase activity (cytotoxicity) (p < 0.001) in both MCF-7 and MDA-MB-231 breast cancer cells when compared with the control condition. Moreover, incubation of breast cancer cells with the post-exercise serum induced apoptosis in MCF-7 and MDA-MB-231 cells, as indicated by increase in DNA damage and the percentage of necrotic cells (p < 0.05) when compared to pre-exercise condition. Similarly, a significant decrease (p < 0.001) was observed in the metabolic activity of MCF-7 cells treated with the SM, along with increased cytotoxicity (p < 0.05), compared to the cells cultured with the regular growth media. Comparable though not as profound effects were observed in MDA-MB-231 cells when treated with the SM secretome. Furthermore, the expression of apoptosis-inducing Caspase-7 (p < 0.001) and Caspase-8 (p < 0.01) proteins was increased, whereas cell survival-regulating factors interleukin-8 (IL-8) (p < 0.001), superoxide dismutase-2 (p < 0.05), Fas cell surface death receptor (p < 0.05), and vascular endothelial growth factor (p < 0.01) were downregulated in the SM-treated MCF-7 cells. In addition, the migrating behavior of MCF-7 cells was diminished, and higher levels of DNA damage were observed in cells treated with either SM or non-stretch media.

Conclusion: Both exercise-conditioned serum of breast cancer patients and skeletal myotubes secretome after mechanical loading can reduce the metabolic activity, promote cell toxicity and DNA damage, modulate the protein expression of crucial cell survival-regulating factors, and lead to apoptosis in breast cancer cells. These findings suggest that even after cancer diagnosis, exercise may exert beneficial effects additive to chemotherapy against breast cancer prognosis.

Purpose: To develop an in vitro model that mimics aspects of corneal healing in humans for uncovering key mechanisms involved in the mechanisms involved in the healing and scarring processes.

Methods: As part of the healing matrix, TGF-β1–induced and corneal-derived myofibroblasts were cultured in fibrin hydrogels with configurations that recapitulate the healthy (aligned) and wounded (random) microenvironment of the cornea.

Results: Evaluation of cellular alpha smooth muscle actin (α-SMA) and collagen hybridizing peptide (CHP) showed cell and matrix alignment, respectively. The aligned compared to the random constructs demonstrated an increased ability to synthesize total soluble proteins, including collagen type V, but collagen type I levels were reduced. This finding reveals a differential pattern for these proteins. Additionally, the collagen fibril diameters were larger in the aligned tissue constructs compared to the random constructs. Fibronectin and CHP colocalization patterns did not differ between groups; however, fibronectin and decorin were increased in the aligned group in contrast to tenascin C, which showed no difference.

Conclusions: These findings suggest that the alignment of the healing microenvironment plays a crucial role in modulating the structural properties of the extracellular matrix (ECM) and regulates the synthesis of key proteins that are closely involved in fibrillogenesis and are indicative of the quality of the deposited ECM.

Translational Relevance: We developed a three-dimensional in vitro model that closely mimics in vivo conditions to investigate the role of corneal myofibroblasts in healing and regeneration. Ultimately, this model can help develop targeted antifibrotic therapies to prevent corneal scarring.

Abdominal hernia is a protruding weakness in the abdominal wall. It affects abdominal strength and life quality and can lead to complications due to intestinal entrapment. Autologous full-thickness skin graft (FTSG) has recently become an alternative material for reinforcement in the surgical repair of large abdominal hernias instead of synthetic mesh. FTSG eventually integrates with the abdominal wall, but the long-term fate of the graft itself is not fully understood. This has implications as to how these grafts should be optimally used and handled intraoperatively. This study investigates the remodeling of FTSG in either the onlay or the intraperitoneal position 8 weeks after FTSG transplantation in an experimental mouse model. There was a significant presence of fibroblasts, indicated by vimentin and S100A4 staining, but there were significant variations among animals as to how much of the graft had been remodeled into dense connective tissue. This correlated significantly with the proportion of vimentin-positive cells in the dense connective tissue. We also found that collagen hybridizing peptide staining intensity, a marker of active remodeling, was significantly associated with the proportion of S100A4-positive cells in the dense connective tissue of the FTSG.

Late-onset Fuchs endothelial corneal dystrophy (FECD) is a disease affecting the corneal endothelium (CE), associated with a cytosine-thymine-guanine repeat expansion at the CTG18.1 locus in the transcription factor 4 (TCF4) gene. It is unknown whether CTG18.1 expansions affect global methylation including TCF4 gene in CE or whether global CE methylation changes at advanced age. Using genome-wide DNA methylation array, we investigated methylation in CE from FECD patients with CTG18.1 expansions and studied the methylation in healthy CE at different ages. The most revealing DNA methylation findings were analyzed by gene expression and protein analysis. 3488 CpGs had significantly altered methylation pattern in FECD though no substantial changes were found in TCF4. The most hypermethylated site was in a predicted promoter of aquaporin 1 (AQP1) gene, and the most hypomethylated site was in a predicted promoter of coagulation factor V (F5 for gene, FV for protein). In FECD, AQP1 mRNA expression was variable, while F5 gene expression showed a ~ 23-fold increase. FV protein was present in both healthy and affected CE. Further gene expression analysis of coagulation factors interacting with FV revealed a ~ 34-fold increase of thrombomodulin (THBD). THBD protein was detected only in CE from FECD patients. Additionally, we observed an age-dependent hypomethylation in elderly healthy CE.Thus, tissue-specific genome-wide and gene-specific methylation changes associated with altered gene expression were discovered in FECD. TCF4 pathological methylation in FECD because of CTG18.1 expansion was ruled out.

