Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

Tbx18 Orchestrates Cytostructural Transdifferentiation of Cardiomyocytes to Pacemaker Cells by Recruiting the Epithelial-Mesenchymal Transition Program.

Previously, we reported that heterologous expression of an embryonic transcription factor, Tbx18, reprograms ventricular cardiomyocytes into induced pacemaker cells (Tbx18-iPMs), though the key pathways are unknown. Here, we have used a tandem mass tag proteomic approach to characterize the impact of Tbx18 on neonatal rat ventricular myocytes. Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited increased expression of known pacemaker ion channels, including Hcn4 and Cx45 as well as upregulation of the mechanosensitive ion channels Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated, as were ion channels associated with ventricular excitation-contraction coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal and extracellular matrix remodeling, including 96 differentially expressed proteins associated with the epithelial-to-mesenchymal transition (EMT). RNAseq extended coverage of low abundance transcription factors, revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2, and Zeb2. Finally, network diffusion mapping of >200 transcriptional regulators indicates EMT and heart development factors occupy adjacent network neighborhoods downstream of Tbx18 but upstream of metabolic control factors. In conclusion, transdifferentiation of cardiac myocytes into pacemaker cells entails massive electrogenic, metabolic, and cytostructural remodeling. Structural changes exhibit hallmarks of the EMT. The results aid ongoing efforts to maximize the yield and phenotypic stability of engineered biological pacemakers.

Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.

Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. Omitting an exogenous Wnt inhibitor, which is routinely added to maximize cardiac myocyte yield during differentiation of mouse and human ESCs, led to increased yield of spontaneously beating cardiomyocytes with action potential properties similar to those of native sinoatrial node pacemaker cells. The pacemaker phenotype was accompanied by enhanced expression of genes and gene products that mark nodal pacemaker cells such as Hcn4, Tbx18, Tbx3, and Shox2. Addition of exogenous Wnt3a ligand, which activates canonical Wnt/ß-catenin signaling, increased the yield of pacemaker-like myocytes while reducing cTNT-positive pan-cardiac differentiation. Conversely, addition of inhibitors of Wnt/ß-catenin signaling led to increased chamber myocyte lineage development at the expense of pacemaker cell specification. The positive impact of canonical Wnt signaling on nodal pacemaker cell differentiation was evidenced in direct differentiation of two human ESC lines and human induced pluripotent stem cells. Our data identify the Wnt/ß-catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: endogenous Wnt signaling favors the pacemaker lineage, whereas its suppression promotes the chamber cardiomyocyte lineage.

Cellular Reprogramming Approaches to Engineer Cardiac Pacemakers.

PURPOSE OF REVIEW: The goal of this paper is to review present knowledge regarding biological pacemakers created by somatic reprogramming as a platform for mechanistic and metabolic understanding of the rare subpopulation of pacemaker cells, with the ultimate goal of creating biological alternatives to electronic pacing devices. RECENT FINDINGS: Somatic reprogramming of cardiomyocytes by reexpression of embryonic transcription factor T-box 18 (TBX18) converts them into pacemaker-like. Recent studies take advantage of this model to gain insight into the electromechanical, metabolic, and architectural intricacies of the cardiac pacemaker cell across various models, including a surgical model of complete atrioventricular block (CAVB) in adult rats. The studies reviewed here reinforce the potential utility of TBX18-induced pacemaker myocytes (iPMS) as a minimally invasive treatment for heart block. Several challenges which must be overcome to develop a viable therapeutic intervention based on these observations are discussed.

Strength-duration relationship as a tool to prioritize cardiac tissue properties that govern electrical excitability.

