AIMS:Adenosine, acting through A1 adenosine receptors (A1ARs), exerts anti-adrenergic effects by inhibiting β1-adrenergic receptor (β1AR)-mediated cyclic adenosine monophosphate (cAMP) production and contractility in the heart. While the functional interaction between A1ARs and β1ARs is well established in both atrial and ventricular myocytes, the subcellular compartmentalization of this crosstalk and how it is disrupted in heart failure (HF) remains incompletely understood. This study investigates the spatial confinement of A1AR-β1AR signalling within atrial microdomains and assesses how structural remodelling in HF alters this regulatory axis. METHODS AND RESULTS:Quantitative polymerase chain reaction (qPCR) analysis revealed that A1AR is the predominant adenosine receptor subtype in both rat and human atrial tissues. In healthy rat and mouse atrial myocytes, A1AR activation reduced β1AR-induced cAMP production and sarcomere shortening, with suppression of cAMP signals at sarcolemmal microdomains enriched in protein kinase A Type II. This was further supported by scanning ion conductance microscopy-guided scanning patch-clamp, which showed that A1AR suppressed β1AR-driven L-type Ca2+ channel activity at both T-tubule and crest membrane domains. In atrial myocytes isolated from failing rat and human hearts, A1AR-mediated inhibition of β1AR-induced cAMP production and contractility was impaired. Caveolar disruption by methyl-β-cyclodextrin in rat atrial myocytes or via cardiac-specific caveolin-3 (Cav3) knockout in mice abolished this A1AR-mediated inhibition. Notably, cholesterol repletion alone did not restore membrane cAMP regulation, whereas Cav3 overexpression rescued A1AR-dependent suppression, supporting a requirement for Cav3-dependent organization. In mouse atrial preparations isolated from failing hearts, high-resolution optical mapping showed that A1AR-mediated anti-adrenergic regulation of Ca2+ cycling was selectively lost in the intercaval region, correlating with the regional absence of T-tubule and downregulation of caveolae structures. CONCLUSION:A1ARs provide anti-adrenergic restraint of β1AR signalling through Cav3-dependent membrane organization. In HF, regional caveolar disorganization uncouples this protective pathway, contributing to spatially heterogeneous Ca2+ dysregulation in the atrium.

BACKGROUND:Atrial fibrillation (AF) is associated by alterations in cardiomyocyte membrane microdomain organization that can modify the distribution and biophysical properties of L-type Ca2+ channels (LTCCs), contributing to downregulation of the L-type Ca2+ current (ICa,L). OBJECTIVE:This study aimed to examine the role of cellular microarchitecture and microdomain-specific changes of single LTCCs in ICa,L remodeling in chronic AF. METHODS:Right atrial (RA) and left atrial (LA) biopsies from patients in sinus rhythm (SR, n = 51) and AF (n = 61) were analyzed to assess cardiomyocyte microarchitecture and microdomain-specific remodeling of single LTCCs and ICa,L. Computational modeling estimated the contributions of different cellular components in ICa,L reduction. RESULTS:In AF, ICa,L was reduced in RA (∼32%, P < .05) and LA (∼71%, P < .01). In RA, this was associated with preserved transverse (T)-tubular LTCCs (T-LTCCs) density and decreased extra-tubular (crest, C-LTCCs) density. In LA-AF, densities of both T- and C-LTCCs were reduced. These changes paralleled T-tubule downregulation in both atria and an LA-specific decrease in caveolin-3 expression. In AF, the open probabilities of T- and C-LTCCs in both atria were 3-5-fold higher than in SR and accompanied by 2-fold increase in protein kinase A and phosphorylated Ca2+/calmodulin kinase II activities. However, computational simulations showed that enhanced LTCC open probability did not compensate for reduced channel density. Caveolin-3 overexpression in LA-AF cardiomyocytes increased CaV1.2 membrane expression, partially restoring ICa,L. CONCLUSION:ICa,L downregulation in AF is driven by chamber-specific degradation of membrane structures and loss of functional LTCCs, not offset by increased channel activity. Caveolin-3 is crucial for maintaining functional LTCCs at the sarcolemma.

