Myocyte Differentiation Research Articles

STEMIN and YAP5SA, the future of heart repair?

This review outlines some of the many approaches taken over a decade or more to repair damaged hearts. We showcase the recent breakthroughs in organ regeneration elicited by reprogramming factors OCT3/4, SOX2, KLF4, and C-MYC (OKSM). Transient OKSM transgene expression rejuvenated senescent organs in mice. OKSM transgenes also caused murine heart cell regeneration. A triplet alanine mutation of the N-terminus of Serum Response Factor's MADS box SRF153(A3), termed STEMIN, and the YAP mutant, YAP5SA synergized and activated OKSM and NANOG in adult rat cardiac myocytes; thus, causing rapid nuclear proliferation and blocked myocyte differentiation. In addition, ATAC seq showed induced expression of growth factor genes FGFs, BMPs, Notchs, IGFs, JAK, STATs and non-canonical Wnts. Injected STEMIN and YAP5SA synthetic modifying mRNA (mmRNA) into infarcted adult mouse hearts, brought damaged hearts back to near normal contractility without severe fibrosis. Thus, STEMIN and YAP5SA mmRNA may exert additional regenerative potential than OKSM alone for treating heart diseases.

Identification and analysis of differentially expressed lncRNAs and their ceRNA networks in DMD/mdx primary myoblasts

This study explored the significance of long non-coding RNAs (lncRNAs), particularly their role in maintaining dystrophin protein stability and regulating myocyte proliferation and differentiation. The investigation focused on DMD/mdx mouse skeletal muscle primary myoblasts, aiming to identify lncRNAs potential as biomarkers and therapeutic targets for Duchenne muscular dystrophy (DMD). Utilizing CLC Genomics Workbench software, 554 differentially expressed lncRNAs were identified in DMD/mdx mice compared to wild-type (WT) control. Among them, 373 were upregulated, and 181 were downregulated. The study highlighted specific lncRNAs (e.g., 5930430L01Rik, Gm10143, LncRNA1490, LncRNA580) and their potential regulatory roles in DMD key genes like IGF1, FN1, TNNI1, and MYOD1. By predicting miRNA and their connections with lncRNA and mRNA (ceRNA network) using tools such as miRNet, miRSYSTEM and miRCARTA, the study revealed potential indirect regulation of Dystrophin, IGF1R and UTRN genes by identified lncRNAs (e.g. 2310001H17Rik-203, C130073E24Rik-202, LncRNA2767, 5930430L01Rik and LncRNA580). These findings suggest that the identified lncRNAs may play crucial roles in the development and progression of DMD through their regulatory influence on key gene expression, providing valuable insights for potential therapeutic interventions.

Adapting cytoskeleton-mitochondria patterning with myocyte differentiation by promyogenic PRR33.

Coordinated cytoskeleton-mitochondria organization during myogenesis is crucial for muscle development and function. Our understanding of the underlying regulatory mechanisms remains inadequate. Here, we identified a novel muscle-enriched protein, PRR33, which is upregulated during myogenesis and acts as a promyogenic factor. Depletion of Prr33 in C2C12 represses myoblast differentiation. Genetic deletion of Prr33 in mice reduces myofiber size and decreases muscle strength. The Prr33 mutant mice also exhibit impaired myogenesis and defects in muscle regeneration in response to injury. Interactome and transcriptome analyses reveal that PRR33 regulates cytoskeleton and mitochondrial function. Remarkably, PRR33 interacts with DESMIN, a key regulator of cytoskeleton-mitochondria organization in muscle cells. Abrogation of PRR33 in myocytes substantially abolishes the interaction of DESMIN filaments with mitochondria, leading to abnormal intracellular accumulation of DESMIN and mitochondrial disorganization/dysfunction in myofibers. Together, our findings demonstrate that PRR33 and DESMIN constitute an important regulatory module coordinating mitochondrial organization with muscle differentiation.

