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

31 Aims 32 Immunoglobulin light chain (AL) amyloidosis is a protein misfolding disease characterized by the 33 systemic deposition of amyloid fibrils derived from monoclonal light chains (LCs). Cardiac 34 involvement is the major determinant of prognosis and mortality, and beyond fibril accumulation, 35 soluble cardiotoxic LCs play a critical role in disease progression. While current in vivo models like 36 C. elegans and murine systems have demonstrated LC toxicity, they lack human relevance or fail to 37 capture soluble LC-induced cardiotoxicity. This study aimed to characterize the electrophysiological 38 effects of cardiotoxic LCs on a human -relevant model using human -induced pluripotent stem cell -39 derived cardiomyocytes (hiPSC-CMs). 40

and Results 41 Two amyloidogenic cardiotoxic LCs (H3 and H6) from AL patients and one non -cardiotoxic LC 42 (M10) from a multiple myeloma patient were biophysically characterized and tested in hiPSC -CMs 43 at clinically relevant concentrations. Electrophysiological recordings revealed that H3 and H6 44 significantly reduced spontaneous action potential (AP) firing frequency and maximal upstroke 45 velocity (dV/dt) in hiPSC-CMs, indicating impaired excitability. H6 also shortened AP duration. H3 46 exposure led to a ~40% reduction in peak sodium current density and altered inactivation kinetics of 47 the L-type calcium current, without affecting major pacemaker or repolarizing potassium (I Kr or IKs) 48 currents. In contrast, M10 had no effect on any measured parameter, validating the model's ability to 49 discriminate toxic from non-toxic LCs. 50

51 This study demonstrates that hiPSC -CMs provide a clinically relevant human model to investigate 52 LC-induced cardiotoxicity. Cardiotoxic LCs exert distinct but converging electrophysiological 53 impairments, including disruption of sodium and L -type calcium currents, contributing to reduced 54 excitability and altered AP morphology. These findings provide mechanistic insights into AL 55 amyloidosis-related cardiac dysfunction and establish a foundation for future therapeutic screening 56 targeting soluble LC toxicity in a human context. 57 58 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 3 1. Introduction 59 Immunoglobulin light chain (AL) amyloidosis is a protein misfolding disorder characterized by the 60 conversion of patient-specific immunoglobulin light chains (LCs) from their native state into highly 61 organized amyloid fibrils1. The disease originates from the hyperproliferation of a plasma cell clone, 62 leading to overproduction of LCs and their secretion into the bloodstream2. The structural details of 63 such amyloids from several patients and from different tissue deposits have recently been reported3-64 8. Clinical manifestations arise from the deposition of fibrillar aggregates in various organs, ultimately 65 causing organ dysfunction 9. However, cardiac involvement is a hallmark of AL amyloidosis, with 66 over 75% of patients presenting significant cardiac manifestations 10. This typically appears as a 67 progressive infiltrative cardiomyopathy characterized by electrical abnormalities and advancing heart 68 failure, and it is the main determinant of morbidity and mortality in these patients 9. Beyond the 69 structural damage caused by LC amyloid deposits in the heart, an additional and crucial pathogenic 70 factor is the direct cardiotoxicity of soluble pre -amyloid LC species 11-16. Clinical observations 71 strongly suggest a relevant role of circulating toxic LC species, as reducing their concentration 72 through anti-clonal chemo/immunotherapy can lead to rapid improvements in cardiac function and 73 patient outcomes, even in the absence of a measurable decrease in amyloid cardiac deposits17-18. 74 Experimental models have further corroborated this concept, showing that cardiotropic 75 amyloidogenic LCs alone, without amyloid fibrils, adversely affect the viability of human and rodent 76 cardiac cells by inducing oxidative stress, disrupting protein homeostasis, and impairing 77 mitochondrial function 16,19-23. The nematode C. elegans is a well -established model to assess LC 78 toxicity in vivo. The administration of aliquots of natively folded LCs derived from AL patients with 79 cardiac involvement resulted in severe structural and functional damage to the worm pharynx, the 80 functional analogue of the vertebrate heart , and is closely associated with increased production of 81 reactive oxygen species (ROS) and mitochondrial injury 24,25. Interestingly, non-amyloidogenic LCs 82 derived from multiple myeloma (MM) patients do not exhibit such significant toxicity in nematodes24. 83 The same concept was corroborated by exploiting a recently developed transgenic nematode 84 expressing human amyloidogenic LCs derived from cardiac AL from an MM patient, further 85 expanding the study of the pathophysiological mechanisms of soluble toxicity in AL amyloidosis in 86 C. elegans26. Martinez-Rivas and colleagues successfully recapitulated the structural damage caused 87 by LC amyloid deposits in a murine model of AL amyloidosis. However, that model lacked the 88 pathogenic profile related to soluble pre-amyloid toxic species since no direct LC cardiotoxicity was 89 observed27. 90 The soluble LC cardiotoxicity correlates well with high fold stability , low flexibility, and 91 conformational variability15,28,29 and specific binders of a non -native open conformation of dimeric 92 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 4 LC efficiently block LC toxicity in vivo30. To date, however, the exact nature of the cardiotoxic species 93 has not been fully elucidated. Moreover, although existing models have been invaluable for 94 demonstrating the cardiotropic nature of AL LCs and their role in cardiac dysfunction, their 95 limitations—particularly their distance from the human system —have hindered detailed studies of 96 the underlying molecular mechanisms of cardiotoxicity and have reduced the translational potential 97 of experimental findings. 98 To address this gap and to gain further insights into the effects of cardiotoxic LCs on a clinically 99 relevant, human cardiac cell s, we employed human -induced pluripotent stem cell -derived 100 cardiomyocytes (hiPSC -CMs), a widely used experimental model for studying cardiomyopathies. 