Cardiomyocyte Network Research Articles

Graphene Field‐Effect Transistors toward Study of Cardiac Ischemia at Early Stage

AbstractIschemia and reperfusion states are studied in a network of cardiomyocytes as a part of real‐state conditions of heart injuries and inflammations, specifically myocardial infractions. Arrays of graphene field‐effect transistors (GFETs) fabricated in this work are used for extracellular recordings of ischemia states of cardiac cells during the external triggering of the ischemia infarction. The low‐concentrated ischemic buffer solution allows to create a cell‐stress condition resulting in the reperfusion process. The results show that the action potentials recorded with the graphene transistors, especially their shape, and duration of the active segment in measured extracellular action potentials, can be used to characterize the real state of the studied cardiac cell culture. The unique property of GFETs to detect such small changes in the action potential of cells in cardiac healthy and unhealthy states provides prospects for building the next generation of ultrasensitive biosensors, enabling the detection of acute ischemic states at an early stage.

DNA damage-associated protein co-expression network in cardiomyocytes informs on tolerance to genetic variation and disease.

Cardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage, which must be repaired by DNA damage response proteins. However, the impact of DNA damage on global cardiomyocyte protein abundance, and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control, and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing, chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD, such as arrhythmia are enriched in specific DOX-correlated modules, DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead, they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD, and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation.

Abstract Mo055: CHD4 Interacts With TBX5 to Maintain the Gene Regulatory Network of Postnatal Atrial Cardiomyocytes

Atrial fibrillation (AF) is the most common sustained arrhythmia in humans, leading to increased mortality due to increased incidence of stroke and heart failure. Over 200 genes are associated with AF by GWAS studies, suggesting a nuanced gene regulatory network (GRN) is required to maintain atrial cardiomyocyte (aCM) function and rhythm. Among these genes is the transcription factor TBX5, which we and others have shown is a master regulator of aCM identity that is essential for atrial rhythm homeostasis. We showed that TBX5 is required to maintain the chromatin looping, accessibility, and activity of atrial enhancers. Chromodomain helicase DNA binding protein 4 (CHD4), a chromatin remodeling protein that interacts with TBX5, is canonically thought to repress transcription as a component of the NuRD complex. Here, we investigated the contribution of CHD4 to TBX5 regulation of aCM gene expression. Methods: We inactivated CHD4 specifically within atrial cardiomyocytes using Cre recombinase driven by the aCM-specific Nppa promoter delivered postnatally with AAV9. Atrial remodeling, transcriptomic changes and AF susceptibility were examined. Results: We show that inactivating CHD4 in postnatal aCMs resulted in fibrotic atrial remodeling, spontaneous AF in a subset of mice, and increased AF susceptibility and burden after electrical pacing. Transcriptomic profiling of CHD4 aCM KO atria revealed gene expression changes that were highly correlated to altered gene expression downstream of TBX5 inactivation. Mechanistically, we show TBX5 recruits CHD4 to 33,170 genomic regions that are important for aCM gene regulation, whose expression is concordantly regulated downstream of TBX5 and CHD4. Together, these data reveal that CHD4 is required by TBX5 to promote accessibility of enhancers and activate many TBX5 target genes to maintain atrial rhythm.

Quercetin inhibits necroptosis in cardiomyocytes after ischemia-reperfusion via DNA-PKcs-SIRT5-orchestrated mitochondrial quality control.

We investigated the mechanism by which quercetin preserves mitochondrial quality control (MQC) in cardiomyocytes subjected to ischemia-reperfusion stress. An enzyme-linked immunosorbent assay was employed in the in vivo experiments to assess myocardial injury markers, measure the transcript levels of SIRT5/DNAPK-cs/MLKL during various time intervals of ischemia-reperfusion, and observe structural changes in cardiomyocytes using transmission electron microscopy. In in vitro investigations, adenovirus transfection was employed to establish a gene-modified model of DNA-PKcs, and primary cardiomyocytes were obtained from a mouse model with modified SIRT5 gene. Reverse transcription polymerase chain reaction, laser confocal microscopy, immunofluorescence localization, JC-1 fluorescence assay, Seahorse energy analysis, and various other assays were applied to corroborate the regulatory influence of quercetin on the MQC network in cardiomyocytes after ischemia-reperfusion. In vitro experiments demonstrated that ischemia-reperfusion injury caused changes in the structure of the myocardium. It was seen that quercetin had a beneficial effect on the myocardial tissue, providing protection. As the ischemia-reperfusion process continued, the levels of DNA-PKcs/SIRT5/MLKL transcripts were also found to change. In vitro investigations revealed that quercetin mitigated cardiomyocyte injury caused by mitochondrial oxidative stress through DNA-PKcs, and regulated mitophagy and mitochondrial kinetics to sustain optimal mitochondrial energy metabolism levels. Quercetin, through SIRT5 desuccinylation, modulated the stability of DNA-PKcs, and together they regulated the "mitophagy-unfolded protein response." This preserved the integrity of mitochondrial membrane and genome, mitochondrial dynamics, and mitochondrial energy metabolism. Quercetin may operate synergistically to oversee the regulation of mitophagy and the unfolded protein response through DNA-PKcs-SIRT5 interaction.

