Myocyte Proliferation Research Articles

Impact of Non-Pharmacological Interventions on the Mechanisms of Atherosclerosis.

Atherosclerosis remains the leading cause of mortality and morbidity worldwide characterized by the deposition of lipids and fibrous elements in the form of atheroma plaques in vascular areas which are hemodynamically overloaded. The global burden of atherosclerotic cardiovascular disease is steadily increasing and is considered the largest known non-infectious pandemic. The management of atherosclerotic cardiovascular disease is increasing the cost of health care worldwide, which is a concern for researchers and physicians and has caused them to strive to find effective long-term strategies to improve the efficiency of treatments by managing conventional risk factors. Primary prevention of atherosclerotic cardiovascular disease is the preferred method to reduce cardiovascular risk. Fasting, a Mediterranean diet, and caloric restriction can be considered useful clinical tools. The protective impact of physical exercise over the cardiovascular system has been studied in recent years with the intention of explaining the mechanisms involved; the increase in heat shock proteins, antioxidant enzymes and regulators of cardiac myocyte proliferation concentration seem to be the molecular and biochemical shifts that are involved. Developing new therapeutic strategies such as vagus nerve stimulation, either to prevent or slow the disease’s onset and progression, will surely have a profound effect on the lives of millions of people.

Abstract P2020: A Novel Molecular Mechanism Of Cardiac Hypertrophy

Proliferation of cardiac myocytes is accompanied by increases in cell mass to maintain myocyte size in fetal heart. In adult cardiac myocytes, cell growth becomes uncoupled from proliferation after growth stimuli, resulting in hypertrophic growth. Both proliferation and hypertrophy growth of cardiac myocytes require more protein synthesis. Brf1 (TF IIB-related factor 1) and Pol III genes (RNA Polymerase III-dependent genes) are the dominant factors of protein produce. Brf1 is a key regulator for Pol III genes. Cardiac hypertrophy is a critical risk factor for cardiovascular disease, including heart failure, arrhythmia, and sudden death. Cardiac hypertrophy involves both the processes of embryonic gene expression and transcriptional reprogramming. The processes are tightly modulated by different signal pathways and epigenetic regulation. Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. To explore the molecular mechanism of cardiac hypertrophy, we investigate the alterations of Brf1 and Pol III gene transcription under glucose treatment by using cell culture model and animal model. We have found that glucose increases Brf1 expression and Pol III gene transcription. We identified that these genes are mediated by JNK1, MSK1, pAMPK and phosphorylated histone H3. inhibiting the pathways decrease Brf1 expression, reduce Pol III gene transcription, consequently repression of myocytes growth. It implies that developing an effective inhibitor to repress Brf1 expression may provide a potential approach for the therapy of cardiac hypertrophy. More studies on cardiac hypertrophy are going in our laboratory.

Abstract P2001: Vessel Normalization By Targeting Endothelial Cell Plasticity To Improve Cardiac Repair After Myocardial Infarction

Ischemic heart disease is a leading cause of death in the world. Formation of new blood vessels is fundamental to cardiac reperfusion and repair after myocardial infarction (MI), and therapeutic promotion of angiogenesis, therefore, has emerged as a promising strategy for treating ischemic heart diseases. However, the therapeutic efficacy is small and transient in most of pro-angiogenic therapy. Here, we characterize unexpectedly prominent vessel aberrancy, albeit a robust angiogenesis, in MI-associated cardiac tissue by 3-dementional light sheet fluorescence imaging. Our single-cell transcriptome analysis of mouse MI tissues with Cdh5-Cre ; Rosa-LSL-tdTomato -mediated endothelial lineage tracing identifies a robust EC plasticity to acquire mesenchymal gene signature in MI-associated ECs, peaked at 1 week and partially retained at 4 and 8 weeks after MI. Moreover, cardiac ECs undergo transcriptional activation to drive mesenchymal phenotypes including over-proliferation, migration and increased permeability, which lead to vessel abnormality after exposure to in vitro ischemic microenvironment. Furthermore, our single-cell and bulk RNA-seq analysis uncovers a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, culminating in aberrant vascularization. Notably, EC-specific knockout of PDGFR-β abrogates mesenchymal gene expression in ECs, attenuates vascular abnormalities in the infarcted tissue, and improves cardiac repair after MI. Interestingly, our single-nuclei analysis uncovers that genetic ablation of PDGFR-β in ECs temporally reprograms cardiomyocyte metabolism and enhances myocyte survival and proliferation. Pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery normalizes vasculature and improves cardiac function recovery after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer exciting opportunities for normalizing vasculature and treating ischemic heart disease.