Placenta‐Derived Mesenchymal Stromal‐Like Cells Promote 3D‐Engineered Muscle Tissue Differentiation and Vessel Network Maturation

Placental‐derived stromal‐like cells (PLX‐PAD) have been shown to facilitate muscle tissue recovery after injury and stimulate angiogenesis. This work assesses the impact of PLX‐PAD cells on the vascularization and maturation of engineered skeletal muscle tissue. Specifically, their effects in direct co‐culture with endothelial cells, pericytes, and myoblasts seeded within microporous 3D scaffolds are characterized. Additionally, the impact of hypoxic PLX‐PAD cell‐conditioned medium (CM) on vascularization and muscle differentiation of engineered tissue is monitored. Co‐culture of PLX‐PAD with myocytes stimulated myocyte differentiation while PLX‐PAD CM promoted the formation of vascular networks. Implantation of a multi‐culture system of vascularized human skeletal muscle tissue and PLX‐PAD into a rectus abdominal defect in nude mice promoted myocyte differentiation, host vessel penetration, and tissue integration. These findings indicate the ability of placenta‐derived cells to induce the formation of vascularized engineered muscle constructs with potential therapeutic applications.

Gene Expression During C2C12 Differentiation

Congenital heart disease (CHD) is one of the most common types of birth defect and is very poorly understood. To comprehend this disease better, the normal genetic expression associated with myocyte differentiation must be mapped out. This gives a deeper understanding of how cardiomyocytes (cardiac muscle cells) divide and specialize, which can contribute to understanding the mechanisms of CHD better. The workflow of this specific project began with culturing C2C12 mouse muscle myoblast cells and harvesting them at six different time points: 50% confluency, 100% confluency, 24-hours postdifferentiation, 48-hour postdifferentiation, 72-hour postdifferentiation, and 96-hour postdifferentiation. The expression levels of MyoD1, Myogenin, MyoM2, MyoZ2, GATA4, TbX5, NKX2.5, Ki67, Tcap, and RBFox1 were then measured and compared with qPCR. It was expected that those gene markers associated with muscle differentiation and structure would increase in expression levels over time while those markers associated with cell proliferation would decrease in expression levels. The qPCR showed the expected induction of myogenic differentiation marker genes, with the following deviation: MyoD1 expression did not increase during differentiation, and cardiogenesis markers did not follow a clear expression pattern during differentiation. Future studies on human-induced pluripotent stem cells differentiation to cardiomyocytes are needed to understand the patterns of gene expression in cardiomyocytes compared to skeletal myocytes. The outcomes of these studies may contribute to a deeper understanding of heart development and abnormalities in patients with CHD, and may improve diagnosis and treatment methods.

#2366 Beneficial effects of TRIM33 on myokine-mediated renal function recovery in diabetic and obese mice

Abstract Background and Aims Myokines secreted from myocytes are related to improving metabolic diseases such as diabetes, obesity, and chronic kidney disease (CKD). Patients with diabetic kidney disease (DKD) have lower irisin levels, suggesting that a low level of irisin is a risk factor for the progression of CKD. However, it is not well known how myokines can alleviate metabolic kidney injury. This study aimed to investigate the effect of TRIM33, a negative regulator of TGF-β1 signaling, as a novel strategy to treat multiple metabolic complications simultaneously in the kidneys and muscles of obese db/db mice. Method For the in vivo experiment, five-week-old male db/db mice were assigned to three groups: db/m+, db/db, and db/db with TRIM33 plasmid mice. TRIM33 plasmid (40 μg/mice) was administered intraperitoneally. Serum glucose and myokine levels, urinary albumin excretion, renal and muscle morphology, and myokine expression in muscles were assessed. In vitro, C2C12 cells were cultivated with 2% horse serum and lipid insult in the absence or presence of TRIM33 plasmid and were analyzed for myocyte differentiation and myokines expression or production. Results TRIM33 treatment showed beneficial effects on tubular injury, tubulointerstitial fibrosis, mesangial expansion, renal dysfunction, variations in skeletal muscle fiber size and arrangement, skeletal muscle activity, and serum myokines levels in db/db mice. During the differentiation of C2C12 cells, myokine mRNA and protein expression and secretion were increased in TRIM33-transfected cells. Reduced levels of myokines expression and secretion in C2C12 cells with palmitate were improved by TRIM33 transfection. In coculture with HK-2 and C2C12 cells, increased EMT transition in TGF-b-treated HK-2 cells by coculture with palmitate-treated C2C12 cells was decreased in coculture with palmitate-treated C2C12 cells with TRIM33 transfection. Conclusion This study showed that TRIM33 is involved in the recovery of renal dysfunction via myokine in mice with diabetes and obesity.