101 hiPSC-CMs offer numerous advantages over primary human cardiomyocytes and animal models, 102 including an almost unlimited supply of cells, retention of the human genetic background, and the 103 circumvention of ethical and technical challenges associated with other systems. These features make 104 them particularly valuable for preclinical research and investigations into cardiac physiology and 105 disease mechanisms31,32. Although hiPSC-CMs remain immature compared to adult cardiomyocytes 106 - exhibiting fetal-like characteristics such as spontaneous pacemaker activity, a depolarized resting 107 membrane potential, elevated I f current expression 33, and reduced I K134 - they still represent a 108 substantial improvement over classical in vitro model. 109 Specifically, we selected two well-characterized cardiotoxic LCs derived from AL patients with 110 severe cardiac involvement (H3 and H6, derived from germline IGLV1-44 and IGLV1-51, 111 respectively) and one non-amyloidogenic LC from a multiple myeloma patient (M10, derived from 112 germline IGLV2-14)28. The toxicity of the soluble forms of purified native H3 and H6 has previously 113 been demonstrated in C. elegans15,30. In this study, hiPSC-CMs were incubated with native H3, H6 114 and M10 under conditions similar to those used in previous C. elegans assays, and their effects on the 115 spontaneous action potentials (APs) were evaluated. 116 Our findings demonstrated that, in contrast to the non -amyloidogenic LC control, cardiotoxic LCs 117 significantly altered AP dynamics by reducing spontaneous firing frequency and disrupting AP 118 morphology. H3, in particular, impaired hiPSC -CMs excitability, likely by affecting ion currents 119 responsible for the rapid AP upstroke. These results indicate that amyloidogenic LCs exert direct and 120 specific toxic effects on hiPSC -CMs, offering valuable insights into the disease’s underlying 121 pathophysiology. 122 2. Materials and Methods 123 2.1 LCs production and purification 124 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 5 Recombinant full-length immunoglobulin LCs from patients with AL amyloidosis or MM were 125 produced according to protocols described in Oberti et al.28. Briefly, heterologous proteins, produced 126 in the E. coli cytoplasm as inclusion bodies, were retrieved and subjected to a renaturation procedure, 127 followed by purification by means of ion exchange and size exclusion chromatography (SEC). 128 2.2 Analytical size exclusion chromatography 129 Analytical SEC was performed using a Superdex 200 increase 10/600 column operated at 4 °C by an 130 Akta purifying system. Samples were injected into the column extensively equilibrated in 50 mM 131 Hepes pH 8.0, 150 mM NaCl. Runs were imported in GraphPad Prism 9.0 software (CA, USA) for 132 data normalization, visualization and graph generation. 133 2.3 Mass photometry experiment 134 Mass photometry experiment was done using a Refeyn OneMP instrument (Oxford, UK). The 135 experiments were performed using microscope coverslips, which were assembled into the flow 136 chamber, and silicone gaskets were positioned on the glass surface for sample loading to hold the 137 sample drops with 4 × 4 wells prior to measurements. Contrast -to-mass calibration was achieved by 138 measuring the contrast of tyroglobuline (660 kDa), beta -amylase (224 kDa, 112 kDa, 56 kDa), and 139 bovine serum albumin (66.5 kDa). Calibration was applied to each sample measurement to calculate 140 the molecular mass of each histogram distribution during analysis. For the experiment, H3 was buffer 141 exchanged to PBS pH 7.4 and diluted to a final concentration of 20 nM prior to sample analysis with 142 3-fold dilution on buffer droplet to a final concentration of 10 nM. For data acquisition, 10 μl of 143 diluted protein was added to the well and mixed, and movies of 60 s duration with 2800 frames were 144 recorded using Refeyn AcquireMP 2023 R1 software in normal measurement mode with regular 145 image acquisition settings. All mass photometry movies of each measurement were processed and 146 analyzed by Refeyn DiscoverMP v2023 R2 software, and Gaussian curves were fit to each histogram 147 distribution, and the mass (kDa), sigma (kDa) and counts were determined. 148 2.4 Circular dichroism spectroscopy 149 Circular dichroism experiments were carried out on a J -1500 spectropolarimeter (JASCO Corp., 150 Tokyo, Japan) equipped with a Peltier system for temperature control. All experiments were carried 151 out in 50 mM sodium phosphate pH 8.0. LC concentration was 0.2 mg/mL in a cuvette with a 152 pathlength of 0.1 cm. Spectra were recorded from 260 to 200 nm. For each measurement, three 153 replicates were recorded and averaged to yield the final CD spectrum. Data were imported in 154 GraphPad Prism 9 software (CA, USA) for data normalization, visualization and graph generation. 155 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 6 2.5 Thermal unfolding ramps 156 Fluorescence-based thermal shift experiments were performed using a Tycho NT.6 device 157 (Nanotemper) following the changes in the intrinsic fluorescence detected at both 350 nm and 330 158 nm. Temperature ramps were performed in 50 mM Hepes, 150 mM NaCl , pH 8.0 from 35 °C to 95 159 °C. Melting temperature is defined as the temperature at which the folding -to-unfolding transition 160 occurs and is the maximum or minimum of the 350/330 nm ratio curve first derivative. Raw data 161 were imported in GraphPad Prism 9.0 software (CA, USA) for data normalization, visualization and 162 graph generation. 163 2.6 hiPSC culture, in vitro cardiac differentiation, and LCs incubation conditions 164 A hiPSC line from a healthy female donor (Thermo Fisher Scientific, cell line: TMOi001 -A) was 165 used and maintained on human Biolaminin 521 LN-coated dishes in TeSR-E8 TM medium (Thermo 166 Fisher Scientific)35. Cardiac differentiation was conducted as previously described 36 using the PSC 167 Cardiomyocytes Differentiation Kit (Thermo Fisher Scientific , Italy ) on monolayer cultured on 168 Matrigel® hESC -qualified Matrix (Corning, Corning, NY , USA) dishes. For electrophysiological 169 experiments, hiPSC-CMs were detached on day 21 of differentiation, purified using magnetic beads 170 (Miltenyi Biotec, Germany) according to the manufacturer’s instructions, and replated as grouped or 171 single cells on Matrigel-coated 35mm dishes (VWR, Italy). Cells were allowed to adhere for at least 172 48 h before conducting experiments. hiPSC-CMs were incubated for 24 h with H3, H6, and M10. LC 173 were diluted in PBS containing Ca++ and Mg++ (Thermo Fisher) and H3 was used at a concentration 174 of 2.5 µM, or 5 µM to evaluate biocompatibility and at 1 µM, 2 .5 µM, or 5 µM to study the 175 electrophysiological impact. These doses were selected based on previous studies 15 and in line with 176 the concentration present in the patients’ serum28. H6 and M10 effects were evaluated only at the 177 concentration of 2.5 µM. 178 2.7 Immunofluorescence staining 179 For the immunofluorescence staining , hiPSC-CMs at day 21 of differentiation were purified as 180 described and plated on a glass slide coated with Matrigel®. After 24 h, cells were fixed with PFA 181 4% in PBS for 15 min at room temperature (RT). Staining was performed as previously described37. 