Monolayers Of Neonatal Rat Ventricular Myocytes Research Articles

Engineered Cardiac Tissues as a Platform for CRISPR-Based Mitogen Discovery.

Improved understanding of cardiomyocyte (CM) cell cycle regulation may allow researchers to stimulate pro-regenerative effects in injured hearts or promote maturation of human stem cell-derived CMs. Gene therapies, in particular, hold promise to induce controlled proliferation of endogenous or transplanted CMs via transient activation of mitogenic processes. Methods to identify and characterize candidate cardiac mitogens in vitro can accelerate translational efforts and contribute to the understanding of the complex regulatory landscape of CM proliferation and postnatal maturation. In this study, A CRISPR knockout-based screening strategy using in vitro neonatal rat ventricular myocyte (NRVM) monolayers is established, followed by candidate mitogen validation in mature 3-D engineered cardiac tissues (ECTs). This screen identified knockout of the purine metabolism enzyme adenosine deaminase (ADA-KO) as an effective pro-mitogenic stimulus. RNA-sequencing of ECTs further reveals increased pentose phosphate pathway (PPP) activity as the primary driver of ADA-KO-induced CM cycling. Inhibition of the pathway's rate limiting enzyme, glucose-6-phosphate dehydrogenase (G6PD), prevented ADA-KO induced CM cycling, while increasing PPPactivityviaG6PD overexpression increased CM cycling. Together, this study demonstrates the development and application of a genetic/tissue engineering platform forin vitrodiscovery and validation of new candidate mitogens affecting regenerative or maturation states of cardiomyocytes.

Abstract P3132: Tgfβ Signaling Triggers Nonmyocyte Gene Expression In Tbx18-induced Pacemaker Myocytes And Abbreviates Their Automaticity

Background: We have previously demonstrated reprogramming of ventricular myocytes into induced pacemaker cells (iPMs) using an embryonic transcription factor TBX18. We have also discovered that Tgfβ pathway is significantly upregulated in TBX18-iPMs, and triggers fibrosis upon gene transfer in vitro and in vivo. Here, we sought to investigate the impact of Tgfβ signaling on iPMs’ automaticity. Methods: Neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno-TBX18 or control Adeno-GFP. Single cell RNAseq was performed at days 3, 6, and 14. Spontaneous field potentials from NRVM monolayers were recorded with microelectrode array. Results: TBX18 gene transfer strongly repressed working cardiomyocyte gene expression, e.g., Gja1, Scn5a, Pln throughout the 14-day period. TBX18-NRVMs progressed toward pacemaker cells, evidenced by increase in nodal pacemaker-related ion channel gene expression. TBX18 also triggered cytoskeletal remodeling, and activated nonmyocyte gene expression throughout the 2-week window. This was illustrated by ECM remodeling gene expression such as Acta2, Col1a1, and Postn. Treatment with A83-01, a small molecule inhibitor of Tgfβ receptors, mitigated the increase in nonmyocyte gene expression. TBX18-NRVMs exhibited spontaneously oscillating field potentials, reaching a peak pacing frequency of 196±36 bpm during week 1, which is comparable to the sinus rhythm of newborn rats. However, the pacing frequency gradually wavered after week 1. In contrast, A83-01-treated TBX18-iPMs showed peak pacing frequency beyond week 1, with the majority of their total beat counts near their sinus rhythm (63.3%) while only 14.9% of total beats were near the sinus rhythm in TBX18-iPMs without Tgfβ inhibition (P<0.001). Interestingly, A83-01 did not impact PM-related ion channel gene expression (such as Hcn4, Gjc1) nor the percentage of TBX18-iPMs (5.1% vs. 5.4%, with or without A83-01, p=0.561), suggesting that improved automaticity may be due to its impact on nonmyocytes. Conclusion: TBX18-iPMs exhibit increased Tgfβ signaling and nonmyocyte gene expression, which negatively impacts their automaticity. Inhibition of Tgfβ signaling modulates nonmyocyte gene expression and enhances automaticity of TBX18-iPMs.

