Hypertrophic cardiomyopathy (HCM) affects approximately 600,000 people in the United States. Loss-of-function mutations in Myosin Binding Protein C3, MYBPC3, are the most common genetic cause of HCM, with the majority of mutations resulting in haploinsufficiency. To restore cardiac MYBPC3, we use an adeno-associated virus (AAV9) vector and engineer an optimized expression cassette with a minimal promoter and cis-regulatory elements (TN-201) to enhance packaging efficiency and cardiomyocyte expression. Rather than simply preventing cardiac dysfunction preclinically, we demonstrate in a symptomatic MYBPC3-deficient murine model the ability of AAV gene therapy to reverse cardiac hypertrophy and systolic dysfunction, improve diastolic dysfunction, and prolong survival. Dose-ranging efficacy studies exhibit restoration of wild-type MYBPC3 protein levels and saturation of cardiac improvement at the clinically relevant dose of 3E13 vg/kg, outperforming a previously published construct. These findings suggest that TN-201 may offer therapeutic benefits in MYBPC3-associated cardiomyopathy, pending further validation in clinical settings.

The I-bar protein MTSS1 has been implicated in heart failure and contractility by multiple genome-wide association studies. Human genetic analyses suggested that a variant lowering cardiac MTSS1 expression was associated with significantly improved survival in individuals with TTN dilated cardiomyopathy (DCM). Experimental knockdown of MTSS1 via small interfering RNA (siRNA) in induced pluripotent stem cell–derived cardiomyocytes deficient in TTN, CSRP3, or RBM20 led to improved increased sarcomere number and enhanced contractility. Engineered heart tissue models confirmed increased twitch force following MTSS1 siRNA knockdown across these genetic forms of DCM. Unbiased mass-spectrometry suggests that MTSS1 was found to interact with MYO18A, a protein critical for sarcomere assembly, and siRNA knockdown of MTSS1 up-regulated MYH7 and other sarcomere-related genes. These findings may suggest that MTSS1 impacts contractility as a negative regulator of sarcomere formation or turnover, and that reduction of MTSS1 may be a therapeutic target in select forms of genetic DCM.

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G>C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.

With current plans for manned missions to Mars and beyond, the need to better understand, prevent, and counteract the harmful effects of long-duration spaceflight on the body is becoming increasingly important. In this study, an automated heart-on-a-chip platform was flown to the International Space Station on a 1-mo mission during which contractile cardiac function was monitored in real-time. Upon return to Earth, engineered human heart tissues (EHTs) were further analyzed with ultrastructural imaging and RNA sequencing to investigate the impact of prolonged microgravity on cardiomyocyte function and health. Spaceflight EHTs exhibited significantly reduced twitch forces, increased incidences of arrhythmias, and increased signs of sarcomere disruption and mitochondrial damage. Transcriptomic analyses showed an up-regulation of genes and pathways associated with metabolic disorders, heart failure, oxidative stress, and inflammation, while genes related to contractility and calcium signaling showed significant down-regulation. Finally, in silico modeling revealed a potential link between oxidative stress and mitochondrial dysfunction that corresponded with RNA sequencing results. This represents an in vitro model to faithfully reproduce the adverse effects of spaceflight on three-dimensional (3D)-engineered heart tissue.

Heart disease is the leading cause of death worldwide and discovering therapies that treat its underlying cause is paramount to improving survivability and patient health. Since heart disease often results in detrimental remodeling of heart tissue, high-throughput screening (HTS) using cardiomyocytes and cardiac fibroblasts is frequently used for drug discovery. A major challenge in analyzing HTS data is ensuring that analysis is both fast and minimally biased. In many forms of heart disease and remodeling, cardiomyocytes will suffer from loss of sarcomeres and sarcomere disarray, while fibroblasts will often express increased α-smooth muscle actin (α-SMA) stress fibers when activated into myofibroblasts. Two custom MATLAB HTS image processing scripts named ‘Tamarack’ and ‘Porcupine’ were developed for quantifying human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) sarcomere morphology and cardiac fibroblast activation, respectively. Both scripts use wavelet transforms to detect subcellular structures that are of interest for analyzing phenotypes at the cellular level. Tamarack enables quantification of sarcomere metrics, including sarcomere count, length, orientation, and α-actinin rich areas. Porcupine enables quantification of fibroblast activation metrics, including α-SMA stress fiber count, length, orientation, nuclei and α-SMA overlap, and F-actin and α-SMA overlap. Both scripts enable analysis of other cellular features, such as nuclei count and stain intensity/protein expression. Tamarack obtained statistically significant results when comparing control hiPSC-CMs with hiPSC-CMs subjected to siRNA knockdowns of genes of interest. Porcupine was validated by analyzing fibroblasts with increasing concentrations of TGF-β which induced increasing activation. By developing technology for analyzing cardiomyocyte and fibroblast imaged-based screens, drug candidates can be efficiently evaluated without bias on cell types that are relevant to human heart disease. This research may help expand drug candidate portfolios and improve the number of treatments available to patients worldwide.