182 The primary antibody (Anti-Cardiac Troponin T antibody [1C11] mouse monoclonal antibody 183 ab8295, Abcam distributed by Prodotti Gianni s.r.l., Italy, diluted 1:800) was incubated overnight at 184 4 °C. The secondary antibody (Donkey Anti -mouse Alexa Fluor 594 A-21203, Thermo Fisher 185 Scientific Italia, Italy, diluted 1:600) w as incubated for 1 h at RT in the dark . To stain nuclei, 4′,6-186 diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific Italia, Italy, diluted 1:5000) was used and 187 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 7 incubated for 10 minutes at RT in the dark . Images were acquired with a LSM710 confocal 188 microscope (Zeiss, Germany), equipped with a 63× oil immersion objective, as single optical section. 189 Image processing was performed using Zeiss ZEN Microscope Lite software version and ImageJ 190 1.48V. 191 2.8 Metabolic activity assay (MTT) 192 hiPSC-CM purified on 21 days were seeded at confluency of 100k cells/cm2 in 96-well Matrigel®-193 coated plates (VWR, Italy). After 48 h, the cells were exposed to culture medium containing H3 (for 194 concentration, refer to paragraph 2.6). Untreated cells and lysed cells (with H2O2) served as negative 195 and positive controls, respectively. After 24 h of incubation, the medium was replaced and 3-(4.5-196 dimethylthiazolo-2-yl)-2.5-diphenytetrasolium bromide (MTT, Sigma-Aldrich, USA) was added at a 197 concentration of 0.5 mg/mL in cardiomyocyte maintenance medium and incubated for 3 h at 37 °C 198 in a 5% CO 2 atmosphere. The resulting formazan crystals were dissolved in a 1:1 solution of EtOH 199 and DMSO, and absorbance was measured at 570 nm and 650 nm using a Varioscan LUX microplate 200 reader (Thermo Fisher Scientific, Italy). The difference in absorbance (570-650 nm) was calculated. 201 As this assay is commonly used as a proxy for cell viability, relative cell viability (%) was determined 202 using untreated cardiomyocytes as reference. Two independent experiments were performed, each 203 with three replicates per condition. 204 2.9 Electrophysiology 205 All experiments on hiPSC-CMs were performed at 37 °C on a manual-patch clamp set-up equipped 206 with a 700B operational amplifier (Molecular Devices , USA ). Patch pipettes were pulled with a 207 P1000 puller (Sutter, USA) to a final resistance of 5-8 MΩ for recording spontaneous APs and to 2-3 208 MΩ for ionic currents. Spontaneous APs were recorded on small groups of beating cells in whole -209 cell, current -clamp gap -free configuration. Despite the protocol applied for differentiating 210 cardiomyocytes from hiPSCs being designed to obtain ventricular -like enriched cell cultures, some 211 heterogeneity was observed in the AP recordings, which is not uncommon in this field 38. Therefore, 212 the ventricular-like APs subpopulation was carefully isolated by applying the method published by 213 Burridge and colleagues to the recorded traces 39. Briefly, cells were classified as ventricular -like if 214 they had a maximal diastolic potential (MDP) 10 215 mV/ms, an AP amplitude (APA) > 90 mV and a ratio between the AP duration at 90% and at 50% of 216 repolarization (APD90/APD50) < 1.4. Any cell that failed to meet just one of these criteria was 217 excluded from the final pool and subsequent analysis. 218 Protocols and solutions for APs recording and ionic currents are detailed in the Supplementary Data. 219 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 8 2.10 Statistical Analysis 220 Since the purpose of the biochemical and biophysical analysis of LCs was to evaluate the quality of 221 the purified light chains rather than to perform comparative analyses among the three proteins, no 222 technical replicates were included and no statistical testing (p -values) was carried out. For the 223 metabolic activity assay and for the electrophysiology, r esults are presented as mean ± SEM, with 224 statistical significance set at P < 0.05. Analyses were performed using unpaired t -tests or One- and 225 Two-Way ANOV A, followed by appropriate post-hoc tests (Fisher for t-tests and Fisher, Bonferroni, 226 or Dunnett for ANOV A). N (number of experiments) and n (number of cells) are detailed in Tables 227 and Figures legends. 228 229 3. Results 230 3.1 Biophysical Characterization of H3, H6, and M10 LC 231 Analytical size exclusion chromatography of H3 coupled to mass photometry analysis revealed the 232 homogeneity and dimeric nature of the LC ( elution volume of 15.5 mL and molecular weight of 47 233 kDa, Fig. 1). Similarly, the size exclusion chromatography profile s of H6 and M10 showed a 234 predominant peak at around 15 .5 mL, revealing their dimeric nature of these LCs in solution (Fig. 235 1C). Circular dichroism (CD) spectra of the three purified LCs display ed a distinct negative peak at 236 218 nm, characteristic of β-sheet enriched proteins (Fig. 1F). Additionally, fluorescence -based 237 thermal unfolding assays demonstrate d a single , cooperative folding transition for each protein, 238 consistent with the behavior of globular and compact proteins (Fig. 1E). Together, these biophysical 239 analyses confirm that H3, H6, and M10 were pure, well-folded proteins, meeting the critical quality 240 requirements for downstream applications. 241 3.2 Assessment of H3 LC Effects on hiPSC-CMs viability 242 We assessed whether H3 at the concentration 2.5 µM and 5 µM, representative of concentrations 243 detected in the serum of patients with AL amyloidosis 28 and previously used in other experimental 244 models15, affected hiPSC-CMs viability. To this aim, the MTT assay, an indirect indicator of cell 245 viability, was initially performed on purified hiPSC -CMs (Supplementary Fig. 1). The level of 246 reducing MTT into formazan by mitochondrial enzymes reflects the level of cell metabolism and can 247 thus be considered as a viability index , assuming that only cells with intact metabolic activity 248 (typically viable cells) can convert MTT in formazan . Results indicated that H3 at both tested 249 concentrations did not significantly impact the level of viability (Supplementary Fig. 1B). Based on 250 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 9 these findings, we concluded that hiPSC-CMs and the selected LC concentrations could represent a 251 reliable model for further studies. 