Sarcomeric Α-actinin Research Articles

S1P-S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial Infarction.

Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3+ cells, are nontumorigenic endogenous pluripotent-like stem cells obtainable from various tissues including the bone marrow. Their therapeutic efficiency has not been validated in acute myocardial infarction. The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair. In vivo dynamics of Nano-lantern-labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]-specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]-introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)-S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of GATA4-gene-silenced Muse cells. Muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression. Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.

The impact of growth factors on human induced pluripotent stem cells differentiation into cardiomyocytes

Human induced pluripotent stem cells (hiPSCs) act as a promising therapeutic alternative for cardiovascular diseases. They yield a large number of functional cardiomyocytes (CMs) from autologous cell sources without ethical or immunological problems. However, significant limitations still remain in terms of line-to-line variability in CM yield and reproducibility. AimTo efficiently enhance NP0040 hiPSCs differentiation into CMs. Main methodsFollowing a standard cardiac differentiation protocol using small molecules targeting the canonical Wnt signaling, growth factors (BMP4 and FGF2) and ascorbic acid were added further in order to increase the cardiac differentiation efficiency. All cultures were conducted in serum-free, feeder-free monolayer system followed by lactate purification. Key findingsUsing NP0040 hiPSCs, the CM yield resulting from modulation of the Wnt signaling pathway alone was inefficient compared to previous studies while the addition of BMP4, FGF2 and ascorbic acid resulted in enhanced cardiac differentiation outcome. The later resulted in a high yield (up to 92%) of cardiac troponin-T (cTnT) + CMs contracting spontaneously as organized sheets in 15 independent experiments. They were validated structurally and functionally using immunofluorescent staining for sarcomeric α-actinin, cTnT, MLC2v and Connexin 43. Reverse-transcriptase PCR revealed cardiac transcription factors and cardiac-specific genes expression. CMs were electrically connected to one another. Recorded action potential (AP) showed waves of relatively mature ventricular-like phenotype. SignificanceWe demonstrated that hiPSC lines respond differently to a standard cardiac differentiation protocol and that a well-orchestrated interplay between Wnt, BMP4, FGF/MEK and Ascorbic acid MEK/ERK1/2 signaling pathways is beneficial in enhancing the differentiation outcome.

A system to monitor statin-induced myopathy in individual engineered skeletal muscle myobundles.

Microphysiological tissue engineering models of human skeletal muscle (myobundles) provide a platform to investigate the mechanism of muscle diseases and to study the response to drugs and toxins in vitro. To examine the dynamic response to drugs, which often take several days to induce responses, we developed a system to monitor the contractile force of the same human skeletal muscle myobundles over time before and after treatment with drugs. Myobundles were formed in series with Ecoflex films (platinum-catalyzed silicones) with embedded microbeads. The displacement of the microbeads in Ecoflex exhibited a linear relation between muscle force production and Ecoflex film stretch. Forces measured with the microbeads embedded in Ecoflex agreed well with simultaneous measurements with a force transducer. Application of the Hill model for the myobundles showed that the Ecoflex affected the magnitude of the response, but not the kinetics. After continuous exposure to 100 nM cerivastatin, both active and passive forces were reduced relative to controls after 2-4 days. The decline in force was associated with a decline in the muscle myofiber organization. The inhibitory effect of cerivastatin was reduced when 0.1-1 mM mevalonate was added with cerivastatin. Although addition of co-enzyme Q10 with cerivastatin inhibited degradation of sarcomeric α-actinin (SAA) in myoblasts, the contractile force still declined, suggesting that statin-induced myopathy was related to mevalonate pathway but the addition of co-enzyme Q10 was insufficient to overcome the effect of statins on the mevalonate pathway. Thus, cerivastatin rapidly induces myopathy which can be reversds with mevalonate but not co-enzyme Q10.

Synergistic role of 5-azacytidine and ascorbic acid in directing cardiosphere derived cells to cardiomyocytes in vitro by downregulating Wnt signaling pathway via phosphorylation of β-catenin.

BackgroundCardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation.MethodsCDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot.ResultsResults revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, β-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho β-catenin while non phospho β-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs.ConclusionCombined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders.

Abstract 24057: Induced Pacemaker Spheroids As A Model To Reverse-Engineer The Native Sinoatrial Node

Background: The sinoatrial node (SAN) has intricate architecture, which facilitates the spontaneous action potentials generated from the SAN to pace and drive the neighboring myocardium. We sought to create an engineered SAN that recapitulates the native SAN’s ability to overcome source-sink mismatch. We hypothesized spheroids consisting of induced pacemaker cells (iPM) can pace and drive surrounding quiescent myocardium. Methods: The iPMs were created by singular expression of a transcription factor, TBX18, to neonatal rat ventricular myocytes as we have previously demonstrated. The iPM or GFP-spheroids (control) were created by subjecting the NRVMs to the AggreWell™ plate at 1000 cells/well, and cultured for three days in suspension. Spheroids consisted of 90% NRVM and 10% cardiac fibroblast. Results: iPM-spheroids demonstrated spontaneous pacing at 145±26 bpm compared to 66±1 bpm (p=6.5770E-9) of control, GFP-spheroids. When one iPM-spheroid was surrounded by a monolayer of quiescent NRVMs (iPM:NRVM=1:100), the iPM-spheroids were able to pace and drive the quiescent ventricular myocardium at a capture rate of 48±7%. In contrast, the random activity of control spheroids captured the myocardium at 7±4% (p=0.0078). A monolayer of TBX18 cells (1:4=TBX18:NRVM) failed to pace and drive the neighboring sheet of ventricular myocytes. iPM-spheroids had a 17-fold increase in SAN-specific gap junction, Cx45, transcripts (p=0.0001) and a 2-fold decrease in myocardial gap junction, Cx43 (p=0.0030), compared to GFP-spheroids. The iPM-spheroids have superior viability compared to control GFP-spheroids, 87±1% and 72±5% respectively (p= 0.0463). TUNEL staining confirmed apoptotic fibroblast in the periphery. When cultured for >2 weeks, iPM-spheroids demonstrated small α-sarcomeric actinin positive cells organized as a mesh in the core, similar to the pacemaker cells in the native SAN. Conclusion: iPM-spheroids can pace and drive surrounding quiescent myocardium, overcoming the source-sink mismatch. The iPM-spheroids are viable in long-term and exhibit native SAN-like pacemaker cell organization. These data provide an in vitro platform on which the design principles of native SAN could be tested.

Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.

Cardiomyocyte coculture on layered fibrous scaffolds assembled from micropatterned electrospun mats

Challenges remain in engineering cardiac tissues with functional and morphological properties similar to those of native myocardium. In the current study, micropatterned fibrous mats are obtained by deposition of electrospun fibers on lithographic collectors to reproduce the anisotropic structure of myocardium, and carbon nanotubes are included in fibers to provide conductivities at the same level of cardiac muscles. The patterned mats are assembled layer-by-layer into patterned scaffolds for coculture of primary cardiomyocytes (CMs) with cardiac fibroblasts (CFs) and endothelial cells (ECs). CMs are organized along the fibers with clear cardiac strips of sarcomeric α-actinin and belt-like connexin-43, showing strong cellular extraction forces and intercellular communications. Compared with square and rectangle patterns, honeycomb (Hc)-patterned scaffolds shows higher ultimate tensile strength and strain to failure. The finite element analysis indicates no apparent stress concentration under stress application in the two orthogonal directions. The Hc-patterned coculture demonstrates significantly higher CM viabilities, deeper penetrations of cells into scaffolds, stronger expression of troponin I, connexin-43 and sarcomeric α-actinin by CMs and more abundant formations of capillary-like networks by ECs than other scaffolds. CMs on Hc-patterned scaffolds display spontaneous beating rates at 101±12times/min after coculture for 5days and remain synchronously beating at 94±8times/min after 15days, which is close to those of adult and neonatal rats. The layered and patterned coculture strategy achieves the spatial arrangement of multiple types of cells and vascularization potential, providing a biomimetic strategy for engineering functional cardiac patches in vitro.

