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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 ofgt;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.

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 via 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 into 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.

This article presents a fully integrated multi-modal CMOS cellular sensor/stimulator array chip with 21 952 pixels and 1568-pixel concurrent readouts, while each array pixel can be independently reconfigured to support three sensing and one stimulation modalities: cellular potential recording, optical-point impedance sensing, and bi-phasic current stimulation. The CMOS sensor/stimulator array chip provides 3.6 mm $\times $ 1.6 mm active field-of-view (FoV). Each pixel contains one 8 $\mu \text{m}\,\,\times $ 8 $\mu \text{m}$ gold deposited electrode, one 6 $\mu \text{m}\,\,\times $ 6 $\mu \text{m}$ photodiode, and in-pixel circuits within 8 $\mu \text{m}\,\,\times $ 11 $\mu \text{m}$ pixel area, while the pixel-to-pixel pitch is 16 $\mu \text{m}\,\,\times $ 16 $\mu \text{m}$ . As a proof of concept, the CMOS array is implemented in a standard 130-nm BiCMOS process. Comprehensive electrical testing (potential/optical/four-point impedance/stimulation) and biological measurements (potential/optical/four-point impedance) with on-chip rat cardiomyocytes demonstrate the functionalities and unique advantages of this multi-modality cellular array. With high throughput multi-modal cellular sensing supported at the pixel level, this array chip enables holistic characterization of on-chip cellular samples with single-cell resolution.

Implantable cardiac pacemaker devices are generally effective for patients with symptomatic bradyarrhythmia. However, device-dependent cardiac pacing is far from ideal and often inadequate, particularly for pediatric patients who need to go through invasive revision of the indwelling hardware. Biological pacemakers have been proposed as device-free alternatives to the current treatment, but sustained, unwavering biological pacing beyond days after the biologic delivery has not been demonstrated. We have previously demonstrated that re-expression of an embryonic transcription factor, TBX18, could reprogram ventricular cardiomyocytes into induced pacemaker myocytes (iPMs). Here, we report that exogenous expression of TBX18 per se leads to severe fibrosis in situ, impairing the iPMs’ ability to pace together. Acute fibrosis is accompanied with proliferation and activation of cardiac fibroblasts via Tgfβ-Smad2/3 pathway. Small molecule inhibition of Tgfβ signaling mitigated the interstitial remodeling, independent from TBX18-induced iPM reprogramming at the single-cell level. Direct and focal gene transfer of TBX18 into the left ventricular myocardium created ventricular pacing in a rat model of chronic atrioventricular block, but such activity began to wane in a week. In contrast, a combination therapy consisting of TBX18 gene transfer and Tgfβ inhibition enabled sustained biological pacing beyond the four-week study period. Our data demonstrate that inhibition of Tgfβ signaling suffices to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

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.

Remarkable progress has been made in the direct differentiation of human pluripotent stem cells (hPSCs) into the major sources of cell types found in the heart. The understanding of cardiovascular development in vivo can provide the guidelines and help us to facilitate the process of generation of cells. In this chapter, we review the state of methods in generating cardiovascular cells from hPSCs according to our understanding of heart development. We also stress the important role of developmental biology during the differentiation from hPSCs. We then highlight the application in disease modeling, drug screening, and cell therapy based on cell transplantation. With the advances of their application, the promise of hPSCs-related products will provide new treatments for degenerative diseases of the heart in the future.

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/or 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.

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.