Engineered cardiac tissue and cardiomyocyte cell cultures offer wide opportunities for improved therapeutic intervention and laboratory heart models. Electrical field excitation is a common intervention in the production of engineered tissue and the investigation of the electrical properties of in vitro cell cultures. In this work, we use strength-duration relationships to investigate systematically factors influencing electrical excitability of two- (2D) and three-dimensional (3D) neonatal rat ventricular myocyte cultures. We find that the strength of the voltage pulse is negatively correlated with the threshold duration, as predicted by the Lapicque-Hill equation, and show that higher pacing frequencies require higher thresholds to capture paced cultures. We also study the impact of properties intrinsic to the 2D and 3D cultures on strength-duration relationships. We show that a smaller culture dimension, perpendicular anisotropic culture orientation with respect to electrical field, higher proportion of added fibroblasts, and TBX18-induced pacemaker reprogramming independently result in higher stimulation thresholds. These properties reflect the characteristics of the well-insulated endogenous pacemaking tissue in the heart (sinoatrial node) and should guide the engineering of biological pacemakers for improved outcomes. NEW & NOTEWORTHY Gaps exist in the availability of in vitro functional assessment tools that can emulate the integration of regenerative cells and tissues to the host myocardium. We use strength-duration relationships of electrically stimulated two- and three-dimensional myocardial constructs to study the effects of pacing frequency, culture dimensions, anisotropic cell alignment, fibroblast content, and pacemaker phenotype on electrical excitability. Our study delivers electrical strength-duration as a quantifiable parameter to evaluate design parameters of engineered cardiac tissue constructs.

DJ-1 protects the heart against ischemia-reperfusion injury by regulating mitochondrial fission.

Recent data indicates that DJ-1 plays a role in the cellular response to stress. Here, we aimed to examine the underlying molecular mechanisms mediating the actions of DJ-1 in the heart following myocardial ischemia-reperfusion (I/R) injury. In response to I/R injury, DJ-1 KO mice displayed increased areas of infarction and worsened left ventricular function when compared to WT mice, confirming a protective role for DJ-1 in the heart. In an effort to evaluate the potential mechanism(s) responsible for the increased injury in DJ-1 KO mice, we focused on SUMOylation, a post-translational modification process that regulates various aspects of protein function. DJ-1 KO hearts after I/R injury were found to display enhanced accumulation of SUMO-1 modified proteins and reduced SUMO-2/3 modified proteins. Further analysis, revealed that the protein expression of the de-SUMOylation enzyme SENP1 was reduced, whereas the expression of SENP5 was enhanced in DJ-1 KO hearts after I/R injury. Finally, DJ-1 KO hearts were found to display enhanced SUMO-1 modification of dynamin-related protein 1, excessive mitochondrial fission, and dysfunctional mitochondria. Our data demonstrates that the activation of DJ-1 in response to myocardial I/R injury protects the heart by regulating the SUMOylation status of Drp1 and attenuating excessive mitochondrial fission.

Wnt signalling suppresses voltage-dependent Na⁺ channel expression in postnatal rat cardiomyocytes.

Wnt signalling plays crucial roles in heart development, but is normally suppressed postnatally. In arrhythmogenic conditions, such as cardiac hypertrophy and heart failure, Wnt signalling is reactivated. To explore the potential role of Wnt signalling in arrhythmogenic electrical remodelling, we examined voltage-dependent ion channels in cardiomyocytes. Treatment of neonatal rat ventricular myocytes with either recombinant Wnt3a protein or CHIR-99021 (CHIR, a glycogen synthase kinase-3ß inhibitor) caused a dose-dependent increase in Wnt target gene expression (Axin2 and Lef1), indicating activation of the Wnt/ß-catenin pathway. Cardiac Na(+) current (INa) density was reduced by Wnt3a (-20 ± 4 vs. control -59 ± 7 pA pF(-1) , at -30 mV) or CHIR (-22 ± 5 pA pF(-1) ), without changes in steady-state activation, inactivation or repriming kinetics. Wnt3a and CHIR also produced dose-dependent reductions in the mRNA level of Scn5a (the cardiac Na(+) channel α subunit gene), as well as a 56% reduction (by Wnt3a) in the Nav 1.5 protein level. Consistent with INa reduction, action potentials in Wnt3a-treated neonatal rat ventricular myocytes had a lower upstroke amplitude (91 ± 3 vs. control 137 ± 2 mV) and decreased maximum upstroke velocity (70 ± 10 vs. control 163 ± 15 V s(-1)). In contrast, inward rectifier K(+) current and L-type Ca(2+) channels were not affected by Wnt3a treatment. Taken together, our data indicate that the Wnt/ß-catenin pathway suppresses INa in postnatal cardiomyocytes and may contribute to ion channel remodelling in heart disease.

Pacing the Heart with Genes: Recent Progress in Biological Pacing.