Abstract 198: Multipotent Placenta-derived Cdx2 Cells Possess in vitro and in vivo Cardiomyogenic Potential

Stem cell-based therapies for cardiac regeneration are of crucial importance and an ideal cell-type is yet to be established. We previously reported that fetal cells from placenta “home” to injured maternal heart and approximately 40% (40/100) of the migrating cells expressed homeodomain protein Cdx2. This interesting observation led us to hypothesize that placental Cdx2 could be a novel cell target for cardiac differentiation. To understand this phenomenon, we employed a cre-lox strategy that labeled Cdx2 cells in placenta with e-GFP and induced myocardial infarction (MI) in pregnant mice at mid-gestation. The maternal heart was analyzed 4 weeks post-MI for the presence of Cdx2-eGFP-derived cardiomyocytes. Additionally, Cdx2 cells were isolated from late-gestation placenta and assayed for cardiac differentiation in vitro followed by live cell imaging. Phenotypic and whole-cell proteomic analysis, clonal and vascular lineage differentiation and immune profiling were carried out subsequently. We observed that Cdx2 cells migrated to injured maternal hearts and differentiated into cardiomyocytes highlighting the functional significance of fetal-maternal stem cell transfer. Additionally, isolated Cdx2 cells from the late placenta differentiated into spontaneously beating cardiomyocytes and expressed structural proteins cardiac troponin T(cTnT), α-sarcomeric actinin and gap junction protein Cx43. These cells underwent clonal expansion and differentiated into endothelial and smooth muscle lineages in culture indicative of their multipotent nature. Low expression of MHC molecules and other components of the immune-response, infer that these cells possess the ability to evade host immune surveillance. Proteomic analysis demonstrated that 145 proteins were uniquely identified in the Cdx2 cells compared to embryonic stem cells. These protein networks reflected an increased activation of functions involving migration, fertility, homing, and chemotaxis. Our study is the first to demonstrate that Cdx2 may play a role in cardiac differentiation and delineate multipotent cells in placenta with an inherent “homing” ability. These findings point to a potential role for Cdx2 cells in cardiac regenerative therapies using allogeneic cells.

Scaffold-in-Scaffold Potential to Induce Growth and Differentiation of Cardiac Progenitor Cells.

The design of reliable biocompatible and biodegradable scaffolds remains one of the most important challenges for tissue engineering. In fact, properly designed scaffolds must display an adequate and interconnected porosity to facilitate cell spreading and colonization of the inner layers, and must release physical signals concurring to modulate cell function to ultimately drive cell fate. In this study, a combination of optimal mechanical and biochemical properties has been considered to design a one-component three-dimensional (3D) multitextured hydrogel scaffold to favor cell-scaffold interactions. A polyethylene glycol diacrylate woodpile (PEGDa-Wp) structure of the order of 100 μm has been manufactured using a microstereolithography process. Subsequently, the PEGDa-Wp has been embedded in a PEGDa hydrogel to obtain a 3D scaffold-in-scaffold (3D-SS) system. Finally, the 3D-SS capability to address cell fate has been assessed using human Lin- Sca-1+ cardiac progenitor cells (hCPCs). Results have shown that a multitextured 3D scaffold represents a favorable microenvironment to promote hCPC differentiation and orientation. In fact, while cultured on 3D-SS, hCPCs adopt an ordered 3D spatial orientation and activate the expression of structural proteins, such as the α-sarcomeric actinin, a specific marker of the cardiomyocyte phenotype, and connexin 43, the principal gap junction protein of the heart. Although preliminary, this study demonstrates that complex multitextured scaffolds closely mimicking the extracellular matrix structure and function are efficient in driving progenitor cell fate. A leap forward will be determined by the use of advanced 3D printing technologies that will improve multitextured scaffold manufacturing and their biological efficiency.

