Optimizing mechanical stretching protocols for hypertrophic and anti-apoptotic responses in cardiomyocyte-like H9C2 cells.

Cardiomyocytes possess the ability to respond to mechanical stimuli by reprogramming their gene expression. This study investigated the effects of different loading protocols on signaling and expression responses of myogenic, anabolic, inflammatory, atrophy and pro-apoptotic genes in cardiomyocyte-like H9C2 cells. Differentiated H9C2 cells underwent various stretching protocols by altering their elongation, frequency and duration, utilizing an in vitro cell tension system. The loading-induced expression changes of MyoD, Myogenin, MRF4, IGF-1 isoforms, Atrogin-1, Foxo1, Fuca and IL-6 were measured by Real Time-PCR. The stretching-induced activation of Akt and Erk 1/2 was also evaluated by Western blot analysis. Low strain (2.7% elongation), low frequency (0.25 Hz) and intermediate duration (12 h) stretching protocol was overall the most effective in inducing beneficial responses, i.e., protein synthesis along with the suppression of apoptosis, inflammation and atrophy, in the differentiated cardiomyocytes. These findings demonstrated that varying the characteristics of mechanical loading applied on H9C2 cells in vitro can regulate their anabolic/survival program.

The Effects of Muscle Cell Aging on Myogenesis.

The process of myogenesis gradually deteriorates as the skeletal muscle ages, contributing to muscle mass loss. The aim of this study is to investigate the effect of senescence/aging on skeletal myogenesis, in vitro. A model of multiple cell divisions of C2C12 myoblasts was used to replicate cell senescence. Control and aged myoblasts were investigated during myogenesis, i.e., at days 0, 2, and 6of differentiation. SA-ß-gal activity and comet assay were used as markers of aging and DNA damage. Flow cytometry was performed to characterize potential differences in cell cycle between control and aged cells. Alterations in the mRNA and/or protein expression of myogenic regulatory factors (MRFs), IGF-1 isoforms, apoptotic, atrophy, inflammatory, metabolic and aging-related factors were evaluated. Compared with the control cells, aged myoblasts exhibited G0/G1 cell cycle arrest, DNA damage, increased SA-ß-gal activity, and increased expression of aging-related factors p16 and p21 during differentiation. Moreover, aged myoblasts showed a reduction in the expression of MRFs and metabolic/anabolic factors, along with an increased expression of apoptotic, atrophy and inflammatory factors. A diminished differentiation capacity characterized the aged myoblasts which, in combination with the induction of apoptotic and atrophy factors, indicated a disrupted myogenic lineage in the senescent muscle cells.

Differential Expression of Insulin Growth Factor 1 (IGF-1) Isoforms in Different Types of Endometriosis: Preliminary Results of a Single-Center Study.

Endometriosis is a benign, estrogen-dependent gynecological condition with an uncertain exact pathogenetic mechanism. The aim of this study was to evaluate the potential differential expression of Insulin Growth Factor 1 (IGF-1) isoforms in deeply infiltrating endometriotic (DIE) lesions, in ovarian endometriomas, and in the eutopic endometrium of the same endometriosis patients and to compare their expression with that in the eutopic endometrium of women without endometriosis. A total of 39 patients were included: 28 with endometriosis, of whom 15 had endometriomas only, 7 had DIE nodules only, and 6 had both DIE and endometriomas, and 11 without endometriosis served as controls. We noticed a similar pattern of expression between IGF-1Ea and IGF-1Ec, which differed from that of the IGF-1Eb isoform, possibly implying differential biological actions of different isoforms in DIE subtypes. We observed a tendency of lower expression of IGF-1Ea and IGF-1Ec in endometriomas without DIE compared to endometriomas with concurrent DIE or in DIE nodules. In conclusion, differential expression of IGF-1 isoforms may indicate that DIE with its associated ovarian lesions and simple ovarian endometriosis should be considered as two forms of the disease developing under different molecular pathways.

Gonadotropin-Releasing Hormone (GnRH)/GnRH Receptors and Their Role in the Treatment of Endometriosis.

Endometriosis, defined as the development of endometrial tissue outside of the uterine cavity, is a common gynecological disorder. The prevalence of pelvic endometriosis approaches 6%-10% in the general female population, and in women with pain, infertility, or both, the frequency is 35%-50%. The gold standard recommended process for diagnosing endometriosis is laparoscopy, an invasive surgical procedure, with or without histologic verification. The currently available nonsurgical treatments include oral contraceptives (estrogen-progestogen preparations), progestogen preparations (containing progesterone derivatives), androgenic hormones (danazol), and gonadotropin-releasing hormone (GnRH) agonists and antagonists. Two GnRH types have been discovered in mammals, GnRH I and GnRH II. In particular, GnRH I is released by the hypothalamus; however, it can be present in various tissues and organs of the body, including neural tissue, where it exerts neuroendocrine, autocrine, and paracrine actions in the peripheral and central nervous system (CNS). Interestingly, another GnRH isoform, GnRH III, has been identified, which has 60% similarity with GnRH I from which it varies by four amino acids. This peptide has been shown to have a significant role in reproduction, specifically in gametogenesis and steroidogenesis. Further research is needed to identify innovative treatment options for endometriosis, such as the therapeutic exogenous administration of GnRH II or antagonists of the GnRH I receptor. In this review, we examined the role of GnRH in endometriosis, outlining the specific actions of GnRH and GnRH receptors (GnRHRs). The innovative use of GnRH analogs and antagonists in the treatment of endometriosis is also discussed.