453 Changed Innervation Upon Loss of Mandibular Division of Trigeminal Nerve to Muscles of Mastication

INTRODUCTION: The gastrulation brain homeobox (Gbx) genes are essential for patterning and maintenance of neurons along the anteroposterior axis of the developing neural tube. Knockout (ko) of Gbx2 results in neonatal lethality associated with neurological and other defects. To understand pathologies, ko studies are not realistic as gene loss usually results in death before birth. Gbx2 neo/neo mutant mice express 6-10% of the wildtype Gbx2 expression levels. They have milder malformations than ko mice but lack the cerebellar vermis and mandibular division of the trigeminal nerve (3rd division of 5th cranial nerve = V3), among other defects. These Gbx2 neo/neo mutant mice die perinatally as they are unable to suckle due to lack of motor innervation via V3 to the muscles of mastication. Muscle cells develop largely without interaction with their motor nerve. However, the final differentiation of myocytes requires interactions with nerve fibers. Unexpectedly, the muscles of mastication appear normal in Gbx2 neo/neo mutant mice, despite the lack of V3. METHODS: We performed microdissections of neonate wildtype and Gbx2neo/neo mutant mice. Additionally, we embedded mutant and wildtype mouse embryos in paraffin, serial sectioned them (7 μm), stained the sections with Azan staining, and analyzed specimen microscopically. RESULTS: Current analysis of the data to identify where nerve fibers in the muscles of mastication originate is in process. We favor the origin of these fibers from the facial nerve (7th cranial nerve), which has several overlapping territories with the trigeminal nerve. CONCLUSIONS: Muscles require innervation for their final differentiation steps. The loss of a nerve can result in the invasion of another nerve into the territory of the lost one, which rescues muscle differentiation but not muscle function. IACUC Ziermann Med-20-02, Funding NSF #2000005 to Ziermann.

Whey Peptide Alleviates Muscle Atrophy by Strongly Regulating Myocyte Differentiation in Mice.

Background and Objectives: Muscle atrophy occurs when protein degradation exceeds protein synthesis, resulting in imbalanced protein homeostasis, compromised muscle contraction, and a reduction in muscle mass. The incidence of muscle atrophy is increasingly recognized as a significant worldwide public health problem. The aim of the current study was to evaluate the effect of whey peptide (WP) on muscle atrophy induced by dexamethasone (DEX) in mice. Materials and Methods: C57BL/6 mice were divided into six groups, each consisting of nine individuals. WPs were orally administered to C57BL/6 mice for 6 weeks. DEX was administered for 5-6 weeks to induce muscle atrophy (intraperitoneal injection, i.p.). Results: Microcomputer tomography (CT) analysis confirmed that WP significantly increased calf muscle volume and surface area in mice with DEX-induced muscle atrophy, as evidenced by tissue staining. Furthermore, it increased the area of muscle fibers and facilitated greater collagen deposition. Moreover, WP significantly decreased the levels of serum biomarkers associated with muscle damage, kidney function, and inflammatory cytokines. WP increased p-mTOR and p-p70S6K levels through the IGF-1/PI3K/Akt pathway, while concurrently decreasing protein catabolism via the FOXO pathway. Furthermore, the expression of proteins associated with myocyte differentiation increased noticeably. Conclusions: These results confirm that WP reduces muscle atrophy by regulating muscle protein homeostasis. Additionally, it is believed that it helps to relieve muscle atrophy by regulating the expression of myocyte differentiation factors. Therefore, we propose that WP plays a significant role in preventing and treating muscle wasting by functioning as a supplement to counteract muscle atrophy.