Heterotypic endosomal fusion as an initial trigger for insulin-induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.

Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot-based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving 'static retention' and 'stimulus-dependent liberation'. Vital imaging analyses and super-resolution microscopy-based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4-containing vesicles in skeletal myofibres. Prior exposure to exercise-mimetic stimuli potentiated this insulin-responsive endomembrane fusion event involving GLUT4-containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin-induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4-containing vesicles with a subset of transferrin receptor-containing endosomes. Importantly, pretreatment with exercise-mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe-like localization close to sarcomeric α-actinin and that insulin induced a reduction of the stripe-like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.

Composite poly(lactic acid)/chitosan nanofibrous scaffolds for cardiac tissue engineering

Fibrous scaffolds with different ratios of poly (lactic acid) (PLA) and chitosan were fabricated by conventional electrospinning. After crosslinking by the glutaraldehyde vapor, the structure, mechanical properties, hydrophilicity, and in-fiber chemical interactions of the scaffolds were investigated. We found that the fiber diameter decreased with the concentration of chitosan, while mechanical properties and hydrophilicity improved. In addition, we found that scaffolds with aligned fibers have higher mechanical strength and biocompatibility than scaffolds with randomly oriented fibers. In particular, scaffolds with aligned fibers with PLA:chitosan ratios of 7:1 was found to support cardiomyocyte viability, elicit cell elongation, and enhance production of sarcomeric α-actinin and troponin I. Collectively, the data indicate that composite scaffolds consisting of PLA/chitosan fibers have great potential for engineering cardiac tissue, and for accelerating the regeneration of myocardia.

Micropatterned co-culture of cardiac myocytes on fibrous scaffolds for predictive screening of drug cardiotoxicities

The spatial arrangement of cardiac myocytes (CMs) and other non-myocytes scaffolds, closely resembling native tissue, is essential to control the CM morphology and function for cardiac tissue regeneration. In the current study, micropatterned fibrous scaffolds were developed to establish a CM co-culture system with cardiac fibroblasts (CFs) and endothelial cells (ECs) as a potential in vitro drug screening model. To pursue a biomimetic approach to influence CM behaviors, strip, oval and wave-patterned mats were constructed by deposition of electrospun fibers on lithographic collectors, followed by precise stacking for cell co-cultures. CMs, CFs, and ECs were located on the patterned scaffolds with controlled cellular distribution in the respective regions and no across condition was found. Compared with those after strip and oval-patterned co-culture, CMs co-cultured on wave-patterned scaffolds displayed significantly greater cell viabilities, larger cell elongation ratios, stronger expressions of cardiac-specific Troponin I, connexin 43 and sarcomeric α-actinin and higher beating rates during 15 days of incubation. The responses of co-cultured CMs to quinidine, erythromycin and sotalol show good correlations with clinical observations in the beating rate and the prolongation of the contraction and relaxation time. The in vivo safety data reflected well with the concentrations for 50% of maximal effect (EC50) after drug treatment on co-cultured CMs, which was determined from the changes in the corrected field potential duration (FPDc) against the drug concentrations. During 15 days of patterned co-culture, the interbeat intervals and fluctuations of the CMs indicated quick changes in response to haloperidol treatment and sufficient restoration of the original beating profiles after drug removal. This study demonstrates the capabilities of micropatterned co-culture of CMs to establish the cardiac function as a reproducible and reliable platform for screening cardiac side effects of drugs.