Tissue engineering with human induced pluripotent stem cell-derived cardiomyocytes enables unique opportunities for creating physiological models of the heart in vitro. However, there are few approaches available that can recapitulate the complex structure-function relationships that govern cardiac function at the macroscopic organ level. Here, we report a down-scaled, conical human 3D ventricular model with controllable cellular organization using multilayered, patterned cardiac sheets. Tissue engineered ventricles whose cardiomyocytes were pre-aligned parallel or perpendicular to the long axis outperformed those whose cardiomyocytes were angled or randomly oriented. Notably, the inner layers of perpendicular cardiac sheets realigned over 4 days into a parallel orientation, creating a helical transmural architecture, whereas minimal remodeling occurred in the parallel or angled sheets. Finite element analysis of engineered ventricles demonstrated that circumferential alignment leads to maximal perpendicular shear stress at the inner layer, whereas longitudinal orientation leads to maximal parallel stress. We hypothesize that cellular remodeling occurs to reduce perpendicular shear stresses in myocardium. This advanced platform provides evidence that physical forces such as shear stress drive self-organization of cardiac architecture.

The availability of human induced pluripotent stem cells (hiPSCs) has offered the possibility to study human-derived models of different genetic strata of cardiomyopathy for mechanistic discovery and therapeutic development. However, the phenotypes derived from cardiomyocytes differentiated from hiPSCs in conventional 2-dimensional culture systems often fail to reproducibly model clinical presentations, thereby reducing the translatability of readouts from these assays. Here we report 3-dimensional engineered heart tissues (EHTs) produced with a variety of cell lines harboring patient-derived mutations (TTN, RBM20, BAG3) known to cause dilated cardiomyopathy (DCM) which recapitulate contractile deficits associated with DCM. Internal development of EHTs also recapitulate contractile defects with small interfering RNA (siRNA) knockdown models of haploinsufficiency across multiple disease states. These observed findings with EHTs suggests improved maturity in cardiomyocytes through the presentation of microenvironmental cues akin to those seen in vivo . With continued advances in EHT technologies and capabilities, this platform may serve to significantly reduce drug candidate attrition, as well as provide new insights into pathology, prototyping of therapeutic approaches, and mechanism of action that were previously difficult to obtain.

In this paper, we report a simple and facile method to fabricate nanomolded Nafion thin films with tunable mechanical, and electrical properties. To achieve this, we combine a novel thermal evaporation-induced capillary force lithography method with swelling process to obtain enhanced pattern fidelity in nanomolded Nafion films. We demonstrate that structural fidelity and mechanical properties of patterned Nafion thin films can be modulated by changing fabrication parameters such as swelling time, Nafion polymer concentration, and curing temperature. Interestingly, we also find that impedance properties of nanomolded Nafion thin films are associated with the Nafion polymer concentration and curing temperature. In particular, 20% Nafion thin films exhibit greater impedance stability and lower impedance values than 5% Nafion thin films at lower frequencies. Moreover, curing temperature-specific impedance changes are observed. These results suggest that capillary lithography can be used to fabricate Nafion nanostructures with high pattern fidelity capable of modifying mechanical and electrical properties of Nafion thin films.