The heartbeat originates within the sinoatrial node (SA node or SAN), a small highly specialized structure containing <10,000 genuine pacemaker cells. The ~5 billion working cardiomyocytes downstream of the SAN remain quiescent when it fails, leading to circulatory collapse and fueling a $6B/year electronic pacemaker industry. The electronic pacemaker devices work quite well. But, device-related problems persist. These include lead failure/repositioning, finite battery life, and infection. For pediatric patients, the children outgrow the length of the leads, necessitating replacement with longer leads. These pitfalls have motivated creation of biological pacing. that are free from all hardware. Toward this goal, we and others have tested the concept of biological pacemakers. Combined with efforts to create clinically relevant, large animal models of biological pacing, the field is moving beyond a conceptual novelty toward a future with clinical reality.

Simulated microgravity improves maturation of cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels.

Transient pacing in pigs with complete heart block via myocardial injection of mRNA coding for the T-box transcription factor 18.

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal's sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers.

AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling.

BACKGROUND: Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation. METHODS: hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli. RESULTS: Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features-including enhanced Ca2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells. CONCLUSION: AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

Transcriptional suppression of connexin43 by TBX18 undermines cell-cell electrical coupling in postnatal cardiomyocytes.

T-box transcription factors figure prominently in embryonic cardiac cell lineage specifications. Mesenchymal precursor cells expressing Tbx18 give rise to the heart's pacemaker, the sinoatrial node (SAN). We sought to identify targets of TBX18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) transduced with GFP showed sarcolemmal, punctate Cx43 expression. In contrast, TBX18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly down-regulated within 2 days of TBX18 transduction. Direct injection of TBX18 in the guinea pig heart in vivo inhibited Cx43 expression. The repressor activity of TBX18 on Cx43 was highly specific; protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by TBX18. A reporter-based promoter assay demonstrated that TBX18 directly represses the Cx43 promoter. Phenotypically, TBX18-NRCMs exhibited slowed intercellular calcein dye transfer kinetics (421 ± 54 versus control 127 ± 43 ms). Intracellular Ca(2+) oscillations in control NRCM monolayers were highly synchronized. In contrast, TBX18 overexpression led to asynchronous Ca(2+) oscillations, demonstrating reduced cell-cell coupling. Decreased coupling led to slow electrical propagation; conduction velocity in TBX18 NRCMs slowed by more than 50% relative to control (2.9 ± 0.5 versus 14.3 ± 0.9 cm/s). Taken together, TBX18 specifically and directly represses Cx43 transcript and protein levels. Cx43 suppression leads to significant electrical uncoupling, but the preservation of other gap junction proteins supports slow action potential propagation, recapitulating a key phenotypic hallmark of the SAN.

Biological therapies for cardiac arrhythmias: can genes and cells replace drugs and devices?

Cardiac rhythm disorders reflect failures of impulse generation and/or conduction. With the exception of ablation methods that yield selective endocardial destruction, present therapies are nonspecific and/or palliative. Progress in understanding the underlying biology opens up prospects for new alternatives. This article reviews the present state of the art in gene- and cell-based therapies to correct cardiac rhythm disturbances. We begin with the rationale for such approaches, briefly discuss efforts to address aspects of tachyarrhythmia, and review advances in creating a biological pacemaker to cure bradyarrhythmia. Insights gained bring the field closer to a paradigm shift away from devices and drugs, and toward biologics, in the treatment of rhythm disorders.

Methodology for Cross-Talk Elimination in Simultaneous Voltage and Calcium Optical Mapping Measurements With Semasbestic Wavelengths.

Most cardiac arrhythmias at the whole heart level result from alteration of cell membrane ionic channels and intracellular calcium concentration ([Ca2+] i ) cycling with emerging spatiotemporal behavior through tissue-level coupling. For example, dynamically induced spatial dispersion of action potential duration, QT prolongation, and alternans are clinical markers for arrhythmia susceptibility in regular and heart-failure patients that originate due to changes of the transmembrane voltage (V m) and [Ca2+] i . We present an optical-mapping methodology that permits simultaneous measurements of the V m - [Ca2+] i signals using a single-camera without cross-talk, allowing quantitative characterization of favorable/adverse cell and tissue dynamical effects occurring from remodeling and/or drugs in heart failure. We demonstrate theoretically and experimentally in six different species the existence of a family of excitation wavelengths, we termed semasbestic, that give no change in signal for one dye, and thus can be used to record signals from another dye, guaranteeing zero cross-talk.