Abstract P2164: Expression Of Engineered Bacterial Sodium Channel Improves Cardiomyocyte Contractility

Introduction: Our recent studies in non-human primates have demonstrated adeno-associated virus (AAV) delivery of prokaryotic sodium channel (BacNa v ) can improve the contractile function of infarcted hearts. We hypothesized that BacNa v expression increases cardiomyocyte (CM) contractile function by indirectly augmenting Ca 2+ transient amplitude via increase in sarcoplasmic reticulum (SR) Ca 2+ stores. Methods: We developed an in vitro model of ischemia-reperfusion (I/R) injury in engineered heart tissues (cardiopatches) made from human iPSC-derived CMs (hiPSC-CMs). Prior to formation, tissues were transduced with lentivirus conferring a doxycycline (Dox)-inducible expression of BacNa v . Upon I/R injury, the tissues were treated with either vehicle or Dox for 48h followed by force testing and Ca 2+ imaging. The effect of BacNa v expression on Ca 2+ handling was additionally studied using neonatal rat ventricular myocytes (NRVMs). Results: The tissue injury from I/R was evident from increases in cleaved-caspase 3 area (0.25% vs 1.53%), ROS generation (1.2 fold), LDH release (4.1 fold), and percent dead cells (0.97% vs 4.67%). Immediately following the I/R injury, tissues showed decrease in contractile force (2.78mN vs 0.74mN) and conduction velocity (20.7cm/s vs 7.9cm/s). At 72hr post-injury, BacNa v -expressing tissues exhibited significantly higher contractile force (1.67mN vs 1.32mN) and Ca 2+ transient (1.8 fold) amplitudes compared to the vehicle control. Furthermore, in uninjured NRVM monolayers, BacNa v expression resulted in higher Ca 2+ transient amplitude (1.31 fold), as well as increased SR calcium stores (1.3 fold) and slower sodium/calcium exchanger (NCX) extrusion rate (1.03s -1 vs 0.82s -1 ) in the presence of caffeine. Blocking NCX with 10M ORM-10962 eliminated the difference in Ca 2+ transient amplitude between BacNa v -expressing and control CMs. Conclusion: Our results suggest BacNa v expression augments CM contractility and Ca 2+ transient amplitude, at least in part through the suppression of Ca 2+ efflux mode of the NCX. In addition to its antiarrhythmic effects, BacNa v gene delivery may represent an effective therapeutic strategy to improve cardiac contractility in congestive heart failure.

Myeloperoxidase causes both arrhythmogenic structural and electrical remodelling in neonatal rat ventricular myocyte monolayers

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This study was supported by Netherlands Organization for Health Research and Development Background Myeloperoxidase (MPO), an enzyme most abundantly expressed by neutrophils, is increased in the atrial tissue of AF patients and is associated with treatment failure. Moreover, MPO increases fibrosis and AF susceptibility in mouse atria. However, how MPO promotes AF has yet to be fully established. Purpose To investigate the role of MPO in electrophysiological and structural remodelling. Methods We incubated cultured neonatal rat ventricular myocyte (NRVM) monolayers with 1 μg/mL MPO or vehicle for 24 hours. We then performed electrophysiological mapping using an 8x8 multielectrode array (MEA) setup with 100 μm-sized electrodes and 700 μm inter-electrode distance (n=8 MPO, n=7 control) and recorded action potentials (AP) using a microelectrode during pacing. Additionally, we quantified the number of fibroblasts using vimentin staining (n=5/group) and the expression of extracellular matrix (ECM) genes (COL1A1, COL8A2, FN1, CTGF, TNC, THBS2, MMP2, MMP12) using qPCR (n=4/group). Multielectrode data were analyzed with custom-made software in MATLAB. Statistical analysis was performed in R. Data presented as median [interquartile range]. Results NRVM monolayers incubated with MPO were unresponsive to pacing (5/8 vs 0/7 control) or had a higher pacing threshold (1300 [1000-2500] vs 500 [400-600] μA) (p=0.02). The percent of excitable tissue was lower in the MPO than in the control group, and the propagation of spontaneous activations was slower and more heterogeneous (index: 5 [3.6-9.5] vs 2 [1.5-3], p=0.004). More fractionated electrograms were present in MPO than in the control (29 [25-40] vs 11 [4-23] electrodes per grid, p=0.02). Re-entry was observed in 3/8 MPO-incubated monolayers vs 0/7 controls. Intracellular recordings revealed a less negative resting membrane potential (-50 [-24- -52] vs -69 [-56- -79] mV, p=0.04) and lower AP upstroke velocity (4 [2.5-14] vs 26 [22-28] V/s, p=0.01) in MPO-incubated cardiomyocytes than in the control. Moreover, relative to the control, MPO significantly increased the number of fibroblasts (126 [120-128] vs 87 [70-106] cells/0.26 mm^2, p=0.02) and the expression of MMP12 (1.17 (fold-change), p=0.04), whereas a trend toward increase was observed for other ECM genes, such as COL8A2 (1.5 (fold change), p=0.1). Conclusion Incubation of NVRM with MPO results in depolarization of cardiomyocytes and an increase in the number of fibroblasts and ECM gene expression. Thereby, MPO increases conduction heterogeneity and susceptibility to re-entry.

