A zebrafish stable model of Galectin-3 to elucidate its role in Arrhythmogenic Cardiomyopathy

A zebrafish stable model of Galectin-3 to elucidate its role in Arrhythmogenic Cardiomyopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A zebrafish stable model of Galectin-3 to elucidate its role in Arrhythmogenic Cardiomyopathy Kalliopi Pilichou, Giovanni Risato, Rudy Celeghin, Marco Cason, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7855683/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Galectin-3 (LGALS3/Gal-3) dysregulation has emerged as a critical mediator of inflammatory processes in arrhythmogenic cardiomyopathy (AC), playing pivotal roles in modulating Wnt/β-catenin signaling and regulating macrophage polarization. AC is a rare genetic disorder, primarily driven by desmosomal gene variants, characterized by fibro-fatty replacement of the ventricular myocardium, progressive ventricular dysfunction, and heightened arrhythmic risk in the young and athletes. To investigate the role of this multifaceted lectin in AC pathogenesis, we developed and characterized a stable lgals3a knock-out zebrafish model. Gal-3 deficiency alone was sufficient to recapitulate hallmark AC features, including ventricular adipose infiltration, chamber dilation, pericardial effusion, and progressive arrhythmias, spanning from larval to adult stages. Ultrastructural analyses revealed disrupted desmosomes, directly implicating Gal-3 in intercellular adhesion independent of other desmosomal gene variants. Transcriptomic analyses demonstrated suppression of both Wnt/β-catenin and TGFβ signaling. Early-stage pharmacological activation of Wnt signaling partially rescued cardiac function, but structural defects persisted in adults, indicating irreversible desmosomal instability. Inflammatory profiling revealed significant immune cell infiltration and upregulation of macrophage-related proinflammatory genes (e.g., MMP12, CCL38, IL16), consistent with AC “hot phases.” This study establishes Gal-3 depletion as a sufficient driver of AC-like pathology and identifies Gal-3–related pathways as promising targets for therapeutic intervention. Health sciences/Cardiology/Cardiovascular biology/Cardiovascular genetics Biological sciences/Biotechnology/Expression systems Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Galectin-3 (Gal-3) dysregulation in cardiac tissue has been linked to a variety of cardiovascular diseases, underlying its involvement in physiological processes such as inflammation, fibrosis, and tissue remodelling 1 , 2 . In 2021, Cason and colleagues advocated a potential pathogenic role of LGALS3 in the myocardial injury of Arrhythmogenic Cardiomyopathy (AC) mediated by destabilization of desmosomes 3 . AC is characterized by the progressive loss of ventricular myocardium due to myocyte death, with subsequent replacement by fibro-fatty scar tissue 4 , 5 , 6 . Rather than being a continuous process, AC progression might occur during periodic bursts (‘hot phases’) in an otherwise stable disease 7 , 8 , 9 . Environmental factors such as physical exercise might facilitate disease progression by worsening cell adhesion disruption. Previous research on AC pathogenesis has shown that myocyte death is the primary trigger, followed by an inflammatory response and subsequent tissue repair through fibrous replacement 10 , 11 . However, more recent studies suggest that inflammation occurs before the manifestation of overt histological abnormalities 11 , 12 , 13 . Specifically, Gal-3 expression has been reported to fluctuate, with a downregulation during the early disease phase, followed by upregulation during acute ‘hot phases’, which are associated with exacerbated inflammation and tissue remodeling 3 . Gal-3 is known to be directly involved in the stabilization of intercellular junctions, through the interaction of N-linked β-galactosides on the extracellular domain of Desmoglein-2 (DSG2) 14 , 15 , 16 and the colocalization with Desmoplakin (DSP) 17 . Pharmacological suppression of Gal-3 in the zebrafish model led to aberrant DSP localisation in epidermal cells, where desmosomes appeared separated from the cytoskeletal complex and unevenly distributed 3 . Gal-3 has a key role in inflammatory response through neutrophil activation and adhesion 18 , chemo-attraction of macrophages 19 , and activation of mast cells 20 . Specifically, Gal-3 is considered a “macrophage activation marker” and is found to be highly expressed and secreted by mononuclear macrophages (CD68- and CD98-positive) 19 , 21 . Therefore, we generated the first stable lgals3a knockout (KO) zebrafish line ( -aa ) designed to mimic the early phases of AC, when Gal-3 is reduced, to allow us to investigate its mechanistic role in initiating disease-related inflammation, cardiac structural changes, and signaling dysregulation. RESULTS Generation of zebrafish lgals3 mutant lines The lgals3 gene is duplicated in zebrafish; therefore, both copies, lgals3a and lgals3b , were targeted to generate lgals3 zebrafish mutants. Accordingly, lgals3 -KO zebrafish lines were generated using the CRISPR/Cas9 approach. The lgals3a (ia305) line carries a 4-bp deletion in exon 3 (Chr 13: g. 36578952¬ 36578957del), causing a frameshift mutation that results in a truncated protein, lacking the C-terminal domain, of 218 amino acids. The synthesis of a non-functional protein, without the carbohydrate recognition domain (CRD), located at the C-terminus as in humans, is expected to cause its premature degradation. The lgals3b (ia306) line carries a 114-bp deletion in exon 3 (Chr 17: g10832046-10832160del), which removes nearly the entire exon. This region encodes the collagen-alpha domain, approximately 110 amino acids in length, which is involved in Gal-3 oligomerization and interactions with other proteins. The deletion removes 38 of these 110 amino acids, which is expected to induce a loss of protein function by disrupting its normal activity. Genotyping of lgals3a and lgals3b zebrafish mutants Amplicons obtained from lgals3a PCR showed the WT single band of 178 bp, while the mutant amplicons displayed a shorter band of 174 bp, reflecting the 4-nucleotide deletion (Supplementary Fig. 1C). This result was confirmed by sequencing analysis: the homozygous mutant sample (Supplementary Fig. 1A) contains the 4-nucleotide deletion (red box), compared to the WT (Supplementary Fig. 1B). For the lgals3b gene, a difference of approximately 100 bp was observed, indicating a clear deletion in exon 3 (Supplementary Fig. 1F). Sanger sequencing further quantified this deletion as 114 bp (red box) (Supplementary Fig. 1D and 1E). Analysis of lgals3a/b mRNA levels in lgals3a mutant larvae To validate the effective genetic KO of the lgals3a gene in our line, we decided to investigate the effect on mRNA expression for both lgals3a and lgals3b by qPCR. The results revealed a statistically significant downregulation of lgals3a mRNA quantity in a whole-body analysis at the larval stage. We confirmed that the mRNA expression of the lgals3b gene was not affected by changes in lgals3a , thus excluding a compensatory effect. At the adult stage, by surgically isolating the heart tissue, we confirmed a strong downregulation of lgals3a , whereas lgals3b showed expression comparable to WT. These results indicate that the 4-nt mutation in lgasl3a triggers the activation of an mRNA decay mechanism, causing the premature degradation of the non-functional mRNA and the subsequent downregulation of the related protein expression (Fig. 1 A). Moreover, re-analysis of data from a recently published study 22 revealed that both lgals3a and lgals3b are expressed across multiple cardiac cell populations, including cardiomyocytes, endocardial and epicardial cells (black boxes), fibroblasts, and macrophages. Notably, lgals3a expression was markedly higher in the heart compared to lgals3b (Fig. 1 B). Fertility analysis of the lgals3b mutant line and selection of the lgals3a line During the generation of stable lgals3a and lgals3b KO zebrafish lines, we observed a strong decrease in fertility in lgals3b homozygous mutants. Therefore, a fertility analysis was conducted by outcrossing male and female lgals3b mutants with WT counterparts. The results underlined that lgals3b homozygous male fish have regular spermatogenesis; in fact, they can fertilize 72% of the total eggs, in line with the percentages obtained by crossing two WT fish. On the other side, lgals3b homozygous female fish exhibited abnormal ovulation, as they tend to release already degrading eggs, with 67% of them remaining unfertilized (Supplementary Fig. 2). Considering that only lgals3b has been previously reported to be expressed in oocytes 23 , together with our fertility analysis and the higher expression of lgals3a in the heart compared to lgals3b (Fig. 1 B), we decided to focus exclusively on the lgals3a homozygous line, hereafter referred to as -aa . Morphological and functional analysis in -aa mutant larvae When compared to WT, approximately 35% of -aa mutant larvae at 3 dpf presented pericardial effusion. Functional imaging analysis, based on Supplementary videos 1–2 and conducted using the pyHeart4Fish imaging software, assessed several cardiac parameters, including ventricular ejection fraction, relative contractility, and heartbeats in both chambers. At 3 dpf, these parameters were slightly but significantly reduced in the mutant ventricles, and both chambers displayed a clear bradycardic phenotype. Although the arrhythmia score at this developmental stage did not differ significantly from controls, a trend toward increased variability was observed (Fig. 2 ). Due to the absence of arrhythmic events and the mild dysregulation of cardiac parameters at 3 dpf, we repeated the analysis at 6 dpf (discussed further on, Supplementary videos 3–4). Survival analysis of mutant lines at juvenile stages We raised equal numbers of WT and mutant larvae (n = 100 each) under identical conditions for one month, recording daily mortality. The survival rate of the mutant line was significantly lower (74%) compared to controls (90%) (Supplementary Fig. 3). Motor behaviour analysis in -aa mutant larvae Locomotion experiments were conducted on zebrafish larvae at 5 dpf to investigate changes in motor behaviour related to the -aa mutant. Prior to these experiments, muscle integrity was assessed via birefringence, which is brighter in well-organized skeletal muscle. This step ensured that any observed issues were due to cardiac dysfunction rather than muscular abnormalities. The analysis, which included two pools of larvae (WT and -aa ), is illustrated in Supplementary Fig. 4A-A’. Mean birefringence intensity, normalized to fish length, showed no significant difference between the two pools (Supplementary Fig. 4A’’), indicating preserved skeletal muscle structure in lgals3a mutant larvae. In contrast, motor behaviour analysis highlighted that while mutation in the lgals3a gene did not affect the larvae's ability to respond to light stimuli (dark/light intervals), it did decrease the normal motor response to these stimuli when compared to WT controls (Supplementary Fig. 4B). Notably, swimming activity during the dark phases was reduced only in the mutant group. However, the most pronounced difference appeared in the second part of the light phases, when the larvae began to recover from stress and start moving again. Here, -aa larvae exhibited greater difficulty initiating movement than WT controls, suggesting increased fatigue. A statistically significant reduction in total distance swum, particularly during light phases, further supported the interpretation of intact sensory perception but impaired motor performance (Supplementary Fig. 4B’). Increased apoptotic and necrotic events in the cardiac region of –aa mutant larvae We investigated whether depletion of lgals3a leads to increased cell death events in the cardiac region of -aa mutant larvae. AO/EB staining was used to discriminate between live, apoptotic and necrotic cells based on differential labelling of DNA and RNA. Our analysis revealed a significant increase in total cell death and necrotic events in the heart tissues of mutant larvae, suggesting that lgals3a plays a crucial role in regulating cell survival in this region. Additionally, dying cells were observed in the skin surrounding the cardiac region of mutant larvae (Fig. 3 ), likely due to tissue stress resulting from cardiac dilation. Pathways analysis in mutant larvae We investigated at larval stage potentially dysregulated pathways relevant to AC. In detail, we examined by qPCR the mRNA levels of two target genes involved in the Wnt/β-catenin signalling pathway, ccnd1 and myca , and found both significantly downregulated. Similarly, expression of the Hippo/YAP-TAZ pathway target genes ccn2a and ccn2b was also reduced. These findings are consistent with observations in human AC patients, where activation of the Hippo cascade leads to YAP phosphorylation, preventing its nuclear translocation and subsequent activation of target gene transcription 24 . Finally, TGFβ signalling appeared only mildly dysregulated (Supplementary Fig. 5). Inflammation and cardiac function rescue after pharmacological treatment The Wnt/β-catenin reporter line Tg(7xTCF-Xla.Siam:EGFP) ia4 , crossed with the lgals3a mutant, revealed a significant tissue-specific reduction of this signal in the epicardium and cardiac valves, structures that rely on this pathway for proper development. This fluorescent mutant line allowed us to validate the efficacy of SB216763 (SB), a well-known agonist of the Wnt/β-catenin pathway, demontrating a clear restoration of reporter expression in the cardiac region after the treatment. (Fig. 4 A). We further characterized the phenotype by investigating potential inflammatory processes in the cardiac region of mutant larvae using an anti-L-plastin antibody as a marker of inflammatory cells. At the larval stage, immunostaining revealed a significant increase in L-plastin-positive cells compared to controls. These cells were not yet localized within heart tissue but accumulated in the surrounding cardiac region, suggesting recruitment potentially triggered by structural damage or functional impairment (Fig. 4 B). Further investigation at the adult stage revealed the presence of inflammatory cells within the epicardial layer (Supplementary Fig. 6). We repeated the cardiac functional analysis at 6 dpf, observing a worsening of the mutant phenotype, compared to the 3 dpf stage previously shown in Fig. 2 . Cardiac parameters were markedly impaired in the mutants, indicating disease progression consistent with the human condition. Episodes of arrhythmia were significantly more severe in the mutant group (Fig. 4 C). Notably, treatment with SB restored the physiological levels of inflammatory cells, improved ventricular contractility, and reduced the incidence of bradycardia and arrhythmic events. (Fig. 4 A-B-C). RNAseq analysis in adult hearts RNAseq analysis identified 813 downregulated and 1,632 upregulated genes in the –aa mutant line compared to WT (Fig. 5 A). The heat map generated from this dataset displays the top significantly dysregulated genes relative to the WT group (Fig. 5 B). Among them, lgals3a was significantly downregulated in mutant samples (highlighted in bold in the heat map), validating the qPCR analysis (Fig. 1 ) and suggesting the premature degradation of the mutated mRNA. Additionally, several genes associated with the Wnt/β-catenin and TGFβ signalling pathways were found downregulated. A significant upregulation of genes related to M1 macrophage polarization, immune response, cell death, and adipogenesis was also observed, further supporting the findings discussed above. A detailed list of dysregulated genes is provided in Table 1. Cardiac dilation and structural changes in -aa mutated adult hearts In 1-year old mutant hearts, we observed significant ventricular dilation, normalized to body size (Fig. 7 A). Histological analysis revealed marked differences between WT and mutant hearts, including accumulation of adipocytes within the myocardial layer (arrowheads), leading to a pseudo-hypertrophy of the ventricular free wall, which measured up to 20 ± 4 µM compared to 10 ± 1 µM in WT hearts (double-headed arrows) (Fig. 7 B). Each heart was serially sectioned, and approximately 50% of the sections from each mutant heart exhibited this pathological phenotype. Notably, myocyte diameters were unaltered (4.2 ± 0.7 µM), and no fibrotic substitution was observed (data not shown). Pharmacological treatment with SB fully restored ventricular dilation to WT-like dimensions (Fig. 7 A). Histological analysis of treated mutants showed hypertrophy of the myocardial wall reaching up to 22 ± 4 µM and a mild increase in cardiomyocyte diameter (6 ± 1.2 µM), while adipocyte accumulation remained prominent (Fig. 7 B). Transmission Electron Microscopy (TEM) revealed disorganization and disruption of "pale" desmosomes at the sarcolemma of cardiomyocytes, accompanied by a significant increase in the extracellular distance between adjacent cardiomyocytes. These findings support a critical role of Gal-3 in maintaining the stability of cell-cell junctions and desmosomal structures. Following SB treatment, desmosome structures remained disrupted, and the extracellular space remained enlarged This suggests that although pathway activation via SB can strengthen the myocardium and reduce the ventricular chamber dilation, it is insufficient to restore the stability of the disrupted cell junctions (Fig. 7 C). Effect of JAK/STAT3 signalling pathway modulation on cardiac electrical activity The JAK/STAT3 pathway has been reported to be hyper-activated in a CS-Dsg2 mouse model 25 , where it modulates pro-inflammatory and fibrotic responses associated with desmosomal dysfunction. Building on this mechanistic insight, we investigated how the absence of lgals3a affects JAK/STAT3 signalling in a genetic background without additional desmosomal abnormalities. The qPCR analysis at the larval stage revealed upregulation of this pathway (Fig. 8 A). Consequently, we pharmacologically inhibited the pathway using a known antagonist, AG490, which partially ameliorated the pathological condition in mutants. Treatment resulted in complete recovery of ventricular contractility and a reduction in arrhythmic episodes. However, we observed a significant reduction in ventricular ejection fraction (Fig. 8 B). DISCUSSION GAL-3: heart involvement Increased Gal-3 expression has been correlated to heart failure and other cardiac conditions 26 , 27 . Specifically, higher Gal-3 plasma levels were observed in AC patients 3 , more often exhibiting ventricular tachycardia/fibrillation 1 . In zebrafish, the Gal-3 encoding gene, lgals3 , has undergone duplication, resulting in two paralogs: lgals3a and lgals3b . Recently, Lgals3a has been linked to zebrafish heart development 28 , with a knockdown model exhibiting cardiomyocyte apoptosis and pericardial effusion. In the present study, we described the first stable lgals3a ( -aa ) KO model, which exhibits approximately a 50% reduction in total Gal-3 ( lgals3a + lgals3b ) expression and associated cardiac dysfunction. Although a lgals3b mutant line was also obtained, it was not included in this study due to its low fertility, a phenotype likely stemming from the pleiotropic role of Gal-3 in the reproductive system, as previously observed in humans and mice 29 , 30 , 31 . Moreover, we leveraged data from a recently published study 22 that provides a comprehensive organ-wide, spatiotemporal transcriptomic and cellular atlas of the regenerating zebrafish heart. This dataset enabled us to further examine the expression profiles of lgals3a and lgals3b at the cellular level. Both genes are expressed across multiple cardiac cell populations, including endocardial cells, smooth muscle cells, fibroblasts, and macrophages. The expression pattern supports our focus on lgals3a in relation to the cardiac phenotype, given its substantially higher expression in the heart compared to lgals3b. Cardiac morphology and function were investigated thoroughly in – aa mutants, showing distinct cardiac abnormalities, including moderate cardiac dilation at 3 and 6 dpf, and pericardial effusion. Survival analysis showed an increase of 16% in mortality rate of -aa compared to controls with alongside reduced motility following light stimulation. Unlike humans, zebrafish larvae rely heavily on spontaneous movement to reach food sources and maintain survival, particularly at early developmental stages when autonomous feeding begins. Moreover, zebrafish are naturally active and highly responsive to environmental stimuli such as changes in light. Therefore, a reduction in motility in this context likely reflects decreased physical performance or increased fatigue, which could be viewed as functionally analogous to exercise intolerance or reduced effort tolerance frequently reported in AC patients, especially during ‘hot phases’ of disease progression. We hypothesize that zebrafish embryos presenting with the most severe cardiac manifestations, including pronounced pericardial and yolk sac edema, are less