Cardiotoxin Injection Research Articles

Statin Therapy Negatively Impacts Skeletal Muscle Regeneration and Cutaneous Wound Repair in Type 1 Diabetic Mice.

Those with diabetes invariably develop complications including cardiovascular disease (CVD). To reduce their CVD risk, diabetics are generally prescribed cholesterol-lowering 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors (i.e., statins). Statins inhibit cholesterol biosynthesis, but also reduce the synthesis of a number of mevalonate pathway intermediates, leading to several cholesterol-independent effects. One of the pleiotropic effects of statins is the reduction of the anti-fibrinolytic hormone plasminogen activator inhibitor-1 (PAI-1). We have previously demonstrated that a PAI-1 specific inhibitor alleviated diabetes-induced delays in skin and muscle repair. Here we tested if statin administration, through its pleiotropic effects on PAI-1, could improve skin and muscle repair in a diabetic rodent model. Six weeks after diabetes onset, adult male streptozotocin-induced diabetic (STZ), and WT mice were assigned to receive control chow or a diet enriched with 600 mg/kg Fluvastatin. Tibialis anterior muscles were injured via Cardiotoxin injection to induce skeletal muscle injury. Punch biopsies were administered on the dorsal scapular region to induce injury of skin. Twenty-four days after the onset of statin therapy (10 days post-injury), tissues were harvested and analyzed. PAI-1 levels were attenuated in statin-treated diabetic tissue when compared to control-treated tissue, however no differences were observed in non-diabetic tissue as a result of treatment. Muscle and skin repair were significantly attenuated in Fluvastatin-treated STZ-diabetic mice as demonstrated by larger wound areas, less mature granulation tissue, and an increased presence of smaller regenerating muscle fibers. Despite attenuating PAI-1 levels in diabetic tissue, Fluvastatin treatment impaired cutaneous healing and skeletal muscle repair in STZ-diabetic mice.

Impaired regeneration in calpain-3 null muscle is associated with perturbations in mTORC1 signaling and defective mitochondrial biogenesis

BackgroundPrevious studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle.MethodsTo gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle.ResultsAt 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers.ConclusionOur findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy.

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle

BackgroundTransplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT.MethodPrimary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury.ResultsLi-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1.ConclusionTreatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro.

Notch1 and Notch2 Coordinately Regulate Stem Cell Function in the Quiescent and Activated States of Muscle Satellite Cells.

Satellite cells, the muscle tissue stem cells, express three Notch receptors (Notch1-3). The function of Notch1 and Notch2 in satellite cells has to date not been fully evaluated. We investigated the role of Notch1 and Notch2 in myogenic progression in adult skeletal muscle using tamoxifen-inducible satellite cell-specific conditional knockout mice for Notch1 (N1-scKO), Notch2 (N2-scKO), and Notch1/Notch2 (scDKO). In the quiescent state, the number of satellite cells was slightly reduced in N2-scKO, but not significantly in N1-scKO, and almost completely depleted in scDKO mice. N1-scKO and N2-scKO mice both exhibited a defect in muscle regeneration induced by cardiotoxin injection, while muscle regeneration was severely compromised with marked fibrosis in scDKO mice. In the activated state, ablation of either Notch1 or Notch2 alone in satellite cells prevented population expansion and self-renewal but induced premature myogenesis. Therefore, our results indicate that Notch1 and Notch2 coordinately maintain the stem-cell pool in the quiescent state by preventing activation and regulate stem-cell-fate decision in the activated state, governing adult muscle regeneration. Stem Cells 2018;36:278-285.