RNA Sequencing of Cardiac in a Rat Model Uncovers Potential Target LncRNA of Diabetic Cardiomyopathy.

Background: Diabetic cardiomyopathy (DCM) is one of the major causes of heart failure in diabetic patients; however, its pathogenesis remains unclear. Long non-coding RNAs (lncRNAs) are involved in the development of various cardiovascular diseases, but little is known in DCM. Objective: The present study was conducted to investigate the altered expression signature of lncRNAs and mRNAs by RNA-sequencing and uncovers the potential targets of DCM. Methods: A DCM rat model was established, and the genome-wide expression profile of cardiac lncRNAs and mRNAs was investigated in the rat model with and without DCM by RNA-sequencing. Bioinformatics analysis included the co-expression, competitive endogenous RNA (ceRNA) network, and functional enrichment analysis of deregulated lncRNAs and mRNAs. Results: A total of 355 lncRNA transcripts and 828 mRNA transcripts were aberrantly expressed. The ceRNA network showed that lncRNA XR_351927.3, ENSRNOT00000089581, XR_597359.2, XR_591602.2, and XR_001842089.1 are associated with the greatest number of differentially expressed mRNAs and AURKB, MELK, and CDK1 may be the potential regulatory targets of these lncRNAs. Functional analysis showed that these five lncRNAs are closely associated with fibration, cell proliferation, and energy metabolism of cardiac myocytes, indicating that these core lncRNAs have high significance in DCM. Conclusions: The present study profiled the DCM-specific lncRNAs and mRNAs, constructed the lncRNA-related ceRNA regulatory network, and identified the potential prognostic biomarkers, which provided new insights into the pathogenesis of DCM.

HLHS: Power of the Chick Model.

Background: Hypoplastic left heart syndrome (HLHS) is a rare but deadly form of human congenital heart disease, most likely of diverse etiologies. Hemodynamic alterations such as those resulting from premature foramen ovale closure or aortic stenosis are among the possible pathways. Methods: The information gained from studies performed in the chick model of HLHS is reviewed. Altered hemodynamics leads to a decrease in myocyte proliferation causing hypoplasia of the left heart structures and their functional changes. Conclusions: Although the chick phenocopy of HLHS caused by left atrial ligation is certainly not representative of all the possible etiologies, it provides many useful hints regarding the plasticity of the genetically normal developing myocardium under altered hemodynamic loading leading to the HLHS phenotype, and even suggestions on some potential strategies for prenatal repair.

Circular RNA ACTA1 Acts as a Sponge for miR-199a-5p and miR-433 to Regulate Bovine Myoblast Development through the MAP3K11/MAP2K7/JNK Pathway.