likely to survive the first month, contributing to the reduced survival rate. Cross-breeding the transgenic zebrafish line Tg(tg:EGFP-myl7) ia300 32 with our - aa model, enabled functional and morphological assessments of atrial and ventricular contractility, ejection fraction, and cardiac rhythm alterations, such as bradycardia. Notably, bradycardia has been reported as a recurrent phenotype in several zebrafish models of AC 33, 34, 35, 36 . In our model, however, we interpret bradycardia as a secondary consequence of early structural and electrical abnormalities, rather than as a core feature of AC. Arrhythmias became pronounced by 6 dpf, paralleling features observed during AC progression in humans, though species-specific differences must be considered. Critically, we acknowledge the challenge of directly linking early-stage morphological defects to long-term outcomes due to the inability to individually track fish over time. Zebrafish must be raised in social groups to preserve normal behavior, and unlike murine models, individuals cannot be permanently marked at early stages (e.g., via ear or tail clipping) without compromising their viability or development. GAL-3: Role in intercellular adhesion and cardiac structure Morphological and functional data obtained at larval stages were further monitored and validated in 1-year-old adult - aa zebrafish, to assess the long-term consequences of lgals3a depletion and to capture any progressive or late-onset cardiac remodelling, in line with the temporal progression of AC in humans. Notably, histological evaluations performed at 6 months of age revealed no structural abnormalities. However, analysis at one year of age revealed significantly increased ventricular dilation compared to WT controls, along with intra-myocardial adipose accumulation and ultrastructural changes, including disrupted and pale desmosomes. It is worth noting that zebrafish possess an innate ability to regenerate cardiac tissue following injury 37 , primarily through cardiomyocyte proliferation and minimal extracellular matrix deposition. As a result, zebrafish hearts typically do not develop persistent fibrotic scars, even in the presence of considerable structural and functional abnormalities. Electron-microscopy findings in - aa mutants show that Gal-3 reduction alone is sufficient to disrupt desmosomal adhesion in the heart . Disruption of intercellular adhesion mediated by desmosomal proteins, such as DSG2, is a hallmark in AC pathogenesis and has also been reported in other epithelial tissues. Notably, Gal-3 associates with the transmembrane cadherin glycoprotein DSG2 via N -linked glycans, resulting in a lactose-sensitive interaction that promotes DSG2 stability at the cell surface and supports epithelial intercellular adhesion 38 . Overall, our findings demonstrate for the first time that Gal-3 alone can regulate intercellular adhesion independently of genetic variants in other desmosomal-encoding genes. GAL-3: Role in intercellular adhesion and inflammation Altered function of intercellular junction proteins contributes not only to compromised tissue barrier but also to disruptions in tissue homeostasis observed in inflammatory disease states. Inflammatory and necroptotic/apoptotic processes 39 , 40 , 41 were active at both larval and adult stages in this stable - aa model, demonstrating that reduction of Gal-3 alone can initiate inflammatory cascades resembling the “hot phases” observed in human AC. L-Plastin was employed as a general marker of inflammatory cells; however it does not discriminate among immune cell subtypes such as neutrophils, macrophages, or lymphocytes. Therefore, while our immunostaining confirms an overall increase in inflammatory cells within cardiac tissue, it cannot specifically attribute this infiltration to macrophages alone. Supporting our previous study hypothesis 3 , the LGALS3 −/− mouse model 42 demonstrates that Gal-3 reduction is associated with inhibition of macrophage polarization, leading to increased expression of proinflammatory genes. Specifically, both our models and the LGALS3 -/- mouse show reduced TGFβ expression, confirming decreased activation of the TGFβ signalling pathway alongside elevated expression of proinflammatory molecules, which ultimately promote accumulation of proinflammatory macrophages. Indeed, Gal-3 knockdown in human macrophages 42 increased the expression of genes commonly associated with macrophage motility, accumulation, and repolarization, including MMP12, which plays a central role in enhancing the invasive capacity of Gal-3-deficient CD68 + macrophage subpopulations; CCL2, which promotes further macrophage recruitment and other proinflammatory molecules such as TNFα, PTSG2 (cyclooxygenase-2), and IL-6 43 . Similarly, RNA sequencing at adult stages revealed increased expression of genes belonging to the MMP family (e.g., MMP25 44 ), the CCL family (e.g., CCL38, an important macrophage-recruiting chemokine 45 ), and the interleukin family (e.g., IL16, which is significantly upregulated in M1-polarized macrophages and modulates macrophage polarization by regulating IL-6 expression 46 ). These findings suggest that Gal-3 deficiency fosters a proinflammatory microenvironment, with macrophages playing a central, though not exclusive, role in the observed cardiac pathology. Our comprehensive experimental data and literature review highlight the role of lgals3a as an antagonist of inflammation and proinflammatory macrophage polarization. Macrophages are dynamic immune responders to diverse environmental signals, and Gal-3 deficiency induces a clear shift toward proinflammatory phenotype. This shift parallels findings reported in AC models across species, including humans and mice, while acknowledging inherent interspecies differences 10 , 11 , 47 . GAL-3: Role in intercellular adhesion and signalling regulation Gal-3 exhibits pleiotropic biologic functions. Extracellularly, it interacts with cell-surface and extracellular matrix glycoproteins and glycolipids; intracellularly, it binds to cytoplasmic and nuclear proteins to modulate signalling pathways 48 . Reduced expression of lgals3 led to suppression of Wnt signalling, as evidenced by decreased levels of phospho-GSK3β at serine 9, resulting in increased GSK3β activity and subsequent β-catenin degradation 3 , 49 . To confirm this mechanism, pharmacological inhibition of GSK3β was achieved using SB216763 (SB), an ATP-competitive inhibitor, which induced β-catenin accumulation, a key downstream effector that activates canonical Wnt signalling. Previous studies have shown that aberrant GSK3β localization and activity contribute to AC progression, and that SB treatment can reverse disease phenotypes in both zebrafish and mouse models of AC. Specifically, SB was found to prevent myocyte injury and cardiac dysfunction in vivo 33 , 50 . The Wnt/β-catenin signalling pathway, which is physiologically active during epicardial and valve formation 51 , 52 , was significantly downregulated at both larval and adult stages. This downregulation may contribute to epicardial and valvular defetcts, including ventricular abnormalities, bradycardia, and arrhythmia. Pharmacological modulation of the Wnt/β-catenin pathway in our model yielded promising results, restoring several cardiac functional parameters and reducing inflammation at the larval stage. However, in adults, ventricular dilation was only partially rescued, and desmosomal structure remained compromised, accompanied by adipocytes accumulation and a hypertrophic myocardial response, potentially driven by prolonged Wnt/β-catenin activation. These findings suggest that, while early-stage intervention with Wnt/β-catenin modulators can mitigate cardiac deterioration, their efficacy declines in advanced disease stages due to persistent desmosomal instability. Thus, although the partial recovery of ventricular dilation is encouraging, Wnt/β-catenin activation alone may be insufficient to stabilize desmosomes, indicating that combined or complementary therapeutic strategies may be necessary. The JAK/STAT3 axis serves as a convergent node for multiple extracellular and intracellular signals, including cytokines (e.g., IL-6, IL-11), growth factors, and Toll-like receptor (TLR) signaling. The sustained proinflammatory state observed in our model may act as an activator of JAK/STAT3 signaling, which was found to be upregulated. However, pharmacological inhibition of the JAK/STAT3 pathway yielded only partial rescue at the larval stage, improving ventricular contractility and reducing arrhythmic events, but failing to achieve substantial functional recovery and ultimately resulting in heart failure. These findings suggest that targeting this pathway alone may be insufficient to reverse disease progression, although it may serve as a valuable adjunct to other therapeutic strategies aimed at improving cardiac outcomes. Study limitations This study has certain limitations that should be acknowledged. The assessment of Gal-3 expression in zebrafish larvae relied on RT-PCR at the cardiac and whole-body levels, leaving room for more anatomically detailed insights that techniques like whole-mount in situ hybridization could offer. Additionally, the RNA-seq data were derived from whole hearts containing a heterogeneous mixture of cardiomyocytes, fibroblasts, endothelial, and immune cells. Consequently, we cannot attribute observed transcriptional changes to specific cell types at this stage. Nonetheless, this tissue-level analysis offers valuable insight into the global transcriptional landscape impacted by the lgals3a depletion. Furthermore, this study did not explore whether simultaneously modulating Wnt/β-catenin and JAK/STAT3 signalling pathways could produce synergistic effects on disease phenotypes, which could represent an intriguing avenue for future research. CONCLUSIONS In conclusion, this study presents a novel and stable zebrafish model of lgals3a deficiency, providing strong evidence that Gal-3 is essential for preserving cardiac desmosome integrity. Through integrated morphological, functional, molecular, and pharmacological analyses, we demonstrate that Gal-3 depletion alone disrupts intercellular adhesion independently of other desmosomal gene variants and drives hallmark features of arrhythmogenic cardiomyopathy (AC), including inflammatory activation, Wnt/β-catenin signaling suppression, and progressive ventricular dysfunction. Emphasizing the importance of therapeutic timing, early pharmacological activation of Wnt signaling partially mitigated these defects. Collectively, our findings