Enhanced Drug Classification Using Machine Learning with Multiplexed Cardiac Contractility Assays.

Cardiac screening of newly discovered drugs remains a longstanding challenge for the pharmaceutical industry. While therapeutic efficacy and cardiotoxicity are evaluated through preclinical biochemical and animal testing, 90% of lead compounds fail to meet safety and efficacy benchmarks during human clinical trials. A preclinical model more representative of the human cardiac response is needed; heart tissue engineered from human pluripotent stem cell derived cardiomyocytes offers such a platform. In this study, three functionally distinct and independently validated engineered cardiac tissue assays are exposed to increasing concentrations of known compounds representing 5 classes of mechanistic action, creating a robust electrophysiology and contractility dataset. Combining results from six individual models, the resulting ensemble algorithm can classify the mechanistic action of unknown compounds with 86.2% predictive accuracy. This outperforms single-assay models and offers a strategy to enhance future clinical trial success align with the recent FDA Modernization Act 2.0.

Adverse effects of hydroxychloroquine and azithromycin on contractility and arrhythmogenicity revealed by human engineered cardiac tissues.

The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic as declared by World Health Organization (WHO). In the absence of an effective treatment, different drugs with unknown effectiveness, including antimalarial hydroxychloroquine (HCQ), with or without concurrent administration with azithromycin (AZM), have been tested for treating COVID-19 patients with developed pneumonia. However, the efficacy and safety of HCQ and/or AZM have been questioned by recent clinical reports. Direct effects of these drugs on the human heart remain very poorly defined. To better understand the mechanisms of action of HCQ +/- AZM, we employed bioengineered human ventricular cardiac tissue strip (hvCTS) and anisotropic sheet (hvCAS) assays, made with human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCMs), which have been designed for measuring cardiac contractility and electrophysiology, respectively. Our hvCTS experiments showed that AZM induced a dose-dependent negative inotropic effect which could be aggravated by HCQ; electrophysiologically, as revealed by the hvCAS platform, AZM prolonged action potentials and induced spiral wave formations. Collectively, our data were consistent with reported clinical risks of HCQ and AZM on QTc prolongation/ventricular arrhythmias and development of heart failure. In conclusion, our study exposed the risks of HCQ/AZM administration while providing mechanistic insights for their toxicity. Our bioengineered human cardiac tissue constructs therefore provide a useful platform for screening cardiac safety and efficacy when developing therapeutics against COVID-19.

Physiologic, Pathologic, and Therapeutic Paracrine Modulation of Cardiac Excitation-Contraction Coupling.

Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral ß-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility.

RATIONALE: The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating heart disease warrant a better understanding of underlying mechanisms of action. hMSC exosomes increase myocardial contractility; however, the exosomal cargo responsible for these effects remains unresolved. OBJECTIVE: This study aims to identify lead cardioactive hMSC exosomal microRNAs to provide a mechanistic basis for optimizing future stem cell-based cardiotherapies. METHODS AND RESULTS: Integrating systems biology and human engineered cardiac tissue (hECT) technologies, partial least squares regression analysis of exosomal microRNA profiling data predicted microRNA-21-5p (miR-21-5p) levels positively correlate with contractile force and calcium handling gene expression responses in hECTs treated with conditioned media from multiple cell types. Furthermore, miR-21-5p levels were significantly elevated in hECTs treated with the exosome-enriched fraction of the hMSC secretome (hMSC-exo) versus untreated controls. This motivated experimentally testing the human-specific role of miR-21-5p in hMSC-exo-mediated increases of cardiac tissue contractility. Treating hECTs with miR-21-5p alone was sufficient to recapitulate effects observed with hMSC-exo on hECT developed force and expression of associated calcium handling genes (eg, SERCA2a and L-type calcium channel). Conversely, knockdown of miR-21-5p in hMSCs significantly diminished exosomal procontractile and associated calcium handling gene expression effects on hECTs. Western blots supported miR-21-5p effects on calcium handling gene expression at the protein level, corresponding to significantly increased calcium transient amplitude and decreased decay time constant in comparison to miR-scramble control. Mechanistically, cotreating with miR-21-5p and LY294002, a PI3K inhibitor, suppressed these effects. Finally, mathematical simulations predicted the translational capacity for miR-21-5p treatment to restore calcium handling in mature ischemic adult human cardiomyocytes. CONCLUSIONS: miR-21-5p plays a key role in hMSC-exo-mediated effects on cardiac contractility and calcium handling, likely via PI3K signaling. These findings may open new avenues of research to harness the role of miR-21-5p in optimizing future stem cell-based cardiotherapies.

Sarco/endoplasmic reticulum Ca2+-ATPase is a more effective calcium remover than sodium-calcium exchanger in human embryonic stem cell-derived cardiomyocytes.

