Proliferation Of Skeletal Myoblasts Research Articles

Skeletal myoblast proliferation and differentiation are reduced in growth restricted fetal sheep

Fetal growth restriction (FGR) increases the risk of type 2 diabetes due in part to deficits in fetal skeletal muscle growth that are not fully compensated after birth. During prenatal development, muscle progenitor cells proliferate, differentiate, and fuse with existing fibers to support myofiber hypertrophy. The total number of myofibers is set in utero, and reductions in myofiber number, size, and myonuclear accretion suggest that myogenesis is impaired in FGR. Previous studies identify similar rates of proliferation and differentiation in FGR and control (CON) myoblasts exposed to enriched growth media in vitro, despite deficits in myoblast proliferation/differentiation in whole muscle of FGR fetuses. Whether muscle progenitor cells of FGR fetuses exhibit impaired myogenic capacity in vivo remains unknown. Using a novel flow cytometry approach to quantify stages of myogenesis in vivo, we aimed to identify the cellular origins of dysregulated skeletal muscle development in FGR due to placental insuffciency. Pregnant sheep were exposed to elevated temperatures (n=15) from early to mid-gestation to induce placental insuffciency and subsequent FGR, defined as birth weight 2 SD below the mean for gestational age. At 90% gestation, fetuses were weighed, and muscle progenitor cells were isolated from biceps femoris of FGR (n=8) and CON (n=5) fetuses for flow cytometry. Muscle cells were identified by anti-CD56, anti-Pax7, and live/dead dye. DAPI was used to quantify resting (G0/1) versus replicating (S/G2) myoblasts. Myogenic regulatory factors, anti-MyoD and anti-myogenin (MyoG), distinguished non-committed (MyoD-/MyoG-) and activated (MyoD+/MyoG-) myoblasts from those beginning (MyoD+/MyoG+) and completing (MyoD-/MyoG+) differentiation. Statistical significance was determined by Student’s t-tests with α=0.05. Percentages are relative to total myoblasts (live, singlets expressing Pax7/CD56). Total myoblasts per live singlets were not different between FGR and CON muscle. FGR fetuses had more muscle progenitors in G0/1 (97% vs 95%, p<0.0001) and fewer replicating myoblasts in S/G2 compared to CON (3% vs 5%, p<0.0001). Myofactor analysis found decreased activated myoblasts (MyoD+/MyoG-) in FGR versus CON (2.5% vs 5%, p<0.005), though the fraction of non-committed progenitors (MyoD-/MyoG-) was similar between groups (14% vs 15%). Despite reduced myogenic induction in FGR, there were no differences between FGR and CON myoblasts expressing MyoG in early (MyoD+, 38% vs 47%) or terminal differentiation (MyoD-, 47% vs 34%). Correlation analyses of all fetuses (n=15) revealed positive relationships between fetal weight and MyoD+/MyoG- (r=0.57) as well as MyoD+/MyoG+ myoblasts (r=0.56), while MyoD-/MyoG+ cells correlated negatively with fetal weight (r=-0.60). FGR fetuses have decreased myoblast proliferation in vivo. Myogenic lineage commitment is reduced in FGR progenitor cells, and positive correlations between fetal weight and myogenic induction suggest that deficits in muscle size and body mass may result from impaired initiation of myogenesis in utero. The elevated percentage of terminally differentiated myocytes in low birthweight fetuses may be a consequence of impaired fusion, which likely contributes to reductions in myonuclei within FGR myofibers. Since the capacity for growth is conserved in FGR skeletal muscle, indicated by retention of myoblast number and proliferation/differentiation capacity in vitro, therapies that stimulate myogenic induction and/or myoblast fusion during fetal development may be necessary/suffcient to restore muscle growth in FGR. NIH R01HD079404; Evonuk Graduate Fellowship; UO VPRI funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sheep recombinant IGF-1 promotes organ-specific growth in fetal sheep.