identify lgals3a as a critical regulator of cardiac structure and function in zebrafish and reinforce its translational value for elucidating AC pathogenesis and guiding treatment strategies. MATERIALS AND METHODS Sex as a biological variable Zebrafish of both sexes were used. Sex was not considered as a biological variable in this study. Zebrafish maintenance Danio rerio (zebrafish) were kept in a temperature-controlled environment (28.5°C), with a 12:12 light-dark cycle, staged and maintained following standard procedures. Wild-type (WT) lines used in this work, including those involved in the generation of the stable lgals3 mutant lines, were the Tuebingen, Giotto, and Umbria strains. The following transgenic lines were used: Wnt/β-catenin reporter line Tg(7xTCF-Xla.Siam:EGFP) ia4 53 and myocardial transgenic line Tg(tg:EGFP-myl7:EGFP) ia300 32 . These reporter lines, with and without mutated genetic backgrounds, were outcrossed with WT fish to obtain a heterozygous fluorescent signal in all analysed embryos. Generation and genetic analysis of lgals3a and lgals3b mutant lines To specifically target an optimal CRISPR sequence in exon 3 of zebrafish lgals3 genes ( lgals3a and lgals3b ), single guide RNAs (sgRNAs) were designed, using the CHOPCHOP software ( https://chopchop.cbu.uib.no/ ), generated according to Gagnon and colleagues 54 , and transcribed in vitro using the MEGAshortscript T7 kit (AM1354, Life Technologies, Milan, Italy) (Supplementary Table 1-A). One-cell stage embryos were injected with 2 nL of a solution containing 280 ng/µL of Cas9 protein (M0646, New England Biolabs, Milan, Italy) and 100 ng/µL of sgRNA; phenol red was used as an injection marker. Injected F0 embryos were raised to adulthood and screened, by F1 genotyping, for germline transmission of the mutation. Heterozygous mutants, harbouring the mutation of choice, were out-crossed 4 times and then incrossed to obtain homozygous mutants (F5 generation), stabilize the line and avoid CRISPR/Cas9-induced off-target effects. The WT fish used as controls in the experiments were siblings, originally obtained by outcrossing the mutants. All zebrafish lines generated in this study are available on request. DNA extraction from zebrafish embryos and adults The HotSHOT protocol 55 was used to prepare genomic DNA from whole embryos at 24–48 hours post fertilization (hpf). Each embryo was placed in a tube with 0.16 mg/mL Tricaine anesthetizing solution in fish water. After 2 min, Tricaine was discarded and the tube was filled with 50 µL of DNA lysis buffer (50 µM NaOH). The tubes were heated at 95°C for 20 min and then cooled at 4°C for 5 min; 5 µL of 1 M Tris HCl (pH 7.5) were added to neutralize the DNA sample. Genomic DNA from zebrafish adults was prepared after caudal fin biopsy (fin clipping). Genotyping of lgals3a and lgals3b mutant lines Homozygous and heterozygous deletions in lgals3a and lgals3b were identified by specific PCR, followed by agarose gel electrophoresis (3.5% w/v) and then by direct sequencing (Supplementary Table 1-B). RNA isolation and quantitative Real-Time Reverse Transcription PCR (qPCR) For gene expression analysis, total RNA was extracted using TRIzol reagent (15596026, Thermo Fisher Scientific, Milan, Italy) from pools of 30 zebrafish embryos at 3 days post-fertilization (dpf) or from hearts of 12-month-old zebrafish. One µg of total RNA was used for cDNA synthesis with M-MLV Reverse Transcriptase RNase H- (06-21-010000, Solis BioDyne, Tartu, Estonia), according to the manufacturer’s protocol. Quantitative PCR (qPCR) was performed in triplicate on batches of 3-dpf mutant larvae, and on pools of 1-year-old isolated mutant hearts, using the 5X HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne) and Light Cycler 480 II (Roche, Basel, Switzerland), following the manufacturer’s protocol. All primers were designed using the Primer3 software ( http://primer3.ut.ee ), with an optimal annealing temperature of 60°C (Supplementary Table 1-C). Analysis of cardiac morphology and activity Whole hearts and cardiac chambers' morphological variations were measured using a bright field microscope (Leica M165FC) equipped with a digital camera (Leica DFC7000T, Leica Microsystems, Milan, Italy), connected to a computer with Leica Software (LAS V4.8) for image acquisition and processing. Embryos anesthetized with 0.16 mg/mL Tricaine solution in fish water were placed on microscope slides with 2% methylcellulose in PBS 1X, oriented laterally. The adult zebrafish were euthanized with an overdose of Tricaine solution (0.30 mg/mL) in fish water to allow for dissection and organ extraction. The morphological abnormalities were quantified with ImageJ software. Cardiac activities including ventricular ejection fraction, relative contractility, heartbeats, and larval ventricular chamber area were measured using the pyHeart4Fish imaging software 56 . This software was rigorously tested and calibrated using pharmacological compounds with well-characterized effects on heart rate and rhythm, providing a reliable framework for arrhythmia detection and classification in zebrafish larvae. “Arrhythmia events” were defined as irregularities in cardiac rhythm, including beat-to-beat interval variability, bradyarrhythmia, and transient pauses, observed during high-speed video acquisition of larval zebrafish cardiac function. Embryos anesthetized with 0.16 mg/mL Tricaine solution in fish water were placed on agarose stamps made for zebrafish larvae (Stampwell, Idylle, Twin Helix, Milan, Italy), oriented ventrally to expose the heart. Parameters were derived from distinct and independent pools of larvae, collected and analyzed separately at 3 dpf and 6 dpf, respectively. Acridine Orange/Ethidium Bromide staining After manually removing the chorion in vivo with needle tools, the 3-dpf embryos were incubated for 30 minutes at RT in Acridine Orange/Ethidium Bromide ( AO/EB) staining solution: 15 µg/mL (5 µg/mL x dpf) of AO (A6014, Sigma-Aldrich, Milan, Italy) and 15 µg/mL (5 µg/mL x dpf) of EB (J62282.AB, Thermo Fisher Scientific, Milan, Italy) in fish water. Three 5-minute washes in fish water at RT were performed to remove the excess staining, with the last wash containing 0.16 mg/mL Tricaine to anesthetize the embryos and allow for easier acquisition. Embryos were then immediately placed on microscope slides with 2% methylcellulose and oriented laterally to acquire the signals as described in the "Fluorescent expression analysis" section in the supplementary materials. Locomotion assay Behavioural experiments were performed using the DanioVision tracking system (Noldus Information Technology, Wageningen, The Netherlands). Zebrafish larvae at 5 dpf were placed in 48-well plates, with one larva per well in 1 mL of fish water. After a 20-min acclimation period, movements of larvae were recorded over three cycles of 10 min light and 10 min dark, as previously described 47 . Pharmacological modulation of the Wnt/β-catenin and JAK/STAT3 signalling pathways Zebrafish mutant embryos were treated for 48 h with 40 µM SB216763 (SB) to activate Wnt/β-catenin signalling (S3442, Sigma-Aldrich, Milan, Italy) 47 . To inhibit JAK/STAT3 overexpression, a 50 µM concentration of AG490 (T3434, Sigma-Aldrich, Milan, Italy) was used 57 , 58 . All drugs administered at the larval stages were directly dissolved in fish water. At the adult stage, the SB drug was mixed with dry food and administered daily for 1 month. The dosage was determined by comparing a single fish with mouse models treated with the same drug by other groups 59 , 60 . The dosage in these studies varied from 0.6 to 4.8 mg/kg; therefore, we decided to use 2.4 mg/kg, the mean value. Fish were weighed (average weight: 380 mg) and the corresponding dose was calculated, resulting in 0.912 µg of drug per fish. This amount was dissolved in 0.123 µL of DMSO and mixed with food prior to administration. Fluorescent expression analysis Embryos anesthetized with 0.16 mg/mL Tricaine solution in fish water were placed on microscope slides with 2% methylcellulose in PBS 1X, oriented laterally. The emitted signals from the fluorescent tissues were acquired under a fluorescence microscope (Leica M165FC, Leica Microsystems, Milan, Italy) equipped with a digital camera (Leica DFC7000T, Leica Microsystems, Milan, Italy) and connected to a computer with Leica Software (LAS V4.8) for image acquisition and processing. The pixel intensity was analysed using ImageJ software. Immunofluorescence Embryos at 3 dpf or small pieces of 12-month-old zebrafish hearts were fixed in 4% PFA/PBS overnight at 4°C and stored at -20°C in 100% MetOH. Samples were rehydrated in MetOH/PBS series (75%, 50%, 25%), 5 minutes each, depigmented using 3% H 2 O 2 and 1% KOH in PBS 1X, and washed 2 times in PBS 1X + 0.5% Triton X. Samples were washed in distilled water for 5 minutes and frozen in acetone at -20°C for 7 minutes for tissue permeabilization. After additional washes for 5 minutes in distilled water and 5 minutes in PBS 1X + 0.5% Triton X, embryos were blocked for 30 minutes in PBDT (PBS 1X + 1% BSA + 1% DMSO + 0.5% Triton X) plus 2% Goat serum at room temperature, and then incubated for 2 days at 4°C in PBDT + 2% Goat serum + Rabbit Anti-L-Plastin antibody (ab210099, Abcam, Cambridge, UK) 1:5000. After four 15-minute washes in PBDT, embryos were incubated in PBDT + Goat-anti-Rabbit AP (Alkaline Phosphatase) conjugated antibodies at 1:1000, overnight at 4°C in the dark. Samples were washed four times for 15 minutes in PBDT and stained with 0.25 mg/mL Fast Blue BB (F3378, Sigma-Aldrich, Milan, Italy) + 0.25 mg/mL Naphthol-AS-MX-phosphate (N5000, Sigma-Aldrich, Milan, Italy) in staining buffer (100 mM Tris-HCl pH 8.2, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20). Finally, samples were stored in 4% PFA at 4°C in the dark or embedded in 1.5% low melting agarose on a glass dish for acquisition with the confocal microscope (Leica SP5, Leica Microsystems, Milan, Italy), exploiting the far-red emission from Fast Blue. The images were analysed using Volocity 6.0 software (Perkin Elmer, Milan, Italy). Transmission Electron Microscopy Small pieces of heart tissue (about 2–3 mm 3 ) were fixed with 2.5% glutaraldehyde (16220, EMS, Hatfield, PA, USA) plus 2% paraformaldehyde (P6148, Sigma-Aldrich, Milan, Italy) in 0.1 M sodium cacodylate buffer pH 7.4 overnight at 4°C. Subsequently, the samples were post-fixed with 1% PFA in 0.1 M sodium cacodylate buffer for 1 hour at 4°C. After three water washes, samples were dehydrated in graded EtOH series and embedded in epoxy resin (46345, Sigma-Aldrich, Milan, Italy). Ultrathin