# L-type Cav1.3 channels as key effectors of heart rate acceleration by catecholamines

Melanie Anna Faure, Eleonora Torre, Isabelle Bidaud, Marvin Gaillardon, Stéphanie Barrère-Lemaire, Joël Nargeot, Angelo Giovanni Torrente, Pietro Mesirca, Matteo Elia Mangoni

► To cite this version:

Melanie Anna Faure, Eleonora Torre, Isabelle Bidaud, Marvin Gaillardon, Stéphanie Barrère-Lemaire, et al.. L-type Cav1.3 channels as key effectors of heart rate acceleration by catecholamines. meeting of the european section of the International Society of Heart Research, ISHR, Jun 2024, Toulouse, France. pp.100106, 10.1016/j.jmccpl.2024.100106. hal-05402526

HAL Id: hal-05402526

https://hal.science/hal-05402526v1

Submitted on 8 Dec 2025

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**Aims**

The present study was double-aimed: i) to investigate whether standard of care medication, i.e., renin-angiotensin-aldosterone system inhibitors (RAASi), betablockers, diuretics, statins, decreases valvular oxidative stress and) to assess the possibility to modulate valvular MAO expression in mitral valve explants.

**Methods**

Thirty surgical patients with severe degenerative mitral valve regurgitation and variable therapeutic schemes, were consecutively included in the study. Mitral valve samples harvested during valvular repair/replacement surgery was analysed for reactive oxygen species (ROS) production and correlations with the aforementioned medication was investigated. Valvular explants were also acutely incubated with angiotensin II (AngII), in the presence RAASi and MAOi, respectively. Spectrophotometry (FOX assay), confocal microscopy, and qRT-PCR techniques were used to evaluate ROS production and MAO isoforms expression.

**Results**

Valvular oxidative stress and the presence of both MAO A and B isoforms was demonstrated in all samples, irrespective of the preoperative patients' treatment. Preoperative use of RAASi and statins was negatively corelated with the amount of ROS production, alone and even more obvious, in combination (the amount of ROS was significantly lower in valvular explants from patients receiving both therapies). The effect was not observed with betablockers and/or diuretics. Acute ex vivo incubation with AngII exacerbated ROS production and increased MAO expression (in the absence of exogenous substrates). Oxidative stress in both treated and non-treated samples was mitigated in the presence of RAASi (AngII receptor 1 blocker -irbesartan) and MAOi (clorgyline for MAO-A and selegiline for MAO-B). Interestingly, the magnitude of ROS decrease was negatively correlated with the use of RAASi and statins (ROS decrease was less important in samples from patients receiving these drugs), pointing to the antioxidant preoperative effect of this medication.

**Conclusions**

In patients with severe degenerative mitral valve regurgitation, valvular oxidative stress is mitigated by standard treatment with RAASi and statins. MAO may contribute to valvular oxidative stress since it was induced by AngII and ROS production was decreased by MAO inhibitors in both stimulated and non-stimulated samples.

doi:10.1016/j.jmccpl.2024.100104

543736

**Increased Atrial Fibrillation burden in Heart Failure is associated with increased vulnerability to atrial alternans**

George Madders¹, Alice Whitley, Mohammed Obeidat, Menglu Li, Agnieszka Swiderskwa, Charlotte Smith, David Eisner, Andrew Trafford, Katharine Dibb

University of Manchester [Manchester] – UK

¹Intervenant

**Introduction:** Heart failure (HF) is a major risk factor for atrial fibrillation (AF), with

≈41% of patients with HF developing newly onset AF, doubling mortality, with limited treatment options available. Recently, a growing body of work has implicated atrial alternans in the promotion of AF. Alternans can be recorded in-vivo as well as in single cells and is a beat-to-beat oscillation in the size or shape of the atrial action potential or Ca2+ transient which has been linked to AF. However, whether atrial alternans promotes AF in HF is unknown.

**Aims:** 1. Does HF increase AF burden in otherwise healthy sheep.
2. Is atrial alternans responsible for an altered AF burden in HF.
3. What cellular mechanism(s) underlie atrial alternans in HF.

**Methods:** HF was induced in healthy adult female sheep by rapidly pacing the right ventricle at 210 bpm. Monophasic action potentials were recorded in subset of 7 sheep, at both implant and at the point of HF, under anaesthesia, through a Blazer electrophysiology catheter positioned on the right atrial posterior wall. Atrial electrophysiology parameters were determined through a series of pacing protocols and alternans identified through discrete Fourier transform. Isolated single atrial myocytes were loaded with Fluo-5F am and patch clamped to understand cellular alternans mechanisms.

**Results:** We first sought to establish if sheep, like humans, were more susceptible to AF in HF. Anaesthetised sheep were paced for 50 beats with a diastolic interval of 400 to 100ms in 10ms decrements. Spontaneous AF episodes were on average 10-fold longer in HF (53.7 vs. 525s) and occurred at lower pacing rates (180 vs. 140ms pacing intervals). AF was preceded in ≈82% of instances by atrial alternans. Alternans, was easier to induce in HF where it was seen at slower pacing rates (350ms vs. 295ms in control). Numerous mechanisms have been shown to influence the ease that alternans develops. Our data suggests alternans occurs more in HF due to alternation of the Ca2+ content of the sarcoplasmic reticulum.

