dual-dye-optical-mapping-hearts-from-ryr2r2474s-knock-mice

Dual-Dye Optical Mapping of Hearts from <em>RyR2</em><sup>R2474S</sup> Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes ...

southwest medical university

BACKGROUND Increased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF. MATERIAL AND METHODS Right atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study. RESULTS Compared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of [i]IKAS[/i] was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on [i]IKAS[/i] was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes. CONCLUSIONS SK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca2+-Activated K+ Current in Patients with Chronic Atrial Fibrillation.

Optogenetic control of the heart is an emergent technology that offers unparalleled spatio-temporal control of cardiac dynamics via light-sensitive ion pumps and channels (opsins). This fast-evolving technique holds broad scope in both clinical and basic research setting. Combination of optogenetics with optical mapping of voltage or calcium fluorescent probes facilitates 'all-optical' electrophysiology, allowing precise optogenetic actuation of cardiac tissue with high spatio-temporal resolution imaging of action potential and calcium transient morphology and conduction patterns. In this review, we provide a synopsis of optogenetics and discuss in detail its use and compatibility with optical interrogation of cardiac electrophysiology. We briefly discuss the benefits of all-optical cardiac control and electrophysiological interrogation compared to traditional techniques, and describe mechanisms, unique features and limitations of optically induced cardiac control. In particular, we focus on state-of-the-art setup design, challenges in light delivery and filtering, and compatibility of opsins with fluorescent reporters used in optical mapping. The interaction of cardiac tissue with light, and physical and computational approaches to overcome the 'spectral congestion' that arises from the combination of optogenetics and optical mapping are discussed. Finally, we summarize recent preclinical work applications of combined cardiac optogenetics and optical mapping approach.

Cardiac Optogenetics and Optical Mapping - Overcoming Spectral Congestion in All-Optical Cardiac Electrophysiology.

In this mini-review, we provide an overview of phenylethanolamine-N-methyl transferase (Pnmt)-derived cardiomyocytes (PdCMs), a recently discovered cardiomyocyte subpopulation. We discuss their anatomical localization, physiological characteristics, possible function, and future perspectives. Their unique distribution in the heart, electrical activity, Ca transient properties, and potential role in localized adrenergic signaling are discussed.

Pnmt-Derived Cardiomyocytes: Anatomical Localization, Function and Future Perspectives.

Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice.

A Protocol for Dual Calcium-Voltage Optical Mapping in Murine Sinoatrial Preparation With Optogenetic Pacing.

The whole world was hit hard by the coronavirus disease-19 (COVID-19). Given that angiotensin I converting enzyme 2 (ACE2) is the viral entry molecule, understanding ACE2 has become a major focus of current COVID-19 research. ACE2 is highly expressed in the gut, but its role has not been fully understood and thus COVID-19 treatments intending to downregulate ACE2 level may cause untoward side effects. Gaining insight into the functions of ACE2 in gut homeostasis therefore merits closer examination, and is beneficial to find potential therapeutic alternatives for COVID-19.

ACE2 contributes to the maintenance of mouse epithelial barrier function.

Effects of hypertrophic challenge on small-conductance, Ca-activated K(SK2) channel expression were explored in intact murine hearts, isolated ventricular myocytes and neonatal rat cardiomyocytes (NRCMs). An established experimental platform applied angiotensin II (Ang II) challenge in the presence and absence of reduced p21-activated kinase (PAK1) (PAK1 vs. PAK1, or shRNA-PAK1 interference) expression. SK2 current contributions were detected through their sensitivity to apamin block. Ang II treatment increased such SK2 contributions to optically mapped action potential durations (APD) and their heterogeneity, and to patch-clamp currents. Such changes were accentuated in PAK1 compared to PAK1, intact hearts and isolated cardiomyocytes. They paralleled increased histological and echocardiographic hypertrophic indices, reduced cardiac contractility, and increased SK2 protein expression, changes similarly greater with PAK1 than PAK1. In NRCMs, Ang II challenge replicated such increases in apamin-sensitive SK patch clamp currents as well as in real-time PCR and western blot measures of SK2 mRNA and protein expression and cell hypertrophy. Furthermore, the latter were enhanced by shRNA-PAK1 interference and mitigated by the PAK1 agonist FTY720. Increased CaMKII and CREB phosphorylation accompanied these effects. These were rescued by both FTY720 as well as the CaMKII inhibitor KN93, but not its inactive analogue KN92. Such CREB then specifically bound to the KCNN2 promoter sequence in luciferase assays. These findings associate Ang II induced hypertrophy with increased SK2 expression brought about by a CaMKII/CREB signaling convergent with the PAK1 pathway thence upregulating the KCNN2 promoter activity. SK2 may then influence cardiac electrophysiology under conditions of cardiac hypertrophy and failure.

Ventricular SK2 upregulation following angiotensin II challenge: Modulation by p21-activated kinase-1.

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models.

A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts.

The nucleus tractus solitarii (NTS) is one of the morphologically and functionally defined centers that engage in the autonomic regulation of cardiovascular activity. Phenotypically-characterized NTS neurons have been implicated in the differential regulation of blood pressure (BP). Here, we investigated whether phenylethanolamine N-methyltransferase (PNMT)-expressing NTS (NTS) neurons contribute to the control of BP. We demonstrate that photostimulation of NTS neurons has variable effects on BP. A depressor response was produced during optogenetic stimulation of NTS neurons projecting to the paraventricular nucleus of the hypothalamus, lateral parabrachial nucleus, and caudal ventrolateral medulla. Conversely, photostimulation of NTS neurons projecting to the rostral ventrolateral medulla produced a robust pressor response and bradycardia. In addition, genetic ablation of both NTS neurons and those projecting to the rostral ventrolateral medulla impaired the arterial baroreflex. Overall, we revealed the neuronal phenotype- and circuit-specific mechanisms underlying the contribution of NTS neurons to the regulation of BP.