Loss of MKP-5 promotes myofiber survival by activating STAT3/Bcl-2 signaling during regenerative myogenesis

BackgroundThe mitogen-activated protein kinases (MAPKs) have been shown to be involved in regulating myofiber survival. In skeletal muscle, p38 MAPK and JNK are negatively regulated by MAPK phosphatase-5 (MKP-5). During muscle regeneration, MKP-5 is downregulated, thereby promoting p38 MAPK/JNK signaling, and subsequent repair of damaged muscle. Mice lacking MKP-5 expression exhibit enhanced regenerative myogenesis. However, the effect of MKP-5 on myofiber survival during regeneration is unclear.MethodsTo investigate whether MKP-5 is involved in myofiber survival, skeletal muscle injury was induced by cardiotoxin injection, and the effects on apoptosis were assessed by TUNEL assay in wild type and MKP-5-deficient mice. The contribution of MKP-5 to apoptotic signaling and its link to this pathway through mitochondrial function were determined in regenerating skeletal muscle of MKP-5-deficient mice.ResultsWe found that loss of MKP-5 in skeletal muscle resulted in improved myofiber survival. In response to skeletal muscle injury, loss of MKP-5 decreased activation of the mitochondrial apoptotic pathway involving the signal transducer and activator of transcription 3 (STAT3) and increased expression of the anti-apoptotic transcription factor Bcl-2. Skeletal muscle of MKP-5-deficient mice also exhibited an improved anti-oxidant capacity as a result of increased expression of catalase further contributing to myofiber survival by attenuating oxidative damage.ConclusionsTaken together, these findings suggest that MKP-5 coordinates skeletal muscle regeneration by regulating mitochondria-mediated apoptosis. MKP-5 negatively regulates apoptotic signaling, and during regeneration, MKP-5 downregulation contributes to the restoration of myofiber survival. Finally, these results suggest that MKP-5 inhibition may serve as an important therapeutic target for the preservation of skeletal muscle survival in degenerative muscle diseases.

Calcium/Calmodulin-Dependent Protein Kinase IV (CaMKIV) Mediates Acute Skeletal Muscle Inflammatory Response

The objective of this study is to investigate the role of Calmodulin-dependent protein kinase IV (CaMKIV) in Cardiotoxin (CTX)-induced mice muscle inflammation. CTX injection i.m. was performed to induce B6 mice acute tibialis anterior (TA) muscle injury. The mice were then injected i.p. with the recombinant CaMKIV protein or its antagonist KN-93. Immunoblotting was used to assess Calmodulin (CaM) and CaMKIV levels. Immunofluorescence was used to detect intramuscular infiltration or major histocompatibility complex (MHC)-I expression in damaged muscle. The extent of infiltration was evaluated by fluorescent intensity analysis. Cytokines/chemokines levels were determined by qPCR. CaMKIV gene knockdown in C2C12 cells was performed in order to evaluate the effects of CaMKIV on immuno-behavior of muscle cells. CTX administration induced a strong up-regulation of CaM and p-CaMKIV levels in infiltrated mononuclear cells and regenerated myofibers. In vivo adding of the recombinant CaMKIV protein enhanced intramuscular infiltration of monocytes/macrophages in damaged muscle and increased the number of proinflammatory Ly-6C+F4/80+ macrophage cells. CaMKIV protein treatment induced a striking up-regulation of mRNA levels of IL-1, IL-6, MCP-1, and MCP-3 in CD45+ cells sorted from damaged muscle; increased the infiltration of CD8+ T cells; and induced the up-regulation of MHC-I in partial regenerated myofibers, which was rarely observed in muscle damage alone. Additionally, CaMKIV protein treatment diminished the regulatory T cells (Tregs) number and led to the damaged TA muscle repair delay. In vitro CaMKIV gene knockdown reversed IFN-γ-induced up-regulation of MHC-I/II and TLR3 in the differentiated C2C12 myotubes. CaMKIV can act as an immunostimulation molecule and enhances the acute muscle inflammatory responses.

Peripheral denervation participates in heterotopic ossification in a spinal cord injury model.

