Light Chains in Cardiac Amyloidosis: Impact on hiPSC-derived Cardiomyocytes Activity and Therapeutic Strategies (CARDIALIGHT)

Immunoglobulin light chain amyloidosis (AL), is a protein misfolding disease and is the predominant type of systemic amyloidosis1. It is characterized by the conversion of patient-specific immunoglobulin light chains (LCs) from their functional state into amyloid fibrils. Every monoclonal LC is unique in its amino acid sequence caused by genetic rearrangement and somatic hypermutation, and the clinical phenotype is variable as well, and often nonspecific, so AL diagnosis is usually delayed2. Involvement of the heart, highlighted by large fibrillar deposits, appears in more than 75% of patients, and is often associated with negative prognosis, especially in advanced cases3. In addition to the fibrils, a direct role in cardiotoxicity is exerted by soluble prefibrillar amyloidogenic LCs. This is in contrast with other conditions where the overproduction of LCs occurs, but it is not cardiotoxic, as in multiple myeloma (MM)4. Clinical observations reveal frequent recurrence of primary cardiac symptoms such as arrhythmias and atrial/ventricular fibrillation, suggesting that soluble LCs compromise the cardiac electrical rhythm and activity, but the precise mechanisms of which remain poorly understood. Traditional interventions like defibrillator implantation may not always be feasible and remedial5. A recently authorized combined therapy6, has proven effective for early-stage patients, but severe AL cases still pose significant risk of morbidity and mortality7. In absence of specific studies, this project question is related to the pathophysiological impact of LCs on the cardiomyocytes activity. The results obtained hold the potential to shed light on this topic, offering insights into cardiac AL pathogenesis and exploring potential therapeutic interventions. Preliminary experiments conducted in Prof. Rivolta's laboratory in collaboration with Prof. Ricagno's group indicated that incubation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with LC H3 (24 hours, 5μM concentration) identified in a patient with LC amyloidosis4,7 led to significant alterations of electrical properties. Manual patch-clamp analysis revealed a 25% decrease in spontaneous action potentials (APs) frequency and, among all APs parameters, a 60% reduction in maximum upstroke velocity (dV/dt), in both condition tested compared to untreated cells. In light of the obtained results, and considering the patient-specific nature of LCs and their potential to variably affect cardiac electrical activity, the proposed project aims to: (A) detail the impact of 3 amyloidogenic (H) and 3 non-amyloidogenic (M) LCs7 on hiPS-CMs activity using patch-clamp and high density-multielectrode array (HD-MEA) approaches. The first allow to complete the characterization of the effect of LCs incubation on the APs and investigate the role of LCs on individual transmembrane currents, particularly on inward ones (If, INa, ICa,), which may contribute significantly to the decrease in APs frequency and dV/dt observed in preliminary experiments. Additionally, the HD-MEA facilitate the comprehensive assessment of LCs’ effects on 2D cardiac tissue-like structures by examining propagation, velocity, and field potential characteristics. For all studies, untreated cells will be used as reference. Given the volume and complexity of the data set coming from HD-MEA experiments, a collaborative effort with Prof. L. Antolini from the University of Milano-Bicocca will be crucial. Her expertise in biostatistics will ensure the implementation of robust methods for data processing and nalysis. (B) Exploring the pathological mechanism of LCs at cellular level using fluorescence microscopy. Thanks to expertise of Prof Ricagno, LCs H and M will be fluorescent labelled without altering their structure. This allow for the first time to follow the LCs once incubated with hiPSC-CMs and monitor their localization respect to the cellula