Abstract 16737: Inhibition of Tgf-β1 Signaling Protect Against Cell-Cell Decoupling and Asynchronous Automaticity of Tbx18-ipms

Background: We have previously demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells (TBX18-iPMs). However, the nascent automaticity appeared to wane over time, characterized by loss of syncytial pacing but preservation of single cell automaticity. Hypothesis: Based on increase in Tgfβ ligands in TBX18-iPMs and the known role of Tgfβ signaling in electrical remodeling, we hypothesized that loss of syncytial automaticity is due to electrical decoupling of the iPMs mediated by Tgfβ signaling. Methods: Adenoviral vectors expressing either human TBX18 or GFP were used for gene transfer into neonatal rat ventricular myocytes (NRVMs) or adult rat ventricular myocardium in vivo. Results: NRVM monolayers transduced with GFP were mostly quiescent, interspersed by paroxysmal contractions. In contrast, TBX18 transduced NRVM monolayers showed spontaneous and rhythmic contractions, but the syncytial pacing wavered over the next 5-7 days although numerous iPMs continued to beat asynchronously. Treatment of TBX18-iPMs with an inhibitor of Tgfβ receptors, A83-01, preserved syncytial pacing, suggesting that electrical remodeling cues from Tgfβ signaling led to loss of iPM-iPM electrical coupling. We examined the molecules that are integral to the function of gap junction in the myocardium, namely Cx43, N-cadherin and β-catenin. Cx43 and N-cadherin were downregulated in TBX18-iPMs compared to control by 77±16% and 43±11% (p<0.01, n=6), respectively. Total β-catenin protein level was unaltered, but its distribution decreased at the sarcolemmal and increased in the nuclei of TBX18-iPMs compared to control. Inhibition of Tgfβ with A83-01 restored Cx43 protein level in TBX18-iPMs (2.2-folds higher) compared to untreated TBX18-iPMs. Similarly, adult rats that received intramyocardial injection of TBX18 showed increased Cx43 expression at the boundary between the iPMs and the host myocardium when the animals were treated with systemic delivery of A83-01 for one week compared to TBX18 animals treated with DMSO. Conclusions: TBX18 activates Tgfβ signaling which suppresses cell-cell electrical coupling. Inhibition of Tgfβ signaling in TBX18-iPMs preserves gap junction components and syncytial pacing.

Abstract 16575: Synthetic TBX18 mRNA Induces Durable Reprogramming of Cardiac Myocytes to Pacemaker Cells

Introduction: Adenoviral gene delivery of transcription factor, TBX18, has been proven to reprogram ordinary cardiomyocytes to pacemaker cells, and provide ventricular pacing in the rodent and porcine heart. Yet host innate immune response to recombinant viral therapies remains a hurdle to overcome for successful translation. Somatic cell reprogramming does not require persistent expression of the reprogramming factors after arriving at the final cell fate. We hypothesized that brief expression of synthetic TBX18 mRNA suffices to convert chamber cardiomyocytes to induced pacemaker cells, thus obviating issues inherent to adenoviral gene therapy. Methods: Synthetic mRNAs were in vitro transcribed as previously described. Neonatal rat ventricular myocytes (NRVMs) were used as an in vitro platform to test pacemaker reprogramming by TBX18 mRNA. GFP or fLuc encoded mRNA was used as controls in all experiments. Results: Three days after synthetic TBX18 mRNA transfection, Hcn4 transcript level increased >6-fold compared to control. After two weeks, genes enriched in the pacemaker tissue, e.g., Shox2, Hcn4, and Tbx3, continued to be expressed higher in TBX18-NRVMs compared to control by >4.2-fold. The level of endogenously expressed, rat Tbx18 increased by 50% in NRVMs transfected with synthetic human TBX18 mRNA, which point to long-term reprogramming. To identify and lineage-trace reprogrammed pacemaker cells in real time, we cultured ventricular cardiomyoytes from transgenic mouse line Hcn4 ( GFP/+ ) , in which de novo pacemaker cells expressing Hcn4 contain eGFP fluorescence. Twelve days after synthetic mRNA or adenoviral gene transfer of TBX18 or GFP control, a significantly higher number of Hcn4 ( GFP/+ ) cells were observed in both mRNA and adenoviral TBX18-treated wells compared to their respective controls. TBX18 mRNA-transfected NRVM monolayers exhibited >2-fold slower conduction velocities compared to fLuc-transfected myocytes. In line with slow conduction velocity in the induced pacemaker myocytes, expression of ventricular gap junction protein, Cx43, decreased significantly compared to control. Conclusions: Together, the data indicate durable nodal pacemaker cell reprogramming with transient expression of synthetic TBX18 mRNA.

