Expression Of Sarcomeric Proteins Research Articles

Rat Cardiac progenitor cells and their application in cell therapy

cells and the subsequent heart failure. When the pharmacological approach no longer complies with the disease evolution, organ transplantation appears to be the only treatment able to rescue the patient life. Cell therapy promises to be clinically efficient and would allow circumventing many limitations of organ transplantation, such as organ low availability, major surgical procedures, high costs and longterm immunosuppression [1]. We designed porous Poly-Lactic Acid (PLLA) and Fibroin scaffolds to deliver CPCs in the heart, we isolated and characterized CPCs for the expression of c-Kit, MDR-1 and Sca-1 by flow cytometry, we tested their degree of differentiation in vitro studying the expression of all known rat sarcomeric proteins by real-time PCR and their differentiated morphology on Electron Microscopy samples [2]. A particular attention was given to the expression of microRNA, because their role in the differentiation process of cardiac precursors is emerging. We also tested the host reaction to scaffolds, CPCs, and CPCs/scaffolds. In vivo, almost all the used scaffolds induced a foreign body reaction in nude mouse and rats, but not in SCID mice. Cardiac stem cells a T cell-mediated immune response induced in nude mice, letting us suppose that, differently from Mesenchymal Stem Cells, they express MHC molecules on their surface. The degree of differentiation, the expression of ECM and integrin proteins, and the expression of several sarcomeric proteins were dependent on the type of scaffold and the polymer used.

Uniaxial cyclic strain of human adipose–derived mesenchymal stem cells and C2C12 myoblasts in coculture

Tissue engineering skeletal muscle in vitro is of great importance for the production of tissue-like constructs for treating tissue loss due to traumatic injury or surgery. However, it is essential to find new sources of cells for muscle engineering as efficient in vitro expansion and culture of primary myoblasts are problematic. Mesenchymal stem cells may be a promising source of myogenic progenitor cells and may be harvested in large numbers from adipose tissue. As skeletal muscle is a mechanically dynamic tissue, we have investigated the effect of cyclic mechanical strain on the myogenic differentiation of a coculture system of murine C2C12 myoblasts and human adipose–derived mesenchymal stem cells. Fusion of mesenchymal stem cells with nascent myotubes and expression of human sarcomeric proteins was observed, indicating the potential for myogenic differentiation of human mesenchymal stem cells. Cyclic mechanical strain did not affect the fusion of mesenchymal stem cells, but maturation of myotubes was perturbed.

Expression of Heat Shock Protein (Hsp90) Paralogues Is Regulated by Amino Acids in Skeletal Muscle of Atlantic Salmon

Heat shock proteins 90 (Hsp90) have an essential role in sarcomere formation and differentiation in skeletal muscle and also act as molecular chaperones during protein folding impacting a wide range of physiological processes. We characterised and provided a phylogenetically consistent nomenclature for the complete repertoire of six Hsp90 paralogues present in duplicated salmonid fish genomes (Hsp90α1a, Hsp90α1b, Hsp90α2a, Hsp90α2b, Hsp90ß1a and Hsp90ß1b). The expression of paralogues in fast skeletal muscle was investigated using in vivo fasting-feeding experiments and primary myogenic cultures. Fasted juvenile Atlantic salmon (Salmo salar) showed a transient 2 to 8-fold increase in the expression of all 4 Hsp90α paralogues within 24h of satiation feeding. Hsp90α1a and hsp90α1b also showed a pronounced secondary increase in expression after 10 days, concomitant with muscle differentiation and the expression of myogenin and sarcomeric proteins (mlc2, myhc). Hsp90ß1b was constitutively expressed whereas Hsp90ß1a expression was downregulated 10-fold between fasted and fed individuals. Hsp90α1a and Hsp90α1b were upregulated 10 to 15-fold concomitant with myotube formation and muscle differentiation in vitro whereas other Hsp90 paralogues showed no change in expression. In cells starved of amino acid (AA) and serum for 72h the addition of AA, but not insulin-like growth factor 1, increased phosphorylation of mTor and expression of all 4 hsp90α paralogues and associated co-chaperones including hsp30, tbcb, pdia4, pdia6, stga and fk504bp1, indicating a general activation of the protein folding response. In contrast, Hsp90ß1a expression in vitro was unresponsive to AA treatment indicating that some other as yet uncharacterised signal(s) regulate its expression in response to altered nutritional state.