Keratin mediated attachment of stem cells to augment cardiomyogenic lineage commitment

The objective of this work was to develop a simple surface modification technique using keratin derived from human hair for efficient cardiomyogenic lineage commitment of human mesenchymal stem cells (hMSCs). Keratin was extracted from discarded human hair containing both the acidic and basic components along with the heterodimers. The extracted keratin was adsorbed to conventional tissue culture polystyrene surfaces at different concentration. Keratin solution of 500μg/ml yielded a well coated layer of 12±1nm thickness with minimal agglomeration. The keratin coated surfaces promoted cell attachment and proliferation. Large increases in the mRNA expression of known cardiomyocyte genes such as cardiac actinin, cardiac troponin and β-myosin heavy chain were observed. Immunostaining revealed increased expression of sarcomeric α-actinin and tropomyosin whereas Western blots confirmed higher expression of tropomyosin and myocyte enhancer factor 2C in cells on the keratin coated surface than on the non-coated surface. Keratin promoted DNA demethylation of the Atp2a2 and Nkx2.5 genes thereby elucidating the importance of epigenetic changes as a possible molecular mechanism underlying the increased differentiation. A global gene expression analysis revealed a significant alteration in the expression of genes involved in pathways associated in cardiomyogenic commitment including cytokine and chemokine signaling, cell–cell and cell-matrix interactions, Wnt signaling, MAPK signaling, TGF-β signaling and FGF signaling pathways among others. Thus, adsorption of keratin offers a facile and affordable yet potent route for inducing cardiomyogenic lineage commitment of stem cells with important implications in developing xeno-free strategies in cardiovascular regenerative medicine.

Electrospun Poly(lactic-co-glycolic acid)/Multiwalled Carbon Nanotube Nanofibers for Cardiac Tissue Engineering

Many conductive scaffolds were fabricated to mimic the topography of the cardiac microenvironment in vitro in order to improve the performance of engineered cardiac tissues. This study fabricated aligned poly(lactic-co-glycolic acid)/multiwalled carbon nanotube fibers by electrospinning. The Young's modulus and conductivity were significantly greater on poly(lactic-co-glycolic acid) fibers with 3% multiwalled carbon nanotubes compared to others. Topographical cues and conductivity were applied to investigate the combined effect on cardiomyocyte behavior. Neonatal rat cardiomyocytes cultured on the conductive fibers maintained their viability, induced cell elongation, and enhanced sarcomeric α-actinin and troponin I production in cardiomyocytes. The results indicate that poly(lactic-co-glycolic acid)/multiwalled carbon nanotube composite fibers have great potential for cardiac tissue engineering.

Tuning the conductivity and inner structure of electrospun fibers to promote cardiomyocyte elongation and synchronous beating

The key to addressing the challenges facing cardiac tissue engineering is the integration of physical, chemical, and electrical cues into scaffolds. Aligned and conductive scaffolds have been fabricated as synthetic microenvironments to improve the function of cardiomyocytes. However, up to now, the influence of conductive capability and inner structure of fibrous scaffolds have not been determined on the cardiomyocyte morphologies and beating patterns. In the current study, highly aligned fibers were fabricated with loaded up to 6% of carbon nanotubes (CNTs) to modulate the electrical conductivity, while blend and coaxial electrospinning were utilized to create a bulk distribution of CNTs in fiber matrices and a spatial embedment in fiber cores, respectively. Conductive networks were formed in the fibrous scaffolds after the inoculation of over 3% CNTs, and the increase in the conductivity could maintain the cell viabilities, induce the cell elongation, enhance the production of sarcomeric α-actinin and troponin I, and promote the synchronous beating of cardiomyocytes. Although the conductivity of blend fibers is slightly higher than that of coaxial fibers with the same CNT loadings, the lower exposures to CNTs resulted in higher cell viability, elongation, extracellular matrix secretion and beating rates for cardiomyocytes on coaxial fibers. Taken altogether, core-sheath fibers with loaded 5% of CNTs in the fiber cores facilitated the cardiomyocyte growth with a production of organized contractile proteins and a pulsation frequency close to that of the atrium. It is suggested that electrospun scaffolds that couple conductivity and fibrous structure considerations may provide optimal stimuli to foster cell morphology and functions for myocardial regeneration or establishment of in vitro cardiomyocyte culture platform for drug screening.