IGF-1 is a critical fetal growth-promoting hormone. Experimental infusion of an IGF-1 analog, human recombinant LR3 IGF-1, into late gestation fetal sheep increased fetal organ growth and skeletal muscle myoblast proliferation. However, LR3 IGF-1 has a low affinity for IGF binding proteins (IGFBP), thus reducing physiologic regulation of IGF-1 bioavailability. The peptide sequences for LR3 IGF-1 and sheep IGF-1 also differ. To overcome these limitations with LR3 IGF-1, we developed an ovine (sheep) specific recombinant IGF-1 (oIGF-1) and tested its effect on growth in fetal sheep. First, we measured in vitro myoblast proliferation in response to oIGF-1. Second, we examined anabolic signaling pathways from serial skeletal muscle biopsies in fetal sheep that received oIGF-1 or saline infusion for 2 hours. Finally, we measured the effect of fetal oIGF-1 infusion versus saline infusion (SAL) for 1 week on fetal body and organ growth, in vivo myoblast proliferation, skeletal muscle fractional protein synthetic rate, IGFBP expression in skeletal muscle and liver, and IGF-1 signaling pathways in skeletal muscle. Using this approach, we showed that oIGF-1 stimulated myoblast proliferation in vitro. When infused for 1 week, oIGF-1 increased organ growth of the heart, kidney, spleen, and adrenal glands and stimulated skeletal myoblast proliferation compared to SAL without increasing muscle fractional synthetic rate or hindlimb muscle mass. Hepatic and muscular gene expression of IGFBPs one to three was similar between oIGF-1 and SAL. We conclude that oIGF-1 promotes tissue and organ-specific growth in the normal sheep fetus.

A cholecystic extracellular matrix-based hybrid hydrogel for skeletal muscle tissue engineering.

Tailoring the properties of extracellular matrix (ECM) based hydrogels by conjugating with synthetic polymers is an emerging method for designing hybridhydrogels for a wide range of tissue engineering applications. In this study, poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer at variable concentrations (ranging from 0.2 to 2% wt/vol) was conjugated with porcine cholecyst derived ECM (C-ECM) (1% wt/vol) and prepared a biosynthetic hydrogel having enhanced physico-mechanical properties, as required for skeletal muscle tissue engineering. The C-ECM was functionalized with acrylate groups using activated N-hydroxysuccinimide ester-based chemistry and then conjugated with PEGDA via free-radical polymerization in presence of ammonium persulfate and ascorbic acid. The physicochemical characteristics of the hydrogels were evaluated by Fourier transform infrared spectroscopy and environmental scanning electron microscopy. Further, the hydrogel properties were studied by evaluating rheology, swelling, gelation time, percentage gel fraction, in vitro degradation, and mechanical strength. Biocompatibility of the gel formulations were assessed using the C2C12 skeletal myoblast cells. The hydrogel formulations containing 0.2 and 0.5% wt/vol of PEGDA were non-cytotoxic and found suitable for growth and proliferation of skeletal myoblasts. The study demonstrated a method for modulating the properties of ECM hydrogels through conjugation with bio-inert polymers for skeletal muscle tissue engineering applications.

MiR-222-3р Regulates the Proliferation and Differentiation of C2C12 Myoblasts by Targeting BTG2

MiR-222-3р has been implicated in tumor cell proliferation and has an important role in the differentiation and maturation of myogenic cells. However, its role in skeletal myoblast proliferation is still unclear. In this study, we found that miR-222-3р expression increases initially and then decreases during C2C12 myoblast proliferation. Using synthetic miRNA mimics and inhibitors in gain- or loss-of-function experiments, we snowed that miR-222-3р overexpression in C2C12 cells promotes myoblast proliferation and represses myofiber formation, while miR-222-3р downregulation has the opposite effect. Using a prediction program, BTG2 was identified as a possible target gene of miR-222-3р. During myogenesis, miR-222-3р mimics repress BTG2 expression, while miR-222-3р inhibitors promote BTG2 expression. Using dual-luciferase reporter assay, we further demonstrated that miR-222-3р specifically targets BTG2. Additionally, we show that siRNA-mediated downregulation of BTG2 expression in C2C12 myoblasts promotes the proliferation and suppresses differentiation. In conclusion, we provide a novel insight into the mechanism by which miR-222-3р regulates the proliferation and differentiation of C2C12 myoblasts by targeting BTG2. This information contributes to our understanding of the role of miRNAs in skeletal muscle development.