252 3.3 Cardiotoxic LCs alter spontaneous action potentials in hiPSC-CMs. 253 hiPSC-CMs exhibit a certain degree of heterogeneity that consists in the simultaneous presence of 254 ventricular-like, atrial-like, and sinoatrial node -like APs39. Since cardiac AL amyloidosis affects the 255 cardiac conduction system 40, we focused our study on the impact of the cardiotoxic H3 using 256 spontaneously beating cells with a ventricular-like AP39. To provide a comparative analysis, we also 257 tested H6, another cardiotoxic LC, and M10, a non -cardiotoxic LC. hiPSC-CMs were incubated for 258 24 h with three concentrations of H3 (1 µM, 2.5 µM, and 5 µM), or with the vehicle. H3 significantly 259 reduced the firing frequency of spontaneous ventricular-like APs (Fig. 2A-B for 2.5 µM, and Table 1 260 for a comparison of 1 µM, 2.5 µM, and 5 µM ) at all tested concentrations , in a concentration-261 dependent manner. Additionally, it markedly decreased the maximal upstroke velocity (dV/dt) ( Fig. 262 2C, Table 1). However, parameters such as maximal diastolic potential (MDP), AP amplitude (APA), 263 and AP durations (APD) measured at 30%, 50%, and 90% of repolarization were unaffected by H3 264 incubation (Fig. 2D, and Table 1). No changes were induced by the presence of the vehicle alone 265 (data not shown). 266 The same parameters related to the spontaneous APs were analyzed as key indicators of the impact 267 (if any) of the other selected LCs. Thus, hiPSC-CMs were also incubated with H6 and M10 at the 268 intermediate concentration used for H3 (2.5 µM). Like H3, H6 significantly reduced the spontaneous 269 AP firing frequency of hiPSC-CMs (Fig. 2A-B, and Table 1) as well as the dV/dt, with values being 270 very close to the statistical significance (Fig. 2C, Table 1). Interestingly, H6 also showed a reduced 271 APD at all percentages of repolarization studied (Fig. 2 D, Table 1). By contrast , in hiPSC-CMs 272 incubated with M10 firing frequency, dV/dt, MDP, APA, and APD, closely aligned with the values 273 obtained with the control (Figure 2A-D, and Table 1). 274 3.4 Cardiotoxic H3 affects inward current in hiPS-CMs. 275 The observed impairment of the fast depolarization phase of the APs could suggest a direct effect of 276 H3 on the hiPSCs inward currents (INa and ICaL). To investigate this, the tetrodotoxin (TTX)-sensitive 277 INa was measured in hiPSC -CMs following a 24 h incubation with H3 at concentrations of 2.5 µM 278 and 5 µM, at which maximal effect was observed . Both concentrations caused a significant ~40% 279 reduction in the peak current density measured at -10 mV (Figure 3A- B, and Table 2). No significant 280 change in the cell capacitance were observed (22.6 ± 1.3 pF, 20.1 ± 0.7 pF e 20.6 ± 0.9 pF in control, 281 H3 2.5 µM and 5 µM, respectively). A small rightward shift in the voltage dependence of channel 282 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 10 activation, significant only at 2.5 µM, was observed (Figure 3C, Table 2), with the magnitude of the 283 shift considered too small to have functional relevance. No significant changes were observed in the 284 fast or slow inactivation time constants at -20 and -10 mV (data not shown) , with a general trend 285 towards slowing the process at -30 mV (Table 2). 286 The L-type calcium current (ICaL) was isolated as nifedipine-sensitive. Incubation with H3 at 2.5 µM 287 or 5 µM did not affect ICaL peak current density (Figure 3D-E, and Table 2). Similar to INa, the voltage 288 dependence of activation of ICaL was slightly right-shifted with both concentrations, with significance 289 reached only at 2.5 µM (Figure 3F, and Table 2). Interestingly, the incubation with H3 significantly 290 modulated the inactivation kinetic of I CaL. When the inactivation decay of the current evoked at -20 291 mV was fitted with a bi-exponential function, both the calculated fast (τFAST) and slow (τSLOW) time 292 constants were altered following exposure to 2.5 µM LCs. Specifically, τFAST approximately doubled 293 and τSLOW significantly increased by 35% compared to control cells. τFAST also rose substantially at 294 other test voltages (data not shown). At 5 µM LC, τSLOW was markedly prolonged by 50%, whereas 295 the increase in τFAST was not statistically significant (Table 2). 296 3.5 Cardiotoxic H3 had no impact on major pacemaker and potassium currents in hiPSC-CMs. 297 To investigate the mechanism underlying the reduced frequency of spontaneous APs observed 298 following incubation with amyloidogenic LCs, the pacemaker current If was assessed. No significant 299 differences in current density amplitude were found between control cells and those treated with H3 300 at 2.5 µM or 5 µM (Fig . 3G-H, and Table 2). Likewise, the voltage dependence of I f activation 301 remained unaffected by the treatment (Fig. 3I). 302 The potential effect of H3 on the two main cardiac repolarizing potassium currents, I Kr and IKs, was 303 also examined. Incubation with either 2.5 µM or 5 µM H3 did not significantly alter current density. 304 (Fig. 3 J-K, and Table 2 ), and the voltage dependence of IKr activation was unchanged across all 305 conditions (Fig. 3L and Table 2). Similarly, IKs was not significantly affected by exposure to H3 (Fig. 306 3 M-O, and Table 2). 307 3.6 Amyloidogenic H6 effect on potassium currents IKr and IKs in hiPSC-CMs. 308 Given that H6 at a concentration of 2.5 µM caused a significant shortening of the late repolarization 309 duration in hiPSC-CMs (Fig 2), we tested its effect on IKr and IKs. Currents were recorded in the same 310 experimental setting employed for LC H3 (see Supplementary data, Materials and Methods section). 311 Incubation of hiPS C-CMs with 2.5 µM H6 did not significantly alter I Kr current density 312 (Supplementary Data Fig. 2A-C). Similarly, IKs was not significantly affected by the incubation with 313 H6 (Supplementary Data Fig. 2D-F). 