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression.

Differential responses of the right ventricle to abnormal loading conditions in vivo: Possible pathophysiologic mechanisms

The right ventricle (RV) demonstrates differential adaptations in response to pressure versus volume loading, a phenomenon that may be important in the management of children and adults with congenital heart disease (CHD). The purpose of this study is to elucidate possible transcriptional mechanisms of the RV response to pressure versus volume loading invivo. Fetal lambs had aortopulmonary shunting or pulmonary artery (PA) banding. Four weeks after spontaneous delivery, ovine hearts were evaluated for hemodynamic changes and changes in expression of sarcomeric gene proteins and transcriptional factors. Western blot densitometry and chromatin immunoprecipitation were applied using standard techniques. Transactivation assays were performed using transient transfections in Schneider's Drosophila line 2 cells in culture. After PA banding, the RV pressure increased from 36±4 mm Hg (n=4) to 96±8 mm Hg (n=4, P<.05). The RVs of shunted and banded animals showed significant increases in the expression levels and promoter binding of activators myocyte enhancer factor 2, GATA-4, Nkx2.5, transcriptional enhancer factor 1, and specificity protein (Sp) 1. The transcriptional repressor Sp3 was downregulated in shunted animals, but its expression was increased paradoxically in the RV of the PA band group. Immunoprecipitation of Sp3 showed posttranslational modification to the acetylated isoform. In transient transfections of Schneider's Drosophila line 2 cells, acetylation of Sp3 converted it from a transcriptional repressor to an activator. Posttranslational modifications of the transcriptional repressor Sp3, by acetylation, may be an important mechanism in the differential response of the RV to abnormal loading conditions. Sp3 may serve as a biomarker for RV failure for various heart defects in children and adults with CHD. These findings may have therapeutic implications in the management of right heart failure.

Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle

Over 190 mutations in the human skeletal muscle α-actin gene, ACTA1 cause congenital actin myopathies. We transgenically expressed six different mutant actins, G15R, I136M, D154N, V163L, V163M and D292V in Drosophila indirect flight muscles and investigated their effects in flies that express one wild type and one mutant actin copy. All the flies were flightless, and the IFMs showed incomplete Z-discs, disorganised actin filaments and 'zebra bodies'. No differences in levels of sarcomeric protein expression were observed, but tropomodulin staining was somewhat disrupted in D164N, V163L, G15R and V163M heterozygotes. A single copy of D292V mutant actin rescued the hypercontractile phenotypes caused by TnI and TnT mutants, suggesting that the D292V mutation interferes with thin filament regulation. Our results show that expression of actin mutations homologous to those in humans in the indirect flight muscles of Drosophila disrupt sarcomere organisation, with somewhat similar phenotypes to those observed in humans. Using Drosophila to study actin mutations may help aid our understanding of congential myopathies caused by actin mutations.

G.P.108 Sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy

Abstract The expression of sarcomeric proteins is impaired in facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy characterized by muscle weakness. In addition, overexpression of DUX4, the leading FSHD candidate gene, has been shown to activate pathways involved in sarcomeric protein degradation. Here we investigated whether sarcomeric dysfunction contributes to muscle weakness, using demembranated single muscle fibers of FSHD patients and control subjects. The force generating capacity of sarcomeres is significantly impaired in FSHD. Sarcomeric weakness was restricted to type II muscle fibers, in which maximum force generation was only 70% of normal strength. In contrast to active force measurements, a 5- to 12-fold increase in passive force was seen in type I and type II fibers respectively, indicating stiffening of titin molecules. Corroborating these findings, we observed a decrease in myofilament lattice spacing and increase in calcium sensitivity, both physiological consequences of titin stiffening. Based on these findings, we propose that sarcomeric dysfunction plays a critical role in muscle weakness in FSHD.