Vagal stimulation is emerging as the next frontier in bioelectronic medicine to modulate peripheral organ health and treat disease. The neuronal molecular phenotypes in the dorsal motor nucleus of the vagus (DMV) remain largely unexplored, limiting the potential for harnessing the DMV plasticity for therapeutic interventions. We developed a mesoscale single-cell transcriptomics data from hundreds of DMV neurons under homeostasis and following physiological perturbations. Our results revealed that homeostatic DMV neuronal states can be organized into distinguishable input-output signal processing units. Remote ischemic preconditioning induced a distinctive shift in the neuronal states toward diminishing the role of inhibitory inputs, with concomitant changes in regulatory microRNAs miR-218a and miR-495. Chronic cardiac ischemic injury resulted in a dramatic shift in DMV neuronal states suggestive of enhanced neurosecretory function. We propose a DMV molecular network mechanism that integrates combinatorial neurotransmitter inputs from multiple brain regions and humoral signals to modulate cardiac health.

The dorsal motor nucleus of the vagus (DMV) is one of the brainstem structures from which arises the vagus nerve, and is responsible for the vagal activity that protects the heart from reperfusion injury during an acute myocardial infarction. Remote ischemic‐reperfusion cardioprotection (RIPC) is a phenomenon known to protect the heart against injury via humoral and vagal mediation (Mastitskaya et al. 2012). It is hypothesized that RIPC cardioprotection may be mediated through changes in the gene expression of DMV neurons. However, the dynamics of gene expression in the DMV following RIPC is not known. Here, we performed RIPC by occluding the femoral artery, and using multiplex qPCR, we examined changes in gene expression of single cells taken from the DMV at several time points following RIPC. We conducted these experiments in both female and male rats and both left and right DMV. We also used a retrograde tracer to identify DMV neurons that travel to the heart and the gut, to identify different transcription profiles between these neuronal subpopulations. Our results show a distinct shift in transcriptomic identity of the neuronal subpopulations in the RIPC group over the sham controls. We conclude that there may be neuronal subpopulations in the DMV that are differentially activated following RIPC, which provides insights for translational applications of RIPC in decreasing reperfusion damage.Support or Funding InformationWe are supported by NIH NHLBI 5U01HL133360‐02 and NIH Director Office 3OT2OD023848. The funding sources played no role in study design, collection nor analysis of the data.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitofusin 2 (MFN2), originally recognized for mediating mitochondrial fusion, has been also established as a major player in the endoplasmic reticulum (ER) and mitochondria interaction. MFN2 ablation reduced the efficacy of interorganelle Ca2+ signalling, which effect has been largely attributed to MFN2's ER- mitochondrial tethering role. However, the mechanism by which MFN2 regulates Ca2+ homeostasis between the organelles remains unclear. Here, we introduced a non-canonical role of MFN2 in ER Ca2+ regulation potentially via an interaction with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To study the effect of MFN2-overexpression (OE) on ER Ca2+ homeostasis two sets of Myc-tagged MFN2 plasmids were used: wild-type present in both ER and mitochondria (WT-MFN2) and MFN2 lacking the mitochondrial targeting sequence (ER-MFN2). Western blotting and confocal microscopy confirmed OE and localization of tagged MFN2. Effect of MFN2 knock-out (KO) and WT-, ER-MFN2-OE on Ca2+ dynamics was studied in permeabilized HEK293T and MEF cells. [Ca2+] in the ER lumen ([Ca2+]ER) was visualized using GEM-CEPIA1er Ca2+-biosensor. Basal [Ca2+]ER was significantly lower in WT- and ER-MFN2-OE cells vs control and MFN2-KO cells where we observed highest level of the basal [Ca2+]ER. Importantly, the rate of [Ca2+]ER re-uptake by SERCA after its depletion by reversible inhibitor cyclopiazonic acid was significantly faster in MFN2-KO cells vs control. In WT- and ER-MFN2-OE [Ca2+]ER re-uptake was significantly slower than in control with more dramatic effect in ER-MFN2-OE. Initial co-immunoprecipitation and FRET experiments suggest SERCA interaction with MFN2. Thus, our data shows that in addition to the ER-mito tethering role, MFN2 might interact with and inhibit SERCA, and potentially regulate ER-mito calcium cross-talk via changing ER Ca2+ handling.