314 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 11 4. Discussion 315 Cardiac AL amyloidosis is associated with severe functional consequences, including heart failure 316 with preserved ejection fraction , tachy- and bradyarrhythmias, and various degrees of conduction 317 disturbances. These include atrial fibrillation, ventricular tachycardia, sinus node dysfunction, 318 atrioventricular block, and bundle branch block 40,41, which are linked to higher rates of ventricular 319 arrhythmias42. Notably, these arrhythmias are not necessarily related to morphologic abnormalities or 320 direct amyloid infiltration of the specialized conduction system 43. To shed light on the cellular 321 mechanisms underlying these clinical manifestations, we investigated the electrophysiological effects 322 of two amyloidogenic and cardiotoxic LCs, namely H3 and H6, on ventricular -like hiPSC-CMs—a 323 powerful model for studying cardiomyopathies that offers critical insights into disease mechanisms 324 while overcoming the limitations of primary human cardiomyocytes. The hiPSC -CM platform 325 employed in this study exhibited electrophysiological properties, including membrane capacitance, 326 upstroke velocity, and resting membrane potential, consistent with those reported for cultures of 327 comparable degree of maturity 31, thereby supporting their reliability and suitability for detailed 328 electrophysiological analyses. 329 Here we show that hiPSC-CM AP dynamics and ionic currents display significant alterations upon 330 incubation with cardiotoxic LCs. In particular, the exposure to H3 resulted in a decrease in both the 331 firing frequency of spontaneous APs and the dV/dt, paralleled by a consistent 40% reduction in I Na 332 current density. The absence of major alterations in INa inactivation kinetics suggests that the primary 333 mechanism of INa impairment is a decrease in current density rather than changes in gating properties. 334 The parameter dV/dt relates to conduction velocity and serves as an index of sodium conductance in 335 isolated myocytes in phase 0 of the AP. A decrease in dV/dt reflects the presence of diseased 336 cardiomyocytes and identifies a potential arrhythmogenic substrate with an increased risk of 337 arrhythmias44. Previous data indicate that there is a strong relationship between dV/dt and diseased 338 myocardium45 and traditionally, a decrease in (dV/dt)max and a reduction in INa have been associated 339 with experimental models of heart failure 46-48. Decreased dV/dt and a lower frequency of action 340 potentials, another effect of incubation with H3, are also found in progressive cardiac conduction 341 disease49, as present in cardiac AL amyloidosis, and may contribute to progressive conduction blocks. 342 However, the observed reduced INa current density well correlating with the decrease in dV/dt does 343 not rule out the possibility that a reduction in dV/dt may also result from decreased gap junctional 344 coupling and structural changes in the cellular architecture of cardiac tissue, which will be the focus 345 of future studies. 346 The effects of H3 on the L-type calcium current (ICaL) were more nuanced. The prolonged inactivation 347 time constants ( τSLOW and τFAST) observed in the presence of H3 may have an impact on calcium 348 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 12 handling50, potentially contributing to the overall effect of H3 on heart function and increasing the 349 risk of arrhythmic events. 350 Surprisingly, the pacemaker current (If), which typically governs spontaneous activity in pacemaker 351 cells, was unaffected by exposure to H3, suggesting that the reduction in spontaneous AP firing 352 frequency is mediated by mechanisms independent of direct modulation of If. 353 AL is a heterogeneous disease, and the vast variability among LCs, due to genetic rearrangement and 354 somatic hypermutation, results in a unique amino acid sequence for each monoclonal LC 28. In fact, 355 from a comprehensive perspective provided by the analysis of the APs properties , the effect s of 356 incubation with H6 led to the reduction of the spontaneous firing frequency of hiPSC -CMs, as 357 observed with H3 with a less pronounced impact on the dV/dt. However, the reduction in APs duration 358 suggested a potential repercussion on potassium outward currents , in terms of an increase in the 359 outward current density, that, however, was not observed. Thus, at the moment, th e mechanism 360 underlying the observed shortening of action potentials thus remains unclear. These results are still 361 consistent with a general remodeling leading to heart failure, but the polymorphic clinical phenotype 362 of cardiac AL may imply that at the cellular level, specific LCs may exert non-identical cardiotoxic 363 effects on cardiomyocytes. 364 Interestingly, the non -cardiotoxic M10 had no measurable adverse effects on any of the 365 electrophysiological parameters tested. This confirms the ability of this hiPSC-CMs-based analysis 366 to discriminate between LCs which display toxic and non-toxic phenotypes in vivo. 367 In conclusion, the present study demonstrates that hiPSC-CMs are a suitable system to model LC 368 cardiotoxicity in relevant human cell types. H3 significantly impairs key ionic currents, including the 369 sodium current and the inactivation kinetics of the calcium current, in ventricular -like hiPSC-CMs. 370 These alterations contribute to reduced spontaneous AP firing frequency and disrupted AP 371 morphology, which are hallmarks of cardiac dysfunction in amyloidosis. These data however suggest 372 that distinct cardiotoxic LCs may exert different effects on cardiomyocytes nevertheless leading to 373 similar heart impairment . Future research will explore the mechanistic basis of these effects on a 374 wider set of patient -derived cardiotoxic LCs and evaluate potential therapeutic strategies to restore 375 normal cardiomyocyte function in the context of amyloid heart disease. 376 Funding 377 This work was supported by FONDAZIONE CARIPLO [grant number 2024-NAZ-0018)]; from 378 Italian Ministry of Health to IRCCS Policlinico San Donato [Ricerca Corrente ]; and by IRCCS 379 Policlinico San Donato own funds ; by Fondazione CARIPLO/Telethon [ Telethon GJC23044]; by 380 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 13 Fondazione AIRC [IG 2024 ID 30307 ]; by Università di Milano, Seed 4 Innovation 2024 grant to 381 Nano-Detox. 382 Authors Contribution 383 S.C., A.F., D.M., L.B., S.R., I.R. Substantial contributions to the conception or design of the work 384 S.C., A.F., D.M., L.B., R.P., F.S. Substantial contributions to the acquisition, analysis, or interpretation 385 of data for the work. 386 S.C., D.M., A.F., L.B., M.N., S.R., I.R. Drafting the work or reviewing it critically for important 387 intellectual content. 388 G.P., M.N., L.A., C.P., S.R., I.R. Final approval of the version to be published. 389 Conflict of Interest 390 Conflict of Interest: none declared. 391