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+-handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights.NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.

Experimental and Computational Insight Into Human Mesenchymal Stem Cell Paracrine Signaling and Heterocellular Coupling Effects on Cardiac Contractility and Arrhythmogenicity.

RATIONALE: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. OBJECTIVE: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. METHODS AND RESULTS: Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). CONCLUSIONS: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.

Dehomogenized Elastic Properties of Heterogeneous Layered Materials in AFM Indentation Experiments.

Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at submicron length scales. However, such samples are often structurally heterogeneous even at the local level, with different regions having distinct mechanical properties. Physical or chemical disruption can isolate individual structural elements but may alter the properties being measured. Therefore, to determine the micromechanical properties of intact heterogeneous multilayered samples indented by AFM, we propose the Hybrid Eshelby Decomposition (HED) analysis, which combines a modified homogenization theory and finite element modeling to extract layer-specific elastic moduli of composite structures from single indentations, utilizing knowledge of the component distribution to achieve solution uniqueness. Using finite element model-simulated indentation of layered samples with micron-scale thickness dimensions, biologically relevant elastic properties for incompressible soft tissues, and layer-specific heterogeneity of an order of magnitude or less, HED analysis recovered the prescribed modulus values typically within 10% error. Experimental validation using bilayer spin-coated polydimethylsiloxane samples also yielded self-consistent layer-specific modulus values whether arranged as stiff layer on soft substrate or soft layer on stiff substrate. We further examined a biophysical application by characterizing layer-specific microelastic properties of full-thickness mouse aortic wall tissue, demonstrating that the HED-extracted modulus of the tunica media was more than fivefold stiffer than the intima and not significantly different from direct indentation of exposed media tissue. Our results show that the elastic properties of surface and subsurface layers of microscale synthetic and biological samples can be simultaneously extracted from the composite material response to AFM indentation. HED analysis offers a robust approach to studying regional micromechanics of heterogeneous multilayered samples without destructively separating individual components before testing.

Functional and transcriptomic insights into pathogenesis of R9C phospholamban mutation using human induced pluripotent stem cell-derived cardiomyocytes.

Dilated cardiomyopathy (DCM) can be caused by mutations in the cardiac protein phospholamban (PLN). We used CRISPR/Cas9 to insert the R9C PLN mutation at its endogenous locus into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. R9C PLN hiPSC-CMs display a blunted ß-agonist response and defective calcium handling. In 3D human engineered cardiac tissues (hECTs), a blunted lusitropic response to ß-adrenergic stimulation was observed with R9C PLN. hiPSC-CMs harboring the R9C PLN mutation showed activation of a hypertrophic phenotype, as evidenced by expression of hypertrophic markers and increased cell size and capacitance of cardiomyocytes. RNA-seq suggests that R9C PLN results in an altered metabolic state and profibrotic signaling, which was confirmed by gene expression analysis and picrosirius staining of R9C PLN hECTs. The expression of several miRNAs involved in fibrosis, hypertrophy, and cardiac metabolism were also perturbed in R9C PLN hiPSC-CMs. This study contributes to better understanding of the pathogenic mechanisms of the hereditary R9C PLN mutation in the context of human cardiomyocytes.

Mechanostimulation Promotes Nuclear and Epigenetic Changes in Oligodendrocytes.

Oligodendrocyte progenitors respond to biophysical or mechanical signals, and it has been reported that mechanostimulation modulates cell proliferation, migration, and differentiation. Here we report the effect of three mechanical stimuli on mouse oligodendrocyte progenitor differentiation and identify the molecular components of the linker of nucleoskeleton and cytoskeleton (LINC) complex (i.e., SYNE1) as transducers of mechanical signals to the nucleus, where they modulate the deposition of repressive histone marks and heterochromatin formation. The expression levels of LINC components increased during progenitor differentiation and silencing the Syne1 gene resulted in aberrant histone marks deposition, chromatin reorganization and impaired myelination. We conclude that spatial constraints, via the actin cytoskeleton and LINC complex, mediate nuclear changes in oligodendrocyte progenitors that favor a default pathway of differentiation. Significance statement: It is recognized that oligodendrocyte progenitors are mechanosensitive cells. However, the molecular mechanisms translating mechanical stimuli into oligodendrocyte differentiation remain elusive. This study identifies components of the mechanotransduction pathway in the oligodendrocyte lineage.

Rho Kinase Regulates Aortic Vascular Smooth Muscle Cell Stiffness Via Actin/SRF/Myocardin in Hypertension.