MiR-499 regulates myoblast proliferation and differentiation by targeting transforming growth factor β receptor 1.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs that play critical roles in muscle cell proliferation and differentiation via post-transcriptional regulation of gene expression. Here, based on our previous high-throughput sequencing results, we evaluated miRNA-499 (miR-499) functions during myoblast proliferation and differentiation. In addition, we analyzed miR-499 expression profiles and characterized the associated functional roles. MiR-499 is known to be a skeletal muscle fiber-type-associated miRNA. However, its roles in skeletal myoblast proliferation and differentiation are poorly understood. MiR-499 overexpression promoted C2C12 cell proliferation and significantly attenuated C2C12 cell myogenic differentiation. Furthermore, miR-499 inhibition enhanced C2C12 cell proliferation and suppressed C2C12 cell differentiation. Using dual-luciferase reporter assays and western blot analysis, we confirmed that miR-499 targeted transforming growth factor β receptor 1 (TGFβR1), a known regulator of skeletal myoblast development. Additionally, our RNA interference analysis, in which TGFβR1 was downregulated, showed that TGFβR1 significantly promoted the differentiation of C2C12 cells and inhibited their proliferation.

Correction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model

[This corrects the article DOI: 10.1371/journal.pone.0175807.].

Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

BackgroundSkeletal myoblast transplantation seems a promising approach for the repair of myocardial infarction (MI). However, the low engraftment efficacy and impaired angiogenic ability limit the clinical efficiency of the myoblasts. Gene engineering with angiogenic growth factors promotes angiogenesis and enhances engraftment of transplanted skeletal myoblasts, leading to improved infarction recovery in myocardial ischemia. The present study evaluated the therapeutic effects of hepatocyte growth factor (HGF) gene-engineered skeletal myoblasts on tissue regeneration and restoration of heart function in a rat MI model.Methods and resultsThe skeletal myoblasts were isolated, expanded, and transduced with adenovirus carrying the HGF gene (Ad-HGF). Male SD rats underwent ligation of the left anterior descending coronary artery. After 2 weeks, the surviving rats were randomized into four groups and treated with skeletal myoblasts by direct injection into the myocardium. The survival and engraftment of skeletal myoblasts were determined by real-time PCR and in situ hybridization. The cardiac function with hemodynamic index and left ventricular architecture were monitored; The adenovirus-mediated-HGF gene transfection increases the HGF expression and promotes the proliferation of skeletal myoblasts in vitro. Transplantation of HGF-engineered skeletal myoblasts results in reduced infarct size and collagen deposition, increased vessel density, and improved cardiac function in a rat MI model. HGF gene modification also increases the myocardial levels of HGF, VEGF, and Bcl-2 and enhances the survival and engraftment of skeletal myoblasts.ConclusionsHGF engineering improves the regenerative effect of skeletal myoblasts on MI by enhancing their survival and engraftment ability.

The Maintenance Ability and Ca2+ Availability of Skeletal Muscle are Enhanced by Sildenafil

Sildenafil relaxes vascular smooth muscle cells, and is used to treat pulmonary artery hypertension as well as erectile dysfunction. However, the effectiveness of sildenafil on skeletal muscle and the benefit of its clinical use have been controversial, and most studies have focused primarily on tissues and organs from disease models, without cellular examinations. Here, the effects of sildenafil on skeletal muscle at the cellular level were examined using mouse primary skeletal myoblasts (the proliferative forms of skeletal muscle stem cells) and myotubes along with single-cell Ca2+ imaging experiments and cellular and biochemical examinations. The proliferation of the skeletal myoblasts was enhanced by sildenafil, without dose-dependency. In the skeletal myotubes, sildenafil enhances the activity of ryanodine receptor 1, an internal Ca2+ channel, and Ca2+ movements that promote skeletal muscle contraction, possibly due to an increase in the resting cytosolic Ca2+ level and a unique microscopic shape in the myotube membranes. Therefore, these results suggest that the maintenance ability of skeletal muscle mass and the contractility of skeletal muscle could be improved by sildenafil via enhancing the proliferation of skeletal myoblasts and increasing the Ca2+ availability of skeletal myotubes, respectively.