Abstract 236: Biphasic Reprogramming of Ventricular Cardiomyocytes to Pacemaker Cells by Tbx18

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.

The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing.

Erythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5-100μM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study, the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45μM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12 ± 9% at concentrations of 15-45μM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28 ± 12% (p < 0.01). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a "window" of concentrations of the drug (15-30μM) at which the probability of re-entry increased.

Fluorescence imaging of the living heart for understanding the basis of arrhythmias

Recent outstanding progress in microscopic imaging technology and the advent of fluorescent probes have enabled us to visualize high spatiotemporal dynamics of intracellular molecules in living tissues. Here I introduce our research outcomes on functional fluorescence imaging of the heart especially for understanding the pathogenesis of cardiac arrhythmias. On the in situ Ca2+ imaging of perfused rat heart by rapid-scanning confocal microscopy, we found that burst emergence of intracellular Ca2+ waves evokes arrhythmogenic triggered activity and subsequent oscillatory depolarizations via the Na+-Ca2+ exchanger. Besides, impairment of Ca2+ release from the sarcoplasmic reticulum leads to emergence of Ca2+ waves and spatiotemporally inhomogeneous Ca2+ dynamics on systole, resulting in beat-to-beat Ca2+ alternans. Such alternating behaviors of Ca2+ dynamics are partly due to poor development of the transverse tubules, which are identified in murine atria and failing ventricular myocytes. In addition, impairment of the gap junctional communication via connexin 43 induced by dominant negative inhibition of neonatal rat ventricular myocyte monolayers results in generation of spiral wave reentry, suggesting the pivotal role of intercellular communications in genesis of arrhythmias. Furthermore, alterations in atrial histoanatomy, e.g., density and arrangements of myocytes and distribution of Cx43, could provide intrinsic arrhythmogenic bases of atrial fibrillation, which was revealed by combined optical imaging of the atria and precise histoanatomical examinations. In combination, fluorescence imaging of the living organisms provides indispensable information for unveiling functions and disease states.

Effect of heptanol and ethanol on excitation wave propagation in a neonatal rat ventricular myocyte monolayer

In this work, the action of heptanol and ethanol was investigated in a two-dimensional (2D) model of cardiac tissue: the neonatal rat ventricular myocyte monolayer. Heptanol is known in electrophysiology as a gap junction uncoupler but may also inhibit voltage-gated ionic channels. Ethanol is often associated with the occurrence of arrhythmias. These substances influence sodium, calcium, and potassium channels, but the complete mechanism of action of heptanol and ethanol remains unknown. The optical mapping method was used to measure conduction velocities (CVs) in concentrations of 0.05–1.8 mM heptanol and 17–1342 mM ethanol. Heptanol was shown to slow the excitation wave significantly, and a mechanism that involves a simultaneous action on cell coupling and activation threshold was suggested. Whole-cell patch-clamp experiments showed inhibition of sodium and calcium currents at a concentration of 0.5 mM heptanol. Computer modeling was used to estimate the relative contribution of the cell uncoupling and activation threshold increase caused by heptanol. Unlike heptanol, ethanol slightly influenced the CV at clinically relevant concentrations. Additionally, the critical concentrations for re-entry formation in ethanol were determined.

Human Cardiomyocyte Progenitor Cells in Co-culture with Rat Cardiomyocytes Form a Pro-arrhythmic Substrate: Evidence for Two Different Arrhythmogenic Mechanisms.