Angiotensin II (ANG II) plays an important role in atrial remodeling associated with atrial fibrillation and heart failure by affecting different processes including activation of abnormal Ca2+ influx. We hypothesized that caveolar localized L-type Ca channels (LTCCs) in atrial myocytes (AMs) are regulated by AngII. To examine thus further, we tested the effect of ANG II on L-type calcium current (ICa,L) in mouse (AMs). In wild type AMs, external application of 1 µM ANG II increased a peak of ICa,L by 122.9±37.4% from baseline (n=7, P<0.01) and accelerated Ca2+-dependent inactivation of ICa,L. Disruption of caveolae by 1-hour pre-treatment with 2 mM methyl-β-cyclodextrin (MbCD) decreased ICa,L density from −15.7±2.6 pA/pF, n=20 to −10.9±1.8 pA/pF, n=9, with no effect on activation and inactivation kinetics. Such ICa,L reduction may indicate the contribution of caveolae-housed channels. Importantly, MbCD prevented the stimulatory effect of ANG II on peak current and Ca2+-dependent inactivation of ICa,L. Similarly, tamoxifen-induced conditional knock-out of caveolin-3 significantly reduced ICa,L density from −15.7±2.6 pA/pF, n=20 to −3.6±0.9 pA/pF, n=7 (P<0.005) and slowed Ca2+-dependent inactivation. Importantly, AMs possess a highly variable membrane capacitance (73.4±8.1 pF, n=20) and ICa,L peak current (−749.9±177.9 pA, n=20) which correlated with the organization of transverse-axial tubular system (r2= 0.56) and possibly anatomical structure of the atria. Our findings indicate that ANG II enhances ICa,L primarily via localized stimulation of caveolae-housed LTCCs highlighting a potential significance of caveolae microdomains in ANG II dependent regulation of the atria.

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures and receptors forming macromolecular signalling complexes. Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiomyocyte structure, may contribute to the pathophysiology of heart failure (HF) altering cardiomyocyte function. Here, we used Ca2+ imaging and super-resolution scanning patch-clamp to examine microdomain-specific regulation of LTCCs by β1 adrenergic (β1ARs) and adenosine A1 receptors (AdoA1Rs) in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1ARs stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ activity which was abolished by AdoA1Rs stimulation (2′-MeCCPA 200 nM). Accordingly, activation of single LTCCs in both t-tubules and on the crest of sarcolemma from non-localized β1ARs stimulation (ISO 2 µM and ICI 50 nM in external solution) was also abolished by local AdoA1R stimulation (10 µM). In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ activities was significantly reduced (28% vs 134% in HF vs control, P<0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P<0.01). In addition, the effects of both β1ARs and AdoA1Rs on normalized cell shortening were significantly reduced in HF. Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment.