BACKGROUND/AIMS: Our previous studies demonstrated that intrinsic aortic smooth muscle cell (VSMC) stiffening plays a pivotal role in aortic stiffening in aging and hypertension. However, the underlying molecular mechanisms remain largely unknown. We here hypothesized that Rho kinase (ROCK) acts as a novel mediator that regulates intrinsic VSMC mechanical properties through the serum response factor (SRF) /myocardin pathway and consequently regulates aortic stiffness and blood pressure in hypertension. METHODS: Four-month old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were studied. Aortic stiffness was measured by echography. Intrinsic mechanical properties of VSMCs were measured by atomic force microscopy (AFM) in vitro. RESULTS: Compared to WKY rats, SHR showed a significant increase in aortic stiffness and blood pressure, which is accompanied by a remarkable cell stiffening and ROCK activation in thoracic aortic (TA) VSMCs. Theses alterations in SHR were abolished by Y-27632, a specific inhibitor of ROCK. Additionally, boosted filamentous/globular actin ratio was detected in TA VSMCs from SHR versus WKY rats, resulting in an up-regulation of SRF and myocardin expression and its downstream stiffness-associated genes including α-smooth muscle actin, SM22, smoothelin and myosin heavy chain 11. Reciprocally, these alterations in SHR TA VSMCs were also suppressed by Y-27632. Furthermore, a specific inhibitor of SRF/myocardin, CCG-100602, showed a similar effect to Y-27632 in SHR in both TA VSMCs stiffness in vitro and aorta wall stiffness in vivo. CONCLUSION: ROCK is a novel mediator modulating aortic VSMC stiffness through SRF/myocardin signaling which offers a therapeutic target to reduce aortic stiffening in hypertension.

Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes.

Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform-such as decreased action potential duration (APD) and plateau height-were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of hEAG1 activity may offer increased safety as a cell delivery cardiotherapy at low levels of hMSC-hCM coupling.

Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression.

Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly efficient in vivo delivery of modified mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injection of FLNP containing 1-10 µg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green fluorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expression kinetics after injecting 5 µg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intracoronary administration of 10 µg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injection, and off-target expression was correspondingly higher. Findings were confirmed in a pilot study in pigs using direct myocardial injection as well as percutaneous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanoparticle-enabled approach for highly efficient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.

Genomic correction of familial cardiomyopathy in human engineered cardiac tissues.

In this study, we used three-dimensional human engineered cardiac tissue technology to directly show that phospholamban (PLN) R14del mutation impairs cardiac contractility and to demonstrate restoration of contractile properties with targeted genetic correction of this inheritable form of dilated cardiomyopathy.

Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.

Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.

Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium.

Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18 ± 0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 µN/mm(2) at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 µM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.

An Evolving Understanding of the Genetic Causes of Abdominal Aortic Aneurysm Disease.

Aneurysms of the abdominal aorta (AAA) are relatively common - affecting as many as 8% of men and 1% of women over the age of 65. AAAs are characterized by a 50% increase in the diameter of the aneurysmal aorta compared with the normal vessel. Degeneration of structural components of the aortic wall is believed to be central in the pathogenesis of AAAs. The exact mechanism of degeneration is not well characterized, although degradation of elastin and collagen has been clearly shown. At least six genetic variants have been associated with AAA in genome-wide association studies: CDKN2BAS1, DAB2IP, LDLR, LRP1, SORT1, and IL6R. These variants reach genome-wide significance; however, they have not been replicated in multiple cohorts, nor have they been clearly shown to be disease causative. AAA is a challenging disease for investigation because it is most often asymptomatic and generally has a late disease onset, making it difficult to diagnose. Determination of the genetic mechanism behind aneurysm formation, progression, and rupture crosses disciplines requiring input from multiple fields of study, larger patient cohorts, and the evolving modalities of genetic testing.

Regenerative potential of mouse neonatal intervertebral disc depends on collagen crosslink density.

Intervertebral disc (IVD) defects heal poorly and can cause back pain and disability. We identified that IVD herniation injury heals regeneratively in neonatal mice until postnatal day 14 (p14) and shifts to fibrotic healing by p28. This age coincides with the shift in expansive IVD growth from cell proliferation to matrix elaboration, implicating collagen crosslinking. ß-aminopropionitrile treatment reduced IVD crosslinking and caused fibrotic healing without affecting cell proliferation. Bulk sequencing on naive IVDs was depleted for matrix structural organization from p14 to p28 to validate the importance of crosslinking in regenerative healing. We conclude that matrix changes are key drivers in the shift to fibrotic healing, and a stably crosslinked matrix is needed for IVD regeneration.

Integrative gene regulatory network analysis discloses key driver genes of fibromuscular dysplasia.