Effect of the community size and spatial arrangement of cardiomyocyte network in regulating cell response to a proarrhythmic compound

Effect of the community size and spatial arrangement of cardiomyocyte network in regulating cell response to a proarrhythmic compound

Importance of Spatial Arrangement of Cardiomyocyte Network for Precise and Stable On-Chip Predictive Cardiotoxicity Measurement

One of the advantages of human stem cell-derived cell-based preclinical screening is the reduction of the false negative/positive misjudgment of lead compounds for predicting their effectiveness and risks during the early stage of development. However, as the community effect of cells was neglected in the conventional single cell-based in vitro screening, the potential difference in results caused by the cell number and their spatial arrangement differences has not yet been sufficiently evaluated. Here, we have investigated the effect of the community size and spatial arrangement difference for cardiomyocyte network response against the proarrhythmic compounds from the viewpoint of in vitro cardiotoxicity. Using three different typical types of cell networks of cardiomyocytes, small cluster, large square sheet, and large closed-loop sheet were formed in shaped agarose microchambers fabricated on a multielectrode array chip simultaneously, and their responses were compared against the proarrhythmic compound, E-4031. The interspike intervals (ISIs) in large square sheets and closed-loop sheets were durable and maintained stable against E-4031 even at a high dose of 100 nM. In contrast, those in the small cluster, which fluctuated even without E-4031, acquired stable beating reflecting the antiarrhythmic efficacy of E-4031 from a 10 nM medium dose administration. The repolarization index, field potential duration (FPD), was prolonged in closed-loop sheets with 10 nM E-4031, even though small clusters and large sheets remained normal at this concentration. Moreover, FPDs of large sheets were the most durable against E-4031 among the three geometries of cardiomyocyte networks. The results showed the apparent spatial arrangement dependence on the stability of their interspike intervals, and FPD prolongation, indicating the importance of the geometry control of cell networks for representing the appropriate response of cardiomyocytes against the adequate amount of compounds for in vitro ion channel measurement.

Active force generation contributes to the complexity of spontaneous activity and to the response to stretch of murine cardiomyocyte cultures.

Cardiomyocyte cultures exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. In such preparations, beat rate variability exhibits features similar to those of heart rate variability in vivo. Mechanical deformations and forces feed back on the electrical properties of cardiomyocytes, but it is not fully elucidated how this mechano‐electrical interplay affects beating variability in such preparations. Using stretchable microelectrode arrays, we assessed the effects of the myosin inhibitor blebbistatin and the non‐selective stretch‐activated channel blocker streptomycin on beating variability and on the response of neonatal or fetal murine ventricular cell cultures against deformation. Spontaneous electrical activity was recorded without stretch and upon predefined deformation protocols (5% uniaxial and 2% equibiaxial strain, applied repeatedly for 1 min every 3 min). Without stretch, spontaneous activity originated from the edge of the preparations, and its site of origin switched frequently in a complex manner across the cultures. Blebbistatin did not change mean beat rate, but it decreased the spatial complexity of spontaneous activity. In contrast, streptomycin did not exert any manifest effects. During the deformation protocols, beat rate increased transiently upon stretch but, paradoxically, also upon release. Blebbistatin attenuated the response to stretch, whereas this response was not affected by streptomycin. Therefore, our data support the notion that in a spontaneously firing network of cardiomyocytes, active force generation, rather than stretch‐activated channels, is involved mechanistically in the complexity of the spatiotemporal patterns of spontaneous activity and in the stretch‐induced acceleration of beating. Key points Monolayer cultures of cardiac cells exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. Beating variability in these preparations recapitulates the power‐law behaviour of heart rate variability in vivo. However, the effects of mechano‐electrical feedback on beating variability are not yet fully understood.Using stretchable microelectrode arrays, we examined the effects of the contraction uncoupler blebbistatin and the non‐specific stretch‐activated channel blocker streptomycin on beating variability and on stretch‐induced changes of beat rate.Without stretch, blebbistatin decreased the spatial complexity of beating variability, whereas streptomycin had no effects.Both stretch and release increased beat rate transiently; blebbistatin attenuated the increase of beat rate upon stretch, whereas streptomycin had no effects.Active force generation contributes to the complexity of spatiotemporal patterns of beating variability and to the increase of beat rate upon mechanical deformation. Our study contributes to the understanding of how mechano‐electrical feedback influences heart rate variability.