3D bioprinting is a powerful technique for engineering tissues used to study cell behavior and tissue properties in vitro. With the right formulation and printing parameters, bioinks can provide native biological and mechanical cues while allowing for versatile 3D structures that recapitulate tissue-level organization. Bio-based materials that support cellular adhesion, differentiation, and proliferation - including gelatin, collagen, hyaluronic acid, and alginate - have been successfully used as bioinks. In particular, decellularized extracellular matrix (dECM) has become a promising material with the unique ability to maintain both biochemical and topographical micro-environments of native tissues. However, dECM has shown technical limitations for 3D printing (3DP) applications posed by its intrinsically low mechanical stability. Herein, we report hydrogel bioinks composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). The Laponite facilitated extrusion-based 3DP, while PEG-DA enabled photo-polymerization after printing. Improving upon previously reported bioinks derived from dECM, our bioinks combine extrudability, shape fidelity, rapid cross-linking, and cytocompatibility in a single formulation (> 97% viability of encapsulated human cardiac fibroblasts and > 94% viability of human induced pluripotent stem cell derived cardiomyocytes after 7 days). The compressive modulus of the cured hydrogel bioinks was tunable from 13.4-89 kPa by changing the concentration of PEG-DA in the bioink formulation. Importantly, this span of mechanical stiffness encompasses ranges of tissue stiffness from healthy (compressive modulus ~5-15 kPa) to fibrotic (compressive modulus ~30-100 kPa) cardiac tissue states. The printed constructs demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) offer tremendous potential when used to engineer human tissues for drug screening and disease modeling; however, phenotypic immaturity reduces assay reliability when translating in vitro results to clinical studies. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cell and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces and had increased expression of genes that regulate contractile function. Improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. dECM-rGO hydrogels could also be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced electrophysiological function of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Recent advancement of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technologies has opened the door to next-generation modeling of human cardiac biology and disease. Not only does this alleviate the need for human primary tissue and compensate for the well documented deficiencies of rodent models for cardiovascular disease, but these technologies may lead to the identification of better candidates for clinical development. Unfortunately, most current 2D hiPSC-CM models lack the biochemical, mechanical, and electrical feedback that cardiomyocytes endure in a multi-cellular aligned tissue, which limits their translatability into clinical settings. To address this, we incorporated hiPSC-CMs modeling various genetic dilated cardiomyopathies (DCM) into 3D engineered heart tissues (EHTs). Here, we show these models develop distinguishable contractile defects recapitulating hallmarks of DCM when compared to biologically relevant controls. Moreover, this distinct phenotype is quantitative, reproducible, and demonstrates utility for drug discovery. As this innovative technology continues to develop, EHTs are on the forefront of emerging biomimetic assays that can be used to prevent drug attrition in the late stages of drug development.

We report on a simple and efficient method for the selective positioning of Au/DNA hybrid nanocircuits using a sequential combination of electron-beam lithography (EBL), plasma ashing, and a molecular patterning process. The nanostructures produced by the EBL and ashing process could be uniformly formed over a 12.6 in 2 substrate with sub-10 nm patterning with good pattern fidelity. In addition, DNA molecules were immobilized on the selectively nanopatterned regions by alternating surface coating procedures of 3-(aminopropyl)triethoxysilane (APS) and diamond like carbon (DLC), followed by deposition of DNA molecules into a well-defined single DNA nanowire. These single DNA nanowires were used not only for fabricating Au/DNA hybrid nanowires by the conjugation of Au nanoparticles with DNA, but also for the formation of Au/DNA hybrid nanocircuits. These nanocircuits prepared from Au/DNA hybrid nanowires demonstrate conductivities of up to 4.3 × 10 5 S/m in stable electrical performance. This selective and precise positioning method capable of controlling the size of nanostructures may find application in making sub-10 nm DNA wires and metal/DNA hybrid nanocircuits.