sections (60–70 nm) were obtained with a Leica Ultracut EM UC7 ultramicrotome, counterstained with uranyl acetate and lead citrate, and viewed with a Tecnai G2 transmission electron microscope (FEI, Hillsboro, OR, USA) operating at 100 kV. Images were captured with a Veleta digital camera (Olympus Soft Imaging System, Olympus, Segrate, Italy). At the desmosome level, the extracellular space distance was measured three times, at five different points, along each desmosome considered in the analysis. Birefringence analysis Anesthetized embryos were placed in 2% methylcellulose in PBS 1X solution and subsequently positioned on a glass slide. The sample was then inserted between two polarizing-rotating filters and analyzed under a Leica M165FC microscope (Leica Microsystems, Milan, Italy). Skeletal muscle birefringence was recorded in a bright field with a DFC7000T digital camera (Leica Microsystems, Milan, Italy), rotating the upper polarizing filter until the light refracted by the skeletal muscles was uniform and homogeneous along the entire larval body. The pixel intensity was then analyzed with ImageJ software. Histology Twelve-month-old adult zebrafish were euthanized and fixed in Bouin's solution (picric acid in milliQ water/formalin/glacial acetic acid) for a minimum of 24 h up to a maximum of 72 h at room temperature, depending on the size of the fish. To allow better fixation, the abdomen was opened along the ventral midline starting from the anal pore. The fixed samples were washed in 70% EtOH plus ammonia solution until the fish appeared with normal colour and the washing solution was clear. After dehydration in a series of graded EtOH and clearing by xylene, samples were embedded in paraffin. Paraffin sectioning (2–3 µm), hematoxylin & eosin (H&E) and Masson's trichrome staining were conducted based on standard procedures, and the sections were photographed under a light microscope. RNA sequencing analysis Zebrafish mutant (n = 6) and WT (n = 6) hearts were immediately minced with a sterile scalpel on ice, submerged in QIAzol (1.0 mL per ~ 10 mg of tissue; Qiagen, Milan, Italy) and homogenized using 5-mm steel beads in a TissueLyser instrument (Qiagen, Milan, Italy), with 2 cycles of 2 min at 30 mHz. The homogenized samples were then incubated with 20 µL of Proteinase K (20 mg/mL; Roche Diagnostics GmbH, Mannheim, Germany) for 10 min at 56°C. Subsequently, 200 µL of chloroform was added to each homogenized sample, which was then centrifuged at 12,000 rcf for 15 min at 4°C. Nucleic acids were purified using spin columns of the RNeasy mini kit with a 20-minute on-column DNase I digestion (Qiagen, Milan, Italy). RNA concentration was determined using a Qubit fluorometer (Life Technologies, Milan, Italy), and integrity was evaluated using a 4200 Tapestation (Agilent Technologies, Milan, Italy). cDNA library preparation and sequencing were performed following standard protocols of the CORALL Total RNA-Seq V2 kit (Lexogen, Vienna, Austria). Specifically, mRNA was obtained from total RNA using the RiboCop HMR plus Globin kit (Lexogen, Vienna, Austria). Individual zebrafish libraries were pooled, and sequencing was carried out for 76 cycles on the NextSeq 550 platform (Illumina, San Diego, CA, USA). Transcriptome assembly was performed on Illumina BaseSpace sequence Hub. Differential gene expression analysis was conducted using DESeq2. Genes were considered differentially expressed if presenting a Log2 FoldChange ± 1 and p-value < 0.05. Enriched pathways were analyzed using NetworkAnalyst ( https://www.networkanalyst.ca/ ) and Ingenuity Pathway Analysis IPA (Qiagen, Milan, Italy). Signal quantification and statistical analysis Signal quantification analysis was performed using the Measurements option of Volocity 6.0 software (Perkin Elmer, Milan, Italy) and ImageJ software. Pairwise analysis was carried out by unpaired t-test. Multiple comparisons were performed by one-way ANOVA followed by Tukey’s test, while survival analysis was conducted using Log-rank (Mantel-Cox) test (GraphPad Prism V7.0 software). The equality of the variances was analyzed by F-test. In the charts, error bars display standard errors of the mean. Asterisks indicate significant differences from controls. The correspondence between asterisks and significance levels is indicated in the figure legends. Sample final sizes were obtained after collection and randomization from multiple mating events, to reduce confounding effects from fish background, and excluding unfertilized eggs or embryos displaying very early developmental defects in all conditions. The sample size was preliminarily calculated using G*Power and Sample Size Calculator analysis. Single-blind experiments were performed at least in duplicate, with at least 10 processed individuals per condition, measured with at least two technical replicates, and with at least three samples per condition acquired for imaging analysis. Declarations Study approval All experiments were performed in accordance with Italian and European Legislation (Directive 2010/63/EU), and with permission for animal experimentation from the Ethics Committee of the University of Padova (OPBA) and the Italian Ministry of Health (Authorization numbers 407/2015-PR and 1111/2024-PR). Data and materials availability All raw data are available upon request to the corresponding author. CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest to declare. FUNDING This work was supported by the Italian Ministry of University and Research (MUR) (PRIN grant 20229FE439 - CUP C53D23004670006 " The mechanistic link between genetic substrate and immune reactions in inflammatory cardiomyopathies "), Rome; the Registry for Cardio-cerebro-vascular Pathology, Veneto region, Venice; and ARCA (Associazione Ricerche Cardiopatie Aritmiche), Padova, Italy. GR is a Postdoc Fellow supported by MUR (PRIN grant 2022WZCXRZ). RC and MBM are Postdoc Fellows supported by the Italian Ministry of Health, PNRR Next-Generation EU grant PNRR-MR1-2022-12376614. RBC is a Postdoc Fellow supported by DeBio, UniPD (grant 2024DIBIO1SIDASSEGNI-00095). NT was supported by the Italian Telethon Foundation (grant GGP19287) and MUR (grant PNRR M4C2 CN00000041). We thank the Zebrafish Facility staff at the Department of Biology, University of Padova, Italy. 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1","display":"","copyAsset":false,"role":"figure","size":389038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003elgals3a\u003c/em\u003eand \u003cem\u003elgals3b\u003c/em\u003e expression analysis. A: qPCR analysis of lgals3a and lgasl3b mRNA expression in WT and lgals3a (–aa) embryos at 3 dpf and adult hearts at 1 years old. The lgals3a mRNA expression decreases significantly in -aa samples, while the expression of lgals3b does not change. Each point on the graph corresponds to a pool of 30 embryos and3 hearts. Sample size larvae: lgasl3b n= 15; lgasl3a n = 24. Sample size adult: n = 6. Log2 FC: Log2 Fold Change. ** = p\u0026lt;0.01; **** = p\u0026lt;0.0001; ns = not significant. Test: Unpaired t-test. B: Single cell lgals3a and lgals3b expression analysis in zebrafish heart obtained from https://db.cngb.org/stomics/zebrafish_VRH/3d.model/. Black boxes highlight the myocardial, endocardial and epicardial populations.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/79d5f888852805d0ebbc8cd4.png"},{"id":96918084,"identity":"b51ed610-eadb-422a-871c-f78c1dbf14d0","added_by":"auto","created_at":"2025-11-27 14:11:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":566158,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and structural alterations of the cardiac region and heart chambers in \u003cem\u003e-aa\u003c/em\u003emutant larvae. Percentage of pericardial effusion. Sample size: WT n = 100; -\u003cem\u003eaa\u003c/em\u003e n = 100. At 3 dpf, mutant ventricle values were slightly but significantly decreased. The arrhythmia score at this developmental stage did not show a significant difference compared to the controls. Sample size: WT n ≈ 150; \u003cem\u003e-aa\u003c/em\u003e n ≈ 150. ** = p\u0026lt;0.01; **** = p\u0026lt;0.0001; ns = not significant. Test: Unpaired t-test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/a18b22eefccd65578b2c319e.png"},{"id":96917103,"identity":"aa9b8c3f-c790-45de-9742-e3efe6253abe","added_by":"auto","created_at":"2025-11-27 14:09:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":350120,"visible":true,"origin":"","legend":"\u003cp\u003eCell death events in the cardiac region of \u003cem\u003elgals3a\u003c/em\u003e mutant larvae. The mutant larvae presented a statistically significant increase in the number of apoptotic and necrotic cells in the heart tissue. Embryos are displayed at 3 dpf, in lateral view, anterior to the left. Sample size: WT: n = 9; \u003cem\u003e-aa\u003c/em\u003e: n = 10. ** = p\u0026lt;0.01; **** = p\u0026lt;0.0001. Test: Unpaired t-test. Red rectangular boxes indicate ROI. R. I. ROI: Relative Intensity ROI.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/f0c1208fa22ceb093de82ca4.png"},{"id":96808579,"identity":"8073eee3-7262-4067-ad24-4aa35dadd25e","added_by":"auto","created_at":"2025-11-26 09:36:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":546441,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Wnt/β-catenin signalling pathway drug modulation on the pathological phenotype of the \u003cem\u003e-aa\u003c/em\u003e mutant line. A: The Wnt/β-catenin-specific transgenic \u003cem\u003e-aa\u003c/em\u003e line showed a significant decrease in pathway activity in the epicardium and cardiac valves, rescued by SB treatment. Embryos are displayed at 3 dpf, in lateral view, anterior to the left. Sample size: WT n = 6; \u003cem\u003e-aa\u003c/em\u003e n = 6; \u003cem\u003e-aa\u003c/em\u003e + SB n = 10. R.I. ROI: Relative Intensity ROI. *** = p\u0026lt;0.001; **** = p\u0026lt;0.0001; ns = not significant. Test: One-way ANOVA followed by Tukey’s test. B: Complete restoration of normal levels of inflammatory cells in the cardiac region of mutant larvae after treatment. Sample size: WT n = 14; \u003cem\u003e-aa\u003c/em\u003e n = 16; \u003cem\u003e-aa\u003c/em\u003e + SB n = 8. **=p\u0026lt;0.01; ns = not significant. Test: One-way ANOVA followed by Tukey’s test. C: Significant rescue of the ejection fraction, with relative contractility becoming stronger and increased compared to the WT. Both chambers showed a complete rescue of the bradycardic phenotype. The arrhythmia episodes, after treatment, were significantly reduced. Sample size: WT n = 70; \u003cem\u003e-aa\u003c/em\u003e n = 70; \u003cem\u003e-aa\u003c/em\u003e + SB n = 70. * = p\u0026lt;0.05; ** = p\u0026lt;0.01; *** = p\u0026lt;0.001; **** = p\u0026lt;0.0001; ns = not significant. Test: One-way ANOVA followed by Tukey’s test\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/79a88990b52d8b9ec3673a1f.png"},{"id":96808603,"identity":"f98504cc-c2cb-4676-a23e-c733f8e79160","added_by":"auto","created_at":"2025-11-26 09:36:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":354136,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis of adult mutant hearts. A: Volcano plot showing genes with a LOG2FC of +/- 1 and p-value \u0026lt;0.05. B: Heat map generated from the volcano plot analysis containing genes significantly dysregulated compared to the WT group. Sample size: n = 3 for each genotype. P-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/063c6f42762c1ac26bca7430.png"},{"id":96808590,"identity":"5a145f84-21fb-44b8-8b65-33f835062534","added_by":"auto","created_at":"2025-11-26 09:36:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":281191,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map containing dysregulated genes and their associated pathways.