Circular RNAs (circRNAs) are a special class of noncoding RNA molecules that regulate many different biological processes. Myogenesis, a complex process, is primarily regulated by myogenic regulatory factors (MRFs) and various noncoding RNAs. However, the functions and regulatory mechanisms of circRNAs in myoblast development are unclear. In this study, we analyzed circRNA sequencing data of bovine myocyte tissues and identified circACTA1. Functional assays showed that circACTA1 could inhibit bovine myocyte proliferation and promote cell apoptosis and cytodifferentiation. In addition, circACTA1 could promote muscle repair in vivo. Mechanistically, luciferase assay and RNA immunoprecipitation were used to examine the interaction between circACTA1, miR-199a-5p, miR-433, and the target genes MAP3K11 and MAPK8. Meanwhile, we found that miR-199a-5p and miR-433 could suppress the expression of MAP3K11 and MAPK8, respectively. However, circACTA1 could mitigate this effect and activate the JNK signaling pathway. In conclusion, our results suggest that circACTA1 regulates the multiplication, apoptosis, and cytodifferentiation of bovine myocytes by competitively combining with miR-199a-5p and miR-433 to activate the mitogen-activated protein kinase kinase kinase 11 (MAP3K11)/mitogen-activated protein kinase kinase 7 (MAP2K7)/JNK signaling pathway.

Heart neurons use clock genes to control myocyte proliferation.

Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neuron–deficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.

Abstract 9369: Nanoparticle-mediated Delivery of TT-10 Promotes Cardiomyocyte Proliferation and Myocardial Repair

The pharmaceutical product TT-10 increases cardiomyocyte proliferation and survival by upregulating nuclear levels of Yes-associated protein (Yap) and the expression of Yap-targeted genes. When evaluated in a mouse model of myocardial infarction (MI), intraperitoneal TT-10 administration was associated with improvements in infarct size and cardiac function 1 week after treatment, but functional parameters progressively declined at later time points [1]. Here, we tested our hypothesis that the potency of TT-10 for myocardial repair could be increased by loading it into poly-lactic-co-glycolic acid nanoparticles (NPs) before intramyocardial administration. Mice were treated with injections of DPBS (n=9), empty nanoparticles (Empty-NPs, n=8), TT-10 solution (TT-10-sol, n=8), or TT-10-loaded NPs (TT-10-NPs, n=9) immediately after MI was induced via permanent ligation of the coronary artery. Cardiac function was assessed via echocardiography, infarct size via histological Fast-Green staining, cardiac hypertrophy via measures of the heart-weight:bodyweight (HW:BW) ratio, cardiomyocyte proliferation via staining for Ki67 and phosphorylated histone 3 (PH3), and apoptosis via TUNEL. In vitro assessments confirmed that TT-10 was released from the TT-10-NPs for up to 4 weeks, and that the NPs were taken up by cultured cardiomyocytes. 1 and 4 weeks after MI induction and treatment, measures of left-ventricular (LV) ejection fraction (EF) and fractional shortening (FS) were significantly greater in TT-10-NP animals than in the TT-10-sol, Empty-NP, or DPBS groups; LV EF and FS also remained stable in the TT-10-NP group, while measurements in all other groups worsened (but not significantly), between the two time points. TT-10-NP treatment was also associated with significantly smaller (by ~20%, p<0.05) infarcts and significantly lower HW:BW ratios at Week 4; with significantly greater proportions of Ki67 + , PH3 + , and nuclear-Yap + cardiomyocytes at Week 1; and with significant declines in TUNEL + cardiomyocytes 72 hours after MI and treatment. Thus, NP-mediated delivery of TT-10 results in the efficacy of both the magnitude and durability of the benefits associated with promotion of myocyte proliferation in a mouse MI model. [1]. PMID: 30456335

The Hippo pathway regulates density-dependent proliferation of iPSC-derived cardiac myocytes

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell–cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition.

Abstract P335: Nanoparticle-mediated Delivery Of TT-10 Promotes Myocardial Repair