Cpxm2 as a novel candidate for cardiac hypertrophy and failure in hypertension

Treatment of hypertension-mediated cardiac damage with left ventricular (LV) hypertrophy (LVH) and heart failure remains challenging. To identify novel targets, we performed comparative transcriptome analysis between genetic models derived from stroke-prone spontaneously hypertensive rats (SHRSP). Here, we identified carboxypeptidase X 2 (Cpxm2) as a genetic locus affecting LV mass. Analysis of isolated rat cardiomyocytes and cardiofibroblasts indicated Cpxm2 expression and intrinsic upregulation in genetic hypertension. Immunostaining indicated that CPXM2 associates with the t-tubule network of cardiomyocytes. The functional role of Cpxm2 was further investigated in Cpxm2-deficient (KO) and wild-type (WT) mice exposed to deoxycorticosterone acetate (DOCA). WT and KO animals developed severe and similar systolic hypertension in response to DOCA. WT mice developed severe LV damage, including increases in LV masses and diameters, impairment of LV systolic and diastolic function and reduced ejection fraction. These changes were significantly ameliorated or even normalized (i.e., ejection fraction) in KO-DOCA animals. LV transcriptome analysis showed a molecular cardiac hypertrophy/remodeling signature in WT but not KO mice with significant upregulation of 1234 transcripts, including Cpxm2, in response to DOCA. Analysis of endomyocardial biopsies from patients with cardiac hypertrophy indicated significant upregulation of CPXM2 expression. These data support further translational investigation of CPXM2.

Abstract 10881: Graph-Based Analysis of Cardiomyocyte Network Connectivity

Introduction: Cardiomyocytes branch and interconnect with one another, allowing for mechanical and electrochemical coupling. Despite being an essential structural feature enabling coordinated contraction, analysis of this branching structure has been limited. Herein, we use graph-based methods to analyze the cardiomyocyte network structure. Hypothesis: Cardiomyocytes are fully-connected, rather than being bundled into discrete, tract-bundles. We therefore expect that the size of connected graph components will increase with graph size, rather than tending towards a constant tract-bundle size. Methods: Rodent myocardium (n = 4) was stained for collagen and imaged using extended volume confocal microscopy. A sheetlet portion was segmented (~150 cells). The centerline of each cardiomyocyte was tracked. Vertices were identified where the cell bodies of adjacent cardiomyocytes merged. From the connected network (Fig 1A), an adjacency matrix was determined and the shortest-paths between all vertices calculated. The largest component was recorded as a function of graph size. Simulated fully-connected and tract-bundle networks (Fig 1B) were then compared to cardiomyocyte networks (Fig 1C). Results: For the simulated fully-connected network, the largest component increased with graph size, while the tract-bundle network levelled-off based on tract size (Fig 1B). In the cardiomyocyte networks (Fig 1C, red squares), the largest component continued to increase with graph size. Myocardial connectivity appears to lie between a tract-bundle and fully-connected simulated networks (Fig 1C). This suggests that the myocardium tends towards being a fully-connected network, although confirmation requires a larger image volume. Conclusion: Graph-based analysis of cardiomyocyte network structure distinguished between simulated fully-connected and tract-bundle networks. Cardiomyocyte networks are more similar to the fully-connected networks.

Aortic stenosis: correlation of prenatal echocardiography to postmortem histology