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/dc1f1bbe4bf5ed9cfcdd7280.png"},{"id":96917586,"identity":"a436ed8b-6e21-4ddd-8eb8-9a681cafccd7","added_by":"auto","created_at":"2025-11-27 14:10:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3150450,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac dilation and structural changes in adult -\u003cem\u003eaa\u003c/em\u003emutant hearts. A: Morphological analysis showing significant ventricular dilation in mutants compared to WT controls, normalized to total fish length. Sample size: n = 15. R.R: Relative Ratio. * = p\u0026lt;0.05; *** = p\u0026lt;0.001; ns = not significant. Test: One-way ANOVA followed by Tukey’s test. B: Histological analysis of 1-year-old \u003cem\u003e-aa\u003c/em\u003emutant zebrafish and SB treatment. Adipocytes within the myocardial layer are indicated by arrowheads, and pseudo-hypertrophy of the ventricular free wall is marked by double-headed arrows. Sample size: n = 3 per condition. Scale bar: 500 μm (4X), 100 μm (20X), 50 μm for (40X). C: TEM analysis showing persistent disorganization and disruption of desmosomes at the sarcolemma after treatment. Sample size: WT = 3 ventricles and ≈ 50 desmosomes each; \u003cem\u003e-aa \u003c/em\u003e= 3 ventricles and ≈ 50 desmosomes each; \u003cem\u003e-aa \u003c/em\u003e+ SB\u003cem\u003e \u003c/em\u003e= 3 ventricles and ≈ 50 desmosomes each. ** = p\u0026lt;0.01; *** = p\u0026lt;0.001; **** = p\u0026lt;0.0001. Test: One-way ANOVA followed by Tukey’s test. Scale bar: 200 nm\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/06c6d95cdb11139f989d0c7c.png"},{"id":96808597,"identity":"d258bc7f-4718-4ceb-816c-e71ea38c977c","added_by":"auto","created_at":"2025-11-26 09:36:10","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":461583,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulation of JAK/STAT3 signalling and effects of drug modulation on cardiac electrical activity. A: qPCR analysis showing upregulation of JAK/STAT3 pathway components in \u003cem\u003elgals3a\u003c/em\u003e mutant larvae. Each data point represents a pool of 30 embryos of the same genotype. Sample size: n ≈ 15. Log2 FC: Log2 Fold Change; ** = p\u0026lt;0.01. Test: Unpaired t-test. B: Significant rescue of the relative contractility and arrhythmia episodes after AG490 treatment. Other cardiac parameters were largely unaffected, while ejection fraction worsened. Sample size: WT n ≈ 70; \u003cem\u003e-aa\u003c/em\u003e n ≈ 70; \u003cem\u003e-aa\u003c/em\u003e + AG490 n ≈ 90. * = p\u0026lt;0.05; ** = p\u0026lt;0.01; **** = p\u0026lt;0.0001; ns = not significant. Test: One-way ANOVA followed by Tukey’s test.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/0024ec1c8c700d2dac833992.jpeg"},{"id":97248712,"identity":"4cfef0ea-5054-4880-9e7e-1be94880a74c","added_by":"auto","created_at":"2025-12-02 13:06:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7176556,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/1b5c4646-84c0-4a2f-83e9-7f8bf764e485.pdf"},{"id":96917561,"identity":"d3c68119-55fd-4379-8c3e-2d79b8399726","added_by":"auto","created_at":"2025-11-27 14:10:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1323724,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"SupplementaryMaterial131025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/fa356ca60785a06debb1fd19.docx"},{"id":96917183,"identity":"40d6ee20-cce3-4b53-8ddf-5876963ab2ba","added_by":"auto","created_at":"2025-11-27 14:09:20","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7557656,"visible":true,"origin":"","legend":"Supplementary videos","description":"","filename":"Supplementaryvideos.rar","url":"https://assets-eu.researchsquare.com/files/rs-7855683/v1/b1c5729065e3a281cc0a4381.rar"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A zebrafish stable model of Galectin-3 to elucidate its role in Arrhythmogenic Cardiomyopathy","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGalectin-3 (Gal-3) dysregulation in cardiac tissue has been linked to a variety of cardiovascular diseases, underlying its involvement in physiological processes such as inflammation, fibrosis, and tissue remodelling \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In 2021, Cason and colleagues advocated a potential pathogenic role of \u003cem\u003eLGALS3\u003c/em\u003e in the myocardial injury of Arrhythmogenic Cardiomyopathy (AC) mediated by destabilization of desmosomes \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. AC is characterized by the progressive loss of ventricular myocardium due to myocyte death, with subsequent replacement by fibro-fatty scar tissue \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Rather than being a continuous process, AC progression might occur during periodic bursts (\u0026lsquo;hot phases\u0026rsquo;) in an otherwise stable disease \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Environmental factors such as physical exercise might facilitate disease progression by worsening cell adhesion disruption.\u003c/p\u003e\u003cp\u003ePrevious research on AC pathogenesis has shown that myocyte death is the primary trigger, followed by an inflammatory response and subsequent tissue repair through fibrous replacement \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, more recent studies suggest that inflammation occurs before the manifestation of overt histological abnormalities \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSpecifically, Gal-3 expression has been reported to fluctuate, with a downregulation during the early disease phase, followed by upregulation during acute \u0026lsquo;hot phases\u0026rsquo;, which are associated with exacerbated inflammation and tissue remodeling \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Gal-3 is known to be directly involved in the stabilization of intercellular junctions, through the interaction of N-linked β-galactosides on the extracellular domain of Desmoglein-2 (DSG2) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and the colocalization with Desmoplakin (DSP) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Pharmacological suppression of Gal-3 in the zebrafish model led to aberrant DSP localisation in epidermal cells, where desmosomes appeared separated from the cytoskeletal complex and unevenly distributed \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Gal-3 has a key role in inflammatory response through neutrophil activation and adhesion \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, chemo-attraction of macrophages \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and activation of mast cells \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Specifically, Gal-3 is considered a \u0026ldquo;macrophage activation marker\u0026rdquo; and is found to be highly expressed and secreted by mononuclear macrophages (CD68- and CD98-positive) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, we generated the first stable \u003cem\u003elgals3a\u003c/em\u003e knockout (KO) zebrafish line (\u003cem\u003e-aa\u003c/em\u003e) designed to mimic the early phases of AC, when Gal-3 is reduced, to allow us to investigate its mechanistic role in initiating disease-related inflammation, cardiac structural changes, and signaling dysregulation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGeneration of zebrafish\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant lines\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003elgals3\u003c/em\u003e gene is duplicated in zebrafish; therefore, both copies, \u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e, were targeted to generate \u003cem\u003elgals3\u003c/em\u003e zebrafish mutants. Accordingly, \u003cem\u003elgals3\u003c/em\u003e-KO zebrafish lines were generated using the CRISPR/Cas9 approach. The \u003cem\u003elgals3a\u003c/em\u003e (ia305) line carries a 4-bp deletion in exon 3 (Chr 13: g. 36578952\u0026not; 36578957del), causing a frameshift mutation that results in a truncated protein, lacking the C-terminal domain, of 218 amino acids. The synthesis of a non-functional protein, without the carbohydrate recognition domain (CRD), located at the C-terminus as in humans, is expected to cause its premature degradation. The \u003cem\u003elgals3b\u003c/em\u003e (ia306) line carries a 114-bp deletion in exon 3 (Chr 17: g10832046-10832160del), which removes nearly the entire exon. This region encodes the collagen-alpha domain, approximately 110 amino acids in length, which is involved in Gal-3 oligomerization and interactions with other proteins. The deletion removes 38 of these 110 amino acids, which is expected to induce a loss of protein function by disrupting its normal activity.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGenotyping of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3a\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3b\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ezebrafish mutants\u003c/span\u003e\u003c/p\u003e\u003cp\u003eAmplicons obtained from \u003cem\u003elgals3a\u003c/em\u003e PCR showed the WT single band of 178 bp, while the mutant amplicons displayed a shorter band of 174 bp, reflecting the 4-nucleotide deletion (Supplementary Fig.\u0026nbsp;1C). This result was confirmed by sequencing analysis: the homozygous mutant sample (Supplementary Fig.\u0026nbsp;1A) contains the 4-nucleotide deletion (red box), compared to the WT (Supplementary Fig.