**Discussion and conclusions:** AF is increased in HF with most AF episodes preceded by atrial alternans. Alternans occurs more readily in HF, we believe, due to a higher SR calcium content that alternates from beat-to-beat. Further work is required to determine the mechanism of transition from alternans to AF.

doi:10.1016/j.jmccpl.2024.100105

543656

**L-type Cav1.3 channels as key effectors of heart rate acceleration by catecholamines**

Melanie Anna Faure¹, Eleonora Torre, Isabelle Bidaud, Marvin Gaillardon, Stéphanie Barrère-Lemaire, Joel Nargeot, Angelo Giovanni Torrente, Pietro Mesirca, Matteo Elia Mangoni

Institute of Functional Genomics – Univ. Montpellier, CNRS, INSERM, Montpellier, France, – France

¹Intervenant

**Introduction**

The ionic mechanism underlying heart rate (HR) acceleration by catecholamines is incompletely understood. It is widely accepted that cAMP-dependent regulation of HCN4 channels and the activation of the Na+/Ca2+exchanger by ryanodine receptors mediated Ca2+ release are the predominant mechanisms of HR acceleration. However, experimental evidences demonstrate that none of these mechanisms is essential for sympathetic regulation of HR, suggesting that other ones could be involved in the positive chronotropic effect of β-adrenoceptors. The sinoatrial node expresses two distinct L-type Ca2+ channel isoforms, Cav1.2, which controls myocardial contractility and Cav1.3.

**Objective**

We previously demonstrated that Cav1.3 channels play a major role in the generation of sinoatrial pacemaker activity and impulse conduction. Here we study the role of Cav1.3 in the acceleration of HR by catecholamines.

**Method**

We crossed mice in which Cav1.2 channels are rendered insensitive to dihydropyridines (DHP) (Cav1.2DHP-/-), with mice lacking cAMP dependent regulation of HCN4 (HCN4-CNBD). Cav1.2DHP-/- HCN4-CNBD mice enable selective inhibition of Cav1.3 by DHP amlodipine (3 µM) or nifedipine (3 µM), or activation by BayK8644 (0.5 µM), in a genetic background lacking cAMP dependent activation of HCN4. We studied HR regulation ex-vivo using the isolated Langendorff-perfused heart. We also used CaV1.3-
flex KI/Hcn4-CreERT2 KI mice, in which CaV1.3 is specifically deleted in HCN4 positive cells. Hearts were perfused under ivabradine (3 µM) to block HCN4 current in presence of the β-adrenergic agonist adrenaline (0.3 µM).

**Results:**

In n=8 Cav1.2DHP-/-/HCN4-CNBD hearts, adrenaline (ADR) failed to significantly increase HR after selective Cav1.3 inhibition by amlodipine (AMLO: 207±19 bpm vs AMLO+ADR: 220±17 bpm n=8 p> 0.05 respectively), showing that inhibition of Cav1.3 in the absence of cAMP-dependent regulation of HCN4 channels prevented the positive chronotropic response of the heart and suggesting that catecholaminergic regulation of HR was reliant on Cav1.3 in this genotype. Moreover, the CaV1.3-flex KI/Hcn4-CreERT2 KI mice confirmed these results (IVA: 113±25 bpm vs IVA+ADR: 118±26 bpm, n=8 p> 0.05 respectively).

Consistently, activation of CaV1.3 by BayK8644 in Cav1.2DHP-/-/HCN4-CNBD hearts in- creased the HR similarly to ADR in the same hearts (ADR: +33.1±7.4% vs BayK: +28.6±4.9% n=8 p> 0.05), suggesting that in the absence of cAMP-dependent regulation of HCN4, β- adrenergic regulation of CaV1.3 channels is sufficient to explain the increase in HR by cate- cholamines.

**Conclusion**

Our study thus identifies, for the first time, Cav1.3 channels as key effectors of β-adrenergic regulation of heart rate acceleration.

doi:10.1016/j.jmccpl.2024.100106

541199

Linking Myocardium Hypercontractility to Microvasculature Dysfunction and Inflammation through NLRP3 Inflammasome Activation

Vasco Sequeira<sup>a,1</sup>, Katharina Ermer<sup>a,1</sup>, Jan M. Federspiel<sup>a</sup>, Mark Waddingham<sup>b</sup>, Jan Dudek<sup>a</sup>, Hirotsugu Tsuchimochi<sup>b</sup>, Christoph Maack<sup>a</sup>, James Pearson<sup>b</sup>

**Background:** Myocardial hypercontractility, a hallmark of various cardiomyopathies, significantly impacts coronary microvasculature function. This study hypothesizes that hypercontractility compresses coronary perfusion during diastole, potentially leading to capillary closure, ischemic rupture, and microvascular endothelial dysfunction. We explored this association, focusing on the role of NLRP3 inflammasome activation as a downstream consequence of this dysfunction, and establish correlations between hyper-contractility and markers for localized ischemia and inflammation.

