Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Background: MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms. Methods: We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice. Results: We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1–Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed I K1 at sub–pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1–M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo. Conclusions: Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs’ biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.

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In vivo ratiometric optical mapping enables high-resolution cardiac electrophysiology in pig models

Cardiovascular Research ◽

10.1093/cvr/cvz039 ◽

2019 ◽

Vol 115 (11) ◽

pp. 1659-1671 ◽

Cited By ~ 11

Author(s):

Peter Lee ◽

Jorge G Quintanilla ◽

José M Alfonso-Almazán ◽

Carlos Galán-Arriola ◽

Ping Yan ◽

...

Keyword(s):

High Resolution ◽

Cardiac Electrophysiology ◽

High Speed ◽

Near Infrared ◽

Optical Mapping ◽

Ex Vivo ◽

Average Rate ◽

Camera System ◽

Epicardial Surface

Abstract Aims Cardiac optical mapping is the gold standard for measuring complex electrophysiology in ex vivo heart preparations. However, new methods for optical mapping in vivo have been elusive. We aimed at developing and validating an experimental method for performing in vivo cardiac optical mapping in pig models. Methods and results First, we characterized ex vivo the excitation-ratiometric properties during pacing and ventricular fibrillation (VF) of two near-infrared voltage-sensitive dyes (di-4-ANBDQBS/di-4-ANEQ(F)PTEA) optimized for imaging blood-perfused tissue (n = 7). Then, optical-fibre recordings in Langendorff-perfused hearts demonstrated that ratiometry permits the recording of optical action potentials (APs) with minimal motion artefacts during contraction (n = 7). Ratiometric optical mapping ex vivo also showed that optical AP duration (APD) and conduction velocity (CV) measurements can be accurately obtained to test drug effects. Secondly, we developed a percutaneous dye-loading protocol in vivo to perform high-resolution ratiometric optical mapping of VF dynamics (motion minimal) using a high-speed camera system positioned above the epicardial surface of the exposed heart (n = 11). During pacing (motion substantial) we recorded ratiometric optical signals and activation via a 2D fibre array in contact with the epicardial surface (n = 7). Optical APs in vivo under general anaesthesia showed significantly faster CV [120 (63–138) cm/s vs. 51 (41–64) cm/s; P = 0.032] and a statistical trend to longer APD90 [242 (217–254) ms vs. 192 (182–233) ms; P = 0.095] compared with ex vivo measurements in the contracting heart. The average rate of signal-to-noise ratio (SNR) decay of di-4-ANEQ(F)PTEA in vivo was 0.0671 ± 0.0090 min−1. However, reloading with di-4-ANEQ(F)PTEA fully recovered the initial SNR. Finally, toxicity studies (n = 12) showed that coronary dye injection did not generate systemic nor cardiac damage, although di-4-ANBDQBS injection induced transient hypotension, which was not observed with di-4-ANEQ(F)PTEA. Conclusions In vivo optical mapping using voltage ratiometry of near-infrared dyes enables high-resolution cardiac electrophysiology in translational pig models.

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Faculty Opinions recommendation of Functional Nav1.8 channels in intracardiac neurons: the link between SCN10A and cardiac electrophysiology.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717953979.793459757 ◽

2012 ◽

Author(s):

David J Adams

Keyword(s):

Cardiac Electrophysiology ◽

Intracardiac Neurons

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In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine

European Journal of Pharmacology ◽

10.1016/j.ejphar.2021.174670 ◽

2021 ◽

pp. 174670

Author(s):

Véronique Ballet ◽

G. Andrees Bohme ◽

Eric Brohan ◽

Rachid Boukaiba ◽

Jean-Marie Chambard ◽

...