Variant to gene mapping for carpal tunnel syndrome risk loci implicates skeletal muscle regulatory elements

Carpal tunnel syndrome (CTS) is a common disorder caused by compression of the median nerve in the wrist, resulting in pain and numbness throughout the hand and forearm. While multiple behavioural and physiological factors influence CTS risk, a growing body of evidence supports a strong genetic contribution. Recent genome-wide association study (GWAS) efforts have reported 53 independent signals associated with CTS. While GWAS can identify genetic loci conferring risk, it does not determine which cell types drive the genetic aetiology of the trait, which variants are "causal" at a given signal, and which effector genes correspond to these non-coding variants. These obstacles limit interpretation of potential disease mechanisms. We analysed CTS GWAS findings in the context of chromatin conformation between gene promoters and accessible chromatin regions across cellular models of bone, skeletal muscle, adipocytes and neurons. We identified proxy variants in high LD with the lead CTS sentinel SNPs residing in promoter connected open chromatin in the skeletal muscle and bone contexts. We detected significant enrichment for heritability in skeletal muscle myotubes, as well as a weaker correlation in human mesenchymal stem cell-derived osteoblasts. In myotubes, our approach implicated 117 genes contacting 60 proxy variants corresponding to 20 of the 53 GWAS signals. In the osteoblast context we implicated 30 genes contacting 24 proxy variants coinciding with 12 signals, of which 19 genes shared. We subsequently prioritized BZW2 as a candidate effector gene in CTS and implicated it as novel gene that perturbs myocyte differentiation invitro. Taken together our results suggest that the CTS genetic component influences the size, integrity, and organization of multiple tissues surrounding the carpal tunnel, in particular muscle and bone, to predispose the nerve to being compressed in this disease setting. This work was supported by NIH Grant UM1 DK126194 (SFAG and WY), R01AG072705 (SFAG & KDH) and the Center for Spatial and Functional Genomics at CHOP (SFAG & ADW). SFAG is supported by the Daniel B. Burke Endowed Chair for Diabetes Research. WY is supported by the Perelman School of Medicine of the University of Pennsylvania.

Hypoxia enhances human myoblast differentiation: involvement of HIF1α and impact of DUX4, the FSHD causal gene

BackgroundHypoxia is known to modify skeletal muscle biological functions and muscle regeneration. However, the mechanisms underlying the effects of hypoxia on human myoblast differentiation remain unclear. The hypoxic response pathway is of particular interest in patients with hereditary muscular dystrophies since many present respiratory impairment and muscle regeneration defects. For example, an altered hypoxia response characterizes the muscles of patients with facioscapulohumeral dystrophy (FSHD).MethodsWe examined the impact of hypoxia on the differentiation of human immortalized myoblasts (LHCN-M2) cultured in normoxia (PO2: 21%) or hypoxia (PO2: 1%). Cells were grown in proliferation (myoblasts) or differentiation medium for 2 (myocytes) or 4 days (myotubes). We evaluated proliferation rate by EdU incorporation, used myogenin-positive nuclei as a differentiation marker for myocytes, and determined the fusion index and myosin heavy chain-positive area in myotubes. The contribution of HIF1α was studied by gain (CoCl2) and loss (siRNAs) of function experiments. We further examined hypoxia in LHCN-M2-iDUX4 myoblasts with inducible expression of DUX4, the transcription factor underlying FSHD pathology.ResultsWe found that the hypoxic response did not impact myoblast proliferation but activated precocious myogenic differentiation and that HIF1α was critical for this process. Hypoxia also enhanced the late differentiation of human myocytes, but in an HIF1α-independent manner. Interestingly, the impact of hypoxia on muscle cell proliferation was influenced by dexamethasone. In the FSHD pathological context, DUX4 suppressed HIF1α-mediated precocious muscle differentiation.ConclusionHypoxia stimulates myogenic differentiation in healthy myoblasts, with HIF1α-dependent early steps. In FSHD, DUX4-HIF1α interplay indicates a novel mechanism by which DUX4 could interfere with HIF1α function in the myogenic program and therefore with FSHD muscle performance and regeneration.