Abstract Background Foetal aortic stenosis (AS) is a challenging congenital heart disease considering its potential to progress during the course of pregnancy. Especially at midgestation, it remains extremely difficult to distinguish the cases that end up biventricular from the cases that will develop into an hypoplastic left heart syndrome. Purpose To test the hypothesis that the degree of myocardial maturation is a possible predictor of biventricular outcome, we present 4 cases of foetal AS with a varying degree of severity and uniquely correlate differences in myocardial function based on prenatal echocardiography to their post-mortem histopathologic maturation. Methods We selected 4 cases with midgestational AS from our tertiary foetal cardiology service between 2018–2020. Speckle tracking recordings of the cardiac four-chamber view were performed during routine foetal echocardiography to quantify myocardial wall motion as a marker for myocardial function. Three cases decided to terminate the pregnancy and donated the cardiac specimen. Immunohistochemical labelling (ICH) against key markers for myocardial maturation (troponin-I, N-cadherin, connexin-43, MLC2A, MLC2V and α-SMA) and fibrosis (Sirius Red) were compared with 2 normal foetal cardiac specimens. Results Two cases with critical AS presented extremely decreased global and segmental longitudinal strain (GLS and SLS) values (GLS −2% and −0.9%) in the left ventricle (LV), indicating an impaired myocardial wall deformation. Post-mortem ICH showed overt endocardial fibro-elastosis (EFE) and pathological fibrosis patterns in the subendocardial layer which was remarkably spatially correlated to the EFE. The cardiomyocytes were disorganised with reduced expression of troponin-I and disturbed expression of connexin-43. The remaining 2 cases had normal LV appearance on foetal echocardiography, showing a mild reduction in left ventricular GLS and SLS (GLS −11.8% and −14.2%). Post-mortem ICH of 1 of these cases showed mild EFE with a milder fibrosis pattern. Cardiomyocytes were less disorganised but also showed a disturbed expression of connexin-43. The 4th case continued the pregnancy and had a biventricular outcome. Conclusions This is a unique case series showing that myocardial function correlates with high extent to histology. The degree of the reduction in myocardial function corresponded with the amount of pathological fibrosis patterns and disorganisation of the cardiomyocyte network. Myocardial wall motion on foetal echocardiography seems to hold promise as a possible marker for cardiac maturation. Funding Acknowledgement Type of funding sources: None. Speckle tracking and fibrosis patterns

Visualization of β-adrenergic receptor dynamics and differential localization in cardiomyocytes

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two β-adrenergic receptor (β-AR) subtypes, β1 and β2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for β1-AR but not for β2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent β-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the β2-AR is confined to and diffuses within the T-tubular network, as opposed to the β1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the β2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the β2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

Microtubules Orchestrate Local Translation to Enable Cardiac Growth

Cardiomyocytes are terminally differentiated cells that respond to stress by regulating their size. During hypertrophy, it is well established that cardiomyocytes activate a hypertrophic transcriptional profile and increase protein translation rates, yet how these changes in transcription and translation are coupled to the sarcomerogenesis remains unclear. Concomitantly, the microtubule network of cardiomyocytes proliferates and is dramatically altered by post-translational modifications and decoration by microtubule-associated proteins, but how the microtubule network may regulate hypertrophy is also unknown. Here, we demonstrate that microtubules are critical for proper localization of the translational machinery (mRNA, ribosomes, and nascent proteins) to the z-disk and intercalated discs of adult rat ventricular myocytes (aRVMs). Microtubule disruption or knockdown of the kinesin-1 motor, but not disruption of the actinomyosin network, causes perinuclear accumulation of the translational machinery in aRVMs. Map4 overexpression or tyrosination of the microtubule network by TTL overexpression or VASH knock down led to similar perinuclear mRNA accumulation. In neonatal rat ventricular myocytes treated with the hypertrophic agonist phenylephrine (PE), mRNA and nascent proteins are redistributed to the periphery during cell growth. Microtubule disruption led to perinuclear accumulation of the translational machinery similar to that observed in aRVMs and prevented the PE-induced hypertrophic growth. Importantly, this lack of hypertrophic growth was not due to a defect in upstream transcriptional or translational regulation as the expression of hypertrophic/fetal gene markers and the enhancement of translation rate was preserved with microtubule disruption, suggesting that localization of the translational machinery is the key factor driving hypertrophic growth. Furthermore, PE-induced hypertrophy in mice is abrograted by colchicine treatment despite augmented expression of hypertrophic/fetal gene markers and increased protein translation rates. Overall, the data demonstrate that microtubule-based trafficking of the translational machinery is critical to couple hypertrophic signaling pathways to productive cardiomyocyte growth.

Geometric Understanding of Local Fluctuation Distribution of Conduction Time in Lined-Up Cardiomyocyte Network in Agarose-Microfabrication Multi-Electrode Measurement Assay

We examined characteristics of the propagation of conduction in width-controlled cardiomyocyte cell networks for understanding the contribution of the geometrical arrangement of cardiomyocytes for their local fluctuation distribution. We tracked a series of extracellular field potentials of linearly lined-up human embryonic stem (ES) cell-derived cardiomyocytes and mouse primary cardiomyocytes with 100 kHz sampling intervals of multi-electrodes signal acquisitions and an agarose microfabrication technology to localize the cardiomyocyte geometries in the lined-up cell networks with 100–300 μm wide agarose microstructures. Conduction time between two neighbor microelectrodes (300 μm) showed Gaussian distribution. However, the distributions maintained their form regardless of its propagation distances up to 1.5 mm, meaning propagation diffusion did not occur. In contrast, when Quinidine was applied, the propagation time distributions were increased as the faster firing regulation simulation predicted. The results indicate the “faster firing regulation” is not sufficient to explain the conservation of the propagation time distribution in cardiomyocyte networks but should be expanded with a kind of community effect of cell networks, such as the lower fluctuation regulation.