DNA Tension Probes Show that Cardiomyocyte Maturation Is Sensitive to the Piconewton Traction Forces Transmitted by Integrins.

Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk extracellular matrix stiffness in mediating the functional fate of CMCs. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (∼12, ∼56, and ∼160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. We show that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements, and protein expression (F-actin, vinculin, α-actinin, YAP, and SERCA2a). Additionally, sarcomeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.

Implementing Biological Pacemakers: Design Criteria for Successful.

Each heartbeat that pumps blood throughout the body is initiated by an electrical impulse generated in the sinoatrial node (SAN). However, a number of disease conditions can hamper the ability of the SAN's pacemaker cells to generate consistent action potentials and maintain an orderly conduction path, leading to arrhythmias. For symptomatic patients, current treatments rely on implantation of an electronic pacing device. However, complications inherent to the indwelling hardware give pause to categorical use of device therapy for a subset of populations, including pediatric patients or those with temporary pacing needs. Cellular-based biological pacemakers, derived in vitro or in situ, could function as a therapeutic alternative to current electronic pacemakers. Understanding how biological pacemakers measure up to the SAN would facilitate defining and demonstrating its advantages over current treatments. In this review, we discuss recent approaches to creating biological pacemakers and delineate design criteria to guide future progress based on insights from basic biology of the SAN. We emphasize the need for long-term efficacy in vivo via maintenance of relevant proteins, source-sink balance, a niche reflective of the native SAN microenvironment, and chronotropic competence. With a focus on such criteria, combined with delivery methods tailored for disease indications, clinical implementation will be attainable.

A CMOS Multi-Modal Electrochemical and Impedance Cellular Sensing Array for Massively Paralleled Exoelectrogen Screening.

The paper presents a 256-pixel CMOS sensor array with in-pixel dual electrochemical and impedance detection modalities for rapid, multi-dimensional characterization of exoelectrogens. The CMOS IC has 16 parallel readout channels, allowing it to perform multiple measurements with a high throughput and enable the chip to handle different samples simultaneously. The chip contains a total of 2 × 256 working electrodes of size 44 µm × 52 µm, along with 16 reference electrodes of dimensions 56 µm × 399 µm and 32 counter electrodes of dimensions 399 µm × 106 µm, which together facilitate the high resolution screening of the test samples. The chip was fabricated in a standard 130nm BiCMOS process. The on-chip electrodes are subjected to additional fabrication processes, including a critical Al-etch step that ensures the excellent biocompatibility and long-term reliability of the CMOS sensor array in bio-environment. The electrochemical sensing modality is verified by detecting the electroactive analyte NaFeEDTA and the exoelectrogenic Shewanella oneidensis MR-1 bacteria, illustrating the chip's ability to quantify the generated electrochemical current and distinguish between different analyte concentrations. The impedance measurements with the HEK-293 cancer cells cultured on-chip successfully capture the cell-to-surface adhesion information between the electrodes and the cancer cells. The reported CMOS sensor array outperforms the conventional discrete setups for exoelectrogen characterization in terms of spatial resolution and speed, which demonstrates the chip's potential to radically accelerate synthetic biology engineering.

Regeneration of infarcted mouse hearts by cardiovascular tissue formed via the direct reprogramming of mouse fibroblasts.

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.

Creation of a biological pacemaker by cell fusion.

As an alternative to electronic pacemakers, we explored the feasibility of converting ventricular myocytes into pacemakers by somatic cell fusion. The idea is to create chemically induced fusion between myocytes and syngeneic fibroblasts engineered to express HCN1 pacemaker channels (HCN1-fibroblasts). HCN1-fibroblasts were fused with freshly isolated guinea pig ventricular myocytes using polyethylene-glycol 1500. In vivo fused myocyte-HCN1-fibroblast cells exhibited spontaneously oscillating action potentials; the firing frequency increased with beta-adrenergic stimulation. The heterokaryons created ectopic ventricular pacemaker activity in vivo at the site of cell injection. Coculture of nonfused HCN1-fibroblasts and myocytes without polyethylene-glycol 1500 revealed no evidence of dye transfer, demonstrating that the I(f)-mediated pacemaker activity arises from heterokaryons rather than electrotonic coupling. This nonviral, non-stem cell approach enables autologous, adult somatic cell therapy to create biopacemakers.