Atrioventricular block (AVB), caused by impairment in the heart conduction system, presents extreme diversity and is associated with other complications. Only half of AVB patients require a permanent pacemaker, and the process determining the pacemaker implantation is associated with an increase in cost and patient morbidity and mortality. Thus, there is a need for models capable of accurately identifying transient or reversible causes for conduction disturbances and predicting the patient risks and the necessity of a pacemaker. Deep learning (DL) is brought to the forefront due to its prediction accuracy, and the DL-based electrocardiogram (ECG) analysis can be a breakthrough to analyze a massive amount of data. However, the current DL models are unsuitable for AVB-ECG, where the P waves are decoupled from the QRS/T waves, and a black-box nature of the DL-based model lowers the credibility of prediction models to physicians. Here, we present a real-time-capable DL-based algorithm that can identify AVB-ECG waves and automate AVB phenotyping for arrhythmogenic risk assessment. Our algorithm can analyze unformatted ECG records with abnormal patterns by integrating the two representative DL algorithms: convolutional neural networks (CNN) and recurrent neural networks (RNN). This hybrid CNN/RNN network can memorize local patterns, spatial hierarchies, and long-range temporal dependencies of ECG signals. Furthermore, by integrating parameters derived from dimension reduction analysis and heart rate variability into the hybrid layers, the algorithm can capture the P/QRS/T-specific morphological and temporal features in ECG waveforms. We evaluated the algorithm using the six AVB porcine models, where TBX18, a pacemaker transcription factor, was transduced into the ventricular myocardium to form a biological pacemaker, and an additional electronic pacemaker was transplanted as a backup pacemaker. We achieved high sensitivity (95% true positive rate) and quantified the potential risks of various pathological ECG patterns. This study may be a starting point in conducting both retrospective and prospective patient studies and will help physicians understand its decision-making workflow and find the incorrect recommendations for AVB patients.

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult., Heart health: understanding pacemaker cells The heart’s pacemaker cells contain mitochondria that are smaller than average and require less energy than other heart cells, properties that help make them naturally resilient to stress. Cardiac pacemaker cells constitute a tiny proportion of the heart’s cells, yet play a critical role in maintaining a steady heartbeat. However, quite how pacemaker cells maintain their automatic rhythm is unclear because their scarcity makes them difficult to study. To examine the cells’ metabolic state further, Hee Cheol Cho at Emory University, Atlanta, and Brian Foster at Johns Hopkins University School of Medicine, Baltimore, and co-workers therefore induced pacemaker cells by adding an embryonic protein to heart muscle cells. The induced pacemaker cells survived well under oxidative stress. The team identified a protein in the pacemakers’ mitochondrial membranes, the expression of which directly influences rhythm responses.

The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.

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.

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. 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.

Cells are complex systems with concurrent multi-physical responses, and cell physiological signals are often encoded with spatiotemporal dynamics and further coupled with multiple cellular activities. However, most existing electronic sensors are only single-modality and cannot capture multi-parametric cellular responses. In this paper, a 1024-pixel CMOS quad-modality cellular interfacing array that enables multi-parametric cell profiling for drug development is presented. The quad-modality CMOS array features cellular impedance characterization, optical detection, extracellular potential recording, and biphasic current stimulation. The fibroblast transparency and surface adhesion are jointly monitored by cellular impedance and optical sensing modalities for comprehensive cell growth evaluation. Simultaneous current stimulation and opto-mechanical monitoring based on cardiomyocytes are demonstrated without any stimulation/sensing dead-zone. Furthermore, drug dose-dependent multi-parametric feature extractions in cardiomyocytes from their extracellular potentials and opto-mechanical signals are presented. The CMOS array demonstrates great potential for fully automated drug screening and drug safety assessments, which may substantially reduce the drug screening time and cost in future new drug development., Lab on a Chip, 18 (19), ISSN:1473-0197, ISSN:1473-0189