Abstract 37: Mir-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Towards a Cardiomyocyte-like Fate by Directly Repressing Specificity Protein 1 (Sp1)

Objective: Transdifferentiation of cardiac fibroblasts into cardiomyocyte-like cells (iCMs) represents a promising strategy in treating human heart disease. However, despite encouraging reprogramming data obtained from rodent models, the transgenes used in those models are not sufficient to reprogram human cells. We therefore sought to optimize a cocktail of factors which can efficiently transdifferentiate human cells. A porcine model was included to test its potential as a surrogate for human reprogramming studies. Methods: Lentivirus expressing Gata4 (G), Mef2c (M), Tbx5 (T), Hand2 (H), Myocardin (My), or two microRNAs miR-590, miR-199, were transduced into cultured porcine cardiac fibroblasts (PCFs) and human cardiac fibroblasts (HCFs) in different combinations. Two weeks after transduction, qRT-PCR, immunofluorescence (IF), and FACS assays were performed, and the efficiency of iCM production was evaluated by the expression of cardiomyocyte markers cardiac troponin T (cTnT) and α-sarcomeric actinin. Results: Combined G, M, T treatment alone was insufficient to transdifferentiate PCFs or HCFs into iCMs, despite the capability of GMT transduction to transdifferentiate up to 7% of treated rat cardiac fibroblasts, as assessed by FACS analysis for cTNT. However, the addition of H, My and miR-590 to GMT resulted in the transdifferentiation of approximately 5% of HCFs and PCFs, as measured by cTnT expression. IF analysis likewise demonstrated high expression of cTNT and α-actinin in these cells. Importantly, the transdifferentiated PCFs exhibited spontaneous contractions when co-cultured with murine cardiomyocytes. qPCR showed that administration of GMT plus either miR-590 or HMy upregulated cardiac genes MYH6 and TNNT2, and downregulated the fibroblast genes Collagen I and Collagen III. Mir-590 also directly suppressed Sp1, a fibrosis inducer and putative inhibitor of cellular reprogramming in the heart. Conclusions: Our data suggest that the porcine model can serve as an appropriate surrogate for human fibroblasts reprogramming studies. Enhanced transdifferentiation associated with miR-590-mediated repression of Sp1 suggests a novel pathway that may be targetable to enhance cardiac cellular reprogramming clinically.

Gold nanorod-incorporated gelatin-based conductive hydrogels for engineering cardiac tissue constructs

The development of advanced biomaterials is a crucial step to enhance the efficacy of tissue engineering strategies for treatment of myocardial infarction. Specific characteristics of biomaterials including electrical conductivity, mechanical robustness and structural integrity need to be further enhanced to promote the functionalities of cardiac cells. In this work, we fabricated UV-crosslinkable gold nanorod (GNR)-incorporated gelatin methacrylate (GelMA) hybrid hydrogels with enhanced material and biological properties for cardiac tissue engineering. Embedded GNRs promoted electrical conductivity and mechanical stiffness of the hydrogel matrix. Cardiomyocytes seeded on GelMA-GNR hybrid hydrogels exhibited excellent cell retention, viability, and metabolic activity. The increased cell adhesion resulted in abundance of locally organized F-actin fibers, leading to the formation of an integrated tissue layer on the GNR-embedded hydrogels. Immunostained images of integrin β-1 confirmed improved cell-matrix interaction on the hybrid hydrogels. Notably, homogeneous distribution of cardiac specific markers (sarcomeric α-actinin and connexin 43), were observed on GelMA-GNR hydrogels as a function of GNRs concentration. Furthermore, the GelMA-GNR hybrids supported synchronous tissue-level beating of cardiomyocytes. Similar observations were also noted by, calcium transient assay that demonstrated the rhythmic contraction of the cardiomyocytes on GelMA-GNR hydrogels as compared to pure GelMA. Thus, the findings of this study clearly demonstrated that functional cardiac patches with superior electrical and mechanical properties can be developed using nanoengineered GelMA-GNR hybrid hydrogels. Statement of SignificanceIn this work, we developed gold nanorod (GNR) incorporated gelatin-based hydrogels with suitable electrical conductivity and mechanical stiffness for engineering functional cardiac tissue constructs (e.g. cardiac patches). The synthesized conductive hybrid hydrogels properly accommodated cardiac cells and subsequently resulted in excellent cell retention, spreading, homogeneous distribution of cardiac specific markers, cell-cell coupling as well as robust synchronized (tissue-level) beating behavior.