Postnatal skeletal muscle myogenesis governed by signal transduction networks: MAPKs and PI3K–Akt control multiple steps

Skeletal muscle myogenesis represents one of the most intensively and extensively examined systems of cell differentiation, tissue formation, and regeneration. Muscle regeneration provides an in vivo model system of postnatal myogenesis. It comprises multiple steps including muscle stem cell (or satellite cell) quiescence, activation, migration, myogenic determination, myoblast proliferation, myocyte differentiation, myofiber maturation, and hypertrophy. A variety of extracellular signaling and subsequent intracellular signal transduction pathways or networks govern the individual steps of postnatal myogenesis. Among them, MAPK pathways (the ERK, JNK, p38 MAPK, and ERK5 pathways) and PI3K–Akt signaling regulate multiple steps of myogenesis. Ca2+, cytokine, and Wnt signaling also participate in several myogenesis steps. These signaling pathways often control cell cycle regulatory proteins or the muscle-specific MyoD family and the MEF2 family of transcription factors. This article comprehensively reviews molecular mechanisms of the individual steps of postnatal skeletal muscle myogenesis by focusing on signal transduction pathways or networks. Nevertheless, no or only a partial signaling molecules or pathways have been identified in some responses during myogenesis. The elucidation of these unidentified signaling molecules and pathways leads to an extensive understanding of the molecular mechanisms of myogenesis.

Myostatin Level in CRF Patients with and without HBV and Its Correlation with Sarcopenia

Abstract Introduction Myostatin (MSTN), a member of TGF-β superfamily, is produced in the skeletal muscle to inhibit myocyte differentiation. MSTN expression is increased in the skeletal muscle in patients with chronic kidney disease (CKD), which may play a role in the pathogenesis of sarcopenia or in the protein energy wasting (PEW). Aim and objectives To assess myostatin level in CRF patients with HBV and its correlation with sarcopenia. Subjects and Methods This study is a cross - sectional study, was carried out at at outpatients' clinics and internal medicine and nephrology department Ain Shams university hospital and Sharq Elmadinah Hospital, Alexandria, on 70 patients on regular hemodialysis divided into 3 groups: (Group A); included 20 patients with HBV infection, (Group B); included 20 patients without HBV infection, (Group C); included 10 normal persons without CRF or HBV, during period from February 2021 till August 2021. Result Myostatin level showed a significant negative correlation with BMI and SMI. Conclusion The serum myostatin level was significantly higher in hemodialysis patients than controls, particularly in post-HCV cirrhotic than non-cirrhotic ones. Serum myostatin level was significantly related to sarcopenia pathogenesis as a predictor marker.

Abstract P3181: A Cyclophilin D-mitochondria-redox Differentiation Pathway Regulates Neonatal Heart Bioenergetics And Function

Background: During birth, demands on the heart increase when exposed to high oxidative stress. Cardiac myocyte differentiation is dependent on changes in mitochondrial function that regulate cell stress pathways to enhance differentiation. We have shown these changes are dependent on closure of the mitochondrial permeability transition pore (mPTP) controlled by the protein cyclophilin D (CypD). These studies aim to determine the effects of independently manipulating CypD, mitochondrial membrane potential, and reactive oxygen species (ROS) production on mitochondrial function, myocyte differentiation, and cardiac function in the neonate. Methods: Neonatal C57Bl6 mice were intraperitoneally injected on postnatal day (P) 1-6 of with mitoparaquat (0.025-0.05 mg/kg), Tempol (0.7 mg/kg), mitoTempo (0.7 mg/kg), 2,4-dinitrophenol (2 mg/kg), or vehicle. Hearts were harvested at P7 and mitochondrial respiration, enzymatic activity of the electron transport chain (ETC) complex (Cx) I and 2, and assembly of mitochondrial supercomplexes were measured by native gel electrophoresis. Results: Treatment with mitoparaquat to increase mitochondrial ROS caused a dose-dependent decrease of Cx I activity measured by oxygen consumption and enzyme assays. This corresponded to decrease in assembly of Cx-I containing supercomplexes. Decreasing mitochondrial ROS using mitoTempo had no effect on Cx I activity but increased assembly of Cx-III containing supercomplexes. Decreasing ROS in the cell with Tempol decreased Cx-I activity but had no effect on supercomplex assembly. Uncoupling at the mitochondrial membrane using 2,4-dinitrophenol increased electron flux from Cx I to Cx III but may decrease supercomplex assembly. Treatment of isolated mitochondria with cyclosporin A to inhibit CypD had no effects on these measures. Conclusions: Changes in ROS and mitochondrial membrane potential have selective effects on ETC assembly, activity in the neonatal period, and are not inhibited by closure of the mPTP. This data suggests a pathway where CypD regulates the mPTP to regulate the ETC, membrane potential, and ROS production to regulate myocyte differentiation; such a pathway could be targeted to increase cardiac maturation in infants with poor cardiac function.