RBM24: a 'Regional Business Manager' in charge of the maintenance of sarcomeric protein expression 24 h a day?

This editorial refers to ‘RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility’ by K.L. Poon et al., doi:10.1093/cvr/cvs095. RNA-binding proteins affect the proteome of a single cell by controlling alternative splicing, transport, localization, and stability of their target RNAs.1 They often display tissue-specific expression, and their crucial influence on the protein set-up especially in muscle cells has become recognized recently.2 A contribution by Poon et al. 3 focuses on RBM24 (also known as Seb-4) and its role in heart development. Their interest was sparked by the observation that Rbm24 is dramatically up-regulated in human embryonic stem cells upon differentiation into cardiomyocytes. They went on to characterize its expression pattern in zebrafish embryos, where its expression was mainly observed in the heart and somites. This fits well with previous data from another group on Xenopus embryos, who had shown a region-specific expression pattern of Rbm24 in the heart and the somites with minor levels in the lens and otic vesicles.4 A major part of the zebrafish genome is duplicated,5 and RMB24 also exists as two variants, Rbm24a and Rbm24b. Poon et al. focused their work on Rbm24a and were able to show that knockdown of its expression by morpholino oligonucleotides in zebrafish leads to impaired contractility of the heart.3 The supplementary data show no discernible effects on the somites, and the vasculature also seems to be normal in the morphants. The heart phenotype is explained by a reduced complexity of the myofibrillar meshwork in the embryonic cardiomyocytes, potentially due to a slight …

Asialoerythropoietin, a Nonerythropoietic Derivative of Erythropoietin, Displays Broad Anti-Heart Failure Activity

We investigated the effects of asialoerythropoietin (asialoEPO), a nonerythrogenic erythropoietin derivative, on 3 murine models of heart failure with different etiologies. Doxorubicin (15 mg/kg) induced heart failure within 2 weeks (toxic cardiomyopathy). Treatment with asialoEPO (6.9 μg/kg) for 2 weeks thereafter attenuated the associated left ventricular dysfunction and dilatation. In addition, the asialoEPO-treated heart showed less myocardial fibrosis, inflammation, and oxidative damage, and diminished atrophic cardiomyocyte degeneration, which was accompanied by restored expression of GATA-4 and sarcomeric proteins. Mice with large 6-week-old myocardial infarctions exhibited marked left ventricular dysfunction with adverse remodeling (ischemic cardiomyopathy). AsialoEPO treatment for 4 weeks significantly mitigated progression of the dysfunction and remodeling and reduced myocardial fibrosis, inflammation, and oxidative damage. Finally, 25-week-old δ-sarcoglycan-deficient mice (genetic cardiomyopathy) were treated with asialoEPO for 5 weeks. AsialoEPO mitigated the progressive cardiac remodeling and dysfunction through cardiomyocyte hypertrophy, and upregulated expression of GATA-4 and sarcomeric proteins. AsialoEPO appears to act by altering the activity of the downstream erythropoietin receptor signals extracellular signal-regulated protein kinase, Akt, signal transducer, and activator of transcription 3 and 5 in a model-specific manner. The findings suggest that asialoEPO exerts broad cardioprotective effects through distinct mechanisms depending on the model, which are independent of the erythrogenic action. This compound may be promising for the treatment of heart failure of various etiologies.

Abstract P158: MicroRNA-22 Modulates Cardiac Gene Expression and Controls Compensation to Hemodynamic Stress in Mice