Background: We have previously demonstrated that an embryonic transcription factor, TBX18, suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells. Here, we sought to gain finer understanding of the reprogramming process by investigating genotype/phenotype of the de novo pacemaker cells in multiple temporal domains. Methods: TBX18-induced pacemaker cells (TBX18-iPMs) were created in vitro by somatic gene transfer of Adeno-TBX18 into neonatal rat ventricular myocytes (NRVMs). Control ventricular myocytes were transduced with Adeno- GFP. To investigate in vivo reprogramming, we delivered AAV9-TBX18 via tail vein of Hcn4(+/eGFP) heterozygous transgenic mice, which expresses eGFP under the Hcn4 promoter, so as to lineage-trace de novo pacemaker cells in the ventricular myocardium. Results: The automaticity of TBX18-iPMs was higher than that of GFP-NRVMs during D1-D3, decreased after D3 and rebound by D14. Monolayers of the iPMs revealed multiple foci of spontaneous field potentials with lower spike amplitude, spike slope, and conduction velocity, demonstrating phenotypic reprogramming to the iPMs by D14. RNAseq and qPCR revealed two distinct phases of reprogramming; an early phase that showed loss of expression of ventricular myocyte-related gene program and a late phase that showed gain of expression of nodal gene program which plateaued by D14. For example, Hcn4 gene expression began increasing during week 1 and continued the pattern during week 2. By week 3, Hcn4-positive TBX18-iPMs appeared in clusters of NRVM monolayers while GFP-NRVMs were spread evenly throughout the monolayers. We lineage-traced de novo, TBX18-reprogrammed pacemaker cells in vivo by delivering AAV9-TBX18 via tail-vein of Hcn4(+/eGFP) mice. In line with our in vitro data, in vivo pacemaker cell lineage tracing experiments indicated a progressive loss of ventricular gene program followed by gain of nodal gene program. Conclusions: Our data indicate that TBX18-induced reprogramming of ventricular myocytes to nodal pacemaker cells undergoes progressive changes in gene expression shedding ventricular gene/phenotype first, and then gaining nodal pacemaker genotype/phenotype, establishing fully functional nodal pacemaker by D14.

This paper presents a fully integrated CMOS multi-modal cellular sensor/stimulator array with 21952 multi-modal pixels, 1568 simultaneous parallel readout channels, 16 μm×16 μm pixel pitch for single cell resolution, and 3.6 mm×1.6 mm tissue-level field-of-view (FoV), achieving high-resolution multi-parametric cellular potential/impedance/optical imaging for holistic cellular characterization and cell-based assays. Moreover, the array system reports the first on-chip true 4-point impedance sensing scheme with 16 parallel impedance sensing channels, which enables precise cellular impedance measurements with aggressively scaled electrodes and large electrode-electrolyte interfacial impedance. The chip also supports concurrent 16-channel 5-bit reconfigurable current-mode cell stimulation. The chip is implemented in a 130 nm low-cost standard CMOS process. Extracellular potentials (700 μV-1.5 mV) from on-chip cultured neonatal rat ventricular myocytes (NRVMs) are successfully measured. With on-chip cultured cardiac fibroblasts, full-chip high-resolution optical images and 4-point impedance mapping precisely capture cell distribution, growth, proliferation, and surface adhesion.

Complete atrioventricular block (CAVB) is a life-threatening arrhythmia. A small animal model of chronic CAVB that properly reflects clinical indices of bradycardia would accelerate the understanding of disease progression and pathophysiology, and the development of therapeutic strategies. We sought to develop a surgical model of CAVB in adult rats, which could recapitulate structural remodeling and arrhythmogenicity expected in chronic CAVB. Upon right thoracotomy, we delivered electrosurgical energy subepicardially via a thin needle into the atrioventricular node (AVN) region of adult rats to create complete AV block. The chronic CAVB animals developed dilated and hypertrophied ventricles with preserved systolic functions due to compensatory hemodynamic remodeling. Ventricular tachyarrhythmias, which are difficult to induce in the healthy rodent heart, could be induced upon programmed electrical stimulation in chronic CAVB rats and worsened when combined with β-adrenergic stimulation. Focal somatic gene transfer of TBX18 to the left ventricular apex in the CAVB rats resulted in ectopic ventricular beats within days, achieving a de novo ventricular rate faster than the slow atrioventricular (AV) junctional escape rhythm observed in control CAVB animals. The model offers new opportunities to test therapeutic approaches to treat chronic and severe CAVB which have previously only been testable in large animal models.

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.

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/β-catenin signaling, increased the yield of pacemaker-like myocytes while reducing cTNT-positive pan-cardiac differentiation. Conversely, addition of inhibitors of Wnt/β-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/β-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.