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures. Such microdomain-specific localization impact channel function and regulation by a variety of neurohormonal pathways, including adrenergic and adenosine. Disruption in their subcellular targeting may contribute to the pathophysiology of cardiac diseases, including heart failure (HF). We used Ca2+ imaging and super-resolution scanning patch clamp to examine microdomain-specific regulation of LTCCs in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1 adrenergic receptors (β1ARs) stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ release events while the following adenosine A1 receptors (AdoA1Rs) stimulation (2'-MeCCPA 200 nM) abolished β1ARs effects. Non-localized β1ARs stimulation (ISO 2 μM and ICI 50 nM in external solution) activated single LTCCs in both t-tubules and on the crest of sarcolemma, increasing LTCC occurrence and enhancing their open probability and amplitude. Local AdoA1R stimulation through the pipette (10 μM) on β1ARs-pretreated cells completely abolished β1ARs effects. In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ release events was significantly reduced in HF (28% vs 134% in HF vs control, P<0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P<0.01), despite up-regulation of β1 and AdoA1Rs mRNA. No changes in caveolae density were observed in HF (4.6±0.4 caveolae/μm vs 3.7±0.5 caveolae/μm for HF vs control, P=0.167). Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment. This extends beyond the classical concept of electrical remodeling, stressing the involvement of alteration of spatial compartmentation of ion channels and receptors in addition to classical concepts of changes in protein expression and post-translational modifications.

Cardiac excitation involves the generation of action potential by individual cells and the subsequent conduction of the action potential from cell to cell through intercellular gap junctions. Excitation of the cellular membrane results in opening of the voltage-gated L-type calcium ion (Ca2+) channels, thereby allowing a small amount of Ca2+ to enter the cell, which in turn triggers the release of a much greater amount of Ca2+ from the sarcoplasmic reticulum, the intracellular Ca2+ store, and gives rise to the systolic Ca2+ transient and contraction. These processes are highly regulated by the autonomic nervous system, which ensures the acute and reliable contractile function of the heart and the short-term modulation of this function upon changes in heart rate or workload. It has recently become evident that discrete clusters of different ion channels and regulatory receptors are present in the sarcolemma, where they form an interacting network and work together as a part of a macro-molecular signalling complex which in turn allows the specificity, reliability and accuracy of the autonomic modulation of the excitation–contraction processes by a variety of neurohormonal pathways. Disruption in subcellular targeting of ion channels and associated signalling proteins may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure and certain arrhythmias. Recent methodological advances have made it possible to routinely image the topography of live cardiomyocytes, allowing the study of clustering functional ion channels and receptors as well as their coupling within a specific microdomain. In this review we highlight the emerging understanding of the functionality of distinct subcellular microdomains in cardiac myocytes (e.g. T-tubules, lipid rafts/caveolae, costameres and intercalated discs) and their functional role in the accumulation and regulation of different subcellular populations of sodium, Ca2+ and potassium ion channels and their contributions to cellular signalling and cardiac pathology.

Background— Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). Methods and Results— Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈40% greater current. Optical mapping of Ca 2+ transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca 2+ release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca 2+ transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈30% whole-cell I Ca,L reduction. Heart failure (16 weeks post–myocardial infarction) in rats resulted in a T-tubule degradation (by ≈40%) and significant elevation of spontaneous Ca 2+ release events. Although heart failure did not affect LTCC occurrence, it led to ≈25% decrease in T-tubule LTCC amplitude. Conclusions— We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.

Background: L-type calcium channels (LTCCs) play a key role in electrical remodeling during atrial fibrillation (AF). AF is associated with increased single LTCC activity and also accompanied by profound changes in myocyte microanatomy. However, it remains unknown how loss of structure affects the activity of LTCCs. Objective: To determine the microdomain-specific location, characteristics and structural composition of LTCCs in myocytes isolated from human right atrium. Methods: Scanning ion conductance and confocal microscopy were used to characterize surface topography and t-tubules (TTs) in myocytes collected from 22 patients with sinus rhythm (AF-) and 28 patients with AF (AF+). Super-resolution scanning patch-clamp was applied to identify LTCCs in subcellular microdomains. qPCR was used to determine mRNA levels of LTCCs forming subunits. Results: The surface topography of myocytes was less organized in AF+ compared to AF- patients with Z-groove index: 0.39± 0.04 vs 0.56 ± 0.02, P Open probability of LTCCs was 2.6-fold higher in AF+ cells. All α1C, β1, β2, β3 and β4 subunits showed transcriptional downregulation in AF+ samples. Conclusion: Significant surface and intercellular structural degradation was shown in human atrial cells collected from AF+ patients. Also single LTCCs located in TTs in AF+ myocytes showed increased open probability and decreased amplitude which correlated with transcriptional downregulation of channel subunits in samples collected from patients with AF. This could underlie their arrhythmogenic phenotype.