MiR-696 Regulates C2C12 Cell Proliferation and Differentiation by Targeting CNTFRα.

Micro-696 (miR-696) has been previously known as an exercise related miRNA, which has a profound role in fatty acid oxidation and mitochondrial biogenesis of skeletal muscle. However, its role in skeletal myoblast proliferation and differentiation is still unclear. In this study, we found that miR-696 expressed highly in skeletal muscle and reduced during C2C12 myoblasts differentiation. MiR-696 overexpression repressed C2C12 myoblast proliferation and myofiber formation, while knockdown of endogenous miR-696 expression showed opposite results. During myogenesis, we observed an inversed expression pattern between miR-696 and CNTFRα in vitro, and demonstrated that miR-696 could specifically target CNTFRα and repress the expression of CNTFRα. Additionally, we further found that knockdown of CNTFRα suppressed the proliferation and differentiation of C2C12 cells. Taking all things together, we propose a novel insight that miR-696 down-regulates C2C12 cell myogenesis by inhibiting CNTFRα expression.

The maintenance ability and Ca2+ availability of skeletal muscle are enhanced by sildenafil.

Sildenafil relaxes vascular smooth muscle cells and is used to treat pulmonary artery hypertension as well as erectile dysfunction. However, the effectiveness of sildenafil on skeletal muscle and the benefit of its clinical use have been controversial, and most studies focus primarily on tissues and organs from disease models without cellular examination. Here, the effects of sildenafil on skeletal muscle at the cellular level were examined using mouse primary skeletal myoblasts (the proliferative form of skeletal muscle stem cells) and myotubes, along with single-cell Ca2+ imaging experiments and cellular and biochemical studies. The proliferation of skeletal myoblasts was enhanced by sildenafil in a dose-independent manner. In skeletal myotubes, sildenafil enhanced the activity of ryanodine receptor 1, an internal Ca2+ channel, and Ca2+ movement that promotes skeletal muscle contraction, possibly due to an increase in the resting cytosolic Ca2+ level and a unique microscopic shape in the myotube membranes. Therefore, these results suggest that the maintenance ability of skeletal muscle mass and the contractility of skeletal muscle could be improved by sildenafil by enhancing the proliferation of skeletal myoblasts and increasing the Ca2+ availability of skeletal myotubes, respectively.

An in silico prediction tool for the expansion culture ofhuman skeletal muscle myoblasts.

Regenerative therapy using autologous skeletal myoblasts requires a large number of cells to be prepared for high-level secretion of cytokines and chemokines to induce good regeneration of damaged regions. However, myoblast expansion culture is hindered by a reduction in growth rate owing to cellular quiescence and differentiation, therefore optimization is required. We have developed a kinetic computational model describing skeletal myoblast proliferation and differentiation, which can be used as a prediction tool for the expansion process. In the model, myoblasts migrate, divide, quiesce and differentiate as observed during in vitro culture. We assumed cell differentiation initiates following cell–cell attachment for a defined time period. The model parameter values were estimated by fitting to several predetermined experimental datasets. Using an additional experimental dataset, we confirmed validity of the developed model. We then executed simulations using the developed model under several culture conditions and quantitatively predicted that non-uniform cell seeding had adverse effects on the expansion culture, mainly by reducing the existing ratio of proliferative cells. The proposed model is expected to be useful for predicting myoblast behaviours and in designing efficient expansion culture conditions for these cells.

Sildenafil is Effective to Enhance the Proliferation of Skeletal Myoblasts

Sildenafil is a specific inhibitor of phosphodiesterase type 5 and is clinically used to treat erectile dysfunction and pulmonary artery hypertension because of its vasodilatation effect due to the relaxation of smooth muscle cells. In cardiac muscle, the cardioprotective effects of sildenafil have been reported. The effectiveness of sildenafil on skeletal muscle has been controversial: sildenafil reduces skeletal muscle fatigue, however, the atrophy of skeletal muscle by sildenafil is also reported. In addition, the effect of sildenafil on skeletal muscle in cellular levels has not been examined. In the present study, sildenafil effect on skeletal muscle cells was examined using mouse primary skeletal myoblasts. Sildenafil was effective to enhance the proliferation of the skeletal myoblasts.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment.