Transcribed ultraconserved region uc.242 is a novel regulator of cardiomyocyte hypertrophy induced by angiotensin II

Cardiomyocyte hypertrophy is a response to stress or hormone stimulation and is characterized by an increase of cardiomyocyte size. Abnormal long non-coding RNA (lncRNA) expression profile has been identified in...

Analyzing the α-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy.

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the α-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Abstract MP115: Transcriptional Patterning of the Ventricular Cardiac Conduction System

The heart beats 2 billion times over the average human lifespan and is the manifestation of a pattern of cardiomyocyte depolarization organized by a specialized network of cardiomyocytes, the cardiac conduction system (CCS). Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat and has been recognized for more than a century. However molecular basis of this regional patterning is not well understood. Using mouse genetics, we found that the ratio between T-box transcriptional activator, Tbx5 , and T-box transcriptional repressor, Tbx3 , determines the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular, cellular and functional criteria. Specifically, the ventricular fast conduction gene expression network was lost whereas the slow conduction nodal gene expression network was retained. Action potentials (APs) of Tbx5 -deficient VCS myocytes adopted nodal-specific characteristics, including increased AP duration and cellular automaticity. Removal of Tbx5 in-vivo precipitated inappropriate depolarizations in the His-bundle that initiated lethal ventricular arrhythmias. A T-box rheostat mechanism for CCS patterning was confirmed by Tbx5/Tbx3 genetic interaction studies. TBX5 bound and directly activated cis -regulatory elements at fast conduction loci genome-wide, defining the identity of the adult VCS. Furthermore, TBX5 bound and activated cis-regulatory elements at Tbx5 itself, describing a multi-tiered T-box-dependent fast conduction gene regulatory network (GRN). The hierarchical GRN established a bi-stable network in mathematical modeling, with only high or low fast conduction gene expression states, suggesting a genomic mechanism for the establishment of VCS versus nodal states in-vivo . Thus, the CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS GRN allowed nodal physiology to emerge, providing a molecular mechanism for some lethal ventricular arrhythmias.

Optical Interrogation of Sympathetic Neuronal Effects on Macroscopic Cardiomyocyte Network Dynamics.

SummaryCardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. We present approaches to study neuron-cardiomyocyte interactions involving optogenetic selective probing and all-optical electrophysiology to measure activity in an automated fashion. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiomyocyte network dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac behavior, and the applicability of optogenetics in mechanistic in vitro studies. As arrhythmias are emergent behaviors that involve the coordinated activity of millions of cells, we image at macroscopic scales to capture complex dynamics. We show that neurons can both decrease and increase wave stability and re-entrant activity in culture depending on their induced activity—a finding that may help us understand the often conflicting results seen in experimental and clinical studies.

Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy.

The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.

Transcriptional Patterning of the Ventricular Cardiac Conduction System.

The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

Dominant rule of community effect in synchronized beating behavior of cardiomyocyte networks.

Exploiting the combination of latest microfabrication technologies and single cell measurement technologies, we can measure the interactions of single cells, and cell networks from "algebraic" and "geometric" perspectives under the full control of their environments and interactions. However, the experimental constructive single cell-based approach still remains the limitations regarding the quality and condition control of those cells. To overcome these limitations, mathematical modeling is one of the most powerful complementary approaches. In this review, we first explain our on-chip experimental methods for constructive approach, and we introduce the results of the "community effect" of beating cardiomyocyte networks as an example of this approach. On-chip analysis revealed that (1) synchronized interbeat intervals (IBIs) of cell networks were followed to the more stable beating cells even their IBIs were slower than the other cells, which is against the conventional faster firing regulation or "overdrive suppression," and (2) fluctuation of IBIs of cardiomyocyte networks decreased according to the increase of the number of connected cells regardless of their geometry. The mathematical simulation of this synchronous behavior of cardiomyocyte networks also fitted well with the experimental results after incorporating the fluctuation-dissipation theorem into the oscillating stochastic phase model, in which the concept of spatially arranged cardiomyocyte networks was involved. The constructive experiments and mathematical modeling indicated the dominant rule of synchronization behavior of beating cardiomyocyte networks is a kind of stability-oriented synchronization phenomenon as the "community effect" or a fluctuation-dissipation phenomenon. Finally, as a practical application of this approach, the predictive cardiotoxicity is introduced.