392 393 1. Merlini G, Dispenzieri A, Sanchorawala V , Schönland SO, Palladini G, Hawkins PN, Gertz MA. 394 Systemic immunoglobulin light chain amyloidosis. Nat Rev Dis Primers. 2018;4(1):38. 395 2. Merlini G. AL amyloidosis: from molecular mechanisms to targeted therapies. Hematology. 396 2017;1:1-12 397 3. Schulte T, Chaves-Sanjuan A, Speranzini V , Sicking K, Milazzo M, Mazzini G, Rognoni P , Caminito 398 S, Milani P, Marabelli C, Corbelli A, Diomede L, Fiordaliso F, Anastasia L, Pappone C, Merlini G, 399 Bolognesi M, Nuvolone M, Fernández-Busnadiego R, Palladini G, Ricagno S. Helical 400 superstructures between amyloid and collagen in cardiac fibrils from a patient with AL amyloidosis. 401 Nat Commun. 2024;15(1):6359. 402 4. Puri S, Schulte T, Chaves-Sanjuan A, Mazzini G, Caminito S, Pappone C, Anastasia L, Milani P, 403 Merlini G, Bolognesi M, Nuvolone M, Palladini G, Ricagno S. The Cryo-EM Structure of Renal 404 Amyloid Fibril Suggests Structurally Homogeneous Multiorgan Aggregation in AL Amyloidosis. J 405 Mol Biol. 2023;435(18) 406 5. Swuec P, Lavatelli F, Tasaki M, Paissoni C, Rognoni P, Maritan M, Brambilla F, Milani P, Mauri P, 407 Camilloni C, Palladini G, Merlini G, Ricagno S, Bolognesi M. Cryo-EM structure of cardiac 408 amyloid fibrils from an immunoglobulin light chain AL amyloidosis patient. Nat Commun. 409 2019;10(1):1269 410 6. Karimi-Farsijani S, Pfeiffer PB, Banerjee S, Baur J, Kuhn L, Kupfer N, Hegenbart U, Schönland SO, 411 Wiese S, Haupt C, Schmidt M, Fändrich M. Light chain mutations contribute to defining the fibril 412 morphology in systemic AL amyloidosis. Nat Commun. 2024;15(1):5121 413 7. Radamaker, L., Karimi-Farsijani, S., Andreotti, G., Baur J., Neumann M., Schreiner S., Berghaus N., 414 Motika R, Haupt C, Walther P, Schmidt V , Huhn S, Hegenbart U, Schönland SO, Wiese S, Read 415 Schmidt MC, Fändrich M. Role of mutations and post-translational modifications in systemic AL 416 amyloidosis studied by cryo-EM. Nat Commun 2021;12:6434 417 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 14 8. Radamaker, L., Baur, J., Huhn, S, Haupt C, Hegenbart U, Schönland S, Bansal A, Schmidt M, 418 Fändrich M. Cryo-EM reveals structural breaks in a patient-derived amyloid fibril from systemic AL 419 amyloidosis. Nat Commun 2021;12:875 420 9. Palladini G, Milani P, Merlini G. Management of AL amyloidosis in 2020. Blood. 421 2020;136(23):2620-2627 422 10. Merlini G, Palladini G. Light chain amyloidosis: the heart of the problem. Haematologica. 2013 423 98:1492-5. 424 11. Brenner DA, Jain M, Pimentel DR, Wang B, Connors LH, Skinner M, Apstein CS, Liao R. Human 425 amyloidogenic light chain directly impair cardiomyocyte function through an increase in cellular 426 oxidant stress. Circ Res. 2004;94(8):1008-10 427 12. Imperlini E, Gnecchi M, Rognoni P, Sabidò E, Ciuffreda MC, Palladini G, Espadas G, Mancuso FM, 428 Bozzola M, Malpasso G, V alentini V , Palladini G, Orrù S, Ferraro G, Milani P , Perlini S, Salvatore F, 429 Merlini G, Lavatelli F. Proteotoxicity in cardiac amyloidosis: amyloidogenic light chains affect the 430 levels of intracellular proteins in human heart cells. Sci Rep. 2017; 7(1):15661. 431 13. Lavatelli F. Mechanisms of Organ Damage and Novel Treatment Targets in AL Amyloidosis Hemato 432 2022;3(1), 47-62 433 14. Marin-Argany M, Lin Y , Misra P, Williams A, Wall JS, Howell KG, Elsbernd LR, McClure M, 434 Ramirez-Alvarado M. Cell Damage in Light Chain Amyloidosis: Fibril Internalization, Toxicity and 435 Cell-Mediated Seeding. J Biol Chem. 2016;291(38):19813-25. 436 15. Maritan M, Romeo M, Oberti L, Sormanni P, Tasaki M, Russo R, Ambrosetti A, Motta P, Rognoni P, 437 Mazzini G, Barbiroli A, Palladini G, Vendruscolo M, Diomede L, Bolognesi M, Merlini G, Lavatelli 438 F, Ricagno S. Inherent Biophysical Properties Modulate the Toxicity of Soluble Amyloidogenic 439 Light Chains. J Mol Biol. 2020;432(4):845-860 440 16. Shi,J. Guan,B. Jiang,D.A. Brenner,F. del Monte,J.E. Ward,L.H. Connors,D.B. Sawyer,M.J. 441 Semigran,T.E. Macgillivray,D.C. Seldin,R. Falk, & R. Liao. Amyloidogenic light chains induce 442 cardiomyocyte contractile dysfunction and apoptosis via a non-canonical p38α MAPK pathway, 443 Proc. Natl. Acad. Sci. U.S.A. 2010;107:4188-4193 444 17. Grogan M, Dispenzieri A, Gertz M. A Light-chain cardiac amyloidosis: strategies to promote early 445 diagnosis and cardiac response Heart 2017;103:1065-1072 446 18. Palladini G, Milani P, Merlini G. In search of the most effective therapy for light chain amyloidosis. 447 Amyloid 2022;29:67-68 448 19. Lavatelli F, Imperlini E, Orrù S, Rognoni P, Sarnataro D, Palladini G, Malpasso G, Soriano ME, Di 449 Fonzo A, Valentini V , Gnecchi M, Perlini S, Salvatore F, Merlini G. Novel mitochondrial protein 450 interactors of immunoglobulin light chains causing heart amyloidosis. F ASEB J. 2015;29:4614-28 451 20. Levinson RT, Olatoye OO, Randles EG, Howell KG, DiCostanzo AC, Ramirez-Alvarado M. Role of 452 mutations in the cellular internalization of amyloidogenic light chains into cardiomyocytes. Sci Rep. 453 2013;3:1278 454 21. Liao R, Jain M, Teller P, Connors LH, Ngoy S, Skinner M, Falk RH, Apstein CS. Infusion of light 455 chains from patients with cardiac amyloidosis causes diastolic dysfunction in isolated mouse hearts. 456 Circulation. 2001;104(14):1594-1597 457 22. McWilliams-Koeppen H, Foster JS, Hackenbrack N, Ramirez-Alvarado M, Donohoe D, Williams A, 458 Macy S, Wooliver C, Wortham D, Morrell-Falvey J, Foster CM, Kennel SJ, Wall JS., Light Chain 459 Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes PLoS ONE 10(9): 460 e0137716 461 23. Sikkink L., Ramirez-Alvarado, M. Cytotoxicity of amyloidogenic immunoglobulin light chains in 462 cell culture. Cell Death Dis 2010;1:e98 463 24. Diomede L, Rognoni P, Lavatelli F, Romeo M, del Favero E, Cantù L, Ghibaudi E, di Fonzo A, 464 Corbelli A, Fiordaliso F, Palladini G, Valentini V , Perfetti V , Salmona M, Merlini G. A 465 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 15 Caenorhabditis elegans-based assay recognizes immunoglobulin light chains causing heart 466 amyloidosis. Blood. 2014;123(23):3543-3552. 467 25. Diomede L, Romeo M, Rognoni P , Beeg M, Foray C, Ghibaudi E, Palladini G, Cherny RA, Verga L, 468 Capello GL, Perfetti V , Fiordaliso F, Merlini G, Salmona M. Cardiac Light Chain Amyloidosis: The 469 Role of Metal Ions in Oxidative Stress and Mitochondrial Damage. Antioxid Redox Signal. 470 2017;27(9):567-582. 471 26. Romeo M, Barzago M, Corbelli A, Maglioni, S, Ventura N, Natale C, Conz A, Salmona M, Palladini 472 G, Nuvolone M, Fiordaliso F, Merlini G, Diomede L. Modeling immunoglobulin light chain 473 amyloidosis in Caenorhabditis elegans Dis Model Mech. 2025;18(7):dmm052230 474 27. Martinez-Rivas G, Ayala MV , Bender S, Codo GR, Swiderska WK, Lampis A, Pedroza L, 475 Merdanovic M, Sicard P, Pinault E, Richard L, Lavatelli F, Giorgetti S, Canetti D, Rinsant A, Kaaki 476 S, Ory C, Oblet C, Pollet J, Naser E, Carpinteiro A, Roussel M, Javaugue V , Jaccard A, Bonaud A, 477 Delpy L, Ehrmann M, Bridoux F, Sirac C. A mouse model of cardiac immunoglobulin light chain 478 amyloidosis reveals insights into tissue accumulation and toxicity of amyloid fibrils. Nat Commun. 