Development of a robust induced pluripotent stem cell atrial cardiomyocyte differentiation protocol to model atrial arrhythmia

BackgroundAtrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably, many therapeutics shown to be efficacious in animal models have not proved effective in humans. Thus, there is a need for a drug screening platform based on human tissue. The aim of this study was to develop a robust protocol for generating atrial cardiomyocytes from human-induced pluripotent stem cells.MethodsA novel protocol for atrial differentiation, with optimized timing of retinoic acid during mesoderm formation, was compared to two previously published methods. Each differentiation method was assessed for successful formation of a contractile syncytium, electrical properties assayed by optical action potential recordings and multi-electrode array electrophysiology, and response to the G-protein-gated potassium channel activator, carbamylcholine. Atrial myocyte monolayers, derived using the new differentiation protocol, were further assessed for cardiomyocyte purity, gene expression, and the ability to form arrhythmic rotors in response to burst pacing.ResultsApplication of retinoic acid at day 1 of mesoderm formation resulted in a robust differentiation of atrial myocytes with contractile syncytium forming in 16/18 differentiations across two cell lines. Atrial-like myocytes produced have shortened action potentials and field potentials, when compared to standard application of retinoic acid at the cardiac mesoderm stage. Day 1 retinoic acid produced atrial cardiomyocytes are also carbamylcholine sensitive, indicative of active Ikach currents, which was distinct from ventricular myocytes and standard retinoic addition in matched differentiations. A current protocol utilizing reduced Activin A and BMP4 can produce atrial cardiomyocytes with equivalent functionality but with reduced robustness of differentiation; only 8/17 differentiations produced a contractile syncytium. The day 1 retinoic acid protocol was successfully applied to 6 iPSC lines (3 male and 3 female) without additional optimization or modification. Atrial myocytes produced could also generate syncytia with rapid conduction velocities, > 40 cm s−1, and form rotor style arrhythmia in response to burst pacing.ConclusionsThis method combines an enhanced atrial-like phenotype with robustness of differentiation, which will facilitate further research in human atrial arrhythmia and myopathies, while being economically viable for larger anti-arrhythmic drug screens.

Iron deficiency is related to lower muscle mass in community-dwelling individuals and impairs myoblast proliferation.

Loss of muscle mass is linked with impaired quality of life and an increased risk of morbidity and premature mortality. Iron is essential for cellular processes such as energy metabolism, nucleotide synthesis and numerous enzymatic reactions. As the effects of iron deficiency (ID) on muscle mass and function are largely unknown, we aimed to assess the relation between ID and muscle mass in a large population-based cohort, and subsequently studied effects of ID on cultured skeletal myoblasts and differentiated myocytes. In a population-based cohort of 8592 adults, iron status was assessed by plasma ferritin and transferrin saturation, and muscle mass was estimated using 24-h urinary creatinine excretion rate (CER). The relationships of ferritin and transferrin saturation with CER were assessed by multivariable logistic regression. Furthermore, mouse C2C12 skeletal myoblasts and differentiated myocytes were subjected to deferoxamine with or without ferric citrate. Myoblast proliferation was measured with a colorimetric 5-bromo-2'-deoxy-uridine ELISA assay. Myocyte differentiation was assessed using Myh7-stainings. Myocyte energy metabolism, oxygen consumption rate and extracellular acidification rate were assessed using Seahorse mitochondrial flux analysis, and apoptosis rate with fluorescence-activated cell sorting. RNA sequencing (RNAseq) was used to identify ID-related gene and pathway enrichment in myoblasts and myocytes. Participants in the lowest age- and sex-specific quintile of plasma ferritin (OR vs middle quintile 1.62, 95% CI 1.25-2.10, P<0.001) or transferrin saturation (OR 1.34, 95% CI 1.03-1.75, P=0.03) had a significantly higher risk of being in the lowest age- and sex-specific quintile of CER, independent of body mass index, estimated GFR, haemoglobin, hs-CRP, urinary urea excretion, alcohol consumption and smoking status. In C2C12 myoblasts, deferoxamine-induced ID reduced myoblast proliferation rate (P-trend <0.001) but did not affect differentiation. In myocytes, deferoxamine reduced myoglobin protein expression (-52%, P<0.001) and tended to reduce mitochondrial oxygen consumption capacity (-28%, P=0.10). Deferoxamine induced gene expression of cellular atrophy markers Trim63 (+20%, P=0.002) and Fbxo32 (+27%, P=0.048), which was reversed by ferric citrate (-31%, P=0.04 and -26%, P=0.004, respectively). RNAseq indicated that both in myoblasts and myocytes, ID predominantly affected genes involved in glycolytic energy metabolism, cell cycle regulation and apoptosis; co-treatment with ferric citrate reversed these effects. In population-dwelling individuals, ID is related to lower muscle mass, independent of haemoglobin levels and potential confounders. ID impaired myoblast proliferation and aerobic glycolytic capacity, and induced markers of myocyte atrophy and apoptosis. These findings suggest that ID contributes to loss of muscle mass.