Background: Cardiomyocyte progenitor cells (CMPCs) are a promising cell source for regenerative cell therapy to improve cardiac function after myocardial infarction. However, it is unknown whether undifferentiated CMPCs have arrhythmogenic risks. We investigate whether undifferentiated, regionally applied, human fetal CMPCs form a pro-arrhythmic substrate in co-culture with neonatal rat ventricular myocytes (NRVMs).Method: Unipolar extracellular electrograms, derived from micro-electrode arrays (8 × 8 electrodes) containing monolayers of NRVMs (control), or co-cultures of NRVMs and locally seeded CMPCs were used to determine conduction velocity and the incidence of tachy-arrhythmias. Micro-electrodes were used to record action potentials. Conditioned medium (Cme) of CMPCs was used to distinguish between coupling or paracrine effects.Results: Co-cultures demonstrated conduction slowing (5.6 ± 0.3 cm/s, n = 50) compared to control monolayers (13.4 ± 0.4 cm/s, n = 26) and monolayers subjected to Cme (13.7 ± 0.6 cm/s, n = 11, all p < 0.001). Furthermore, co-cultures had a more depolarized resting membrane than control monolayers (−47.3 ± 17.4 vs. −64.8 ± 7.7 mV, p < 0.001) and monolayers subjected to Cme (−64.4 ± 8.1 mV, p < 0.001). Upstroke velocity was significantly decreased in co-cultures and action potential duration was prolonged. The CMPC region was characterized by local ST-elevation in the recorded electrograms. The spontaneous rhythm was faster and tachy-arrhythmias occurred more often in co-cultured monolayers than in control monolayers (42.0 vs. 5.4%, p < 0.001).Conclusion: CMPCs form a pro-arrhythmic substrate when co-cultured with neonatal cardiomyocytes. Electrical coupling between both cell types leads to current flow between a, slowly conducting, depolarized and the normal region leading to local ST-elevations and the occurrence of tachy-arrhythmias originating from the non-depolarized zone.

Recombinant human collagen-based microspheres mitigate cardiac conduction slowing induced by adipose tissue-derived stromal cells.

BackgroundStem cell therapy to improve cardiac function after myocardial infarction is hampered by poor cell retention, while it may also increase the risk of arrhythmias by providing an arrhythmogenic substrate. We previously showed that porcine adipose tissue-derived-stromal cells (pASC) induce conduction slowing through paracrine actions, whereas rat ASC (rASC) and human ASC (hASC) induce conduction slowing by direct coupling. We postulate that biomaterial microspheres mitigate the conduction slowing influence of pASC by interacting with paracrine signaling.AimTo investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM).MethodUnipolar extracellular electrograms, derived from microelectrode arrays (8x8 electrodes) containing NRVM, co-cultured with ASC or ASC loaded microspheres, were used to determine conduction velocity (CV) and conduction heterogeneity. Conditioned medium (Cme) of (co)cultures was used to assess paracrine mechanisms.ResultsMicrospheres did not affect CV in control (NRVM) monolayers. In co-cultures of NRVM and rASC, hASC or pASC, CV was lower than in controls (14.4±1.0, 13.0±0.6 and 9.0± 1.0 vs. 19.5±0.5 cm/s respectively, p<0.001). Microspheres loaded with either rASC or hASC still induced conduction slowing compared to controls (13.5±0.4 and 12.6±0.5 cm/s respectively, p<0.001). However, pASC loaded microspheres increased CV of NRVM compared to pASC and NRMV co-cultures (16.3±1.3 cm/s, p< 0.001) and did not differ from controls (p = NS). Cme of pASC reduced CV in control monolayers of NRVM (10.3±1.1 cm/s, p<0.001), similar to Cme derived from pASC-loaded microspheres (11.1±1.7 cm/s, p = 1.0). The presence of microspheres in monolayers of NRVM abolished the CV slowing influence of Cme pASC (15.9±1.0 cm/s, p = NS vs. control).ConclusionThe application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through interference with a secondary autocrine myocardial factor.

Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived from Different Species.

Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose‐derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction. Stem Cells Translational Medicine 2017;6:22–30

Allosteric Modulation of Kv11.1 (hERG) Channels Protects Against Drug-Induced Ventricular Arrhythmias.