479 2025;16(1):2992 480 28. Oberti L, Rognoni P, Barbiroli A, Lavatelli F, Russo R, Maritan M, Palladini G, Bolognesi M, 481 Merlini G, Ricagno S. Concurrent structural and biophysical traits link with immunoglobulin light 482 chains amyloid propensity. Sci Rep. 2017;7(1):16809 483 29. Paissoni C, Puri S, Broggini L, Sriramoju MK, Maritan M, Russo R, Speranzini V , Ballabio F, 484 Nuvolone M, Merlini G, Palladini G, Hsu SD, Ricagno S, Camilloni C. A conformational fingerprint 485 for amyloidogenic light chains. Elife. 2025;13:RP102002; 486 30. Broggini L, Barzago MM, Speranzini V , Schulte T, Sonzini F, Giono M, Romeo M, Milani P , 487 Caminito S, Mazzini G, Rognoni P , Merlini G, Pappone C, Anastasia L, Nuvolone M, Palladini G, 488 Diomede L, Ricagno S. Nanobodies counteract the toxicity of an amyloidogenic light chain by 489 stabilizing a partially open dimeric conformation. J Mol Biol. 2023;435(24):168320 490 31. Li J, Hua Y , Miyagawa S, Zhang J, Li L, Liu L, Sawa Y . hiPSC-Derived Cardiac Tissue for Disease 491 Modeling and Drug Discovery. Int J Mol Sci. 2020;21(23):8893. 492 32. Zhu K, Bao X, Wang Y , Lu T, Zhang L. Human induced pluripotent stem cell (hiPSC)-derived 493 cardiomyocyte modelling of cardiovascular diseases for natural compound discovery. Biomed 494 Pharmacother. 2023;157:113970. 495 33. Giannetti F, Benzoni P, Campostrini G, Milanesi R, Bucchi A, Baruscotti M, Dell'Era P, Rossini A, 496 Barbuti A. A detailed characterization of the hyperpolarization-activated "funny" current (If) in 497 human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes with pacemaker activity. 498 Pflugers Arch. 2021;473(7):1009-1021. 499 34. Goversen B, van der Heyden MAG, van Veen TAB, de Boer TP. The immature electrophysiological 500 phenotype of iPSC-CMs still hampers in vitro drug screening: special focus on IK1. Pharmacol 501 Ther. 2018;183:127-136. 502 35. Calamaio S, Serzanti M, Boniotti J, Fra A, Garrafa E, Cominelli M, Verardi R, Poliani PL, Dotti S, 503 Villa R, Mazzoleni G, Dell'Era P, Steimberg N. Human iPSC-Derived 3D Hepatic Organoids in a 504 Miniaturized Dynamic Culture System. Biomedicines. 2023;11(8):2114. 505 36. Tarantino A, Ciconte G, Melgari D, Frosio A, Ghiroldi A, Piccoli M, Villa M, Creo P, Calamaio S, 506 Castoldi V , Coviello S, Micaglio E, Cirillo F, Locati ET, Negro G, Boccellino A, Mastrocinque F, 507 Ćalović Z, Ricagno S, Leocani L, Vicedomini G, Santinelli V , Rivolta I, Anastasia L , and Pappone 508 C. NaV1.5 autoantibodies in Brugada syndrome: pathogenetic implications. Eur Heart J. 509 2024;45(40):4336-4348 510 37. Frosio A, Micaglio E, Polsinelli I, Calamaio S, Melgari D, Prevostini R, Ghiroldi A, Binda A, 511 Carrera P , Villa M, Mastrocinque F, Presi S, Salerno R, Boccellino A, Anastasia L, Ciconte G, 512 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 16 Ricagno S, Pappone C, Rivolta I. Unravelling Novel SCN5A Mutations Linked to Brugada 513 Syndrome: Functional, Structural, and Genetic Insights. Int J Mol Sci. 2023;24(20):15089 514 38. Barbuti A, Benzoni P, Campostrini G, Dell'Era P. Human derived cardiomyocytes: A decade of 515 knowledge after the discovery of induced pluripotent stem cells. Dev Dyn. 2016;245(12):1145-1158 516 39. Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S, Huber B, 517 Mordwinkin NM, Plews JR, Abilez OJ, Cui B, Gold JD, Wu JC. Chemically defined generation of 518 human cardiomyocytes. Nat Methods. 2014;11(8):855-60. 519 40. Hartnett J, Jaber W, Maurer M, Sperry B, Hanna M, Collier P, Patel DR, Wazni OM, Donnellan E. 520 Electrophysiological Manifestations of Cardiac Amyloidosis: JACC: CardioOncology State-of-the-521 Art Review. JACC CardioOncol. 2021;3(4):506-515 522 41. Yu J, Kong F, Gao P, Chen T, Liu Y , Cheng Z, Deng H, Lai J, Zhang L, Fan J, Wang J, Qin X, Sun K, 523 Li J, Fang Q, Yang D, Cheng K. Case Report: Left bundle branch pacing in an amyloid light-chain 524 cardiac amyloidosis patient with atrioventricular block. Front Cardiovasc Med. 2024;10:1333484. 525 42. Lopez J, Duarte G, Mark JD, Vergara Sanchez C, De Camps Martinez ER, Black AL, Perez A, 526 Zachariah A, Elshazly M, Colombo RA, Losiniecki F, Chait R, Jimenez M, Baez-Escudero J. Impact 527 of cardiac conduction disease in patients with cardiac amyloidosis: A national retrospective cohort 528 study. Int J Cardiol. 2024;417:132468. 529 43. Ridolfi RL, Bulkley BH, Hutchins GM. The conduction system in cardiac amyloidosis. Clinical and 530 pathologic features of 23 patients. Am J Med. 1977;62(5):677-86. 531 44. Temma T, Lux RL, Yoshie K, Hayase J, Bradfield JS, Shivkumar K, Ajijola OA. The derivative of 532 tissue activation as a marker of arrhythmogenic myocardium. Heart Rhythm. 2023;20(2):261-271 533 45. Marchlinski FE, Callans DJ, Gottlieb CD, Zado E. Linear ablation lesions for control of unmappable 534 ventricular tachycardia in patients with ischemic and nonischemic cardiomyopathy Circulation, 535 2000;101:1288-1296 536 46. Gelband, H., and Bassett, A. L. Depressed transmembrane potentials during experimentally induced 537 ventricular failure in cats. Circ. Res. 1973;32, 625-634. 538 47. Kuryshev YA, Brittenham GM, Fujioka H, Kannan P, Shieh CC, Cohen SA, Brown AM. Decreased 539 sodium and increased transient outward potassium currents in iron-loaded cardiac myocytes. 540 Implications for the arrhythmogenesis of human siderotic heart disease. Circulation. 541 1999;100(6):675-683 542 48. Ufret-Vincenty, C. A., Baro, D. J., Lederer, W. J., Rockman, H. A, Quinones, L. E., and Santana, L. 543 F. Role of sodium channel deglycosylation in the genesis of cardiac arrhythmias in heart failure. J. 544 Biol. Chem. 2001;276, 28197-28203. 545 49. Rohr S, Kucera JP, Kléber AG. Slow conduction in cardiac tissue, I: effects of a reduction of 546 excitability versus a reduction of electrical coupling on microconduction. Circ Res. 1998;83(8):781-547 794 548 50. Bézard M, Vartanian-Grimaldi JS, Henri J, Calin D, Zaroui A, Kharoubi M, Damy T, Agbulut O, 549 Kordeli E. Amyloidogenic immunoglobulin light chains disturb contractile function and calcium 550 transients in a human cardiac spheroid model of light chain (AL) amyloidosis. Sci Rep. 551 2025;15(1):4292. 