Matrix metalloproteinase-2 in oncostatin M-induced sarcomere degeneration in cardiomyocytes.

Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration.

Cell Density and Joint microRNA-133a and microRNA-696 Inhibition Enhance Differentiation and Contractile Function of Engineered Human Skeletal Muscle Tissues

To utilize three-dimensional (3D) engineered human skeletal muscle tissue for translational studies and in vitro studies of drug toxicity, there is a need to promote differentiation and functional behavior. In this study, we identified conditions to promote contraction of engineered human skeletal muscle bundles and examined the effects of transient inhibition of microRNAs (miRs) on myogenic differentiation and function of two-dimensional (2D) and 3D cultures of human myotubes. In 2D cultures, simultaneously inhibiting both miR-133a, which promotes myoblast proliferation, and miR-696, which represses oxidative metabolism, resulted in an increase in sarcomeric α-actinin protein and the metabolic coactivator PGC-1α protein compared to transfection with a scrambled miR sequence (negative control). Although PGC-1α was elevated following joint inhibition of miRs 133a and 696, there was no difference in myosin heavy chain (MHC) protein isoforms. 3D engineered human skeletal muscle myobundles seeded with 5 × 10(6) human skeletal myoblasts (HSkM)/mL and cultured for 2 weeks after onset of differentiation consistently did not contract when stimulated electrically, whereas those seeded with myoblasts at 10 × 10(6) HSkM/mL or higher did contract. When HSkM were transfected with both anti-miRs and seeded into fibrin hydrogels and cultured for 2 weeks under static conditions, twitch and tetanic specific forces after electrical stimulation were greater than for myobundles prepared with HSkM transfected with scrambled sequences. Immunofluorescence and Western blots of 3D myobundles indicate that anti-miR-133a or anti-miR-696 treatment led to modest increases in slow MHC, but no consistent increase in fast MHC. Similar to results in 2D, only myobundles prepared with myoblasts treated with anti-miR-133a and anti-miR-696 produced an increase in PGC-1α mRNA. PGC-1α targets were differentially affected by the treatment. HIF-2α mRNA showed an expression pattern similar to that of PGC-1α mRNA, but COXII mRNA levels were not affected by the anti-miRs. Overall, joint inhibition of miR-133a and miR-696 accelerated differentiation, elevated the metabolic coactivator PGC-1α, and increased the contractile force in 3D engineered human skeletal muscle bundles.

Fullerene mediates proliferation and cardiomyogenic differentiation of adipose-derived stem cells via modulation of MAPK pathway and cardiac protein expression.

Zero-dimensional fullerenes can modulate the biological behavior of a variety of cell lines. However, the effects and molecular mechanisms of proliferation and cardiomyogenic differentiation in brown adipose-derived stem cells (BADSCs) are still unclear. In this study, we report the initial biological effects of fullerene-C60 on BADSCs at different concentrations. Results suggest that fullerene-C60 has no cytotoxic effects on BADSCs even at a concentration of 100 μg/mL. Fullerene-C60 improves the MAPK expression level and stem cell survival, proliferation, and cardiomyogenesis. Further, we found that the fullerene-C60 modulates cardiomyogenic differentiation. Fullerene-C60 improves the expression of cardiomyocyte-specific proteins (cTnT and α-sarcomeric actinin). At elevated concentration, fullerene-C60 reduces the incidence of diminished spontaneous cardiac differentiation of BADSCs with time. At the genetic level, fullerene-C60 (5 μg/mL) also improves the expression of cTnT. In addition, fullerene-C60 promotes the formation of gap junction among cells. These findings have important implications for clinical application of fullerenes in the treatment of myocardial infarction.