Ventricular arrhythmias as a result of unintentional blockade of the Kv11.1 (hERG [human ether-à-go-go-related gene]) channel are a major safety concern in drug development. In past years, several highly prescribed drugs have been withdrawn for their ability to cause such proarrhythmia. Here, we investigated whether the proarrhythmic risk of existing drugs could be reduced by Kv11.1 allosteric modulators. Using [(3)H]dofetilide-binding assays with membranes of human Kv11.1-expressing human embryonic kidney 293 cells, 2 existing compounds (VU0405601 and ML-T531) and a newly synthesized compound (LUF7244) were found to be negative allosteric modulators of dofetilide binding to the Kv11.1 channel, with LUF7244 showing the strongest effect at 10 μmol/L. The Kv11.1 affinities of typical blockers (ie, dofetilide, astemizole, sertindole, and cisapride) were significantly decreased by LUF7244. Treatment of confluent neonatal rat ventricular myocyte (NRVM) monolayers with astemizole or sertindole caused heterogeneous prolongation of action potential duration and a high incidence of early afterdepolarizations on 1-Hz electric point stimulation, occasionally leading to unstable, self-terminating tachyarrhythmias. Pretreatment of NRVMs with LUF7244 prevented these proarrhythmic effects. NRVM monolayers treated with LUF7244 alone displayed electrophysiological properties indistinguishable from those of untreated NRVM cultures. Prolonged exposure of NRVMs to LUF7244 or LUF7244 plus astemizole did not affect their viability, excitability, and contractility as assessed by molecular, immunological, and electrophysiological assays. Allosteric modulation of the Kv11.1 channel efficiently suppresses drug-induced ventricular arrhythmias in vitro by preventing potentially arrhythmogenic changes in action potential characteristics, raising the possibility to resume the clinical use of unintended Kv11.1 blockers via pharmacological combination therapy.

Standing & walking posture in children with bilateral spastic cerebral palsy: Are they related?

Regional depolarization of the mitochondrial network can alter cellular electrical excitability and increase the propensity for reentry, in part, through the opening of sarcolemmal KATP channels. Mitochondrial inner membrane potential (ΔΨm) instability or oscillation can be induced in myocytes by exposure to reactive oxygen species (ROS), laser excitation, or glutathione depletion, and is thought to be a major factor in arrhythmogenesis during ischemia–reperfusion. Nevertheless, the correlation between ΔΨm recovery kinetics and reperfusion-induced arrhythmias has been difficult to demonstrate experimentally. Here, we investigate the relationship between subcellular changes in ΔΨm, cellular glutathione redox potential, electrical excitability, and wave propagation during coverslip-induced ischemia–reperfusion (IR) in neonatal rat ventricular myocyte (NRVM) monolayers. Ischemia led to decreased action potential amplitude and duration followed by electrical inexcitability after ~ 15 min of ischemia. ΔΨm depolarization occurred in two phases during ischemia: in phase 1 (< 30 min ischemia), mitochondrial clusters within individual NRVMs depolarized, while phase 2 ΔΨm depolarization (30–60 min) was characterized by global functional collapse of the mitochondrial network across the whole ischemic region of the monolayer, typically involving a propagating metabolic wave. Oxidation of the glutathione (GSSG:GSH) redox potential occurred during ischemia, followed by recovery upon reperfusion (i.e., lifting the coverslip). ΔΨm recovered in the mitochondria of individual myocytes quite rapidly upon reperfusion (< 5 min), but was highly unstable, characterized by subcellular oscillations or flickering of clusters of mitochondria in NRVMs across the reperfused region. Electrical excitability also recovered in a heterogeneous manner, providing an arrhythmogenic substrate which led to formation of sustained reentry. Treatment with 4′-chlorodiazepam, a peripheral benzodiazepine receptor ligand, prevented ΔΨm oscillation, improved GSH recovery rate, and prevented reentry during reperfusion, indicating that stabilization of mitochondrial network dynamics is important for preventing post-ischemic arrhythmias. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Multicellular automaticity of cardiac cell monolayers: effects of density and spatial distribution of pacemaker cells