We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles in NHO mouse model. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signaling pathways. Twenty one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (Bone Volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery (n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm3 +/- 0.5) compared to contralateral sides (0.56 mm3 +/-0.4), p = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery (n = 6), bilateral HO were present in three mice at day 28. Quantitative PCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice (shamSCI). Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

The Chemokine Cxcl10 is Not Needed For Normal Skeletal Muscle Regeneration Following a Toxin-induced Injury

PURPOSE: The CCL2/CCR2 chemokine receptor axis is necessary for successful muscle regeneration following injury. Recently, another chemokine known as CXCL10 (or IP-10) was found to be elevated in human skeletal muscle following muscle-damaging eccentric contractions. This finding may indicate that CXCL10, like CCL2, is important for muscle regeneration. METHODS:To test this, we measured functional and histological markers of muscle regeneration in mice out to 14 days post muscle injury (DPI) in wild type (WT) mice and CXCL10 knockout (KO) mice (n = 13 and 12, respectively). The muscle injury was induced by cardiotoxin (CTX) injection into the tibialis anterior (TA) muscle of a hind limb of each mouse. The opposite limb was injected with saline to serve as a within-animal sham control. At 2, 7 and 14 DPI muscle function of both the CTX- and saline-injected TA muscles was assessed using an in situ contraction preparation. After the functional testing, the TA muscles were excised, weighed, frozen, cut by cross section and mounted on glass slides for histological studies. Mounted muscle samples were stained using hematoxylin and eosin to identify central nucleated fibers and quantify cross sectional area. Samples were also stained immunohistochemically for embryonic myosin heavy chain (eMyHC) to identify the number of fibers undergoing regeneration. RESULTS: Relative to the saline-injected TA, strength significantly decreased (p<0.05) at 2 (WT=46.7±15.9%, KO=44±9.4%) and 7 (WT=65±8.3%, KO= 66±15.9%) DPI, and was recovered by 14 DPI. No significant differences in muscle strength were found between genotypes. Compared to the control TA, the CTX-injected TA muscle mass was significantly reduced (p<0.05) at 7 DPI (WT=86±3%, KO=91±9%) compared to 14 DPI (WT=105±8.3%, KO= 105±11%). No significant differences in muscle mass were found between genotypes. Cross-sectional area of regenerating myofibers was greater at 14 DPI compared to 7 (p<0.05), yet again no significant differences were found between genotypes. The number of myofibers expressing eMyHC was greater at 7 DPI than 14 (p<0.05), with no significant difference between genotypes. CONCLUSIONS: We conclude that CXCL10 is not necessary for normal muscle regeneration from a toxin-induced muscle injury in mice.

The Consumption of Plasminogen Following Severe Burn and Its Implications in Muscle Calcification

INTRODUCTIONBurn and blast injuries present with serious risk for complications including fibrosis and calcification of the soft tissues. Plasmin, the active form of plasminogen, is the key serine protease responsible for the degradation of fibrin (fibrinolysis). Our lab has previously shown that plasmin is essential for the prevention of muscle fibrosis and calcification following injury. We hypothesized that 1) plasminogen is consumed in response to severe burn, and 2) this transient plasminogen deficiency impairs proper muscle regeneration.METHODSTo test our first hypothesis in humans, we collected blood from 10 control subjects at one time‐point and 20 burn patients with burns ranging from 25–75% total body surface area (TBSA) at 0, 3, 7, and 14 days post‐admission. Plasma plasminogen levels were assessed by human plasminogen ELISA. Methods including human subjects were pre‐approved by Vanderbilt's IRB.Because our lab has demonstrated that plasmin activity prevents muscle calcification, we used the formation of dystrophic calcification and as an output measure for impaired muscle regeneration. To assess muscle regeneration following severe burn in mice, we induced bilateral muscle injuries in the mice by injecting cardiotoxin (CTX) into the calf muscles and then we administered a 30% TBSA burn to the dorsum by scalding in 100°C water. Control groups received either the burn alone or the CTX injection alone. Another group of mice received the same injury and burn concurrent with a subcutaneous injection of either an antisense oligonucleotide (ASO) against plasmin's inhibitor, α2‐antiplasmin, or a control ASO, beginning 2 weeks prior to injury. Following the burn injury, weekly radiographs of the lower extremities were taken up to 28 days post‐injury (DPI) to assess dystrophic calcification. Following sacrifice at 28 DPI, ex vivo micro‐computed tomography (μCT) was performed on the lower extremities to further assess muscle calcification.To assess plasminogen consumption following severe burn in mice, we collected blood from animals with either CTX‐induced injury, injury with burn (as described above) or no injury following sacrifice at 3 DPI. Plasma plasminogen levels were assessed by mouse plasminogen ELISA. Vanderbilt IACUC pre‐approved all animal experiments.RESULTSBurn patients exhibited a significant plasminogen deficit compared to control subjects at 0 and 3 days post‐admission, followed by a return to control plasminogen levels between 7 and 14 days (Figure 1).Consistent with the human subjects, mice exhibited a significant deficit in circulating plasminogen following burn when compared to both uninjured mice and mice given CTX injection alone.In the animal models assessing muscle regeneration, following the CTX‐induced injury alone, mice did not form significant dystrophic calcification in the injured muscles, but the mice that received the CTX injection and the burn developed dystrophic calcification in the injured muscle at 7 DPI. This phenotype in the burn mice was rescued by a systemic increase in plasmin activity through weekly administration of α2AP ASO (Figure 2).DISCUSSIONWhile many response mechanisms following severe burn have been implicated in impaired healing, the loss of plasminogen and the consequences of this have not been well characterized. Conclusively, this study suggests that severe burn increases the risk of the formation of calcification in injured muscle due to consumption of plasminogen.Support or Funding InformationNIH T32 AR059039‐06A1 Rheumatology Training Grant, Vanderbilt Department of Orthopaedics and Rehabilitation, Caitlin Lovejoy Fund

