Time-dependent effects of BRAF-V600E on cell cycling, metabolism, and function in engineered myocardium.

Candidate cardiomyocyte (CM) mitogens such as those affecting the extracellular signal-regulated kinase (ERK) signaling pathway represent potential targets for functional heart regeneration. We explored whether activating ERK via a constitutively active mutant of B-raf proto-oncogene (BRAF), BRAF-V600E (caBRAF), can induce proproliferative effects in neonatal rat engineered cardiac tissues (ECTs). Sustained CM-specific caBRAF expression induced chronic ERK activation, substantial tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. caBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a promigratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid and precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase is sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.

Prokaryotic voltage-gated sodium channels are more effective than endogenous Nav1.5 channels in rescuing cardiac action potential conduction: an in silico study.

Methods to augment Na+ current in cardiomyocytes hold potential for the treatment of various cardiac arrhythmias involving conduction slowing. Because the gene coding cardiac Na+ channel (Nav1.5) is too large to fit in a single adeno-associated virus (AAV) vector, new gene therapies are being developed to enhance endogenous Nav1.5 current (by overexpression of chaperon molecules or use of multiple AAV vectors) or to exogenously introduce prokaryotic voltage-gated Na+ channels (BacNav) whose gene size is significantly smaller than that of the Nav1.5. In this study, based on experimental measurements in heterologous expression systems, we developed an improved computational model of the BacNav channel, NavSheP D60A. We then compared in silico how NavSheP D60A expression vs. Nav1.5 augmentation affects the electrophysiology of cardiac tissue. We found that the incorporation of BacNav channels in both adult guinea pig and human cardiomyocyte models increased their excitability and reduced action potential duration. When compared with equivalent augmentation of Nav1.5 current in simulated settings of reduced tissue excitability, the addition of the BacNav current was superior in improving the safety of conduction under conditions of current source-load mismatch, reducing the vulnerability to unidirectional conduction block during premature pacing, preventing the instability and breakup of spiral waves, and normalizing the conduction and ECG in Brugada syndrome tissues with mutated Nav1.5. Overall, our studies show that compared with a potential enhancement of the endogenous Nav1.5 current, expression of the BacNav channels with their slower inactivation kinetics can provide greater anti-arrhythmic benefits in hearts with compromised action potential conduction.NEW & NOTEWORTHY Slow action potential conduction is a common cause of various cardiac arrhythmias; yet, current pharmacotherapies cannot augment cardiac conduction. This in silico study compared the efficacy of recently proposed antiarrhythmic gene therapy approaches that increase peak sodium current in cardiomyocytes. When compared with the augmentation of endogenous sodium current, expression of slower-inactivating bacterial sodium channels was superior in preventing conduction block and arrhythmia induction. These results further the promise of antiarrhythmic gene therapies targeting sodium channels.

Conserved chamber-specific polyploidy maintains heart function in Drosophila.

Developmentally programmed polyploidy (whole-genome duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, in both Drosophila larvae and human organ donors, we reveal distinct polyploidy levels in cardiac organ chambers. In Drosophila, differential growth and cell cycle signal sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume and cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic human cardiomyopathies. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest that precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila.

Developmentally programmed polyploidy (whole-genome-duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, we reveal roles for precise polyploidy levels in cardiac tissue. We highlight a conserved asymmetry in polyploidy level between cardiac chambers in Drosophila larvae and humans. In Drosophila , differential Insulin Receptor (InR) sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume, cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic systemic human heart failure. Using human donor hearts, we reveal asymmetry in nuclear volume (ploidy) and insulin signaling between the left ventricle and atrium. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Targeted Delivery for Cardiac Regeneration: Comparison of Intra-coronary Infusion and Intra-myocardial Injection in Porcine Hearts.