Indoxyl sulfate inhibits muscle cell differentiation via Myf6/MRF4 and MYH2 downregulation.

Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)-myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6/MRF4)-and expression of myosin heavy chain, Myh2. C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6/MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)-the cell receptor of IS-was studied by adding an AHR inhibitor into the cell culture milieu. In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6/MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6/MRF4 gene was down-regulated in striated muscles. In conclusion, IS inhibits Myf6/MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD.

Cardiac Hamartomas: Report of Two Rare Primary Cardiac Tumors (Rhabdomyoma and Hamartoma of Mature Cardiomyocytes) with Review of the Literature

Abstract The most common primary cardiac tumors are myxomas, while cardiac hamartomas are rare. Cardiac hamartomas may constitute manifestation of a genetic disorder such as tuberous sclerosis, or could be diagnosed independently as a solitary lesion. We present two rare cases of primary cardiac tumors detected via diagnostic imaging and subsequent histopathology examination. The first described case is that of a male preterm foetus with multiple rhabdomyomas in the heart, discovered during foetal echocardiography. The finding was later confirmed by autopsy, which also revealed presence of cortical tubers with typical balloon cells constituting well known epileptogenic lesions in the foetal brain. The second case describes an even rarer benign cardiac lesion – a hamartoma of mature cardiomyocytes – which was discovered by chance in a 49-year old female patient during echocardiography. The performed CT scan revealed a 37 x 16 x 12 mm tumor in the right atrium obstructing the superior vena cava inlet. The malformation was surgically removed and examined histologically revealing disorganized hypertrophic mature cardiomyocytes, partly separated by fibrous strands and admixed with mature adipocytes. Differential diagnosis was performed to rule out other benign tumors with myocyte differentiation – cardiac rhabdomyoma, histiocytoid cardiomyopathy and adult cellular rhabdomyoma. Conclusion The diagnosis of cardiac hamartomas was possible only after pathomorphological examination of material from the tumors. Reporting of rare neoplasms is crucial in order to determine their prevalence and to inform clinical practice.