Introduction: Important differences in Ca2+ signaling occurred between ventricular and atrial cardiomyocytes has been attributed to the lack of a regular T-tubular system and distinct distribution of atrial L-type Ca2+ channels (LTCCs). In addition to critical subpopulation of LTCCs localized to dyadic junctions, extradyadic channels associated with district regions of surface membrane have been distinguished. Hypothesis: Here, we hypothesise that a subpopulation of LTCCs housed in caveolae microdomains, could play an important role in modulation of Ca2+ signaling, particularly in cells lacking T-tubules such as atrial cardiomyocytes. Methods: Scanning ion conductance, confocal, and electron microscopy were used to characterize membrane topography, caveolae and T-tubular network in adult rat atrial cardiomyocytes. Ca2+-sensitive fluorescent dye Fluo-4AM was used to monitor changes in [Ca2+]i. Super-resolution scanning patch-clamp was applied to identify distribution of functional LTCCs, before and after caveolae depletion by methyl-β-cyclodextrin (MβCD). Results: MβCD abolished ~60% caveolae and significantly decreased occurrence of spontaneous calcium events (from 1.64±0.22 events/cell at baseline to 0.57±0.15 events/cell after MβCD treatment, P<0.001). At baseline, functional atrial LTCCs were found both in the T-tubules and in the crest areas of the sarcolemma with similar occurrence. While MβCD did not affect LTCCs occurrence in the T-tubules (29% vs. 33%, before and after MβCD treatment, NS), it completely abolished occurrence of LTCCs on the crest of sarcolemma (33% vs. 0% before and after MβCD treatment, P<0.001). No changes in cell topography and T-tubule openings were observed after MβCD treatment. Along with changes in LTCCs distribution, MβCD decreased response to both β1- (6.52±1.42 events/cell vs. 2.66±0.59 events/cell before and after MβCD, P<0.05) and β2-ARs stimulation (3.20±0.58 events/cell vs. 1.45±0.21 events/cell before and after MβCD, P<0.05). Conclusions: Our results provide the first direct evidence of caveolae specific localization and regulation of functional LTCCs in atrial cardiomyocytes and suggest their possible role in the mechanism of unique atrial calcium cycling.

Background: Specific spatial distribution of L-type calcium channels (LTCCs) in ventricular myocytes is known to be crucial for EC coupling. However little is known about the spatial distribution of functional LTCCs in atria. Also the extent of the t-tubular (TT) network in atrial cardiomyocytes in comparison with the well-developed TT network in ventricle remains debatable. Methods: Confocal microscopy and scanning ion conductance microscopy were used to characterize cell surface topography and TTs in isolated rat atrial myocytes. Ventricular myocytes were used as control cells as they have a well-studied TT system. Super-resolution scanning patch-clamp was applied to identify distribution of functional LTCCs in different subcellular domains. Spontaneous Ca2+ transient events were promoted by Ca2+ loading by 1 min of 4-Hz pacing and optically visualized by Ca2+-sensitive fluorescent dye Fluo-4 AM. Results: About one half of the studied atrial myocytes had TTs, most of them located in the left atrium. Myocytes with TTs (density: 17.3±0.5%) and well-developed surface topography had a larger mean diameter (19.4±1.3μm) than cells with disorganized TTs (density: 12.7±0.5%, P=0.006) or absent TTs and non-structured areas on the surface (16.2±0.6μm, P=0.021, and 12.2±0.4μm, P Conclusions: This study provides the first direct evidence of distinct distribution of functional LTCCs within the specific, subcellular compartments of atrial versus ventricular myocytes and offers new insights into the molecular mechanisms of unique atrial myocyte calcium cycling.