Delayed Reinnervation During Muscle Regeneration After NO‐Donor Pre‐Treatment

Activation of satellite cells (SCs) toward myogenesis leads to structural regeneration of damaged myofibers and reinnervation is essential to muscle function. SC activation is mediated by NO (a signaling and neurotransmitter molecule) and manipulation of the NO signal is shown to enhance muscle repair and promote growth in older mice in combination with exercise. SCs up‐regulate mRNA expression of a neural chemorepellent, semaphorin 3A (Sema3A) that peaks 6–8 days after muscle injury. Since Sema3A expression is implicated in regulating the timing of axon growth toward regenerating fibers, we hypothesized that premature activation of SC before an injury would advance Sema3A synthesis and disrupt the process of reinnervation. The time‐ course of reinnervation in regenerating tibialis anterior muscle (TA) of 6–10‐week‐old C57BL6 mice was examined after injury by crush (a large traumatic injury) or injection of cardiotoxin (CTX, which damages only fibers). We tested whether early activation of SC before injury using a 2‐day daily pre‐treatment with isosorbide dinitrate (ISDN, an NO‐donor drug) affects the process of new‐fiber innervation post‐injury. Muscle was collected 0, 4, 6, 8, 10, and 14 days after unilateral TA injury (n=4–5 per time point in crush, CTX, and respective control groups). The position of the nuclei and the length of the newly‐formed myotubes were used to track muscle regeneration on coded slides. CTX‐injured muscles had noticeably more advanced formation of neuromuscular junctions (NMJs) and faster fiber regeneration compared to crush‐ injured muscles at the same time point post‐injury. Combined staining of NMJs for acetylcholinesterase (AchE, silver staining) and acetylcholine receptors (AchRs, α‐bungarotoxin epifluorescence) on coded slides, revealed immature or mature NMJs (positive for AchE and AchRs) by day 14 in untreated crush‐ or CTX‐injured muscles, respectively. ISDN pre‐treatment in both crush‐ and CTX‐injured muscles delayed NMJ development on myotubes up to day 14 post‐injury. Results are consistent with the action of Sema3A as a neural chemorepellent, since SC activation before injury resulted in delayed myotube reinnervation. Therapeutic manipulation of Sema3A during regeneration, in time and/or concentration, may have potential to improve reinnervation and muscle function in conditions where nerve‐muscle interactions are disrupted by injury or disease.Support or Funding InformationSupported by a NSERC Discovery grant (to JEA) and a University of Manitoba Graduate Fellowship (to ND)

Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness

BackgroundThe Ste20-like kinase, SLK, plays an important role in cell proliferation and cytoskeletal remodeling. In fibroblasts, SLK has been shown to respond to FAK/Src signaling and regulate focal adhesion turnover through Paxillin phosphorylation. Full-length SLK has also been shown to be essential for embryonic development. In myoblasts, the overexpression of a dominant negative SLK is sufficient to block myoblast fusion.MethodsIn this study, we crossed the Myf5-Cre mouse model with our conditional SLK knockout model to delete SLK in skeletal muscle. A thorough analysis of skeletal muscle tissue was undertaken in order to identify defects in muscle development caused by the lack of SLK. Isometric force analysis was performed on adult knockout mice and compared to age-matched wild-type mice. Furthermore, cardiotoxin injections were performed followed by immunohistochemistry for myogenic markers to assess the efficiency muscle regeneration following SLK deletion.ResultsWe show here that early deletion of SLK from the myogenic lineage does not markedly impair skeletal muscle development but delays the regenerative process. Interestingly, adult mice (~6 months) display an increase in the proportion of central nuclei and increased p38 activation. Furthermore, mice as young as 3 months old present with decreased force generation, suggesting that the loss of SLK impairs myofiber stability and function. Assessment of structural components revealed aberrant localization of focal adhesion proteins, such as FAK and paxillin. Our data show that the loss of SLK results in unstable myofibers resulting in a progressive myopathy. Additionally, the loss of SLK resulted in a delay in muscle regeneration following cardiotoxin injections.ConclusionsOur results show that SLK is dispensable for muscle development and regeneration but is required for myofiber stability and optimal force generation.

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin & Eosin staining that is widely used for subsequent morphological and morphometric analysis.

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin & Eosin staining that is widely used for subsequent morphological and morphometric analysis.

Prenatal myonuclei play a crucial role in skeletal muscle hypertrophy in rodents.

Multinucleated muscle fibers are formed by the fusion of myogenic progenitor cells during embryonic and fetal myogenesis. However, the role of prenatally incorporated myonuclei in the skeletal muscle fibers of adult animals is poorly understood. We demonstrated, using muscle-specific reporter mice, that the prenatal myonuclei remained in the adult soleus muscle, although cardiotoxin injection caused the loss of prenatal myonuclei. Overloading by the tendon transection of synergists failed to induce compensatory hypertrophy in regenerated soleus muscle fibers of adult rats, whereas unloading by tail suspension normally induced the fiber atrophy. Loss of hypertrophying function correlated with the lowered histone acetylation at the transcription start site of Igf1r gene, which was one of the genes that did not respond to the overloading. These parameters were improved by the transplantation of cells harvested from the juvenile soleus muscles of neonatal rats in association with enhanced histone acetylation of Igf1r gene. These results indicated that the presence of prenatal myonuclei was closely related to the status of histone acetylation, which could regulate the responsiveness of muscle fibers to physiological stimuli.

Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice.

Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.

Muscle and Bone: Combating the Evil Side of the Connection.

Muscle and Bone: Combating the Evil Side of the Connection.

PDGFRα+ Progenitor Cells Contribute to Muscle Fibroadipogensis Following Massive Rotator Cuff Tears in a Mouse Model