Recent evidence suggests that miRNAs play an important role in cardiac morphogenesis and pathophyiology of heart failure. To explore the role of miR-22 in the mouse heart physiology, we generated miR-22 null (KO) mice. Although, miR-22 KO mice showed normal cardiac structure and function at baseline, these mice are sensitized to maladaptive remodeling (cardiac dilation) and decompensation in response to pressure overload by transverse aortic constrictions (TAC) stimulation. Genome-wide molecular analysis of KO hearts revealed attenuated expression of numerous CarG-dependent genes encoding proteins that reside at the sarcomeric Z-disc (including Myh7, Acta1, Mlp, Melusin, MyoZ2) indicating that miR-22 is required for optimum muscle gene expression. Alterations in sarcomeric gene expression is especially interesting as this suggests a primary role of miR-22 in controlling cardiac contractility and adaptation to stress. Targetomics analysis revealed that mechanistically this effect could be modulated in part by miR-22 target PURB (Purine Rich element binding protein B), a transcriptional/translational repressor. In conclusion we define a critical role of miR-22 in cardiac adaptation to hemodynamic stress. Furthermore, these data provides a previously unseen essential molecular mechanism that underlies homeostatic control of sarcomeric protein expression in the heart.

Abstract 8771: Asialoerythropoietin, a Nonerythropoietic Derivative of Erythropoietin, Displays Broad Anti-Heart Failure Activity

Accumulating evidence suggests cardioprotective effect of erythropoietion (EPO) in experiments, but EPO has some unwanted effects. We investigated effect of asialoerythropoietin (AEPO), a nonerythrogenic derivative of EPO, on 3 different models of heart failure: doxorubicin (DOX)-induced cardiomyopathy, post-large myocardial infarction (MI), and dilated cardiomyopathy in δ-sarcoglycan deficient mouse (Sgcd-null). Accoding to echocardiography and cardiac catheterization, a single intraperitoneal injection of DOX (15 mg/kg) induced an established heart failure in mice 2 weeks later, to which AEPO (1500 U/kg, twice a week, for 2 weeks) was administered. Left ventricular dysfunction was attenuated in the AEPO-treated group (ejection fraction: 71±3% vs. 50±1% in controls). The AEPO-treated heart showed less myocardial fibrosis, inflammatory cell infiltration, and atrophic degeneration of cardiomyocytes accompanying restored expression of GATA-4 and sarcomeric proteins. Among downstream signals of EPO receptor, DOX inactivated ERK in the heart, of which activity AEPO restored. Next, in the mice with large sized, 6-week-old MI, a marked left ventricular dysfunction with adverse remodeling was apparent. AEPO treatment for the subsequent 4 weeks mitigated the further progression of dysfunction and remodeling (ejection fraction: 37±2% vs. 26±3% in controls), accompanying reduced myocardial fibrosis. ERK and Akt were activated in the MI bearing hearts, which were furthermore augmented by AEPO. AEPO activated STAT3, too. Finally, AEPO was administered to 25 weeks old Sgcd-null mice with decreased left ventricular contractility, for the subsequent 5 weeks. Left ventricular dysfunction and dilatation were mitigated (ejection fraction: 71±1% vs. 52±0.4% in controls), accompanying reduced myocardial fibrosis and furthermore compensative hypertrophy. ERK was inactivated, and Akt was activated in the heart of Sgcd null mice. AEPO restored the ERK activity, and furthermore activated Akt. Neither polycythemia nor splenomegaly was noted in any groups. In conclusion, AEPO displays broad anti-heart failure activity regardless of the etiology of heart failure through distinct molecular signals.

Abstract 16798: MicroRNA 142-5p Targets Myocardial Hypertrophy and Cytokine Response