Keyword(s):

Ion Channel ◽

Cardiac Electrophysiology ◽

Ex Vivo

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Bridging Organ- and Cellular-Level Behavior in Ex Vivo Experimental Platforms Using Populations of Models of Cardiac Electrophysiology

Journal of Engineering and Science in Medical Diagnostics and Therapy ◽

10.1115/1.4040589 ◽

2018 ◽

Vol 1 (4) ◽

Cited By ~ 1

Author(s):

Carlos A. Ledezma ◽

Benjamin Kappler ◽

Veronique Meijborg ◽

Bas Boukens ◽

Marco Stijnen ◽

...

Keyword(s):

Cardiac Electrophysiology ◽

Experimental Approach ◽

Potassium Concentration ◽

Ex Vivo ◽

Cellular Level ◽

Proof Of Concept ◽

Physiological Variability ◽

Novel Method ◽

Organ Level ◽

Pathological Behavior

The inability to discern between pathology and physiological variability is a key issue in cardiac electrophysiology since this prevents the use of minimally invasive acquisitions to predict early pathological behavior. The goal of this work is to demonstrate how experimentally calibrated populations of models (ePoM) may be employed to inform which cellular-level pathologies are responsible for abnormalities observed in organ-level acquisitions while accounting for intersubject variability; this will be done through an exemplary computational and experimental approach. Unipolar epicardial electrograms (EGM) were acquired during an ex vivo porcine heart experiment. A population of the Ten Tusscher 2006 model was calibrated to activation–recovery intervals (ARI), measured from the electrograms, at three representative times. The distributions of the parameters from the resulting calibrated populations were compared to reveal statistically significant pathological variations. Activation–recovery interval reduction was observed in the experiments, and the comparison of the calibrated populations of models suggested a reduced L-type calcium conductance and a high extra-cellular potassium concentration as the most probable causes for the abnormal electrograms. This behavior was consistent with a reduction in the cardiac output (CO) and was confirmed by other experimental measurements. A proof of concept method to infer cellular pathologies by means of organ-level acquisitions is presented, allowing for an earlier detection of pathology than would be possible with current methods. This novel method that uses mathematical models as a tool for formulating hypotheses regarding the cellular causes of observed organ-level behaviors, while accounting for physiological variability has been unexplored.

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Cardiac electrophysiology catheters for electrophysiological assessments of the lower urinary tract-A proof of concept ex vivo study in viable ureters

Neurourology and Urodynamics ◽

10.1002/nau.23816 ◽

2018 ◽

Vol 38 (1) ◽

pp. 87-96 ◽

Cited By ~ 1

Author(s):

Andreas Haeberlin ◽

Klaus Schürch ◽

Thomas Niederhauser ◽

Romy Sweda ◽

Marc P. Schneider ◽

...

Keyword(s):

Urinary Tract ◽

Cardiac Electrophysiology ◽

Lower Urinary Tract ◽

Ex Vivo ◽

Proof Of Concept ◽

Ex Vivo Study ◽

Lower Urinary

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Abstract 15963: Microrna Biophysically Modulates Cardiac Physiology via Directly Binding to Ion Channel

Circulation ◽

10.1161/circ.142.suppl_3.15963 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Dandan Yang ◽

Xiaoping Wan ◽

Adrienne T Dennis ◽

Emre Bektik ◽

Zhihua Wang ◽

...

Keyword(s):

