Biology subjects

Melgari, D.

Publications and source records attributed to Melgari, D..

5 recordsLinked to original sources

bioRxiv2025

Exploring light chain cardiotoxicity in AL amyloidosis: Impact on hiPSC-derived Cardiomyocyte Activity

AimsImmunoglobulin light chain (AL) amyloidosis is a protein misfolding disease characterized by the systemic deposition of amyloid fibrils derived from monoclonal light chains (LCs). Cardiac involvement is the major determinant of prognosis and mortality, and beyond fibril accumulation, soluble cardiotoxic LCs play a critical role in disease progression. While current in vivo models like C. elegans and murine systems have demonstrated LC toxicity, they lack human relevance or fail to capture soluble LC-induced cardiotoxicity. This study aimed to characterize the electrophysiological effects of cardiotoxic LCs on a human-relevant model using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and ResultsTwo amyloidogenic cardiotoxic LCs (H3 and H6) from AL patients and one non-cardiotoxic LC (M10) from a multiple myeloma patient were biophysically characterized and tested in hiPSC-CMs at clinically relevant concentrations. Electrophysiological recordings revealed that H3 and H6 significantly reduced spontaneous action potential (AP) firing frequency and maximal upstroke velocity (dV/dt) in hiPSC-CMs, indicating impaired excitability. H6 also shortened AP duration. H3 exposure led to a [~]40% reduction in peak sodium current density and altered inactivation kinetics of the L-type calcium current, without affecting major pacemaker or repolarizing potassium (IKr or IKs) currents. In contrast, M10 had no effect on any measured parameter, validating the models ability to discriminate toxic from non-toxic LCs. ConclusionThis study demonstrates that hiPSC-CMs provide a clinically relevant human model to investigate LC-induced cardiotoxicity. Cardiotoxic LCs exert distinct but converging electrophysiological impairments, including disruption of sodium and L-type calcium currents, contributing to reduced excitability and altered AP morphology. These findings provide mechanistic insights into AL amyloidosis-related cardiac dysfunction and establish a foundation for future therapeutic screening targeting soluble LC toxicity in a human context.

pathology↗

bioRxiv2024

Lysosomal signalling pathways influence heart rhythm, and regulate atrial function

In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium (Ca2+) release to augment sarcoplasmic reticulum (SR) Ca2+ sequestration, producing larger Ca2+ transients. However, the role of lysosomal Ca2+ signals in pacemaker activity, a distinct Ca2+-operated function of the sinoatrial node (SAN), or in the atrial myocardium has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits the spontaneous beating rate response to beta-adrenergic stimulation in intact SAN. We found intracellular signaling microdomains between lysosomes and neighboring SR or mitochondria in mouse, and goat tissue. The spatial relationship between lysosomes and other Ca2+-handling organelles are altered in goat atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce cAMP in response to lysosomal signaling, adding a novel trigger for cyclic nucleotide signaling. Our findings support the hypothesis that lysosomal Ca2+ signaling contributes to regulation of cardiomyocyte cAMP levels and pacemaker activity.

physiology↗

bioRxiv2019

Distinct CASK domains control cardiac sodium channel membrane expression and focal adhesion anchoring

Membrane-associated guanylate kinase (MAGUK) proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein CASK (Calcium/CAlmodulin-dependent Serine protein Kinase) negatively regulates the main cardiac sodium channel, NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex, and like syntrophin, binds to the C-terminal domain of the channel. Here we show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. Thus, the multi-modular structure of CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocyte.\n\nSUMMARYSequential functional domain deletion approach identifies three critical domains of CASK in cardiomyocytes. CASK binds the cell adhesion dystrophin-glycoprotein complex through HOOK domain and inhibits NaV1.5 channel membrane expression by impeding trafficking through L27B and GUK domains.

physiology↗

bioRxiv2019

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusturization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule-dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.

cell biology↗

bioRxiv2019

Rapid characterisation of hERG channel kinetics II: temperature dependence

Ion channel behaviour can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing and inactivating) are commonly represented with Q10 coefficients or an Eyring relationship. In this paper we assess the validity of these representations by characterising channel kinetics at multiple temperatures. We focus on the hERG channel, which is important in drug safety assessment and commonly screened at room temperature, so that results require extrapolation to physiological temperature. In Part I of this study we established a reliable method for high-throughput characterisation of hERG1a (Kv11.1) kinetics, using a 15 second information-rich optimised protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using CHO cells over-expressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch clamp platform, with temperature control. We characterise the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37 {degrees}C, and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients: it broadly follows a generalised, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol-dependent. Our results show that a direct fit using our 15 second protocol best represents hERG kinetics at any given temperature, and suggests that predictions from the Generalised Eyring theory may be preferentially used if no experimentally-derived data are available.\n\nStatement of SignificanceIon channel currents are highly sensitive to temperature changes. Yet because many experiments are performed more easily at room temperature, it is common to extrapolate findings to physiological temperatures through the use of Q10 coefficients or Eyring rate theory. By applying short, information-rich protocols that we developed in Part I of this study we identify how kinetic parameters change over temperature. We find that the commonly-used Q10 and Eyring formulations are incapable of describing the parameters temperature dependence, a more Generalised Eyring relationship works well, but remeasuring kinetics and refitting a model is optimal. The findings have implications for the accuracy of the many applications of Q10 coefficients in electrophysiology, and suggest that care is needed to avoid misleading extrapolations in their many scientific and industrial pharmaceutical applications.

biophysics↗