Background: p300 is a dose-dependent and limiting regulator of cardiac hypertrophy. p300 regulates hypertrophy by controlling the expression of specific microRNAs. Objective: To dissect the role of miR142 during hypertrophic growth and to verify its targets. Methods and Results: In mice with myocardial overexpression (OE) of p300 (p300tg) and in acute myocardial ischemia, we observed altered expression of microRNAs miR142-5p and -3p (miR142). Induction of myocyte hypertrophy in vitro and in vivo were associated with downregulation of miR142. Induction of miR142 by serum was sensitive to inhibitors of ERK/MAPK and JNK signaling pathways, establishing an inverse relationship between hypertrophic signaling and miR142 levels. Adenovirus-mediated OE of p300 in NRVM caused a steep reduction in miR142, while siRNA-mediated KD of p300 significantly upregulated miR142. Thus, p300 is both necessary and sufficient for miR142 downregulation. MiR142 altered the expression of many sarcomeric proteins including Tni2, Tnt2, Myh3 and Mlc1. MiR142 directly inhibited the translation of ACTN4 through a conserved 3’UTR binding site. MiR142 potently repressed multiple components of the NF-κB, TNF-α and interleukin signaling pathways and abrogated the upregulation of iNOS mRNA and NO production in myocytes exposed to cytokines. We identified IL6st/gp130 as a direct target of miR142 through paired 3’UTR 142-5p and -3p binding sites, implicating miR142 in myocyte survival signalling. Lentiviral transduction of miR142 confirmed the same gene targets in myocardium in vivo, associated with development of systolic dysfunction and extensive apoptosis at 2 months. Finally, miR142 inhibited the expression of p300 both in vitro and in vivo, through a miR142-5p 3’UTR binding site. Altered immune response gene expression in p300tg mice was restored to near-wt levels by miR142 OE, indicating that p300 and miR142 are mutually inhibitory. Conclusion: MiR142 is a potent inhibitor of myocardial cytokine signaling through repression of IL6st/gp130 expression. Induction of hypertrophy involves the MAPK-activated, p300-mediated inhibition of miR142, which removes a block to sarcomeric protein expression, gp130-mediated survival signaling, and sustained expression of p300.

P5.77 Sarcomeric gene and protein expression during human myogenesis in vitro

In vertebrates, skeletal muscle is found throughout the body and forms during the entire life span. Adult skeletal muscle displays a high regenerative capacity, with satellite cells as the primary source of this remarkable ability. Satellite cells are quiescent muscle progenitors located between myofibers and their surrounding basal lamina. The formation of skeletal muscle cells, myogenesis, is a complex process, which takes place during embryonic development, postnatal growth and adult muscle regeneration. Myogenesis is marked by specific changes in muscle cell morphology and cytoarchitecture through the fusion of mononucleated myoblasts into multinucleated myotubes and the assembly of contractile sarcomeric myofibrils. The formation of contractile myofibrils involves the transition of myoblast cytoarchitecture, which requires a change in the expression of proteins from non-muscle-specific to muscle-specific isoforms. In this study, combined molecular and immunocytochemistry methods were used to investigate the expression profile of sarcomeric genes and proteins during myoblast proliferation and differentiation in human muscle cell line. We observed the expression of a panel of sarcomeric gene transcripts together with the early expression of a number of the investigated sarcomeric proteins at the proliferated myoblasts. In addition, after 6 days of differentiation, the majority of cells expressed all the investigated sarcomeric proteins. The results presented here provide novel insights into the sequential onset of the sarcomeric genes and proteins and support the previous notion that non-committed stem cells exist in the adult muscle satellite cells during myogenesis in vitro. In addition, our study demonstrates the effectiveness of a differentiation system, which transforms undifferentiated and star-shaped myoblasts, into long multinucleated myotubes, displaying the features of contractile muscle cells.

Asialoerythropoietin, a Nonerythropoietic Derivative of Erythropoietin, Exerts Broad Anti-Heart Failure Actions Regardless of the Etiology of Heart Failure

We investigated effect of asialoerythropoietin (AEPO), a nonerythrogenic derivative of EPO, on 3 different models of heart failure. A single intraperitoneal injection of doxorubicin (15 mg/kg) induced an established heart failure in mice 2 weeks later, to which AEPO (1500 U/kg, twice a week, for 2 weeks) was administered. Left ventricular dysfunction was attenuated by AEPO. The AEPO-treated heart showed less myocardial fibrosis, inflammatory cell infiltration, and atrophic degeneration of cardiomyocytes accompanying restored expression of GATA-4 and sarcomeric proteins.