Bradycardia arising from pacemaker dysfunction can be debilitating and life threatening. Electronic pacemakers serve as effective treatment options for pacemaker dysfunction. They however present their own limitations and complications. This has motivated research into discovering more effective and innovative ways to treat pacemaker dysfunction. Gene therapy is being explored for its potential to treat various cardiac conditions including cardiac arrhythmias. Gene transfer vectors with increasing transduction efficiency and biosafety have been developed and trialed for cardiovascular disease treatment. With an improved understanding of the molecular mechanisms driving pacemaker development, several gene therapy targets have been identified to generate the phenotypic changes required to correct pacemaker dysfunction. This review will discuss the gene therapy vectors in use today along with methods for their delivery. Furthermore, it will evaluate several gene therapy strategies attempting to restore biological pacing, having the potential to emerge as viable therapies for pacemaker dysfunction.

Understanding the factors that influence the electrical excitability is important for successful engineering and therapeutic translation of cardiac tissue. Electric field excitation is widely utilized in vitro and in vivo to assess the response of cardiomyocytes to electrical excitation. The excitability of cardiac tissue is influenced by a variety of factors, including anatomic location in the heart, disease status, and age. We dissect properties that alter the electrical excitability of ventricular myocardial tissue constructs in vitro in 2D and 3D models of neonatal rat ventricular myocytes, using a custom-made integrated live-cell imaging and pacing setup. We find that for both 2D and 3D constructs, the electrical signal strength correlates negatively with the threshold stimulation duration required for pacing and driving the engineered tissue, and the resulting strength-duration curve is well approximated by the Lapicque-Hill model of electrically excitable membranes. By comparing strength-duration relationships, we find that pacing 2D constructs at 2 Hz requires 1.7 to 1.9 times the stimulus strength compared to pacing at 1 Hz (n=3, p

This paper presents a fully integrated CMOS multimodality joint sensor/stimulator array with 1024 pixels for real-time holistic cellular characterization and drug screening. The proposed system consists of four pixel groups and four parallel signal-conditioning blocks. Every pixel group contains 16 × 16 pixels, and each pixel includes one 28 μm × 28 μm gold-plated electrode, four 12 μm × 12 μm photodiodes, and in-pixel circuits, within a 58 μm × 58 μm pixel footprint. Each pixel supports real-time extracellular potential recording, optical detection, charge-balanced biphasic current stimulation, and cellular impedance measurement for the same cellular sample. The proposed system is fabricated in a standard 130-nm CMOS process. Rat cardiomyocytes are successfully cultured on-chip. Measured high-resolution optical opacity images, extracellular potential recordings, biphasic current stimulations, and cellular impedance images demonstrate the unique advantages of the system for holistic cell characterization and drug screening. Furthermore, this paper demonstrates the use of optical detection on the on-chip cultured cardiomyocytes to real-time track their cyclic beating pattern and beating rate.

Background Despite growing evidence regarding the role of osteopathic manipulative treatment (OMT) for the management of low back pain, there is little evidence to support the use of OMT as a post-operative rehabilitation to improve the functional outcomes of lumbar disc surgery. Objective To assess the feasibility for a future definitive randomised control trial that would indicate whether OMT improves post-operative outcomes after lumbar microdiscectomy compared to a standard exercise programme. Design Randomised controlled pilot study. Setting Department of Spinal Surgery and Department of Spinal Rehabilitation at a major metropolitan spine surgery hospital, Seoul, South Korea. Methods Patients who underwent lumbar microdiscectomy due to low back pain with referred leg pain resulting from a herniated disc were enrolled in the study. Thirty-three patients aged 25–65 years were randomly assigned using a random number table to the OMT ( n = 16) group or exercise group ( n = 17). Patients received the allocated intervention twice a week for 4 weeks. Each session was 30 min. Primary outcomes were post-surgical functional disability and intensity of low back and leg pain. Outcome measures were assessed at baseline (2–3 weeks after surgery) and post-intervention (7–8 weeks after surgery). Double blinding was not feasible in the study setting. Results Thirty-three participants were analysed. Both rehabilitation interventions improved all primary and secondary outcomes. Post-surgical physical disability improved more with OMT rehabilitation than the exercise programme (54% vs. 26%, P Conclusion The current pilot study shows the feasibility of a future definitive randomised control trial investigating whether rehabilitation with OMT is a viable approach for post-operative management of a lumbar microdiscectomy.