The pharmaceutical product TT-10 increases cardiomyocyte proliferation and survival by upregulating nuclear levels of Yes-associated protein (Yap) and the expression of Yap-targeted genes. When evaluated in a mouse model of myocardial infarction (MI), intraperitoneal TT-10 administration was associated with improvements in infarct size and cardiac function one week after treatment, but functional parameters progressively declined at later time points [1]. Here, we tested our hypothesis that the potency of TT-10 for myocardial repair could be increased by loading it into poly-lactic-co-glycolic acid nanoparticles (NPs) before intramyocardial administration. Mice were treated with injections of DPBS (n=9), empty nanoparticles (Empty-NPs, n=8), TT-10 solution (TT-10-sol, n=8), or TT-10-loaded NPs (TT-10-NPs, n=9) immediately after MI was induced via permanent ligation of the coronary artery. Cardiac function was assessed via transthoracic echocardiography, infarct size via histological Fast-Green staining, cardiac hypertrophy via measures of the heart-weight:bodyweight (HW:BW) ratio, cardiomyocyte proliferation via staining for Ki67 and phosphorylated histone 3 (PH3), and apoptosis via TUNEL. In vitro assessments confirmed that TT-10 was released from the TT-10-NPs for up to four weeks, and that the NPs were taken up by cultured cardiomyocytes. One and four weeks after MI induction and treatment, measures of left-ventricular (LV) ejection fraction (EF) and fractional shortening (FS) were significantly greater in TT-10-NP animals than in the TT-10-sol, Empty-NP, or DPBS groups; LV EF and FS also remained stable in the TT-10-NP group, while measurements in all other groups worsened (but not significantly) (LV EF at Week 4: TT-10-sol vs TT-10-NPs; 22.25 ± 1.474 vs 43.95 ± 1.884, p<0.05), between the two time points. TT-10-NP treatment was also associated with significantly smaller (by ~20%, p<0.05) infarcts and significantly lower HW:BW ratios at Week 4; with significantly greater proportions of Ki67 + , PH3 + , and nuclear-Yap + cardiomyocytes at Week 1; and with significant declines in TUNEL + cardiomyocytes 72 hours after MI and treatment. Thus, NP-mediated delivery of TT-10 results in the efficacy of both the magnitude and durability of the benefits associated with promotion of myocyte proliferation in a mouse MI model. [1] H. Hara, N. Takeda, M. Kondo, M. Kubota, T. Saito, J. Maruyama, et al., Discovery of a Small Molecule to Increase Cardiomyocytes and Protect the Heart After Ischemic Injury, JACC Basic Transl Sci, 3 (2018) 639-653.

Abstract MP213: Activation Of Biosynthetic Pathways Regulates Cardiomyocyte Cell Cycle Induction In Human Cardiomyocyte

Induction of cardiomyocyte proliferation is a promising therapeutic approach to treat heart failure. Several studies have identified metabolism as an important regulator of myocyte proliferation; however, the changes in metabolism during cardiomyocyte division remain unclear. Here, we use ectopic expression of cyclin B1, Cyclin D1, CDK1, and CDK4 (termed 4F) as a tool for understanding how metabolism influences cardiomyocyte proliferation. Mature hiPS-CMs stimulated to proliferate by 4F expression showed significant downregulation of oxidative phosphorylation genes, decreased glucose oxidation, and upregulation of genes that regulate biosynthetic pathways of glucose metabolism such as those involved in NAD(P) + synthesis ( NAMPT, NADK1, NNT ), the hexosamine biosynthetic pathway (HBP) and protein O-GlcNAcylation ( GFPT1 , OGT, OGA ), and the serine biosynthesis pathway (SBP; PHGDH , PSAT1 , SHMT2 ). In 4F-expressing hiPSC-CMs, stable isotope tracing indicated higher enrichment of glucose-derived 13 C in pentose phosphate intermediates, UDP-hexose, phospholipid precursors, NAD + , pyrimidines, UDP-HexNAc, and products of the serine biosynthesis pathway and one-carbon metabolism, suggesting that cell cycle induction activates biosynthetic pathways in cardiomyocytes. Knocking down nicotinamide phosphoribosyltransferase (NAMPT), a critical enzyme in the NAD + salvage pathway, 2 days before 4F overexpression significantly inhibited cell cycle progression in 4F-transduced hiPS-CMs. OGA overexpression, which catalyzes the hydrolytic cleavage of O-GlcNAc from post-transitionally modified proteins, completely abolished 4F-mediated cell cycle induction. Furthermore, NCT503, an inhibitor of the rate-limiting step in the serine biosynthesis pathway, abolished 4F-mediated increases in cell cycle markers. In a gain-of-function approach, we overexpressed phosphoenolpyruvate carboxykinase 2 (PCK2), which can drive carbon from the Krebs cycle to the glycolytic intermediate pool. PCK2 overexpression significantly augmented 4F-mediated cell cycle entry. These findings suggest that a metabolic shift from catabolic to anabolic activity is a critical step for cardiomyocyte cell cycle entry and is required to facilitate proliferation.