Fibromuscular dysplasia (FMD) is a poorly understood disease affecting 3-5% of adult females. The pathobiology of FMD involves arterial lesions of stenosis, dissection, tortuosity, dilation and aneurysm, which can lead to hypertension, stroke, myocardial infarction and even death. Currently, there are no animal models for FMD and few insights as to its pathobiology. In this study, by integrating DNA genotype and RNA sequence data from primary fibroblasts of 83 patients with FMD and 71 matched healthy controls, we inferred 18 gene regulatory co-expression networks, four of which were found to act together as an FMD-associated supernetwork in the arterial wall. After in vivo perturbation of this co-expression supernetwork by selective knockout of a top network key driver, mice developed arterial dilation, a hallmark of FMD. Molecular studies indicated that this supernetwork governs multiple aspects of vascular cell physiology and functionality, including collagen/matrix production. These studies illuminate the complex causal mechanisms of FMD and suggest a potential therapeutic avenue for this challenging disease.

Rianú: Multi-tissue tracking software for increased throughput of engineered cardiac tissue screening.

Background: The field of tissue engineering has provided valuable three-dimensional species-specific models of the human myocardium in the form of human Engineered Cardiac Tissues (hECTs) and similar constructs. However, hECT systems are often bottlenecked by a lack of openly available software that can collect data from multiple tissues at a time, even in multi-tissue bioreactors, which limits throughput in phenotypic and therapeutic screening applications. Methods: We developed Rianú, an open-source web application capable of simultaneously tracking multiple hECTs on flexible end-posts. This software is operating system agnostic and deployable on a remote server, accessible via a web browser with no local hardware or software requirements. The software incorporates object-tracking capabilities for multiple objects simultaneously, an algorithm for twitch tracing analysis and contractile force calculation, and a data compilation system for comparative analysis within and amongst groups. Validation tests were performed using in-silico and in-vitro experiments for comparison with established methods and interventions. Results: Rianú was able to detect the displacement of the flexible end-posts with a sub-pixel sensitivity of 0.555 px/post (minimum increment in post displacement) and a lower limit of 1.665 px/post (minimum post displacement). Compared to our established reference for contractility assessment, Rianú had a high correlation for all parameters analyzed (ranging from R2=0.7514 to R2=0.9695), demonstrating its high accuracy and reliability. Conclusions: Rianú provides simultaneous tracking of multiple hECTs, expediting the recording and analysis processes, and simplifies time-based intervention studies. It also allows data collection from different formats and has scale-up capabilities proportional to the number of tissues per field of view. These capabilities will enhance throughput of hECTs and similar assays for in-vitro analysis in disease modeling and drug screening applications.

Computational design of custom therapeutic cells to correct failing human cardiomyocytes.

Background: Myocardial delivery of non-excitable cells-namely human mesenchymal stem cells (hMSCs) and c-kit+ cardiac interstitial cells (hCICs)-remains a promising approach for treating the failing heart. Recent empirical studies attempt to improve such therapies by genetically engineering cells to express specific ion channels, or by creating hybrid cells with combined channel expression. This study uses a computational modeling approach to test the hypothesis that custom hypothetical cells can be rationally designed to restore a healthy phenotype when coupled to human heart failure (HF) cardiomyocytes. Methods: Candidate custom cells were simulated with a combination of ion channels from non-excitable cells and healthy human cardiomyocytes (hCMs). Using a genetic algorithm-based optimization approach, candidate cells were accepted if a root mean square error (RMSE) of less than 50% relative to healthy hCM was achieved for both action potential and calcium transient waveforms for the cell-treated HF cardiomyocyte, normalized to the untreated HF cardiomyocyte. Results: Custom cells expressing only non-excitable ion channels were inadequate to restore a healthy cardiac phenotype when coupled to either fibrotic or non-fibrotic HF cardiomyocytes. In contrast, custom cells also expressing cardiac ion channels led to acceptable restoration of a healthy cardiomyocyte phenotype when coupled to fibrotic, but not non-fibrotic, HF cardiomyocytes. Incorporating the cardiomyocyte inward rectifier K+ channel was critical to accomplishing this phenotypic rescue while also improving single-cell action potential metrics associated with arrhythmias, namely resting membrane potential and action potential duration. The computational approach also provided insight into the rescue mechanisms, whereby heterocellular coupling enhanced cardiomyocyte L-type calcium current and promoted calcium-induced calcium release. Finally, as a therapeutically translatable strategy, we simulated delivery of hMSCs and hCICs genetically engineered to express the cardiomyocyte inward rectifier K+ channel, which decreased action potential and calcium transient RMSEs by at least 24% relative to control hMSCs and hCICs, with more favorable single-cell arrhythmia metrics. Conclusion: Computational modeling facilitates exploration of customizable engineered cell therapies. Optimized cells expressing cardiac ion channels restored healthy action potential and calcium handling phenotypes in fibrotic HF cardiomyocytes and improved single-cell arrhythmia metrics, warranting further experimental validation studies of the proposed custom therapeutic cells.