Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes.

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and ß-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (ß1 + ß2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (ß1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (ß2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating ß1- and α1-ARs in both human and rabbit, with a ß2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.

Consecutive-Day Ventricular and Atrial Cardiomyocyte Isolations from the Same Heart: Shifting the Cost-Benefit Balance of Cardiac Primary Cell Research.

Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4-8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.

Region-specific distribution of transversal-axial tubule system organization underlies heterogeneity of calcium dynamics in the right atrium.

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

A library of induced pluripotent stem cells from clinically well-characterized, diverse healthy human individuals.

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.

Chamber-Specific Protein Expression during Direct Cardiac Reprogramming.

Forced expression of core cardiogenic transcription factors can directly reprogram fibroblasts to induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. This cardiac reprogramming approach provides a proof of concept for induced heart regeneration by converting a fibroblast fate to a cardiomyocyte fate. However, it remains elusive whether chamber-specific cardiomyocytes can be generated by cardiac reprogramming. Therefore, we assessed the ability of the cardiac reprogramming approach for chamber specification in vitro and in vivo. We found that in vivo cardiac reprogramming post-myocardial infarction exclusively induces a ventricular-like phenotype, while a major fraction of iCMs generated in vitro failed to determine their chamber identities. Our results suggest that in vivo cardiac reprogramming may have an inherent advantage of generating chamber-matched new cardiomyocytes as a potential heart regenerative approach.

Characterization of Ventricular and Atrial Cardiomyocyte Subtypes from Human-Induced Pluripotent Stem Cells.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) are expected to be used in regenerative therapies and drug discovery for heart failure. hiPSC-CMs are a mixture of mainly ventricular CMs (VCMs) and also of atrial CMs (ACMs) and pacemaker cells. Here we describe a method to enrich VCM and ACM differentiation and to characterize these subtypes by gene expression analysis using qRT-PCR and by electrophysiological properties using the patch-clamp method. The differentiated VCMs and ACMs highly express VCM and ACM marker genes, respectively. Furthermore, both subtypes show specific properties of action potentials.

Analysis of Transcriptional Profiling of Chamber-Specific Human Cardiac Myocytes Derived from Pluripotent Stem Cells.

Differentiation protocols to direct cell fate decision from pluripotent stem cells to cardiac myocytes normally achieve high purity and quality of cells. Nonetheless, the highly specialized anatomy of the heart enables the possibility that acquisition of terminal somatic differentiation from pluripotency might imply heterogeneity of non-desire cell lineages. Directed cardiac differentiation empowers differentiation of pool of cells commonly reported to contain different proportions of ventricular, atrial, and nodal-like cells. RNA sequencing (RNA-Seq) allows a precise transcriptional profiling, ensuring a quality checking of the cell identity our protocol has derived as a main outcome. Here we describe a workflow methodology on how to adapt RNA sequencing analysis for integration into the R analysis pipeline in order to characterize chamber-specific gene signatures of the major cardiac lineages of myocytes in the heart.

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects.

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. IKs (KCNQ1+KCNE1), a slowly activating K+ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the IKs channel. Here, we show that C28 specifically shifted IKs VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of IKs VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes.

BACKGROUND: There is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (ICa) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of ß1-adrenoceptor (ß1-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on ICa and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes. METHODS: Adult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). ICa was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 µM) were used to inhibit PDE3 and PDE4, respectively. ß1-AR were stimulated with the physiological agonist norepinephrine (100 µM). RESULTS: In adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and ICa by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and ICa (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and ICa + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and ICa + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on ICa. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and ICa, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and ICa + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on ICa (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET. CONCLUSION: Our results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller ICa responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes.

Cyclic stretching boosts microRNA-499 to regulate Bcl-2 via microRNA-208a in atrial fibroblasts.

MicroRNAs that modulate transcription can regulate other microRNAs and are also up-regulated under pathological stress. MicroRNA-499 (miR-499), microRNA-208a (miR-208a) and B-cell lymphoma 2 (Bcl-2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl-2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts-adult atrial (HCF-aa) was investigated. Using negative pressure, HCF-aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up-regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl-2 was up-regulated by longer stretching times. Pre-treatment with antagomir-499 reversed the miR-208a down-regulation, whereas Bcl-2 expression could be suppressed by miR-208a overexpression. In the HCF-aa under stretching for 1 h, miR-499 overexpression decreased pri-miR-208a luciferase activity; this inhibition of pri-miR-208a luciferase activity with stretching was reversed when the miR-499-5p binding site in pri-miR-208a was mutated. The addition of antagomir-208a reversed the Bcl-2-3'UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre-treatment with miR-499 or antagomir-208a inhibited cellular apoptosis in stretched HCF-aa. In hearts with volume overload, miR-499 overexpression inhibited myocardial miR-208a expression, whereas Bcl-2 expression could be suppressed by the addition of miR-208a. In conclusion, miR-208a mediated the regulation of miR-499 on Bcl-2 expression in stretched HCF-aa and hearts with volume overload.

CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Neonatal hyperoxia inhibits proliferation and survival of atrial cardiomyocytes by suppressing fatty acid synthesis.

Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes, and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death, while overexpressing these genes maintained their expansion in hyperoxia, suggesting that oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1, and other fatty acid synthesis genes in atrial cardiomyocytes may, thus, provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.

Action potential propagation and block in a model of atrial tissue with myocyte-fibroblast coupling.

The electrical coupling between myocytes and fibroblasts and the spacial distribution of fibroblasts within myocardial tissues are significant factors in triggering and sustaining cardiac arrhythmias, but their roles are poorly understood. This article describes both direct numerical simulations and an asymptotic theory of propagation and block of electrical excitation in a model of atrial tissue with myocyte-fibroblast coupling. In particular, three idealized fibroblast distributions are introduced: uniform distribution, fibroblast barrier and myocyte strait-all believed to be constituent blocks of realistic fibroblast distributions. Primary action potential biomarkers including conduction velocity, peak potential and triangulation index are estimated from direct simulations in all cases. Propagation block is found to occur at certain critical values of the parameters defining each idealized fibroblast distribution, and these critical values are accurately determined. An asymptotic theory proposed earlier is extended and applied to the case of a uniform fibroblast distribution. Biomarker values are obtained from hybrid analytical-numerical solutions of coupled fast-time and slow-time periodic boundary value problems and compare well to direct numerical simulations. The boundary of absolute refractoriness is determined solely by the fast-time problem and is found to depend on the values of the myocyte potential and on the slow inactivation variable of the sodium current ahead of the propagating pulse. In turn, these quantities are estimated from the slow-time problem using a regular perturbation expansion to find the steady state of the coupled myocyte-fibroblast kinetics. The asymptotic theory gives a simple analytical expression that captures with remarkable accuracy the block of propagation in the presence of fibroblasts.

Regulatory role of cardiomyocyte metabolism via AMPK activation in modulating atrial structural, contractile, and electrical properties following atrial fibrillation.

The number of patients diagnosed with atrial fibrillation (AF) has been rising due to increased incidence, enhanced detection methods, and greater survival rates following diagnosis. Due to this increase, AF is now the most commonly diagnosed arrhythmia in clinical practice. AF is characterized by irregular, high-frequency contractions of atrial myocytes that lead to turbulent blood flow and the potential for thrombus formation, stroke, or heart failure. These high-frequency contractions of the atrial myocytes cause an imbalance between metabolic supply and demand. Although advances have been made in understanding the pathophysiology of AF, the etiology and underlying pathogenic mechanism remain unknown. However, recent evidence suggests that cardiomyocyte metabolism involving 5' AMP-activated protein kinase (AMPK) activation is altered in patients with AF. Here, we critically reviewed the current understanding of AMPK activation in AF and how it could affect structural, contractile, and electrophysiological cellular properties in the pathogenesis of AF.

IP3-mediated Ca2+ release regulates atrial Ca2+ transients and pacemaker function by stimulation of adenylyl cyclases.

Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.

Drug screening platform using human induced pluripotent stem cell-derived atrial cardiomyocytes and optical mapping.

Current drug development efforts for the treatment of atrial fibrillation are hampered by the fact that many preclinical models have been unsuccessful in reproducing human cardiac physiology and its response to medications. In this study, we demonstrated an approach using human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes (hiPSC-aCMs and hiPSC-vCMs, respectively) coupled with a sophisticated optical mapping system for drug screening of atrial-selective compounds in vitro. We optimized differentiation of hiPSC-aCMs by modulating the WNT and retinoid signaling pathways. Characterization of the transcriptome and proteome revealed that retinoic acid pushes the differentiation process into the atrial lineage and generated hiPSC-aCMs. Functional characterization using optical mapping showed that hiPSC-aCMs have shorter action potential durations and faster Ca2+ handling dynamics compared with hiPSC-vCMs. Furthermore, pharmacological investigation of hiPSC-aCMs captured atrial-selective effects by displaying greater sensitivity to atrial-selective compounds 4-aminopyridine, AVE0118, UCL1684, and vernakalant when compared with hiPSC-vCMs. These results established that a model system incorporating hiPSC-aCMs combined with optical mapping is well-suited for preclinical drug screening of novel and targeted atrial selective compounds.