Recent Advance on Vitamin D in Athletes

PURPOSE: Vitamin D plays important roles in calcium homeostasis and bone metabolism. Since vitamin D receptors (VDRs) are located in a variety of organs, including skeletal muscle, vitamin D has potentially widespread effects. The purpose of this review was to summarize the current understanding of the effects of vitamin D on muscle function and exercise performance in athletes.METHODS: In this narrative review, we summarized previous studies by searching the literature in the PubMed, Google Scholar, and Science Direct databases.RESULTS: Vitamin D has been shown to regulate multiple actions in skeletal muscle tissue, such as myocyte proliferation and growth via genomic and non-genomic molecular pathways. Higher levels of vitamin D are associated with improved skeletal muscle function and exercise performance. Moreover, in some studies, vitamin D supplementation has beneficial effects on muscle strength in athletes, especially those who are vitamin D-deficient.CONCLUSIONS: Vitamin D appears to have beneficial effects on muscle and exercise performance in athletes. However, more studies are needed to clarify the action and dosage of vitamin D in athletes.

Cell cycle induction in human cardiomyocytes is dependent on biosynthetic pathway activation

AimsThe coordinated gene and metabolic programs that facilitate cardiomyocyte entry and progression in the cell cycle are poorly understood. The purpose of this study was to identify the metabolic changes that influence myocyte proliferation. Methods and resultsIn adult mouse cardiomyocytes and human induced pluripotent stem cell cardiomyocytes (hiPS-CMs), cell cycle initiation by ectopic expression of Cyclin B1, Cyclin D1, CDK1, and CDK4 (termed 4F) downregulated oxidative phosphorylation genes and upregulated genes that regulate ancillary biosynthetic pathways of glucose metabolism. Results from metabolic analyses and stable isotope tracing experiments indicate that 4F-mediated cell cycle induction in hiPS-CMs decreases glucose oxidation and oxidative phosphorylation and augments NAD+, glycogen, hexosamine, phospholipid, and serine biosynthetic pathway activity. Interventions that diminish NAD+ synthesis, serine synthesis, or protein O-GlcNAcylation decreased 4F-mediated cell cycle entry. In a gain of function approach, we overexpressed phosphoenolpyruvate carboxykinase 2 (PCK2), which can drive carbon from the Krebs cycle to the glycolytic intermediate pool, and found that PCK2 augments 4F-mediated cell cycle entry. ConclusionsThese findings suggest that a metabolic shift from catabolic to anabolic activity is a critical step for cardiomyocyte cell cycle entry and is required to facilitate proliferation.

Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction.

Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2WTCMs or CCND2OECMs, respectively) and injected into infarcted pig hearts. Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2WTCMs or CCND2OECMs (3×107 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. The number of the engrafted CCND2OECMs exceeded that of the engrafted CCND2WTCMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2WTCMs or the delivery vehicle, the administration of CCND2OECM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2OECM-treated than from the CCND2WTCM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2OECMs (CCND2OEExos) than from the CCND2WTCMs (CCND2WTExos). As demonstrated by our study, CCND2OEExos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2OEExos versus CCND2WTExos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. We demonstrated that enhanced potency of CCND2OECMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2OECMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

Ablation of VDR Induced Atrophy of Skeletal Muscle via Locally Affecting the Renin‐Angiotensin System