Ion Channels ◽

Ion Channel ◽

Cardiac Arrhythmias ◽

Cardiac Electrophysiology ◽

Ex Vivo ◽

Resting Membrane Potential ◽

Seed Region ◽

Cardiac Physiology ◽

Long Term Effect ◽

Evolutionarily Conserved

Background: Cardiac arrhythmias are a leading cause of morbidity and mortality. MicroRNAs (miRs) regulate the (electro) physiology of the heart and remodeling by canonical RNAi mechanism. Hypothesis: miRs maintain cardiac physiology also through noncanonical mechanisms. Methods: The physical binding between cardiac predominant miR--miR1 and ion channels was explored by EMSA, in situ PLA, RNA pull down and RIP assays. Electrophysiology of cardiomyocytes (CMs) and ex vivo miR1-deficient hearts were studied to reveal the functional outcome and the pathophysiological significance. Results: miR1 physically binds with an inward rectifier K + channel Kir2.1, which exists endogenously in CMs. This miR1-Kir2.1 binding is evolutionarily conserved. Functionally, miR1, at sub-pmol/L concentration, significantly suppresses IK1, depolarizes resting membrane potential, and prolongs final repolarization of action potentials in CMs. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 though the core sequence AAGAAG, which is outside the seed region. This biophysical modulation is involved in the dysregulation of a gain-of-function mutation Kir2.1-M301K in short-QT/AF patients. An AF-associated miR1-hSNP14A/G specifically disrupts the biophysical modulation while maintains miR1’s RNAi function. Significantly, miR1 but not hSNP14A/G eliminates the high inducibility of arrhythmia in miR1-deficient hearts. Conclusion: We reveal a novel function of miRs and develop a ground-breaking concept that endogenous miRs can physically bind with ion channels and rapidly modulate cardiac electrophysiology before its long-term effect of conventional RNAi mechanism. Our study provides more comprehensive understanding of ion-channel dysregulation associated with cardiac arrhythmias.

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Adult zebrafish ventricular electrical gradients as tissue mechanisms of ECG patterns under baseline vs. oxidative stress

Cardiovascular Research ◽

10.1093/cvr/cvaa238 ◽

2020 ◽

Author(s):

Yali Zhao ◽

Nicholas A James ◽

Ashraf R Beshay ◽

Eileen E Chang ◽

Andrew Lin ◽

...

Keyword(s):

Oxidative Stress ◽

Cardiac Electrophysiology ◽

Ex Vivo ◽

Mechanical Efficiency ◽

Adult Zebrafish ◽

Qrs Complex ◽

Electrical Stability ◽

T Wave ◽

Zebrafish Heart

Abstract Aims In mammalian ventricles, electrical gradients establish electrical heterogeneities as essential tissue mechanisms to optimize mechanical efficiency and safeguard electrical stability. Electrical gradients shape mammalian electrocardiographic patterns; disturbance of electrical gradients is proarrhythmic. The zebrafish heart is a popular surrogate model for human cardiac electrophysiology thanks to its remarkable recapitulation of human electrocardiogram and ventricular action potential features. Yet, zebrafish ventricular electrical gradients are largely unexplored. The goal of this study is to define the zebrafish ventricular electrical gradients that shape the QRS complex and T wave patterns at baseline and under oxidative stress. Methods and results We performed in vivo electrocardiography and ex vivo voltage-sensitive fluorescent epicardial and transmural optical mapping of adult zebrafish hearts at baseline and during acute H2O2 exposure. At baseline, apicobasal activation and basoapical repolarization gradients accounted for the polarity concordance between the QRS complex and T wave. During H2O2 exposure, differential regional impairment of activation and repolarization at the apex and base disrupted prior to baseline electrical gradients, resulting in either reversal or loss of polarity concordance between the QRS complex and T wave. KN-93, a specific calcium/calmodulin-dependent protein kinase II inhibitor (CaMKII), protected zebrafish hearts from H2O2 disruption of electrical gradients. The protection was complete if administered prior to oxidative stress exposure. Conclusions Despite remarkable apparent similarities, zebrafish and human ventricular electrocardiographic patterns are mirror images supported by opposite electrical gradients. Like mammalian ventricles, zebrafish ventricles are also susceptible to H2O2 proarrhythmic perturbation via CaMKII activation. Our findings suggest that the adult zebrafish heart may constitute a clinically relevant model to investigate ventricular arrhythmias induced by oxidative stress. However, the fundamental ventricular activation and repolarization differences between the two species that we demonstrated in this study highlight the potential limitations when extrapolating results from zebrafish experiments to human cardiac electrophysiology, arrhythmias, and drug toxicities.

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DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology

Cardiovascular Research ◽

10.1093/cvr/cvy251 ◽

2018 ◽

Vol 115 (6) ◽

pp. 1052-1066 ◽

Cited By ~ 6

Author(s):

Elisabeth Kaiser ◽

Qinghai Tian ◽

Michael Wagner ◽

Monika Barth ◽

Wenying Xian ◽

...