What is the role of CHCHD2 in adrenal tumourigenesis?

PURPOSE: CHCHD2 is an antiapoptotic mitochondrial protein acting through the BCL2/BAX pathway in various cancers. However, data on the regulatory role of CHCHD2 in adrenal tumourigenesis are scarce. METHODS: We studied the expression of CHCHD2, BCL2, and BAX in human adrenocortical tissues and SW13 cells. mRNA and protein levels were analyzed through qPCR and immunoblotting, respectively, in 16 benign adrenocortical neoplasms (BANs), along with their adjacent normal adrenal tissues (controls), and 10 adrenocortical carcinomas (ACCs). BCL2/BAX mRNA expression was also analyzed in SW13 cells after CHCHD2 silencing. MTS, flow cytometry and scratch assays were performed to assess cell viability, apoptosis, and invasion, respectively. RESULTS: BCL2 and CHCHCD2 mRNA and protein expression was increased in BANs compared to normal adrenal tissues whereas BAX was decreased. BAX and CHCHD2 mRNA and protein levels were significantly downregulated and upregulated, respectively, in ACCs compared with either BANs or controls. Expression of the studied genes was not different among cortisol-secreting and nonfunctional ACAs. No significant association was found between genes' expression and other established prognostic markers of ACCs patients. In vitro analysis showed that CHCHD2 silencing resulted in reduced cell viability and invasion as well as increased SW13 cells apoptosis. CONCLUSIONS: CHCHD2 expression seems to be implicated in adrenal tumourigenesis and its absence resulted to increased apoptosis in vitro. However, the exact mechanism of action and particularly its association with the BAX/BCL2 pathway needs to be further studied and evaluate whether it could be a protentional therapeutic target.

The Effects of Mechanical Loading Variations on the Hypertrophic, Anti-Apoptotic, and Anti-Inflammatory Responses of Differentiated Cardiomyocyte-like H9C2 Cells.

Cardiomyocytes possess the ability to respond to mechanical stimuli by adapting their biological functions. This study investigated cellular and molecular events in cardiomyocyte-like H9C2 cells during differentiation as well as the signalling and gene expression responses of the differentiated cells under various mechanical stretching protocols in vitro. Immunofluorescence was used to monitor MyHC expression and structural changes during cardiomyoblast differentiation. Moreover, alterations in the expression of cardiac-specific markers, cell cycle regulatory factors, MRFs, hypertrophic, apoptotic, atrophy and inflammatory factors, as well as the activation of major intracellular signalling pathways were evaluated during differentiation and under mechanical stretching of the differentiated H9C2 cells. Compared to undifferentiated cells, advanced-differentiation cardiomyoblasts exhibited increased expression of cardiac-specific markers, MyHC, MRFs, and IGF-1 isoforms. Moreover, differentiated cells that underwent a low strain/frequency mechanical loading protocol of intermediate duration showed enhanced expression of MRFs and hypertrophic factors, along with a decreased expression of apoptotic, atrophy, and inflammatory factors compared to both high-strain/frequency loading protocols and to unloaded cells. These findings suggest that altering the strain and frequency of mechanical loading applied on differentiated H9C2 cardiomyoblasts can regulate their anabolic/survival program, with a low-strain/frequency stretching being, overall, most effective at inducing beneficial responses.

Effect of Mechanical Loading of Senescent Myoblasts on Their Myogenic Lineage Progression and Survival.

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.

Characterization of Optimal Strain, Frequency and Duration of Mechanical Loading on Skeletal Myotubes' Biological Responses.

BACKGROUND/AIM: Mechanical loading of differentiated myoblasts in vitro may mimic loading patterns of skeletal muscle in vivo. However, it is still uncharacterized the loading conditions that can produce the most effective muscle cells' biological responses, in vitro. This study investigated the effects of different loading protocols on the expression of myogenic regulatory factors, anabolic, atrophy and pro-apoptotic factors in skeletal myotubes. MATERIALS AND METHODS: C2C12 myoblasts were differentiated and underwent various stretching protocols by altering their elongation, frequency and duration, utilizing an in vitro cell tension system. The loading-induced expression changes of MyoD, Myogenin, MRF4, IGF-1 isoforms, Murf1, Atrogin, Myostatin, Foxo and Fuca were measured by Real Time-PCR. RESULTS: Stretching by 2% elongation at 0.25 Hz for 12 h was overall the most effective in inducing beneficial responses. CONCLUSION: A low strain, low frequency intermediate duration stretching can most effectively up-regulate myogenic/anabolic factors and down-regulate pro-apoptotic and atrophy genes in myotubes.