Objective Our previous studies demonstrated that vitamin D is a vital protector against tissue injuries by locally suppressing renin-angiotensin system (RAS). It is known that vitamin D deficiency is associated with a range of muscle disorders, such as muscle atrophy. This study aimed to investigate the biological role of vitamin D receptor (VDR) in skeletal muscle and the underlying mechanism using a mice model with deletion of VDR associated with dexamethasone-induced muscle atrophy. Materials and Methods The wild type and VDR(-/-) mice were treated with vehicle and dexamethasone (i.p., once daily) for 10 consecutive days. The murine myoblast cell line C2C12 and the skeletal muscle satellite cells were applied as in vitro models in this study. Results Treatment with the angiotensin II (Ang II) type 1 receptor blocker olmesartan eliminated Ang II-induced atrophy of myotube cells, reflected by alleviating myotube cell atrophy, suggesting that Ang II could aggravate atrophy of skeletal muscle. The ablation of VDR in muscle satellite cells worsened the phenotype of muscle atrophy induced by Ang II. After challenging to dexamethasone, VDR-null mice exhibited more severe amyotrophy compared with wild-type counterparts, manifested by significant decrease in muscle wet weight coefficient of the tibialis anterior muscle and gastrocnemius as well as the reduction of limb grip and weight-loading swimming time. Similarly, the dystrophin fluorescence staining and the haematoxylin eosin (H&E) staining showed much more decrease in cross-sectional area of skeletal muscle in the VDR-null mice in a comparison with the counterparts. MAFbx and MuRF1 are two E3 ubiquitin ligases that are important regulators for ubiquitin-mediated protein degradation in skeletal muscle, while myogenic differentiation-1 (MyoD) and myosin heavy chain (MHC) are necessary to maintain myocytes proliferation and differentiation. VDR ablation significantly accelerated the up-regulation of MAFbx and MuRF1 proteins and down-regulated protein expression of MyoD and MHC in skeletal muscle tissue of mice with muscle atrophy. Conclusion These results provided evidences that VDR might attenuate skeletal muscle atrophy at least in part by suppressing the renin-angiotensin system.

MiR-130b inhibits proliferation and promotes differentiation in myocytes via targeting Sp1.

Muscle regeneration after damage or during myopathies requires a fine cooperation between myoblast proliferation and myogenic differentiation. A growing body of evidence suggests that microRNAs play critical roles in myocyte proliferation and differentiation transcriptionally. However, the molecular mechanisms underlying the orchestration are not fully understood. Here, we showed that miR-130b is able to repress myoblast proliferation and promote myogenic differentiation via targeting Sp1 transcription factor. Importantly, overexpression of miR-130b is capable of improving the recovery of damaged muscle in a freeze injury model. Moreover, miR-130b expression is declined in the muscle of muscular dystrophy patients. Thus, these results indicated that miR-130b may play a role in skeletal muscle regeneration and myopathy progression. Together, our findings suggest that the miR-130b/Sp1 axis may serve as a potential therapeutic target for the treatment of patients with muscle damage or severe myopathies.

Platelet-derived growth factor-BB induces pulmonary venous smooth muscle cells proliferation by upregulating calcium sensing receptor under hypoxic conditions.