Abstract 2260: Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma

Abstract Background: Neoadjuvant anti-PD-1-based therapies have shown promise in several cancers, including Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. We have shown that on-treatment specimens from patients responding to anti-PD-1 are characterized histologically by exuberant proliferative fibrosis, neovascularization, and robust inflammation including diverse immune cell types. Understanding how the stroma participates in the process of tumor regression may open therapeutic opportunities by redirecting stromal remodeling in treatment-refractory tumors. Methods: To evaluate stromal changes in neoadjuvant treated MCC, 13 on-treatment FFPE tumor specimens from patients with resectable MCC who received anti-PD-1 for ~4 weeks before surgery (NCT02488759) were subjected to laser capture microdissection to collect stromal tissue and isolate RNA. We conducted multiplex qRT-PCR targeting 125 unique mRNAs representing TME-relevant immune cell subsets, immune checkpoint pathways, and immune-modulating transcription factors and cytokines, followed by whole transcriptome RNA sequencing. Differential gene expression was considered significant if the magnitude of the signed fold-change was ≥2 and the p-value was ≤0.1 (Benjamini-Hochberg adjusted for RNAseq). Gene set enrichment analysis (GSEA) was performed with R Topper. Pathologic response was scored on H&amp;E staining as pathologic complete response (pCR, n=6), major pathologic response (MPR, ≤10% residual viable tumor [RVT]; n=4), or non-MPR/pCR (&amp;gt;10% RVT; n=3). Results: Stratifying by response status (6 pCR vs 3 non-MPR/pCR) demonstrated that pCR stroma in MCC is distinguished by upregulation of molecules associated with neovascularization (VEGFA, EGR2) and M1 and M2 macrophages (IL1A, ARG1) via qRT-PCR, and mesenchymal cells (fibroblasts, myocytes, adipocytes) and extracellular matrix formation via RNAseq. GSEA demonstrated a strong association of pCR with both myocyte and fibroblast differentiation and functional pathways, suggesting a role for myofibroblasts in tissue remodeling during anti-PD-1-mediated tumor regression. In contrast, the stroma of non-MPR/pCR tumors showed evidence for ongoing IFNg-mediated inflammation, as evidenced by upregulation of IFNg, IL-27, STAT1, CXCL10 and LAG-3 via qRT-PCR; these findings were supported by RNAseq, which also showed upregulation of genes associated with inflammation (e.g., PLXNC1, TRPV2, LYZ, LAIR1, HLA-A, B2M, CD4). Similar results were obtained when MPRs were grouped with pCRs (n=10) vs non-MPR/pCR (n=3). Conclusion: These findings demonstrate that improved response to anti-PD-1 therapy is associated with a shift in the tumor stroma from an inflammatory to a proliferative myofibroblastic phenotype, recapitulating the stages of normal wound healing. This suggests a potential functional role for myofibroblasts in that critical transition. Citation Format: Joel C. Sunshine, Tracee L. McMiller, Alyza Skaist, Yan Zhang, Alan Berger, Kornel Schuebel, Jennifer Meyers, Julie S. Deutsch, Aleksandra Ogurtsova, Elizabeth L. Engle, Leslie Cope, Janis M. Taube, Suzanne L. Topalian. Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2260.

HDAC8 regulates protein kinase D phosphorylation in skeletal myoblasts in response to stress signaling

Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.

Cyclophilin D inhibition rescues cardiac function in neonatal hypoxia.

The heart develops in utero under hypoxic conditions, and exposure to higher oxygen levels at birth activates changes in the heart to allow survival ex-utero. Mitochondria are critical in terminal cardiac myocyte differentiation after birth, and inhibition of the mitochondrial chaperone cyclophilin D (CypD) enhances this process by regulating the electron transport chain (ETC) to meet increased metabolic demands. Disruption of this process may play a role in neonatal cardiomyopathy and congenital heart disease. We tested the hypothesis that inhibition of CypD with cyclosporin A (CsA) or N-methyl-4-isoleucine cyclosporin (NIM811) will rescue the effects of hypoxia on cardiac and mitochondrial function. Mice were exposed to hypoxia (12% oxygen) immediately before birth (gestational age E19.5) to postnatal day 7 (P7). Hypoxic mice received no treatment (NoTx) or intraperitoneal injections 10mg/kg of vehicle, CsA, or NIM811 from P1 to P6. Room air neonates served as controls. On P7, mice underwent echocardiography and hearts were harvested for mitochondrial isolation for various assays of mitochondrial structure and function. Hypoxia decreased survival and body weight, caused cardiomegaly, decreased cellular organization, and decreased cardiac function in the NoTx and vehicle groups when compared to room air controls, while the enzymatic function of ETC complexes I and III (P<0.01) and complex I-mediated respiration (P<0.0001) was decreased. Treatment with CsA and NIM811 normalized cardiomegaly, cardiac function, cellular structure, ETC activity of complexes I and III and the mitochondrial respiratory capacity for complex I. Thus, pharmacologic inhibition of CypD reversed the effects of hypoxia on ETC activity and overall cardiac function and structurein the neonatal heart. Our study may help develop therapies to treat cardiomyopathies and the effects of hypoxia on the neonatal heart.