552 553 554 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 17 Figure Legends 555 Figure 1. Biochemical and biophysical features of LCs. (A) Alignment of LCs amino acid 556 sequence. (B) Typical immunoglobulin LC dimeric fold with the variable domain (VL) VL-VL and the 557 constant domain (C L) CL-CL interfaces stabilizing the homo -dimer. (C) Analytical size exclusion 558 chromatography of LC H3 (red), H6 (blue), and M10 (green) showing a single peak at around 15.5 559 mL. (D) Mass photometry analysis of LC H3 showing the dimeric nature of the protein. (E) Thermal 560 unfolding ramps of LC H3 (red), H6 (blue), and M10 (green) indicating a single folded -to-unfolded 561 transition. (F) Circular dichroism spectrum of LC H3 (red), H6 (blue), and M10 (green) showing a 562 single negative peak at 218 nm. As stated in the Materials and Methods section, no technical replicates 563 were included and no statistical testing (p-values) was carried out. 564 Figure 2. Electrophysiological effects of amyloidogenic and non -amyloidogenic LCs on 565 ventricular-like hiPSC-CMs. (A) Representative traces of spontaneous action potentials (APs) 566 recorded in hiPSC-CMs after 24 h incubation with LC H3 (N=6, n=11), LC H6 (N=5, n=20), LC M10 567 (N=5, n=31) (2.5 µM each), or vehicle control (N=16, n=54). (B) Quantitative analysis of firing 568 frequency across conditions. LC H3 and LC H6 significantly reduced the firing frequency of 569 spontaneous APs compared to vehicle control, while LC M10 had no effect. (C) Quantitative analysis 570 of the maximal upstroke velocity (dV/dt). Both LC H3 and LC H6 decreased the dV/dt, with LC H6 571 showing values close to statistical significance, whereas LC M10 exhibited no impact. (D) Analysis 572 of the Minimal Diastolic Potential (MDP), Amplitude of Action Potential (APA), and Action Potential 573 Duration (APD) at 30%, 50%, and 90% of repolarization (APD30, APD50, and APD90). LC H6 574 exhibited significantly reduced APD at all measured percentages of repolarization, while LC H3 and 575 LC M10 did not alter any of the parameters analyzed . 576 Data are presented as mean ± SEM (N=number of experiments, n=number of cells) . Statistical 577 significance was determined using appropriate statistical test, i.e. ANOV A with Fisher’s multiple 578 comparisons test or t-test, *p < 0.05 compared to vehicle control. See Table 1 for detailed quantitative 579 values. 580 Figure 3. Electrophysiological effects of amyloidogenic LCs H3 (2.5 µM and 5 µM) on ionic 581 currents in hiPSC-CMs after 24 h treatment. (A, B) Sodium current (INa) density showing that LC 582 H3 significantly reduced I Na peak density of about 40% at both concentrations tested. (C) V oltage 583 dependence of INa activation, showing a minor rightward shift only at 2.5 µM (CTR N=5, n=15; LC 584 H3 2.5 µM N=5, n=14; LC H3 5 µM N=5, n=11). (D, E) L-type calcium current (I CaL) density that 585 remained unchanged after incubation with LC H3. (F) A slight rightward shift in voltage dependence 586 of activation observed with LC H3 2.5 µM treatment (CTR N=5, n=29; LC H3 2.5 µM N=5, n=37; 587 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 18 LC H3 5 µM N=5, n=51). (G) Pacemaker current (If): neither the amplitude of If density (H) nor the 588 voltage dependence of channel activation (I) was affected by LC H3 treatment (CTR N=4, n=32; LC 589 H3 2.5 µM N=4, n=27; LC H3 5 µM N=4, n=25). (J) IKr current: LC H3 at both concentrations did 590 not significantly alter the current density (K) or voltage dependence of activation (L) (CTR N=6, 591 n=23; LC H3 2.5 µM N=6, n=15; LC H3 5 µM N=6, n=21). (M) IKs current: no change in the current 592 density (N) or in the voltage dependence of activation (O) was observed following the treatment (CTR 593 N=6, n=9; LC H3 2.5 µM N=6, n=9; LC H3 5 µM N=6, n=9). Data are presented as mean ± SEM 594 (N=number of experiments, n=number of cells,) . Statistical significance was determined using 595 ANOV A test with Dunnet’s multiple comparisons test , *p < 0.05 compared to vehicle control. See 596 Table 2 for detailed quantitative results. 597 598 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 19 Frequency (Hz) MDP (mV) APA (mV) dV/dt APD30 (ms) APD50 (ms) APD90 (ms) CTR (N=16, n=54) 2.2 ± 0.1 -54.8±0.5 102.2±0.9 32.2±3.2 118.0±7.0 151.6±8.8 183.9±10.5 H3 1 µM (N=5, n=28) 1.2± 0.1 (p= 6.5×10⁻⁷)* -56.4±0.9 103.1±1.6 23.7±2.5 (p= 0,046)* 107.4±7.7 142.0±10.3 172.7±11.7 H3 2.5 µM (N=6, n=11) 1.7 ± 0.1 (p= 0,049)* -56.9±1.3 104.3±1.8 12.3±0.8 (p= 0,0012)* 142.9±18 184.28±21.6 212.1±23.0 H3 5 µM (N=5, n=18) 1.8 ± 0.1 (p= 0,048)* -56.1±0.9 103.3±1.8 14.3±1.0 (p=4.03x10-4)* 128.3±13.6 161.0±15.7 183.1±16.9 H6 2.5 µM (N=5, n=20) 1.6±0.2 (p= 0,028)# -55.6±1.0 100.8±1.7 21.7±3.5 (p=0.08) 85.4±9.0 (p= 0,014)# 112.7±11.2 (p= 0,019)# 140.2±13.1 (p= 0,027)# M10 2.5 µM (N=5, n=31) 2.5±0.2 -54.4±0.8 100.8±0.9 35.7±4.8 115.5±9.8 144.3±12.1 177.6±14.0 Table 1. Effect of amyloidogenic light-chains on the parameters of spontaneous APs in hiPS-CMs treated with LC vs 599 untreated CTR (N=number of experiments, n=number of cells, *p<0.05 One-Way ANOV A, Fisher test; # p<0.05 unpaired t-test;) 600 601 CTR H3 2.5µM H3 5µM INa parameters Current density @-10 mV (pA/pF) -49.8±6.7 (N=5, n=15) -29.5±4.2 (N=5, n=14) (p= 9×10⁻5)* -31.0±5.8 (N=5, n=11) (p=3x10-4)* Activation V1/2 (mV) -25±0.6 -22.0±0.8 -23.9±0.8 Activation slope (mV) 6.4±0.4 6.3±0.5 7.1±0.6 Tau fast @ -30mV (ms) 1.4±0.2 2.1±0.6 (p=0.02)* 1.8±0.3 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint 20 Tau slow @ -30mV (ms) 8.6±1.7 12.6±2.1 14.7±3.6 ICaL parameters Current density @ 0 mV (pA/pF) -19.1±1.0 (N=5, n=29) -17.9±1.0 (N=5, n=37) 19.2±0.9 (N=5, n=51) Activation V1/2 (mV) -14.3±0.6 -12.6±0.4 (p=0.02)* -13.2±04 Activation slope (mV) 7.1±0.2 6.8±0.1 6.7±0.1 Tau fast @ -20mV (ms) 5.2±0.6 10.0±1.9 (p=4.3x10-4)* 7.9±0.9 Tau slow @ -20mV (ms) 52.4±4.6 71.1±7.6 (p=0.02)* 79.4±7.9 (p=3x10-4)* If parameters Current density @ -125 mV (pA/pF) -3.1±0.3 (N=4, n=32) -2.9±0.2 (N=4, n=27) -3.2±0.4 (N=4, n=25) Activation V1/2 (mV) -83.2±1.0 -80.5±1.1 -80.7±1.8 Activation slope (mV) 8.4±0.6 6.8±0.5 8.2±1.0 IKr parameters Current density @ +40 mV (pA/pF) 0.94±0.08 (N=6, n=23) 0.86±0.11 (N=6, n=15) 1.00±0.1 (N=6, n=21) Activation V1/2 (mV) -27.7±1.2 28.6±4.5 -28.0±4.3 Activation slope (mV) 6.6±0.7 7.0±1.9 5.5±1.9 IKs parameters Current density @ +40 mV (pA/pF) 0.95±0.2 (N=6, n=9) 1.06±0.18 (N=6, n=9) 1.53±0.35 (N=6, n=9) Table 2. Parameters of the ion currents tested in hiPS-CMs exposed to LC H3 (N= number of experiments, n=number of cells, * One or Two-602 Way ANOV A) 603 604 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 24, 2025. ; https://doi.org/10.1101/2025.10.23.684089doi: bioRxiv preprint