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-3β inhibitor) caused a dose-dependent increase in Wnt target gene expression (Axin2 and Lef1), indicating activation of the Wnt/β-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/β-catenin pathway suppresses INa in postnatal cardiomyocytes and may contribute to ion channel remodelling in heart disease.

Summary When pluripotency factors are removed, embryonic stem cells (ESCs) undergo spontaneous differentiation, which, among other lineages, also gives rise to cardiac sublineages, including chamber cardiomyocytes and pacemaker cells. Such heterogeneity complicates the use of ESC-derived heart cells in therapeutic and diagnostic applications. We sought to direct ESCs to differentiate specifically into cardiac pacemaker cells by overexpressing a transcription factor critical for embryonic patterning of the native cardiac pacemaker (the sinoatrial node). Overexpression of SHOX2 during ESC differentiation upregulated the pacemaker gene program, resulting in enhanced automaticity in vitro and induced biological pacing upon transplantation in vivo. The accentuated automaticity is accompanied by temporally evolving changes in the effectors and regulators of Wnt signaling. Our findings provide a strategy for enriching the cardiac pacemaker cell population from ESCs., Highlights • SHOX2 accentuates the molecular profile of pacemaker cells in differentiating ESCs • SHOX2 increases the frequency and rate of spontaneously active cardiac derivatives • SHOX2-overexpressing EBs function as biopacemakers when transplanted in vivo • Wnt signaling underlies SHOX2-mediated pacemaker cell specification, Cho and colleagues show that heterologous SHOX2 expression boosts the differentiation of embryonic stem cells to cardiac pacemaker cells from embryonic stem cells by enhancing pacemaker cell-specific electrophysiology. Implantation of SHOX2-overxpressing embryoid bodies to the rat heart in vivo creates biological pacing. The SHOX2-facilitated pacemaker cell phenotype correlates with specific Wnt modulation, suggesting that Wnt may be a negotiator for cardiac subtype specification.

Background Cardiac electrical conduction delays and blocks cause rhythm disturbances such as complete heart block, which can be fatal. Standard of care relies on electronic devices to artificially restore synchrony. We sought to create a new modality for treating these disorders by engineering electrical conduction tracts designed to propagate electrical impulses. Objectives This study sought to create a new approach for treating cardiac conduction disorders by using engineered electrical conduction tracts (EECTs). Methods Paramagnetic beads were conjugated with an antibody to gamma-sarcoglycan, a cardiomyocyte cell surface antigen, and mixed with freshly isolated neonatal rat ventricular cardiomyocytes. A magnetic field was used to pattern a linear EECT. Results In an in vitro model of conduction block, the EECT was patterned so that it connected 2 independently beating neonatal rat ventricular cardiomyocyte monolayers; it achieved coordinated electrical activity, with action potentials propagating from 1 region to the other via EECT. Spiking the EECT with heart-derived stromal cells yielded stable structures with highly reproducible conduction velocities. Transplantation of EECTs in vivo restored atrioventricular conduction in a rat model of complete heart block. Conclusions An EECT can re-establish electrical conduction in the heart. This novel approach could, in principle, be used not only to treat cardiac arrhythmias but also to repair other organs.