Intermittent Optogenetic Tachypacing of Atrial Engineered Heart Tissue Induces Only Limited Electrical Remodelling.

ABSTRACT: Atrial tachypacing is an accepted model for atrial fibrillation (AF) in large animals and in cellular models. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) provide a novel human source to model cardiovascular diseases. Here, we investigated whether optogenetic tachypacing of atrial-like hiPSC-CMs grown into engineered heart tissue (aEHT) can induce AF-remodeling. After differentiation of atrial-like cardiomyocytes from hiPSCs using retinoic acid, aEHTs were generated from ∼1 million atrial-like hiPSC-CMs per aEHT. AEHTs were transduced with lentivirus expressing channelrhodopsin-2 to enable optogenetic stimulation by blue light pulses. AEHTs underwent optical tachypacing at 5 Hz for 15 seconds twice a minute over 3 weeks and compared with transduced spontaneously beating isogenic aEHTs (1.95 ± 0.07 Hz). Force and action potential duration did not differ between spontaneously beating and tachypaced aEHTs. Action potentials in tachypaced aEHTs showed higher upstroke velocity (138 ± 15 vs. 87 ± 11 V/s, n = 15-13/3; P = 0.018), possibly corresponding to a tendency for more negative diastolic potentials (73.0 ± 1.8 vs. 68.0 ± 1.9 mV; P = 0.07). Tachypaced aEHTs exhibited a more irregular spontaneous beating pattern (beat-to-beat scatter: 0.07 ± 0.01 vs. 0.03 ± 0.004 seconds, n = 15-13/3; P = 0.008). Targeted expression analysis showed higher RNA levels of KCNJ12 [Kir2.2, inward rectifier (IK1); 69 ± 7 vs. 44 ± 4, P = 0.014] and NPPB (NT-proBNP; 39,690 ± 4834 vs. 23,671 ± 3691; P = 0.024). Intermittent tachypacing in aEHTs induces some electrical alterations found in AF and induces an arrhythmic spontaneous beating pattern, but does not affect resting force. Further studies using longer, continuous, or more aggressive stimulation may clarify the contribution of different rate patterns on the changes in aEHT mimicking the remodeling process from paroxysmal to persistent atrial fibrillation.

Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Combination of Cardiac Progenitor Cells From the Right Atrium and Left Ventricle Exhibits Synergistic Paracrine Effects In Vitro.

Cardiovascular diseases, such as ischemic heart disease, remain the most common cause of death worldwide. Regenerative medicine with stem cell therapy is a promising tool for cardiac repair. Combination of different cell types has been shown to improve the therapeutic potential, which is thought to be due to synergistic or complimentary reparative effects. We investigated if the combination of cardiac progenitor cells (CPCs) of right atrial appendage (RAA) and left ventricle (LV) that are isolated from the same patient exert synergistic or complimentary paracrine effects for apoptotic cell death and angiogenesis in an in vitro model. Flow cytometry analysis showed that both RAA and LV CPCs expressed the mesenchymal cell markers CD90 and CD105, and were predominantly negative for the hematopoietic cell marker, CD34. Analysis of conditioned media (CM) collected from the CPCs cultured either alone or in combination in serum-deprived hypoxic conditions to simulate ischemia showed marked increase in the level of pro-survival hepatocyte growth factor and pro-angiogenic vascular endothelial growth factor-A in the combined RAA and LV CPC group. Next, to determine the therapeutic potential of CM, AC16 human ventricular cardiomyocytes and human umbilical vein endothelial cells (HUVECs) were treated with CM. Results showed a significant reduction in hypoxia-induced apoptosis of human cardiomyocytes treated with CM collected from combined RAA and LV CPC group. Similarly, matrigel assay showed a significantly increased tube length formed by HUVECs when treated with CM from combined RAA and LV CPC group. Our study provided evidence that the combination of RAA CPCs and LV CPCs may have superior therapeutic effects due to synergistic paracrine effects for cardiac repair. Therefore, in vivo studies are warranted to determine if a combination of different stem cell types have greater therapeutic potential than single-cell therapies.