Self-organization of pacemaker (PM) activity of interconnected elements is important to the general theory of reaction–diffusion systems as well as for applications such as PM activity in cardiac tissue to initiate beating of the heart. Monolayer cultures of neonatal rat ventricular myocytes (NRVMs) are often used as experimental models in studies on cardiac electrophysiology. These monolayers exhibit automaticity (spontaneous activation) of their electrical activity. At low plated density, cells usually show a heterogeneous population consisting of PM and quiescent excitable cells (QECs). It is therefore highly probable that monolayers of NRVMs consist of a heterogeneous network of the two cell types. However, the effects of density and spatial distribution of the PM cells on spontaneous activity of monolayers remain unknown. Thus, a simple stochastic pattern formation algorithm was implemented to distribute PM and QECs in a binary-like 2D network. A FitzHugh–Nagumo excitable medium was used to simulate electrical spontaneous and propagating activity. Simulations showed a clear nonlinear dependency of spontaneous activity (occurrence and amplitude of spontaneous period) on the spatial patterns of PM cells. In most simulations, the first initiation sites were found to be located near the substrate boundaries. Comparison with experimental data obtained from cardiomyocyte monolayers shows important similarities in the position of initiation site activity. However, limitations in the model that do not reflect the complex beat-to-beat variation found in experiments indicate the need for a more realistic cardiomyocyte representation.

Mitochondrial instability during regional ischemia–reperfusion underlies arrhythmias in monolayers of cardiomyocytes

Regional depolarization of the mitochondrial network can alter cellular electrical excitability and increase the propensity for reentry, in part, through the opening of sarcolemmal KATP channels. Mitochondrial inner membrane potential (ΔΨm) instability or oscillation can be induced in myocytes by exposure to reactive oxygen species (ROS), laser excitation, or glutathione depletion, and is thought to be a major factor in arrhythmogenesis during ischemia–reperfusion. Nevertheless, the correlation between ΔΨm recovery kinetics and reperfusion-induced arrhythmias has been difficult to demonstrate experimentally. Here, we investigate the relationship between subcellular changes in ΔΨm, cellular glutathione redox potential, electrical excitability, and wave propagation during coverslip-induced ischemia–reperfusion (IR) in neonatal rat ventricular myocyte (NRVM) monolayers. Ischemia led to decreased action potential amplitude and duration followed by electrical inexcitability after ~15min of ischemia. ΔΨm depolarization occurred in two phases during ischemia: in phase 1 (<30min ischemia), mitochondrial clusters within individual NRVMs depolarized, while phase 2 ΔΨm depolarization (30–60min) was characterized by global functional collapse of the mitochondrial network across the whole ischemic region of the monolayer, typically involving a propagating metabolic wave. Oxidation of the glutathione (GSSG:GSH) redox potential occurred during ischemia, followed by recovery upon reperfusion (i.e., lifting the coverslip). ΔΨm recovered in the mitochondria of individual myocytes quite rapidly upon reperfusion (<5min), but was highly unstable, characterized by subcellular oscillations or flickering of clusters of mitochondria in NRVMs across the reperfused region. Electrical excitability also recovered in a heterogeneous manner, providing an arrhythmogenic substrate which led to formation of sustained reentry. Treatment with 4′-chlorodiazepam, a peripheral benzodiazepine receptor ligand, prevented ΔΨm oscillation, improved GSH recovery rate, and prevented reentry during reperfusion, indicating that stabilization of mitochondrial network dynamics is important for preventing post-ischemic arrhythmias. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Electrotonic suppression of early afterdepolarizations in the neonatal rat ventricular myocyte monolayer

Pathologies that result in early afterdepolarizations (EADs) are a known trigger for tachyarrhythmias, but the conditions that cause surrounding tissue to conduct or suppress EADs are poorly understood. Here we introduce a cell culture model of EAD propagation consisting of monolayers of cultured neonatal rat ventricular myocytes treated with anthopleurin-A (AP-A). AP-A-treated monolayers display a cycle length dependent prolongation of action potential duration (245 ms untreated, vs. 610 ms at 1 Hz and 1200 ms at 0.5 Hz for AP-A-treated monolayers). In contrast, isolated single cells treated with AP-A develop prominent irregular oscillations with a frequency of 2.5 Hz, and a variable prolongation of the action potential duration of up to several seconds. To investigate whether electrotonic interactions between coupled cells modulates EAD formation, cell connectivity was reduced by RNA silencing gap junction Cx43. In contrast to well-connected monolayers, gap junction silenced monolayers display bradycardia-dependent plateau oscillations consistent with EADs. Further, simulations of a cell displaying EADs electrically connected to a cell with normal action potentials show a coupling strength-dependent suppression of EADs consistent with the experimental results. These results suggest that electrotonic effects may play a critical role in EAD-mediated arrhythmogenesis.