This paper presents a single-cell-resolution multimodality cellular interfacing array for holistic cell characterization and high-throughput drug screening. It contains 22,528 multimodality sensing pixels with a pixel size of 17μm×17μm. Each pixel contains a 4μm×4μm gold-plated electrode for extracellular potential recording and a 3μm×6μm photodiode for optical detection. To compensate for the degraded signal to noise ratio (SNR) due to the large electrode-electrolyte impedance at the cell-sensor interface, we employ a DC-coupled chopper amplifier when performing extracellular potential recording. A proof-of-concept design is implemented in a standard 130nm CMOS process. It achieves a measured input-referred noise of 2.46μVrms integrated from 1Hz to 4kHz with a chopping frequency of 20kHz, and cell-based measurement results demonstrate real-time recorded action potentials with high SNR. Measured high-resolution optical shadow images provide the detailed cell and tissue morphology as well as the 2D distribution of on-chip cultured cells. Furthermore, this paper presents the first CMOS sensing array to successfully capture on-chip rat cardiomyocyte contraction using multiple sensing modalities, specifically optical detection and potential recording.

Cardiosphere-derived cells (CDCs) effect therapeutic regeneration after myocardial infarction (MI) both in animal models and in humans. Here, we test the hypothesis that cell-cell contact plays a role in mediating the observed therapeutic benefits of CDCs, above and beyond conventional paracrine effects. Human CDCs or vehicle were injected into immunodeficient (SCID) mouse hearts during acute MI. CDC transplantation augmented the proportion of cycling (Ki67+) cardiomyocytes and improved ventricular function. CDC-conditioned media only modestly augmented the percentage of Ki67+ cardiomyocytes (>control but

Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl currents (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown.In the present study, we found that the specific ICl,swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50 = 2.7 μmol/l) ICl,swell and inhibited (IC50 = 6.9 μmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl channel genes underlying ICl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to ICl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by45%) compared with control-shRNA, it did not alter ICl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution.Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.

Intracellular action potential signals reveal enriched physiological information. Patch clamp techniques have been widely used to measure intracellular potential. Despite their high signal fidelity, they suffer from low throughput. Recently, 3D nanoelectrodes have been developed for intracellular potential recording. However, they are limited by scalability, yield, and cost, directly constraining their use in monitoring large number of cells and high throughput applications. In this paper, we demonstrate intracellular potential monitoring of cardiomyocytes using simple 2D planar electrode array in a standard CMOS process without patch clamps or post fabricated 3D nanoelectrodes. This is enabled by our unique cardiomyocytes/fibroblasts co-culturing technique and electroporation. The co-cultured fibroblasts promote tight sealing of cardiomyocytes on electrodes and enable high-fidelity intracellular potential monitoring based on 2D planar electrode. Compared to existing technologies, our platform has a unique potential to achieve an unprecedented combination of throughput, spatiotemporal resolution, and a tissue-level field-of-view for cellular electrophysiology monitoring.

Every heartbeat originates from a tiny tissue in the heart called the sinoatrial node (SAN). The SAN harbors only ≈10 000 cardiac pacemaker cells, initiating an electrical impulse that captures the entire heart, consisting of billions of cardiomyocytes for each cardiac contraction. How these rare cardiac pacemaker cells (the electrical source) can overcome the electrically hyperpolarizing and quiescent myocardium (the electrical sink) is incompletely understood. Due to the scarcity of native pacemaker cells, this concept of source–sink mismatch cannot be tested directly with live cardiac tissue constructs. By exploiting TBX18 induced pacemaker cells by somatic gene transfer, 3D cardiac pacemaker spheroids can be tissue‐engineered. The TBX18 induced pacemakers (sphTBX18) pace autonomously and drive the contraction of neighboring myocardium in vitro. TBX18 spheroids demonstrate the need for reduced electrical coupling and physical separation from the neighboring ventricular myocytes, successfully recapitulating a key design principle of the native SAN. β‐Adrenergic stimulation as well as electrical uncoupling significantly increase sphTBX18s' ability to pace‐and‐drive the neighboring myocardium. This model represents the first platform to test design principles of the SAN for mechanistic understanding and to better engineer biological pacemakers for therapeutic translation., The engineering sinoatrial node (eSAN) in a dish design is inspired by the remarkable structure of the native SAN exit pathways. The architecture of the native SAN reduces the need for large number of pacemaker cells to spontaneously and autonomously pace‐and‐drive every single heartbeat.