Nitrite circumvents canonical cGMP signaling to enhance proliferation of myocyte precursor cells.

Skeletal muscle tissue has a remarkable high regenerative capacity. The underlying cellular events are governed by complex signaling processes, and the proliferation of skeletal myoblasts is a key initial event. The role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, an NO oxidation product, is a stable source for NO-like bioactivity particularly in cases when oxygen shortage compromises NO-synthases activity. Although numerous studies suggest that nitrite effects are largely related to NO-dependent signaling, emerging evidence also implicates that nitrite itself can activate protein pathways albeit under physiological, normoxic conditions. This includes a recently demonstrated cyclic guanosine monophosphate-(cGMP)-independent enhancement of endothelial cell proliferation. Whether nitrite itself has the potential to affect myoblast proliferation and metabolism with or without activation of the canonical NO/cGMP pathway to subsequently support muscle cell regeneration is not known. Here we show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimida-zoline-1-oxyl-3 oxide and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP.

P29: Nitrite influences proliferation of myoblasts via its reduction to nitric oxide in skeletal muscle tissue

Skeletal muscle regeneration is governed by complex cellular signaling processes. Proliferation of skeletal myoblasts is a key initial event in muscle regeneration and has been largely related to mammalian target of rapamycin (mTOR) signal transduction mechanisms. Although the precise underlying signaling remains incompletely understood, the role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, the oxidation product of NO, is regarded as a stable source for NO-like bioactivity in cases when the oxygen-dependent NO-synthases become dysfunctional. Whether nitrite itself has the potential to affect myoblast proliferation and metabolic activity under normoxic conditions with or without activation of the NO/cyclic guanosine monophosphate (cGMP) pathway is not known. The influence of nitrite and NO on C 2 C 12 myoblast proliferation was observed in cultures grown in DMEM + 10% FCS at 37 °C/5% CO 2 , with or without sodium nitrite, NO-donor DETA-NONOate, NO-scavenger cPTIO, soluble guanylatecyclase (sGC) inhibitor ODQ, or mTOR inhibitor rapamycin. Neutral red staining, propodium iodide staining for cell cycle analysis, WST-8 viability assay, mTOR phosphorylation activity assay and intracellular cGMP measuerements were performed according to modified manufacturer’s protocols. We here show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently in normoxia. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR phosphorylation activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP. Nothing to disclose.

Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.

Endogenous erythropoietin signaling facilitates skeletal muscle repair and recovery following pharmacologically induced damage

Erythropoietin acts by binding to its cell surface receptor on erythroid progenitor cells to stimulate erythrocyte production. Erythropoietin receptor expression in nonhematopoietic tissue, including skeletal muscle progenitor cells, raises the possibility of a role for erythropoietin beyond erythropoiesis. Mice with erythropoietin receptor restricted to hematopoietic tissue were used to assess contributions of endogenous erythropoietin to promote skeletal myoblast proliferation and survival and wound healing in a mouse model of cardiotoxin induced muscle injury. Compared with wild-type controls, these mice had fewer skeletal muscle Pax-7(+) satellite cells and myoblasts that do not proliferate in culture, were more susceptible to skeletal muscle injury and reduced maximum load tolerated by isolated muscle. In contrast, mice with chronic elevated circulating erythropoietin had more Pax-7(+) satellite cells and myoblasts with increased proliferation and survival in culture, decreased muscle injury, and accelerated recovery of maximum load tolerated by isolated muscle. Skeletal muscle myoblasts also produced endogenous erythropoietin that increased at low O(2). Erythropoietin promoted proliferation, survival, and wound recovery in myoblasts via the phosphoinositide 3-kinase/AKT pathway. Therefore, endogenous and exogenous erythropoietin contribute to increasing satellite cell number following muscle injury, improve myoblast proliferation and survival, and promote repair and regeneration in this mouse induced muscle injury model independent of its effect on erythrocyte production.