Statement of Purpose: Cardiac fibrosis is a devastating form of pathological remodeling in the myocardium, which results in progressive heart failure. Therefore, there is need for an in vitro platform that mimics the structural and mechanical properties of in vivo conditions to study disease mechanisms. We hypothesize that 3D-printed, cell-laden constructs derived from the cardiac ECM can be used to recapitulate the in vivo properties of both healthy and fibrotic cardiac tissues. In this work, we report bioinks composed of decellularized ECM (dECM), Laponite, and poly(ethylene glycol)-diacrylate (PEG-DA) that are extrudable, photo-crosslinkable, and demonstrate good printing fidelity. Furthermore, the shear storage modulus (G’) after photocuring can be tuned to model the stiffness of both healthy and fibrotic cardiac tissue.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for use in engineering human tissues for regenerative therapy and drug screening. However, differentiated cardiomyocytes are phenotypically immature, reducing assay reliability when translating in vitro results to clinical studies and precluding hiPSC-derived cardiac tissues from therapeutic use in vivo . To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cellular and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces at 14 days of culture and had increased the expression of genes that regulate contractile function. Similar improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. We also demonstrate that dECM-rGO hydrogels can be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced maturation of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Bio-sourced and biodegradable polymers for additive manufacturing couldenable the rapid fabrication of parts for a broad spectrum of applicationsranging from healthcare to aerospace. However, a limited number of thesematerials are suitable for vat photopolymerization processes. Herein, we report a two-step additivemanufacturing process to fabricate robust protein-based constructs using acommercially available laser-based SLA printer. Methacrylated bovine serumalbumin (MA-BSA) was synthesized and formulated into aqueous resins that were usedto print complex 3D objects with a resolution comparable to a commerciallyavailable resin. The MA-BSA resins were characterized by rheometry to determinethe viscosity and the cure rate, as both of these parameters can ultimately beused to predict the printability of the resin. In the first step of patterningthese materials, the MA-BSA resin was 3D printed, and in the second step, theprinted construct was thermally cured to denature the globular protein andincrease the intermolecular noncovalent interactions. Thus, the final 3Dprinted part was comprised of both chemical and physical cross-links. Compressionstudies of hydrated and dehydrated constructs demonstrated a broad range ofcompressive strengths and Young’s moduli that could be further modulated byadjusting the type and amount of co-monomer. The printed hydrogel constructsdemonstrated good cell viability (> 95%) after a 21-day culture period. TheseMA-BSA resins are expected to be compatible with other vat photopolymerizationtechniques including digital light projection (DLP) and continuous liquidinterface production (CLIP).

We report on the development of bioinspired cardiac scaffolds made from electroconductive acid-modified silk fibroin-poly(pyrrole) (AMSF+PPy) substrates patterned with nanoscale ridges and grooves reminiscent of native myocardial extracellular matrix (ECM) topography to enhance the structural and functional properties of cultured human pluripotent stem cells (hPSC)-derived cardiomyocytes. Nanopattern fidelity was maintained throughout the fabrication and functionalization processes, and no loss in conductive behavior occurred due to the presence of the nanotopographical features. AMSF+PPy substrates were biocompatible and stable, maintaining high cell viability over a 21-day culture period while displaying no signs of PPy delamination. The presence of anisotropic topographical cues led to increased cellular organization and sarcomere development, and electroconductive cues promoted a significant improvement in the expression and polarization of connexin 43 (Cx43), a critical regulator of cell-cell electrical coupling. The combination of biomimetic topography and electroconductivity also increased the expression of genes that encode key proteins involved in regulating the contractile and electrophysiological function of mature human cardiac tissue.

Intravenous regional anesthesia (IVRA; Bier block) is commonly used to anesthetize an extremity for surgery. Limitations of the procedure include pain from the required tourniquet, the toxicity that can occur from systemic release of local anesthetics, and the lack of postoperative pain relief. We hypothesized that the nanoencapsulation of the local anesthetic would prolong local anesthesia and enhance safety. Here, we developed an ∼15 nm micellar bupivacaine formulation (M-Bup) and tested it in a rat tail vein IVRA model, in which active agents were restricted in the tail by a tourniquet for 15 min. After tourniquet removal, M-Bup provided local anesthesia for 4.5 h, which was two times longer than that from a larger dose of free bupivacaine. Approximately 100 nm liposomal bupivacaine (L-Bup) with the same drug dose as M-Bup did not cause anesthesia. Blood levels of bupivacaine after tourniquet removal were lower in animals receiving M-Bup than L-Bup or free bupivacaine, demonstrating enhanced safety. Tissue reaction to M-Bup was benign.