**Methods** We utilized synchrotron microangiography at the Spring-8 Synchrotron Radiation Research Institute in Japan to observe coronary microvessels (30-100 µm) in wild-type C57BL6 mice. The effect of the calcium-sensitizer EMD-57033 (1.6 mg/kg, 10 µM) and the control vehicle on coronary function was assessed by jugular vein infusion. A baseline image was recorded 10 min after administration of the vehicle solution and a repeat recording made 10 min after EMD-57033 administration. Visualised vessel area was quantified during diastole and systole using Fiji/ImageJ 2.9.0/1.53t. Additionally, in separate experiments, hearts were stress-paced at 1 Hz for 20 minutes ex vivo with or without EMD-57033 and Isoprenaline (ISO, 30 nM), followed by rapid cooling in liquid nitrogen for Western blot analysis. Western blotting analysis focused on NLRP3 inflammasome components including NLRP3, CASPASE-1 (p50 and p20 fragments), and pro-IL-1β to assess the inflammatory response induced by hypercontractility.

**Results** Under baseline conditions, coronary vascular area reduced by 30±13% from diastole to systole, as previously reported

with normal coronary artery physiology. EMD-57033 perfusion resulted in a substantial decrease in diastolic coronary vessel area by 82±16% from vehicle conditions, consistent with net hypercontractility-induced coronary artery constriction. Western blot analysis revealed activation of the NLRP3 inflammasome including the presence of pro-IL1b translation in ISO, EMD, or ISO+EMD treated hearts.

**Conclusions** This study indicates that myocardial hypercontractility leads to a reduction in diastolic coronary vessel area, suggesting impaired coronary flow. The corresponding rise in NLRP3 inflammasome components in hypercontractile conditions links myocardial hypercontractility with microvascular dysfunction and inflammation. We propose that NLRP3 inflammasome activation is a reactive mechanism to localized myocardial ischemia triggered by hypercontractility. These findings are particularly relevant to cardiac conditions characterized by hypercontractility, microvascular dysfunction, and ischemia.

doi:10.1016/j.jmccpl.2024.100107

543624

Mitochondrial metabolism is dynamically and reversibly modulated by cytosolic Na elevation in the heart

Yu Jin Chung<sup>a</sup>, Zoe Hoare<sup>a</sup>, Friedrich Baark<sup>b</sup>, Chak Shun Yu<sup>c</sup>, Jia Guo<sup>d</sup>, William Fuller<sup>e</sup>, Richard Southworth<sup>b</sup>, Doerthe Katschinski<sup>d</sup>, Michael P. Murphy<sup>c</sup>, Thomas R. Eykyn<sup>b</sup>, Michael J. Shattock<sup>a,1</sup>

**Introduction:** Elevated intracellular sodium (Na) and altered energy metabolism are common features of heart failure. Increased intracellular Na has been shown to adversely affect mitochondrial metabolism, to promote the well-known substrate preference shift away from fatty acids towards glucose, and to contribute to systolic and diastolic function, and arrhythmia susceptibility.

**Aim:** To establish whether the adverse metabolic effects of Na elevation, once established, can be reversed by lowering Na, and to further investigate the underlying mechanisms.

**Materials and Methods:** Intracellular Na was elevated and lowered in Langendorff perfused rat hearts by the transient exposure to the Na/K ATPase inhibitor ouabain in the presence of the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Intracellular Na and cardiac energetics were measured using 23Na and 31P NMR spectroscopy, respectively. Steady-state metabolic fluxes were assessed by perfusing hearts with 1-13C glucose and then subjecting the extracted metabolites to 13C or 1H NMR spectroscopy.

**Results:** Elevated intracellular Na decreased Gibb's free energy of ATP hydrolysis, acutely reduced ATP in the absence of altered PCR, caused accumulation of the TCA cycle intermediates succinate and fumarate and decreased electron transport chain activity at Complexes I, II and III, which were all restored upon lowering Na to baseline levels. This was accompanied by a state of pseudo hypoxia and the stabilization of HIF-1α under conditions of high Na de- spite normal tissue oxygenation (confirmed using the PET hypoxia tracer (64Cu)Cu-CTS). Inhibition of the mitochondrial Na/ Ca-exchanger (NCLX) with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevented the metabolic alterations observed during Na elevation. Elevated Na caused a

<footnote><sup>a</sup>comprehensive heart failure center Würzburg – Germany</footnote>

<footnote><sup>b</sup>National Cerebral and Cardiovascular Center Research Institute – Japon</footnote>

<footnote n="1"><sup>1</sup>Intervenant</footnote>

<footnote><sup>a</sup>School of Cardiovascular Medicine and Metabolic Sciences, King's College London – UK</footnote>

<footnote><sup>b</sup>School of Biomedical Engineering and Imaging Sciences, King's College London – UK</footnote>

<footnote><sup>c</sup>MRC Mitochondrial Biology Unit, University of Cambridge – UK</footnote>

<footnote><sup>d</sup>Georg-August-University, Gottingen, Germany</footnote>

<footnote><sup>e</sup>University of Glasgow – UK</footnote>

<footnote n="1"><sup>1</sup>Intervenant</footnote>