Erythropoietin Receptor Signaling Mitigates Renal Dysfunction-Associated Heart Failure by Mechanisms Unrelated to Relief of Anemia

We examined the effect of asialoerythropoietin (asialoEPO), a nonerythrogenic derivative of erythropoietin (EPO), on renal dysfunction-associated heart failure. Although EPO is known to exert beneficial effects on cardiac function, the clinical benefits in patients with chronic kidney disease are controversial. It remains to be addressed whether previously reported outcomes were the result of relief of the anemia, adverse effects of EPO, or direct cardiovascular effects. Mice underwent 5/6 nephrectomy to cause renal dysfunction. Eight weeks later, when renal dysfunction was established, anemia and cardiac dysfunction and remodeling were apparent. Mice were then assigned to receive saline (control), recombinant human erythropoietin (rhEPO) at 5,000 IU (714 pmol)/kg, or asialoEPO at 714 pmol/kg, twice/week for 4 weeks. Although only rhEPO relieved the nephrectomy-induced anemia, both rhEPO and asialoEPO significantly and similarly mitigated left ventricular dilation and dysfunction. The hearts of rhEPO- or asialoEPO-treated mice showed less hypertrophy, reflecting decreases in cardiomyocyte hypertrophy and degenerative subcellular changes, as well as significant attenuation of fibrosis, leukocyte infiltration, and oxidative deoxyribonucleic acid damage. These phenotypes were accompanied by restored expression of GATA-4, sarcomeric proteins, and vascular endothelial growth factor and decreased inflammatory cytokines and lipid peroxidation. Finally, myocardial activation was observed of extracellular signal-regulated protein kinase and signal transducer and activator of transcription pathways in the treated mice. EPO receptor signaling exerts direct cardioprotection in an animal model of renal dysfunction-associated heart failure, probably by mitigating degenerative, pro-fibrosis, inflammatory, and oxidative processes but not through relief of anemia.

Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes

Heart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation. We treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)-polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation. We observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium. These studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy.

Induced differentiation of adipose-derived stromal cells into myoblasts

This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.

Calcium sensitivity and the Frank–Starling mechanism of the heart are increased in titin N2B region-deficient mice

Previous work suggests that titin-based passive tension is a factor in the Frank–Starling mechanism of the heart, by increasing length-dependent activation (LDA) through an increase in calcium sensitivity at long sarcomere length. We tested this hypothesis in a mouse model (N2B KO model) in which titin-based passive tension is elevated as a result of the excision of the N2B element, one of cardiac titin's spring elements. LDA was assessed by measuring the active tension–pCa (− log[Ca 2+]) relationship at sarcomere length (SLs) of 1.95, 2.10, and 2.30 µm in WT and N2B KO skinned myocardium. LDA was positively correlated with titin-based passive tension due to an increase in calcium sensitivity at the longer SLs in the KO. For example, at pCa 6.0, the KO:WT tension ratio was 1.28 ± 0.07 and 1.42 ± 0.04 at SLs of 2.1 and 2.3 µm, respectively. There was no difference in protein expression or total phosphorylation of sarcomeric proteins. We also measured the calcium sensitivity after PKA treating the skinned muscle and found that titin-based passive tension was also now correlated with LDA, with a slope that was significantly increased compared to no PKA treatment. Finally, we performed isolated heart experiments and measured the Frank–Starling relation (slope of developed wall stress–LV volume relation) as well as diastolic stiffness (slope of diastolic wall stress–volume relation). The FSM was more pronounced in the N2B KO hearts and the slope of the FSM correlated with diastolic stiffness. These findings support that titin-based passive tension triggers an increase in calcium sensitivity at long sarcomere length, thereby playing an important role in the Frank–Starling mechanism of the heart.

Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.

Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.

Myofibrillar Protein Expression and Contractility in Neonates and Infants with Congenital Right Ventricular Outflow Obstruction

In this study we investigated the postnatal developmental changes in sarcomeric protein expression in parallel with contractile parameters in myofibrils isolated from small resections from the right ventricular (RV) outflow tract in 25 patients with Tetralogy of Fallot and related congenital heart diseases (CHD). These CHDs are associated with RV hypertrophy and outflow tract obstruction. The age of the patients ranged from 4 days to 38 months. The resections were procured during surgical correction of the cardiac malformation and would have been otherwise discarded.