Abstract 070: Interaction between Mena, Connexin43, and Rac1 at the Intercalated Disc

Background: We previously reported that Mena, a member of Ena/VASP family of actin regulatory protein, is associated with heart failure (HF). Mena is localized to the intercalated disc (ICD) and interacts with ICD proteins implicated in HF. The ICD mediates force transmission and electrical coupling between cardiomyocytes. Slowed conduction in HF is associated with Cx43 remodeling, the predominant connexin isoform in the heart. Mena-/- mice develop cardiac dysfunction, conduction abnormalities, ICD disorganization, and Cx43 lateralization. We hypothesized that: 1) Mena’s interaction with the cytoskeleton is critical for ICD stabilization and maintenance of electrical function; 2) Mena may modulate signal(s) from the cardiac sarcolemma to the ICD for control of expression and localization of Cx43 through the regulation of the small GTPase Rac1. Methods and Results: Interaction of Mena with Cx43 and Rac1 was evaluated in neonatal rat ventricular myocytes (NRVM), HeLa cells, and in transgenic mice overexpressing constitutively active V12Rac1 (RacET). Pull down assay using GST-PAK1 demonstrated a direct association of Mena with active Rac1-GTP in vitro. In NRVMs, siRNA knockdown of Mena significantly increased activity of Rac1-GTPase by 2-fold (p=0.05, n=5). Western blot analysis revealed increased total Cx43 expression in Mena siRNA (1.11±0.2) compared to scramble (0.33±0.1, p&lt;0.01, n=5). Enhanced Cx43 expression was associated with increased rate of recovery after photobleaching between cardiomyocytes by gap-FRAP analysis (Tau 153.0±4.8 vs. 73.0±1.8 sec, p&lt;0.0001). Ongoing experiments with optical mapping of NRVM monolayers will determine whether conduction velocity may be enhanced after Mena knockdown. Confocal imaging revealed altered Cx43 trafficking and localization with Mena knockdown. Additionally in ventricles of RacET hearts, Mena expression was increased by 59% (p=0.01, n=4) compared to control, along with lateral redistribution of Cx43. Conclusions: Our results suggest that Mena is a critical regulator of the ICD at the intersection of Cx43 and Rac1. Increased Rac1 activation is associated with lateralized Cx43, and enhanced cellular coupling. Mena may modulate Cx43 localization through the regulation of Rac1 activity.

Spatial gradients in action potential duration created by regional magnetofection of hERG are a substrate for wavebreak and turbulent propagation in cardiomyocyte monolayers

Spatial dispersion of action potential duration (APD) is a substrate for the maintenance of cardiac fibrillation, but the mechanisms are poorly understood. We investigated the role played by spatial APD dispersion in fibrillatory dynamics. We used an in vitro model in which spatial gradients in the expression of ether-à-go-go-related (hERG) protein, and thus rapid delayed rectifying K(+) current (I(Kr)) density, served to generate APD dispersion, high-frequency rotor formation, wavebreak and fibrillatory conduction. A unique adenovirus-mediated magnetofection technique generated well-controlled gradients in hERG and green fluorescent protein (GFP) expression in neonatal rat ventricular myocyte monolayers. Computer simulations using a realistic neonatal rat ventricular myocyte monolayer model provided crucial insight into the underlying mechanisms. Regional hERG overexpression shortened APD and increased rotor incidence in the hERG overexpressing region. An APD profile at 75 percent repolarization with a 16.6 ± 0.72 ms gradient followed the spatial profile of hERG-GFP expression; conduction velocity was not altered. Rotors in the infected region whose maximal dominant frequency was 12.9 Hz resulted in wavebreak at the interface (border zone) between infected and non-infected regions; dominant frequency distribution was uniform when the maximal dominant frequency was <12.9 Hz or the rotors resided in the uninfected region. Regularity at the border zone was lowest when rotors resided in the infected region. In simulations, a fivefold regional increase in I(Kr) abbreviated the APD and hyperpolarized the resting potential. However, the steep APD gradient at the border zone proved to be the primary mechanism of wavebreak and fibrillatory conduction. This study provides insight at the molecular level into the mechanisms by which spatial APD dispersion contributes to wavebreak, rotor stabilization and fibrillatory conduction.