Circuit-Specific Control of Blood Pressure by PNMT-Expressing Nucleus Tractus Solitarii Neurons.

The X-linked inhibitor of apoptosis (XIAP) protein is encoded by the XIAP gene and is critical for multiple cell responses and plays a role in preventing cell death. XIAP mutations are associated with several diseases, primarily including hemophagocytic lymphohistiocytosis and inflammatory bowel disease (IBD). We report the clinical features and results associated with hemizygous mutation of the XIAP gene in a young male with Crohn's disease complicated with acute heart failure.This 16-year-old patient ultimately died of heart failure.

Novel XIAP mutation with early-onset Crohn's disease complicated with acute heart failure: a case report.

The development of the cardiac conduction system (CCS) is essential for correct heart function. However, critical details on the cell types populating the CCS in the mammalian heart during the development remain to be resolved. Using single-cell RNA sequencing, we generated a large dataset of transcriptomes of ~0.5 million individual cells isolated from murine hearts at six successive developmental corresponding to the early, middle and late stages of heart development. The dataset provides a powerful library for studying the development of the heart's CCS and other cardiac components. Our initial analysis identified distinct cell types between 20 to 26 cell types across different stages, of which ten are involved in forming the CCS. Our dataset allows researchers to reuse the datasets for data mining and a wide range of analyses. Collectively, our data add valuable transcriptomic resources for further study of cardiac development, such as gene expression, transcriptional regulation and functional gene activity in developing hearts, particularly the CCS.

Single-cell RNA sequencing of murine hearts for studying the development of the cardiac conduction system.

Arrhythmia is one of the most common cardiovascular diseases. The search for new drugs to suppress various types of cardiac arrhythmias has always been the focus of attention. In the past decade, the screening of antiarrhythmic active substances from plants has received extensive attention. These natural compounds have obvious antiarrhythmic effects, and chemical modifications based on natural compounds have greatly increased their pharmacological properties. The chemical modification of botanical antiarrhythmic drugs is closely related to the development of new and promising drugs. Therefore, the structural characteristics and action targets of natural compounds with antiarrhythmic effects are reviewed in this paper, so that pharmacologists can select antiarrhythmic lead compounds from natural compounds based on the disease target - chemical structural characteristics.

The characteristics and molecular targets of antiarrhythmic natural products.

The heterogeneity of functional cardiomyocytes arises during heart development, which is essential to the complex and highly coordinated cardiac physiological function. Yet the biological and physiological identities and the origin of the specialized cardiomyocyte populations have not been fully comprehended. Here we report a previously unrecognised population of cardiomyocytes expressing Dbhgene encoding dopamine beta-hydroxylase in murine heart. We determined how these myocytes are distributed across the heart by utilising advanced single-cell and spatial transcriptomic analyses, genetic fate mapping and molecular imaging with computational reconstruction. We demonstrated that they form the key functional components of the cardiac conduction system by using optogenetic electrophysiology and conditional cardiomyocyte Dbh gene deletion models. We revealed their close relationship with sympathetic innervation during cardiac conduction system formation. Our study thus provides new insights into the development and heterogeneity of the mammalian cardiac conduction system by revealing a new cardiomyocyte population with potential catecholaminergic endocrine function.

Article	Year
Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca2+-Activated K+ Current in Patients with Chronic Atrial Fibrillation. Medical science monitor : international medical journal of experimental and clinical research\| PubMed ID: 29737974	2018
Cardiac Optogenetics and Optical Mapping - Overcoming Spectral Congestion in All-Optical Cardiac Electrophysiology. Frontiers in physiology\| PubMed ID: 30899227	2019
Pnmt-Derived Cardiomyocytes: Anatomical Localization, Function and Future Perspectives. Frontiers in physiology\| PubMed ID: 31354504	2019
A Protocol for Dual Calcium-Voltage Optical Mapping in Murine Sinoatrial Preparation With Optogenetic Pacing. Frontiers in physiology\| PubMed ID: 31456689	2019
ACE2 contributes to the maintenance of mouse epithelial barrier function. Biochemical and biophysical research communications\| PubMed ID: 33097186	2020
Ventricular SK2 upregulation following angiotensin II challenge: Modulation by p21-activated kinase-1. Journal of molecular and cellular cardiology\| PubMed ID: 34774547	2022
A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts. Scientific data\| PubMed ID: 34916511	2021
Circuit-Specific Control of Blood Pressure by PNMT-Expressing Nucleus Tractus Solitarii Neurons. Neuroscience bulletin\| PubMed ID: 36588135	2023
Novel XIAP mutation with early-onset Crohn's disease complicated with acute heart failure: a case report. BMC cardiovascular disorders\| PubMed ID: 37479963	2023
Single-cell RNA sequencing of murine hearts for studying the development of the cardiac conduction system. Scientific data\| PubMed ID: 37666871	2023
The characteristics and molecular targets of antiarrhythmic natural products. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie\| PubMed ID: 37897974	2023
Dbh catecholaminergic cardiomyocytes contribute to the structure and function of the cardiac conduction system in murine heart. Nature communications\| PubMed ID: 38016975	2023

Xianhong Ou

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