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, which exists in both pulmonary arteries and pulmonary veins. Pulmonary vascular remodeling stems from excessive proliferation of pulmonary vascular myocytes. Platelet-derived growth factor-BB (PDGF-BB) is a vital vascular regulator whose level increases in PH human lungs. Although the mechanisms by which pulmonary arterial smooth muscle cells respond to PDGF-BB have been studied extensively, the effects of PDGF-BB on pulmonary venous smooth muscle cells (PVSMCs) remain unknown. We herein examined the involvement of calcium sensing receptor (CaSR) in PDGF-BB-induced PVSMCs proliferation under hypoxic conditions. In PVSMCs isolated from rat intrapulmonary veins, PDGF-BB increased the cell number and DNA synthesis under normoxic and hypoxic conditions, which was accompanied by upregulated CaSR expression. The influences of PDGF-BB on proliferation and CaSR expression in hypoxic PVSMCs were greater than that in normoxic PVSMCs. In hypoxic PVSMCs superfused with Ca2+-free solution, restoration of extracellular Ca2+ induced an increase of [Ca2+]i, which was significantly smaller than that in PDGF-BB-treated hypoxic PVSMCs. The positive CaSR modulator spermine enhanced, whereas the negative CaSR modulator NPS2143 attenuated, the extracellular Ca2+-induced [Ca2+]i increase in PDGF-BB-treated hypoxic PVSMCs. Furthermore, the spermine enhanced, whereas the NPS2143 inhibited, PDGF-BB-induced proliferation in hypoxic PVSMCs. Silencing CaSR with siRNA attenuated the extracellular Ca2+-induced [Ca2+]i increase in PDGF-BB-treated hypoxic PVSMCs and inhibited PDGF-BB-induced proliferation in hypoxic PVSMCs. In conclusion, these results demonstrated that CaSR mediating PDGF-BB-induced excessive PVSMCs proliferation is an important mechanism involved in the initiation and progression of PVSMCs proliferation under hypoxic conditions.

The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

Cardiac Morphology and Collagen Deposition in A Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3) Knockout Mouse model

Background: Glucose-6-phosphatase-- β (3), one of multiple isoforms of glucose-6-phosphatase, catalyzes the final step in gluconeogenesis. It is known that mutated G6P3 is associated with severe neutropenia in addition to congenital heart defects, but little is known about the histological changes in cardiac tissue as a result of mutated or deleted G6PC34. Objectives: We sought to further characterize the histological alterations caused by deleted G6PC3 and determine the role of collagen deposition, myocyte proliferation and apoptosis in these changes. Methods: Cardiac tissue from G6PC3 knockout mice and WT mice were harvested, imbedded and stained for markers of collagen (Trichrome), proliferation (KI-67), apoptosis (caspase 3) and hematopoietic stem cells (CD34). Slides were digitally uploaded, and Leica stain quantification was calculated. Results: We demonstrated that in G6PC3 knock out adult mice have significant differences in heart morphology including decreased left ventricular collagen, decreased cellular proliferation and increased apoptosis histologically. Conclusions: As compared to wild type, the hearts of G6PC3 knockout mice demonstrated significantly decreased collagen globally, a crucial component for adequate strength and contractility of myocardial tissue. More investigation should be done to further explore the functional effects of such alterations via echocardiograms.

A loss-of-function mutation p.T256M in NDRG4 is implicated in the pathogenesis of pulmonary atresia with ventricular septal defect (PA/VSD) and tetralogy of Fallot (TOF).

Pulmonary atresia with ventricular septal defect (PA/VSD) is a rare congenital heart disease (CHD) characterized by a lack of luminal continuity and blood flow from either the right ventricle or the pulmonary artery, together with VSDs. The prevalence of PA/VSD is about 0.2% of live births and approximately 2% of CHDs. PA/VSD is similar to tetralogy of Fallot (TOF) in terms of structural and pathological characteristics. The pathogenesis of these two CHDs remains incompletely understood. It was previously reported that N-myc downstream-regulated gene (NDRG)4 is required for myocyte proliferation during early cardiac development. In the present study, we enrolled 80 unrelated patients with PA/VSD or TOF and identified a probably damaging variant p.T256M of NDRG4. The p.T256M variant impaired the proliferation ability of human cardiac myocytes (hCM). Furthermore, the p.T256M variant resulted in G1 and G2 arrest of hCM, followed by an increase in p27 and caspase-9 expression. Our results provide evidence that the p.T256M variant in NDRG4 is a pathogenic variant associated with impaired hCM proliferation and cell-cycle arrest and likely contributes towards the pathogenesis of PA/VSD and TOF.