Objectives:Rotator cuff tears affect an estimated 10% of patients over the age of 60, leading to significant activity related pain and decreased quality of life. After sustaining a tear, the rotator cuff musculature often undergoes muscle atrophy and fatty degeneration, which have been shown to result in poor clinical outcomes and high failure rates for surgical repairs. The cellular processes underlying these fibroadipogenic changes remain unknown although PDGFRβ+ cells have previously been implicated. Recently, it has been demonstrated in hind limb muscle that following acute, reversible injury via Cardiotoxin injection, PDGFRα-expressing cells are responsible for fibrotic and adipogenic muscle degeneration. Our study aimed to distinguish between the contribution of PDGFRα+ PDGFRβ+ cells and PDGFRα- PDGFRβ+ cells to fibroadipogenesis following massive rotator cuff tears.Methods:We performed supraspinatus tenotomy and denervation (TT+DN) operations on 8-10 week-old PDGFRβ-Cre x mTmG mice. These transgenic mice express GFP in cells co-expressing PDGFRβ, which allows PDGFRβ+ cell populations to be tracked via their GFP expression. At 5 days, 2-, 4- and 6-weeks postop, these mice and sham surgery controls were sacrificed and their supraspinatus muscles were harvested. We stained tissue sections with Oil Red O and Picro Sirius to compare levels of adipogenesis and fibrosis after TT+DN, respectively. In addition, sectioned muscle tissues were immuno-labeled with anti-PDGFRα and anti-PDGFRβ to assess differences in PDGFRα+ PDGFRβ+ cell localization. GFP expression was used to trace PDGFRβ cells and their progenies after TT+DN. Dissociated cells from supraspinatus tissue of TT+DN versus sham mice were either analyzed by flow cytometry for PDGFRα and GFP expression at established postop time points or sorted and cultured for in vitro differentiation experiments to assess the fibroadipogenic potential of these cell populations.Results:Oil Red O and Picro Sirius staining demonstrated that supraspinatus tissue reliably underwent significant fibroadipogenic changes that increased over time and were more pronounced 6 weeks following TT+DN. At 6 weeks following massive rotator cuff tears, PDGFRα+ GFP+ cells, GFP+ fibrotic tissue, and GFP+ adipocytes were detected in interstitial scar tissue (Fig. 1), indicating a contribution of PDGFRα+ PDGFRβ+ cells to fibroadipogenesis. Flow cytometry analyses revealed that the frequency of adipogenic PDGFRα+ sub-population was significantly increased 5 days postop and subsequently decreased to basal levels within 2 weeks. PDGFRα+ GFP+ cell populations were significantly more fibrogenic than other cell populations and increased fibrogenic potential was detected in PDGFRα cells from TT+DN mice in vitro (Table 1).Conclusion:PDGFRα+ fibroadipogenic progenitor cells directly contribute to post-injury rotator cuff fibroadipogenesis in a mouse model of massive rotator cuff tears. This cell population likely contributes to muscle atrophy and fatty degeneration of the rotator cuff in human pathology as well. With further development of clinical protocols, perioperative inhibition of this cell population or depletion of PDGFRα+ cells prior to cell transplantation in a regenerative treatment strategy may diminish fatty degeneration and improve clinical outcomes associated with rotator cuff tears.

Customized Platelet-Rich Plasma for Skeletal Muscle Injuries

Objectives:Skeletal muscle injuries are among the most common sports-related trauma. Current treatment strategies result in formation of fibrous tissue that hinders the healing process before complete recovery. Incomplete recovery impairs muscle function and predisposes to re-injury. Platelet-Rich-Plasma (PRP) contains a multitude of growth factors and is an autologous source of growth factors for various tissue repairs. It is well established that PRP contains beneficial growth factors for muscle repair; however, it also contains high concentrations of deleterious growth factors for optimal muscle healing, such as transforming growth factor-beta 1 (TGF-β1). TGF-β1 leads to increased fibrosis impeding muscle healing. We therefore hypothesized that neutralization of TGF-β1’s action within PRP could improve PRP’s beneficial effect on skeletal muscle repair.Methods:Sixteen week old in-bred Fisher rats were used. Three rats were used for PRP isolation. 10 ml of blood were extracted from abdominal aorta and mixed with citrate phosphate dextrose solution. PRP were isolated by twice centrifugation. 24 rats were randomly assigned to four groups. A small incision was made along the tibialis anterior (TA) muscle; 50 µl cardiotoxin (CTX) (0.15ug/ul) was injected intramuscularly to the TA. One day after CTX injection, the animals were treated with PBS (control), plain PRP (PRP group), customized PRP+Ab-1x, and PRP+Ab-5x. Animals were sacrificed, and TA muscles were dissected on week 1 and 2 for assessment of muscle regeneration, fibrosis, macrophage infiltration, and satellite cell activation.Results:We observed significantly more regenerative myofibers in the PRP and customized PRP groups compared to control (Fig 1A-C). Collagen deposition (fibrosis) was detected in all groups at week 1 and week 2 after injury; while customized PRP group showed significantly decreased collagen deposition at week 1 and week 2 when compare to control and PRP groups (Fig. 1D-F). PRP was also shown to activate satellite cells at day 7 while customized PRP extended satellite cell activation through day 14 (Fig. 1G, H). Macrophage infiltration was significantly decreased in PRP group when compared to control at week 1 after injury; however the macrophage infiltration was increased in customized PRP group when compare with PRP group, specifically, we observed an increased in the percentage of the M2 macrophage subtype in the customized PRP group at week 1 but not week 2(Fig. 1I, J).Conclusion:This study demonstrates that substantial fibrosis occurs in skeletal muscle following CTX injury. PRP treatment significantly increases muscle regeneration by promoting angiogenesis, satellite cell activation, and reducing the infiltration of inflammatory macrophages. However, augmented fibrosis was also observed. Neutralizing TGF-β1 within PRP also significantly promotes muscle regeneration while significantly reducing fibrosis. It has been suggested that M2 macrophages are involved in the suppression, resolution and reversal of fibrosis. Hence, the increase in M2 macrophages in the customized PRP group may have contributed to the observed decrease in fibrosis. Our results demonstrate that neutralization of TGF-β1 within PRP is a promising approach to improve PRP’s beneficial effect on skeletal muscle repair while decreasing deleterious fibrosis.