Keyword(s):

Cardiac Function ◽

Cardiac Electrophysiology ◽

Striated Muscle ◽

Ex Vivo ◽

Isolated Heart ◽

Phosphorylation Site ◽

Muscle Creatine Kinase ◽

Electrophysiological Properties ◽

Transgenic Mouse Line

Abstract Aims Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. Methods and results We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. Conclusion Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes’ Ca2+ handling but also directly alters the heart’s electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.

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Aging Disrupts Normal Time-of-Day Variation in Cardiac Electrophysiology

Circulation Arrhythmia and Electrophysiology ◽

10.1161/circep.119.008093 ◽

2020 ◽

Vol 13 (9) ◽

Cited By ~ 1

Author(s):

Zhen Wang ◽

Srinivas Tapa ◽

Samantha D. Francis Stuart ◽

Lianguo Wang ◽

Julie Bossuyt ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Electrophysiology ◽

Ex Vivo ◽

Time Of Day ◽

Male Mice ◽

Time Points ◽

Isolated Hearts ◽

Gene And Protein Expression ◽

Gated Channel ◽

Cardiac Gene

Background: Cardiac gene expression and arrhythmia occurrence have time-of-day variation; however, daily changes in cardiac electrophysiology, arrhythmia susceptibility, and Ca 2+ handling have not been characterized. Furthermore, how these patterns change with age is unknown. Methods: Hearts were isolated during the light (zeitgeber time [ZT] 4 and ZT9) and dark cycle (ZT14 and ZT21) from adult (12–18 weeks) male mice. Hearts from aged (18–20 months) male mice were isolated at ZT4 and ZT14. All hearts were Langendorff-perfused for optical mapping with voltage- and Ca 2+ -sensitive dyes (n=4–7/group). Cardiac gene and protein expression were assessed with real-time polymerase chain reaction (n=4–6/group) and Western blot (n=3–4/group). Results: Adult hearts had the shortest action potential duration (APD) and Ca 2+ transient duration (CaTD) at ZT14 (APD 80 : ZT4: 45.4±4.1 ms; ZT9: 45.1±8.6 ms; ZT14: 34.7±4.2 ms; ZT21: 49.2±7.6 ms, P <0.05 versus ZT4 and ZT21; and CaTD 80 : ZT4: 70.1±3.3 ms; ZT9: 72.7±2.7 ms; ZT14: 64.3±3.3 ms; ZT21: 74.4±1.2 ms, P <0.05 versus other time points). The pacing frequency at which CaT alternans emerged was faster, and average CaT alternans magnitude was significantly reduced at ZT14 compared with the other time points. There was a trend for decreased spontaneous premature ventricular complexes and pacing-induced ventricular arrhythmias at ZT14, and the hearts at ZT14 had diminished responses to isoproterenol compared with ZT4 (ZT4: 49.5.0±5.6% versus ZT14: 22.7±9.5% decrease in APD, P <0.01). In contrast, aged hearts exhibited no difference between ZT14 and ZT4 in nearly every parameter assessed (except APD 80 : ZT4: 39.7±1.9 ms versus ZT14: 33.8±3.1 ms, P <0.01). Gene expression of KCNA5 (potassium voltage-gated channel subfamily A member 5; encoding Kv1.5) was increased, whereas gene expression of ADRB1 (encoding β1-adrenergic receptors) was decreased at ZT14 versus ZT4 in adult hearts. No time-of-day changes in expression or phosphorylation of Ca 2+ handling proteins (SERCA2 [sarco/endoplasmic reticulum Ca 2+ -ATPase], RyR2 [ryanodine receptor 2], and PLB [phospholamban]) was found in ex vivo perfused adult isolated hearts. Conclusions: Isolated adult hearts have strong time-of-day variation in cardiac electrophysiology, Ca 2+ handling, and adrenergic responsiveness, which is disrupted with age.

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