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.

An enhancer-based gene-therapy strategy for spatiotemporal control of cargoes during tissue repair.

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.

Fibroblast Growth Factor (FGF) 23 and FGF Receptor 4 promote cardiac metabolic remodeling in chronic kidney disease.

Chronic kidney disease (CKD) is a global health epidemic that significantly increases mortality due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiac injury in CKD. High serum levels of fibroblast growth factor (FGF) 23 in patients with CKD may contribute mechanistically to the pathogenesis of LVH by activating FGF receptor (FGFR) 4 signaling in cardiac myocytes. Mitochondrial dysfunction and cardiac metabolic remodeling are early features of cardiac injury that predate development of hypertrophy, but these mechanisms of disease have been insufficiently studied in models of CKD. Wild-type mice with CKD induced by adenine diet developed LVH that was preceded by morphological changes in mitochondrial structure and evidence of cardiac mitochondrial and metabolic dysfunction. In bioengineered cardio-bundles and neonatal rat ventricular myocytes grown in vitro, FGF23-mediated activation of FGFR4 caused a mitochondrial pathology, characterized by increased bioenergetic stress and increased glycolysis, that preceded the development of cellular hypertrophy. The cardiac metabolic changes and associated mitochondrial alterations in mice with CKD were prevented by global or cardiac-specific deletion of FGFR4. These findings indicate that metabolic remodeling and eventually mitochondrial dysfunction are early cardiac complications of CKD that precede structural remodeling of the heart. Mechanistically, FGF23-mediated activation of FGFR4 causes mitochondrial dysfunction, suggesting that early pharmacologic inhibition of FGFR4 might serve as novel therapeutic intervention to prevent development of LVH and heart failure in patients with CKD.

Meteorin-like is an injectable peptide that can enhance regeneration in aged muscle through immune-driven fibro/adipogenic progenitor signaling.

Pathologies associated with sarcopenia include decline in muscular strength, lean mass and regenerative capacity. Despite the substantial impact on quality of life, no pharmacological therapeutics are available to counteract the age-associated decline in functional capacity and/or, resilience. Evidence suggests immune-secreted cytokines can improve muscle regeneration, a strategy which we leverage in this study by rescuing the age-related deficiency in Meteorin-like through several in vivo add-back models. Notably, the intramuscular, peptide injection of recombinant METRNL was sufficient to improve muscle regeneration in aging. Using ex vivo media exchange and in vivo TNF inhibition, we demonstrate a mechanism of METRNL action during regeneration, showing it counteracts a pro-fibrotic gene program by triggering TNFα-induced apoptosis of fibro/adipogenic progenitor cells. These findings demonstrate therapeutic applications for METRNL to improve aged muscle, and show Fibro/Adipogenic Progenitors are viable therapeutic targets to counteract age-related loss in muscle resilience.

Translating musculoskeletal bioengineering into tissue regeneration therapies.

Musculoskeletal injuries and disorders are the leading cause of physical disability worldwide and a considerable socioeconomic burden. The lack of effective therapies has driven the development of novel bioengineering approaches that have recently started to gain clinical approvals. In this review, we first discuss the self-repair capacity of the musculoskeletal tissues and describe causes of musculoskeletal dysfunction. We then review the development of novel biomaterial, immunomodulatory, cellular, and gene therapies to treat musculoskeletal disorders. Last, we consider the recent regulatory changes and future areas of technological progress that can accelerate translation of these therapies to clinical practice.

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle.

Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle ("myobundles"). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p < 0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo.

BRG1 is a biomarker of hypertrophic cardiomyopathy in human heart specimens.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the sarcomere that causes otherwise unexplained cardiac hypertrophy and is associated with sudden death. While previous studies showed the role of the epigenetic modifier Brg1 in mouse models of HCM, additional work is needed to identify its role in humans. We tested the hypothesis that BRG1 expression is increased in periods of cardiac remodeling during fetal growth and in development of HCM. We employed immunohistochemical staining to evaluate protein expression of BRG1 in 796 human cardiac specimens (81 from patients with HCM) and describe elevated BRG1 expression in human fetal hearts in early development. In addition, we not only demonstrate increased expression of BRG1 in HCM, but we also show that other diseases that lead to heart failure have similar BRG1 expression to healthy controls. Inhibition of BRG1 in human induced pluripotent stem cell-derived cardiomyocytes significantly decreases MYH7 and increases MYH6, suggesting a regulatory role for BRG1 in the pathological imbalance of the two myosin heavy chain isoforms in human HCM. These data are the first demonstration of BRG1 as a specific biomarker for human HCM and provide foundation for future studies of epigenetics in human cardiac disease.

CRISPR Library Screening in Cultured Cardiomyocytes.

CRISPR-Cas9-based screening technologies enable precise, high-throughput genetic and epigenetic manipulation to study mechanisms of development and disease and identify new therapeutic targets. Here, we describe a general protocol for the generation of custom, pooled CRISPR sgRNA libraries for screening in cardiomyocyte cultures. This methodology can address a variety of lab-specific research questions in cardiomyocytes and other cell types, as the genes to be modified can be curated or whole genomes can be investigated. The use of lentiviral sgRNA delivery followed by high-throughput sequencing allows for rapid comparison and identification of candidate genes and epigenetic modifiers, which can be further validated individually or in sub-pooled libraries following screening.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells.

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues ("myobundles"). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.

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.

Hif-1a suppresses ROS-induced proliferation of cardiac fibroblasts following myocardial infarction.

We report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was ∼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues. CF proliferation was associated with higher reactive oxygen species (ROS) as occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ). The mitochondrial-targeted antioxidant MitoTEMPO rescued Hif-1a mutant phenotypes. Thus, HIF-1α in CFs provides a critical braking mechanism against excessive post-ischemic CF activation and proliferation through regulation of mitochondrial ROS. CFs are potential cellular targets for designer antioxidant therapies in cardiovascular disease.

Neuromuscular Development and Disease: Learning From in vitro and in vivo Models.

The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.

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.