Establishing a Functional Model of Mouse Muscle Injury and Recovery

PURPOSE: To establish an in vivo mouse model of muscle injury and recovery. METHODS: Sixteen genetically identical female mice were randomized to two 8-mouse injury cohorts (either Phosphate Buffered Saline (PBS) (control) or cardiotoxin (injured)). Injections were performed unilaterally to the right tibialis anterior. One protocol (nocturnal wheel running) was evaluated longitudinally throughout the 17 day study period, while the remaining 5 protocols (dynamic weight bearing, treadmill gait analysis, Rota-Rod performance, open field testing, and grip strength) were evaluated as one-time assays over post-injury days 5-6. All experiments were conducted at Jackson Laboratory, Bar Harbor, Maine. RESULTS: Of the six protocols examined, only the Rota-Rod assay demonstrated a statistically significant difference between injured and control mice (p<0.0018). The Rota-Rod is a rotating rod that increases velocity until the mouse is no longer able to stay on and falls to a soft surface for a maximum trial duration of 300 seconds. The mean latency (seconds until falling) was 298 seconds for control mice and 186 seconds for cardiotoxin-injured mice, with a significant difference of 111 seconds (95% CI 17.0-205.5). There was no significant difference in the two groups in terms of wheel running, weight bearing, gait, open field, or grip strength. CONCLUSION: The Rota-Rod consistently and significantly delineated between cardiotoxin-injured mice and placebo mice at day 5 post-injury. It is likely that even greater statistical significance would result from Rota-Rod trials lasting greater than 300 seconds. It is unknown how Rota-Rod would perform at other time points. Unilateral cardiotoxin injection to the tibialis anterior and subsequent Rota-Rod trial provides a low cost easy to perform in vivo murine model of muscle injury functional assessment within which novel therapies can be assessed.

Serum Osteopontin as a Novel Biomarker for Muscle Regeneration in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is a lethal X-linked muscle disorder. We have already reported that osteopontin (OPN), an inflammatory cytokine and myogenic factor, is expressed in the early dystrophic phase in canine X-linked muscular dystrophy in Japan, a dystrophic dog model. To further explore the possibility of OPN as a new biomarker for disease activity in Duchenne muscular dystrophy, we monitored serum OPN levels in dystrophic and wild-type dogs at different ages and compared the levels to other serum markers, such as serum creatine kinase, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Serum OPN levels in the dystrophic dogs were significantly elevated compared with those in wild-type dogs before and 1 hour after a cesarean section birth and at the age of 3 months. The serum OPN level was significantly correlated with the phenotypic severity of dystrophic dogs at the period corresponding to the onset of muscle weakness, whereas other serum markers including creatine kinase were not. Immunohistologically, OPN was up-regulated in infiltrating macrophages and developmental myosin heavy chain-positive regenerating muscle fibers in the dystrophic dogs, whereas serum OPN was highly elevated. OPN expression was also observed during the synergic muscle regeneration process induced by cardiotoxin injection. In conclusion, OPN is a promising biomarker for muscle regeneration in dystrophic dogs and can be applicable to boys with Duchenne muscular dystrophy.