Engraftment Is Optimal When Myoblasts Are Transplanted Early: The Role of Hepatocyte Growth Factor

In a recent clinical trial, skeletal myoblast (SKMB) transplantation performed late after myocardial infarction (MI) did not improve left ventricular function. We hypothesized that (1) delaying SKMB transplantation until a chronic infarct scar has developed reduces engraftment, and (2) hepatocyte growth factor (HGF), a main regulator of SKMBs, is present in acute but not chronic MI, potentially influencing engraftment. Rats underwent coronary artery ligation followed by SKMB transplantation immediately (n = 12) or delayed by 5 weeks (n = 11). The volume of engrafted SKMBs was quantified 6 weeks later. Hepatocyte growth factor was evaluated by computerized analysis of immunohistochemical labeling of rat heart sections 48 hours, 1 week, 2 weeks, and 5 weeks after coronary artery ligation. The impact of HGF on SKMB proliferation and its ability to protect against oxidative stress and hypoxia was evaluated in vitro. Skeletal myoblast transplantation immediately after MI resulted in an engraftment volume of 29.1 +/- 2.9 mm(3). However, delaying SKMB transplantation 5 weeks caused a 95% drop in engraftment (1.4 +/- 0.3 mm(3); p < 0.001). Hepatocyte growth factor labeling in MIs 48 hours after coronary artery ligation was similar to control myocardium (18.0 +/- 2.0 versus 16.8 +/- 1.3 units). However, HGF declined progressively at 1, 2, and 5 weeks after MI (9.1 +/- 1.4, 4.2 +/- 0.4, and 3.1 +/- 0.6 units, respectively; p < 0.05 versus 48 hours). Hepatocyte growth factor caused a dose-dependent increase in SKMB proliferation in vitro and protected against oxidative stress and hypoxia. These results demonstrate that engraftment of SKMBs is impaired when transplantation is delayed until a chronic infarct has developed. Hepatocyte growth factor in MI declines with time and may enhance engraftment of SKMBs transplanted early after MI. Delivery of exogenous HGF to enhance SKMB engraftment in chronic infarcts warrants further investigation.

Dynamics and mediators of acute graft attrition after myoblast transplantation to the heart

Survival and proliferation of skeletal myoblasts within the cardiac environment are crucial to the therapeutic efficacy of myoblast transplantation to the heart. We have analyzed the early dynamics of myoblasts implanted into the myocardium and investigated the mechanisms underlying graft attrition. At 10 min after implantation of [14C]thymidine-labeled male myoblasts into female mice hearts, 14C measurement showed that 39.2 +/- 3.0% of the grafted cells survived, and this steadily decreased to 16.0 +/- 1.7% by 24 h and to 7.4 +/- 0.9% by 72 h. PCR of male-specific Smcy gene calculated that the total (surviving plus proliferated) number of donor-derived cells was 18.3 +/- 1.6 and 23.3 +/- 1.3% at 24 and 72 h, respectively, indicating that proliferation of the surviving cells began after 24 h. Acute inflammation became prominent by 24 h and was reduced by 72 h as indicated by myeloperoxidase activity and histological findings. Multiplex RT-PCR revealed corresponding changes in IL-1beta, TGF-beta, IL-6, and TNF-alpha expression. Treatment with CuZn-superoxide dismutase attenuated the initial rapid death and resulted in enhanced cell numbers afterward, giving a twofold increased total number at 72 h compared with the nontreatment. This effect was associated with reduced inflammatory response, suggesting a causative role for superoxide in the initial rapid graft death and subsequent inflammation. These data describe the early dynamics of myoblasts implanted into the myocardium and suggest that initial oxidative stress and following inflammatory response may be important mechanisms contributing to acute graft attrition, both of which could be potential therapeutic targets to improve the efficiency of cell transplantation to the heart.

Overexpression of Interleukin-15 Induces Skeletal Muscle Hypertrophy in Vitro: Implications for Treatment of Muscle Wasting Disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and α-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.