Sarcopenic obesity (SO) constitutes the coexistence of skeletal muscle mass loss (sarcopenia) and excess adiposity (obesity). It is mainly considered as a condition in the elderly with health-threatening impacts ranging from frailty to mortality. Mitochondrial dysfunction consists one of the basic pathophysiological mechanisms leading to the development of SO and its consequences. Indirect indicators of mitochondrial function, such as VO2max and exercise capacity, have been demonstrated to be negatively affected in individuals with SO, while the positive effect of exercise on mitochondrial function has been widely proved; thus, in this review, we aimed at investigating the effects of endurance, resistance, and concurrent exercise training on indexes of mitochondrial dysfunction in SO patients. The results of the clinical trials evaluated reveal positive effects of chronic exercise on VO2max and physical capacity, as well as mitochondrial biogenesis and activity. It has been concluded that utilizing a systematic exercise training program that includes both aerobic and strength exercises can be an effective strategy for managing SO and promoting overall health in these patients.

One of the most common adverse effects of cancer and its therapeutic strategies is sarcopenia, a condition which is characterised by excess muscle wasting and muscle strength loss due to the disrupted muscle homeostasis. Moreover, cancer-related sarcopenia may be combined with the increased deposition of fat mass, a syndrome called cancer-associated sarcopenic obesity. Both clinical conditions have significant clinical importance and can predict disease progression and survival. A growing body of evidence supports the claim that physical exercise is a safe and effective complementary therapy for oncology patients which can limit the cancer- and its treatment-related muscle catabolism and promote the maintenance of muscle mass. Moreover, even after the onset of sarcopenia, exercise interventions can counterbalance the muscle mass loss and improve the clinical appearance and quality of life of cancer patients. The aim of this narrative review was to describe the various pathophysiological mechanisms, such as protein synthesis, mitochondrial function, inflammatory response, and the hypothalamic–pituitary–adrenal axis, which are regulated by exercise and contribute to the management of sarcopenia and sarcopenic obesity. Moreover, myokines, factors produced by and released from exercising muscles, are being discussed as they appear to play an important role in mediating the beneficial effects of exercise against sarcopenia.

Background: During aging, muscle cell apoptosis increases and myogenesis gradually declines. The impaired myogenic and survival potential of the aged skeletal muscle can be ameliorated by its mechanical loading. However, the molecular responses of aged muscle cells to mechanical loading remain unclear. This study examined the effect of mechanical loading of aged, proliferating, and differentiated myoblasts on the gene expression and signaling responses associated with their myogenic lineage progression and survival. Methods: Control and aged C2C12 cells were cultured on elastic membranes and underwent passive stretching for 12 h at a low frequency (0.25 Hz) and different elongations, varying the strain on days 0 and 10 of myoblast differentiation. Activation of ERK1/2 and Akt, and the expression of focal adhesion kinase (FAK) and key myogenic regulatory factors (MRFs), MyoD and Myogenin, were determined by immunoblotting of the cell lysates derived from stretched and non-stretched myoblasts. Changes in the expression levels of the MRFs, muscle growth, atrophy, and pro-apoptotic factors in response to mechanical loading of the aged and control cells were quantified by real-time qRT-PCR. Results: Mechanical stretching applied on myoblasts resulted in the upregulation of FAK both in proliferating (day 0) and differentiated (day 10) cells, as well as in increased phosphorylation of ERK1/2 in both control and aged cells. Moreover, Akt activation and the expression of early differentiation factor MyoD increased significantly after stretching only in the control myoblasts, while the late differentiation factor Myogenin was upregulated in both the control and aged myoblasts. At the transcriptional level, mechanical loading of the proliferating myoblasts led to an increased expression of IGF-1 isoforms and MRFs, and to downregulation of muscle atrophy factors mainly in control cells, as well as in the upregulation of pro-apoptotic factors both in control and aged cells. In differentiated cells, mechanical loading resulted in an increased expression of the IGF-1Ea isoform and Myogenin, and in the downregulation of atrophy and pro-apoptotic factors in both the control and aged cells. Conclusions: This study revealed a diminished beneficial effect of mechanical loading on the myogenic and survival ability of the senescent muscle cells compared with the controls, with a low strain (2%) loading being most effective in upregulating myogenic/anabolic factors and downregulating atrophy and pro-apoptotic genes mainly in the aged myotubes.

It is known that mechanical loading of muscles increases the strength of healing tendon tissue, but the mechanism involved remains elusive. We hypothesized that the secretome from myoblasts in co-culture with tenocytes affects tenocyte migration, cell phenotype, and collagen (Col) production and that the effect is dependent on different types of mechanical loading of myoblasts. To test this, we used an in vitro indirect transwell co-culture system. Myoblasts were mechanically loaded using the FlexCell® Tension system. Tenocyte cell migration, proliferation, apoptosis, collagen production, and several tenocyte markers were measured. The secretome from myoblasts decreased the Col I/III ratio and increased the expression of tenocyte specific markers as compared with tenocytes cultured alone. The secretome from statically loaded myoblasts significantly enhanced tenocyte migration and Col I/III ratio as compared with dynamic loading and controls. In addition, the secretome from statically loaded myoblasts induced tenocytes towards a myofibroblast-like phenotype. Taken together, these results demonstrate that the secretome from statically loaded myoblasts has a profound influence on tenocytes, affecting parameters that are related to the tendon healing process.