BACKGROUND: The optimal delivery route to enhance effectiveness of regenerative therapeutics to the human heart is poorly understood. Direct intra-myocardial (IM) injection is the gold standard, however, it is relatively invasive. We thus compared targeted IM against less invasive, catheter-based intra-coronary (IC) delivery to porcine myocardium for the acute retention of nanoparticles using cardiac magnetic resonance (CMR) imaging and viral vector transduction using qPCR. METHODS: Ferumoxytol iron oxide (IO) nanoparticles (5 ml) were administered to Yorkshire swine (n = 13) by: (1) IM via thoracotomy, (2) catheter-based IC balloon-occlusion (BO) with infusion into the distal left anterior descending (LAD) coronary artery, (3) IC perforated side-wall (SW) infusion into the LAD, or (4) non-selective IC via left main (LM) coronary artery infusion. Hearts were harvested and imaged using at 3T whole-body MRI scanner. In separate Yorkshire swine (n = 13), an adeno-associated virus (AAV) vector was similarly delivered, tissue harvested 4-6 weeks later, and viral DNA quantified from predefined areas at risk (apical LV/RV) vs. not at risk in a potential mid-LAD infarct model. Results were analyzed using pairwise Student's t-test. RESULTS: IM delivery yielded the highest IO retention (16.0 ± 4.6% of left ventricular volume). Of the IC approaches, BO showed the highest IO retention (8.7 ± 2.2% vs. SW = 5.5 ± 4.9% and LM = 0%) and yielded consistent uptake in the porcine distal LAD territory, including the apical septum, LV, and RV. IM delivery was limited to the apex and anterior wall, without septal retention. For the AAV delivery, the BO was most efficient in the at risk territory (Risk: BO = 6.0 × 10-9, IM = 1.4 × 10-9, LM = 3.2 × 10-10 viral copies per µg genomic DNA) while all delivery routes were comparable in the non-risk territory (BO = 1.7 × 10-9, IM = 8.9 × 10-10, LM = 1.2 × 10-9). CONCLUSIONS: Direct IM injection has the highest local retention, while IC delivery with balloon occlusion and distal infusion is the most effective IC delivery technique to target therapeutics to a heart territory most in risk from an infarct.

Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy.

Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders.

Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in CMs as a potential strategy for regenerative heart repair.

In vitro discovery of novel prokaryotic ion channel candidates for antiarrhythmic gene therapy.

Sudden cardiac death continues to have a devastating impact on public health prompting the continued efforts to develop more effective therapies for cardiac arrhythmias. Among different approaches to normalize function of ion channels and prevent arrhythmogenic remodeling of tissue substrate, cardiac cell and gene therapies are emerging as promising strategies to restore and maintain normal heart rhythm. Specifically, the ability to genetically enhance electrical excitability of diseased hearts through voltage-gated sodium channel (VGSC) gene transfer could improve velocity of action potential conduction and act to stop reentrant circuits underlying sustained arrhythmias. For this purpose, prokaryotic VGSC genes are promising therapeutic candidates due to their small size (<1kb) and potential to be effectively packaged in adeno-associated viral (AAV) vectors and delivered to cardiomyocytes for stable, long-term expression. This article describes a versatile method to discover and characterize novel prokaryotic ion channels for use in gene and cell therapies for heart disease including cardiac arrhythmias. Detailed protocols are provided for: (1) identification of potential ion channel candidates from large genomic databases, (2) candidate screening and characterization using site-directed mutagenesis and engineered human excitable cell system and, (3) candidate validation using electrophysiological techniques and an in vitro model of impaired cardiac impulse conduction.

Three-dimensional tissue-engineered human skeletal muscle model of Pompe disease.

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA-/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.

Engineering human pluripotent stem cells into a functional skeletal muscle tissue.

The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force-frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.

Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue.

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.

An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis.

The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic "on" switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells. Illumination of cells with blue light activated myogenic differentiation, including upregulation of myogenic markers and fusion into multinucleated myotubes. Cell differentiation was spatially patterned by illumination of cell cultures through a photomask. To demonstrate the application of the system to controlling in vivo tissue development, the light inducible switch was used to control the expression of VEGF and angiopoietin-1, which induced angiogenic sprouting in a mouse dorsal window chamber model. Live intravital microscopy showed illumination-dependent increases in blood-perfused microvasculature. This optogenetic switch is broadly useful for applications in which sustained and patterned gene expression is desired following transient induction, including tissue engineering, gene therapy, synthetic biology, and fundamental studies of morphogenesis.

Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells.

Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Adjunctive ß2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, ß2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.

Size and ionic currents of unexcitable cells coupled to cardiomyocytes distinctly modulate cardiac action potential shape and pacemaking activity in micropatterned cell pairs.

BACKGROUND: Cardiac cell therapies can yield electric coupling of unexcitable donor cells to host cardiomyocytes with functional consequences that remain unexplored. METHODS AND RESULTS: We micropatterned cell pairs consisting of a neonatal rat ventricular myocyte (NRVM) coupled to an engineered human embryonic kidney 293 (HEK293) cell expressing either connexin-43 (Cx43 HEK) or inward rectifier potassium channel 2.1 (Kir2.1) and Cx43 (Kir2.1+Cx43 HEK). The NRVM-HEK contact length was fixed yielding a coupling strength of 68.9±9.7 nS, whereas HEK size was systematically varied. With increase in Cx43 HEK size, NRVM maximal diastolic potential was reduced from -71.7±0.6 mV in single NRVMs to -35.1±1.3 mV in pairs with an HEK:NRVM cell surface area ratio of 1.7±0.1, whereas the action potential upstroke ([dV(m)/dt](max)) and duration decreased to 1.6±0.7% and increased to 177±32% in single NRVM values, respectively (n=21 cell pairs). Pacemaking occurred in all NRVM-Cx43 HEK pairs with cell surface area ratios of 1.1 to 1.9. In contrast, NRVMs, coupled with Kir2.1+Cx43 HEKs of increasing size, had similar maximal diastolic potentials, exhibited no spontaneous activity, and showed a gradual decrease in action potential duration (n=23). Furthermore, coupling single NRVMs to a dynamic clamp model of HEK cell ionic current reproduced the cardiac maximal diastolic potentials and pacemaking rates recorded in cell pairs, whereas reproducing changes in (dV(m)/dt)(max) and action potential duration required coupling to an HEK model that also included cell membrane capacitance. CONCLUSIONS: Size and ionic currents of unexcitable cells electrically coupled to cardiomyocytes distinctly affect cardiac action potential shape and initiation with important implications for the safety of cardiac cell and gene therapies.

Colonizing the heart from the epicardial side.

The clinical use of stem cells, such as bone marrow-derived and, more recently, resident cardiac stem cells, offers great promise for treatment of myocardial infarction and heart failure. The epicardium-derived cells have also attracted attention for their angiogenic paracrine actions and ability to differentiate into cardiomyocytes and vascular cells when activated during cardiac injury. In a recent study, Chong and colleagues have described a distinct population of epicardium-derived mesenchymal stem cells that reside in a perivascular niche of the heart and have a broad multilineage potential. Exploring the therapeutic capacity of these cells will be an exciting future endeavor.

X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector.

Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or ß-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector.

Soluble miniagrin enhances contractile function of engineered skeletal muscle.

Neural agrin plays a pleiotropic role in skeletal muscle innervation and maturation, but its specific effects on the contractile function of aneural engineered muscle remain unknown. In this study, neonatal rat skeletal myoblasts cultured within 3-dimensional engineered muscle tissue constructs were treated with 10 nM soluble recombinant miniagrin and assessed using histological, biochemical, and functional assays. Depending on the treatment duration and onset time relative to the stage of myogenic differentiation, miniagrin was found to induce up to 1.7-fold increase in twitch and tetanus force amplitude. This effect was associated with the 2.3-fold up-regulation of dystrophin gene expression at 6 d after agrin removal and enhanced ACh receptor (AChR) cluster formation, but no change in cell number, expression of muscle myosin, or important aspects of intracellular Ca(2+) handling. In muscle constructs with endogenous ACh levels suppressed by the application of α-NETA, miniagrin increased AChR clustering and twitch force amplitude but failed to improve intracellular Ca(2+) handling and increase tetanus-to-twitch ratio. Overall, our studies suggest that besides its synaptogenic function that could promote integration of engineered muscle constructs in vivo, neural agrin can directly promote the contractile function of aneural engineered muscle via mechanisms distinct from those involving endogenous ACh.