\u0026nbsp;1B). For the \u003cem\u003elgals3b\u003c/em\u003e gene, a difference of approximately 100 bp was observed, indicating a clear deletion in exon 3 (Supplementary Fig.\u0026nbsp;1F). Sanger sequencing further quantified this deletion as 114 bp (red box) (Supplementary Fig.\u0026nbsp;1D and 1E).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAnalysis of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3a/b\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emRNA levels in\u003c/span\u003e \u003cem\u003elgals3a\u003c/em\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant larvae\u003c/span\u003e\u003c/p\u003e\u003cp\u003eTo validate the effective genetic KO of the \u003cem\u003elgals3a\u003c/em\u003e gene in our line, we decided to investigate the effect on mRNA expression for both \u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e by qPCR. The results revealed a statistically significant downregulation of \u003cem\u003elgals3a\u003c/em\u003e mRNA quantity in a whole-body analysis at the larval stage. We confirmed that the mRNA expression of the \u003cem\u003elgals3b\u003c/em\u003e gene was not affected by changes in \u003cem\u003elgals3a\u003c/em\u003e, thus excluding a compensatory effect.\u003c/p\u003e\u003cp\u003eAt the adult stage, by surgically isolating the heart tissue, we confirmed a strong downregulation of \u003cem\u003elgals3a\u003c/em\u003e, whereas \u003cem\u003elgals3b\u003c/em\u003e showed expression comparable to WT. These results indicate that the 4-nt mutation in \u003cem\u003elgasl3a\u003c/em\u003e triggers the activation of an mRNA decay mechanism, causing the premature degradation of the non-functional mRNA and the subsequent downregulation of the related protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, re-analysis of data from a recently published study \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e revealed that both \u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e are expressed across multiple cardiac cell populations, including cardiomyocytes, endocardial and epicardial cells (black boxes), fibroblasts, and macrophages. Notably, \u003cem\u003elgals3a\u003c/em\u003e expression was markedly higher in the heart compared to \u003cem\u003elgals3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFertility analysis of the\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3b\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant line and selection of the\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3a\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eline\u003c/span\u003e\u003c/p\u003e\u003cp\u003eDuring the generation of stable \u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e KO zebrafish lines, we observed a strong decrease in fertility in \u003cem\u003elgals3b\u003c/em\u003e homozygous mutants. Therefore, a fertility analysis was conducted by outcrossing male and female \u003cem\u003elgals3b\u003c/em\u003e mutants with WT counterparts. The results underlined that \u003cem\u003elgals3b\u003c/em\u003e homozygous male fish have regular spermatogenesis; in fact, they can fertilize 72% of the total eggs, in line with the percentages obtained by crossing two WT fish. On the other side, \u003cem\u003elgals3b\u003c/em\u003e homozygous female fish exhibited abnormal ovulation, as they tend to release already degrading eggs, with 67% of them remaining unfertilized (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eConsidering that only \u003cem\u003elgals3b\u003c/em\u003e has been previously reported to be expressed in oocytes \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, together with our fertility analysis and the higher expression of \u003cem\u003elgals3a\u003c/em\u003e in the heart compared to \u003cem\u003elgals3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), we decided to focus exclusively on the \u003cem\u003elgals3a\u003c/em\u003e homozygous line, hereafter referred to as \u003cem\u003e-aa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMorphological and functional analysis in\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e-aa\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant larvae\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhen compared to WT, approximately 35% of \u003cem\u003e-aa\u003c/em\u003e mutant larvae at 3 dpf presented pericardial effusion. Functional imaging analysis, based on Supplementary videos 1\u0026ndash;2 and conducted using the pyHeart4Fish imaging software, assessed several cardiac parameters, including ventricular ejection fraction, relative contractility, and heartbeats in both chambers. At 3 dpf, these parameters were slightly but significantly reduced in the mutant ventricles, and both chambers displayed a clear bradycardic phenotype. Although the arrhythmia score at this developmental stage did not differ significantly from controls, a trend toward increased variability was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Due to the absence of arrhythmic events and the mild dysregulation of cardiac parameters at 3 dpf, we repeated the analysis at 6 dpf (discussed further on, Supplementary videos 3\u0026ndash;4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSurvival analysis of mutant lines at juvenile stages\u003c/h2\u003e\u003cp\u003eWe raised equal numbers of WT and mutant larvae (n\u0026thinsp;=\u0026thinsp;100 each) under identical conditions for one month, recording daily mortality. The survival rate of the mutant line was significantly lower (74%) compared to controls (90%) (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMotor behaviour analysis in\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e-aa\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant larvae\u003c/span\u003e\u003c/p\u003e\u003cp\u003eLocomotion experiments were conducted on zebrafish larvae at 5 dpf to investigate changes in motor behaviour related to the \u003cem\u003e-aa\u003c/em\u003e mutant. Prior to these experiments, muscle integrity was assessed via birefringence, which is brighter in well-organized skeletal muscle. This step ensured that any observed issues were due to cardiac dysfunction rather than muscular abnormalities. The analysis, which included two pools of larvae (WT and \u003cem\u003e-aa\u003c/em\u003e), is illustrated in Supplementary Fig.\u0026nbsp;4A-A\u0026rsquo;. Mean birefringence intensity, normalized to fish length, showed no significant difference between the two pools (Supplementary Fig.\u0026nbsp;4A\u0026rsquo;\u0026rsquo;), indicating preserved skeletal muscle structure in \u003cem\u003elgals3a\u003c/em\u003e mutant larvae. In contrast, motor behaviour analysis highlighted that while mutation in the \u003cem\u003elgals3a\u003c/em\u003e gene did not affect the larvae's ability to respond to light stimuli (dark/light intervals), it did decrease the normal motor response to these stimuli when compared to WT controls (Supplementary Fig.\u0026nbsp;4B). Notably, swimming activity during the dark phases was reduced only in the mutant group. However, the most pronounced difference appeared in the second part of the light phases, when the larvae began to recover from stress and start moving again. Here, \u003cem\u003e-aa\u003c/em\u003e larvae exhibited greater difficulty initiating movement than WT controls, suggesting increased fatigue. A statistically significant reduction in total distance swum, particularly during light phases, further supported the interpretation of intact sensory perception but impaired motor performance (Supplementary Fig.\u0026nbsp;4B\u0026rsquo;).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIncreased apoptotic and necrotic events in the cardiac region of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e\u0026ndash;aa\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant larvae\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWe investigated whether depletion of \u003cem\u003elgals3a\u003c/em\u003e leads to increased cell death events in the cardiac region of \u003cem\u003e-aa\u003c/em\u003e mutant larvae. AO/EB staining was used to discriminate between live, apoptotic and necrotic cells based on differential labelling of DNA and RNA. Our analysis revealed a significant increase in total cell death and necrotic events in the heart tissues of mutant larvae, suggesting that \u003cem\u003elgals3a\u003c/em\u003e plays a crucial role in regulating cell survival in this region. Additionally, dying cells were observed in the skin surrounding the cardiac region of mutant larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), likely due to tissue stress resulting from cardiac dilation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePathways analysis in mutant larvae\u003c/h3\u003e\n\u003cp\u003eWe investigated at larval stage potentially dysregulated pathways relevant to AC. In detail, we examined by qPCR the mRNA levels of two target genes involved in the Wnt/β-catenin signalling pathway, \u003cem\u003eccnd1\u003c/em\u003e and \u003cem\u003emyca\u003c/em\u003e, and found both significantly downregulated. Similarly, expression of the Hippo/YAP-TAZ pathway target genes \u003cem\u003eccn2a\u003c/em\u003e and \u003cem\u003eccn2b\u003c/em\u003e was also reduced. These findings are consistent with observations in human AC patients, where activation of the Hippo cascade leads to YAP phosphorylation, preventing its nuclear translocation and subsequent activation of target gene transcription \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Finally, TGFβ signalling appeared only mildly dysregulated (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e\n\u003ch3\u003eInflammation and cardiac function rescue after pharmacological treatment\u003c/h3\u003e\n\u003cp\u003eThe Wnt/β-catenin reporter line \u003cem\u003eTg(7xTCF-Xla.Siam:EGFP)\u003c/em\u003e\u003csup\u003e\u003cem\u003eia4\u003c/em\u003e\u003c/sup\u003e, crossed with the \u003cem\u003elgals3a\u003c/em\u003e mutant, revealed a significant tissue-specific reduction of this signal in the epicardium and cardiac valves, structures that rely on this pathway for proper development. This fluorescent mutant line allowed us to validate the efficacy of SB216763 (SB), a well-known agonist of the Wnt/β-catenin pathway, demontrating a clear restoration of reporter expression in the cardiac region after the treatment. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further characterized the phenotype by investigating potential inflammatory processes in the cardiac region of mutant larvae using an anti-L-plastin antibody as a marker of inflammatory cells. At the larval stage, immunostaining revealed a significant increase in L-plastin-positive cells compared to controls. These cells were not yet localized within heart tissue but accumulated in the surrounding cardiac region, suggesting recruitment potentially triggered by structural damage or functional impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Further investigation at the adult stage revealed the presence of inflammatory cells within the epicardial layer (Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eWe repeated the cardiac functional analysis at 6 dpf, observing a worsening of the mutant phenotype, compared to the 3 dpf stage previously shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Cardiac parameters were markedly impaired in the mutants, indicating disease progression consistent with the human condition. Episodes of arrhythmia were significantly more severe in the mutant group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eNotably, treatment with SB restored the physiological levels of inflammatory cells, improved ventricular contractility, and reduced the incidence of bradycardia and arrhythmic events. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B-C).\u003c/p\u003e\n\u003ch3\u003eRNAseq analysis in adult hearts\u003c/h3\u003e\n\u003cp\u003eRNAseq analysis identified 813 downregulated and 1,632 upregulated genes in the \u003cem\u003e\u0026ndash;aa\u003c/em\u003e mutant line compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The heat map generated from this dataset displays the top significantly dysregulated genes relative to the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Among them, \u003cem\u003elgals3a\u003c/em\u003e was significantly downregulated in mutant samples (highlighted in \u003cb\u003ebold\u003c/b\u003e in the heat map), validating the qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and suggesting the premature degradation of the mutated mRNA. Additionally, several genes associated with the Wnt/β-catenin and TGFβ signalling pathways were found downregulated. A significant upregulation of genes related to M1 macrophage polarization, immune response, cell death, and adipogenesis was also observed, further supporting the findings discussed above. A detailed list of dysregulated genes is provided in Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCardiac dilation and structural changes in\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e-aa\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutated adult hearts\u003c/span\u003e\u003c/p\u003e\u003cp\u003eIn 1-year old mutant hearts, we observed significant ventricular dilation, normalized to body size (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Histological analysis revealed marked differences between WT and mutant hearts, including accumulation of adipocytes within the myocardial layer (arrowheads), leading to a pseudo-hypertrophy of the ventricular free wall, which measured up to 20\u0026thinsp;\u0026plusmn;\u0026thinsp;4 \u0026micro;M compared to 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026micro;M in WT hearts (double-headed arrows) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Each heart was serially sectioned, and approximately 50% of the sections from each mutant heart exhibited this pathological phenotype. Notably, myocyte diameters were unaltered (4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 \u0026micro;M), and no fibrotic substitution was observed (data not shown). Pharmacological treatment with SB fully restored ventricular dilation to WT-like dimensions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Histological analysis of treated mutants showed hypertrophy of the myocardial wall reaching up to 22\u0026thinsp;\u0026plusmn;\u0026thinsp;4 \u0026micro;M and a mild increase in cardiomyocyte diameter (6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026micro;M), while adipocyte accumulation remained prominent (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTransmission Electron Microscopy (TEM) revealed disorganization and disruption of \"pale\" desmosomes at the sarcolemma of cardiomyocytes, accompanied by a significant increase in the extracellular distance between adjacent cardiomyocytes. These findings support a critical role of Gal-3 in maintaining the stability of cell-cell junctions and desmosomal structures. Following SB treatment, desmosome structures remained disrupted, and the extracellular space remained enlarged This suggests that although pathway activation via SB can strengthen the myocardium and reduce the ventricular chamber dilation, it is insufficient to restore the stability of the disrupted cell junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eEffect of JAK/STAT3 signalling pathway modulation on cardiac electrical activity\u003c/h3\u003e\n\u003cp\u003eThe JAK/STAT3 pathway has been reported to be hyper-activated in a CS-Dsg2 mouse model \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, where it modulates pro-inflammatory and fibrotic responses associated with desmosomal dysfunction. Building on this mechanistic insight, we investigated how the absence of \u003cem\u003elgals3a\u003c/em\u003e affects JAK/STAT3 signalling in a genetic background without additional desmosomal abnormalities. The qPCR analysis at the larval stage revealed upregulation of this pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Consequently, we pharmacologically inhibited the pathway using a known antagonist, AG490, which partially ameliorated the pathological condition in mutants. Treatment resulted in complete recovery of ventricular contractility and a reduction in arrhythmic episodes. However, we observed a significant reduction in ventricular ejection fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eGAL-3: heart involvement\u003c/h2\u003e\u003cp\u003eIncreased Gal-3 expression has been correlated to heart failure and other cardiac conditions \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Specifically, higher Gal-3 plasma levels were observed in AC patients \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, more often exhibiting ventricular tachycardia/fibrillation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In zebrafish, the Gal-3 encoding gene, \u003cem\u003elgals3\u003c/em\u003e, has undergone duplication, resulting in two paralogs: \u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e. Recently, Lgals3a has been linked to zebrafish heart development \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, with a knockdown model exhibiting cardiomyocyte apoptosis and pericardial effusion. In the present study, we described the first stable \u003cem\u003elgals3a\u003c/em\u003e (\u003cem\u003e-aa\u003c/em\u003e) KO model, which exhibits approximately a 50% reduction in total Gal-3 (\u003cem\u003elgals3a\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003elgals3b\u003c/em\u003e) expression and associated cardiac dysfunction. Although a \u003cem\u003elgals3b\u003c/em\u003e mutant line was also obtained, it was not included in this study due to its low fertility, a phenotype likely stemming from the pleiotropic role of Gal-3 in the reproductive system, as previously observed in humans and mice \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMoreover, we leveraged data from a recently published study \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e that provides a comprehensive organ-wide, spatiotemporal transcriptomic and cellular atlas of the regenerating zebrafish heart. This dataset enabled us to further examine the expression profiles of \u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e at the cellular level. Both genes are expressed across multiple cardiac cell populations, including endocardial cells, smooth muscle cells, fibroblasts, and macrophages. The expression pattern supports our focus on \u003cem\u003elgals3a\u003c/em\u003e in relation to the cardiac phenotype, given its substantially higher expression in the heart compared to \u003cem\u003elgals3b.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCardiac morphology and function were investigated thoroughly in \u0026ndash;\u003cem\u003eaa\u003c/em\u003e mutants, showing distinct cardiac abnormalities, including moderate cardiac dilation at 3 and 6 dpf, and pericardial effusion. Survival analysis showed an increase of 16% in mortality rate of \u003cem\u003e-aa\u003c/em\u003e compared to controls with alongside reduced motility following light stimulation. Unlike humans, zebrafish larvae rely heavily on spontaneous movement to reach food sources and maintain survival, particularly at early developmental stages when autonomous feeding begins. Moreover, zebrafish are naturally active and highly responsive to environmental stimuli such as changes in light. Therefore, a reduction in motility in this context likely reflects decreased physical performance or increased fatigue, which could be viewed as functionally analogous to exercise intolerance or reduced effort tolerance frequently reported in AC patients, especially during \u0026lsquo;hot phases\u0026rsquo; of disease progression. We hypothesize that zebrafish embryos presenting with the most severe cardiac manifestations, including pronounced pericardial and yolk sac edema, are less likely to survive the first month, contributing to the reduced survival rate.\u003c/p\u003e\u003cp\u003eCross-breeding the transgenic zebrafish line \u003cem\u003eTg(tg:EGFP-myl7)\u003c/em\u003e\u003csup\u003e\u003cem\u003eia300\u003c/em\u003e 32\u003c/sup\u003e with our -\u003cem\u003eaa\u003c/em\u003e model, enabled functional and morphological assessments of atrial and ventricular contractility, ejection fraction, and cardiac rhythm alterations, such as bradycardia. Notably, bradycardia has been reported as a recurrent phenotype in several zebrafish models of AC \u003csup\u003e33, 34, 35, 36\u003c/sup\u003e. In our model, however, we interpret bradycardia as a secondary consequence of early structural and electrical abnormalities, rather than as a core feature of AC. Arrhythmias became pronounced by 6 dpf, paralleling features observed during AC progression in humans, though species-specific differences must be considered. Critically, we acknowledge the challenge of directly linking early-stage morphological defects to long-term outcomes due to the inability to individually track fish over time. Zebrafish must be raised in social groups to preserve normal behavior, and unlike murine models, individuals cannot be permanently marked at early stages (e.g., via ear or tail clipping) without compromising their viability or development.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGAL-3: Role in intercellular adhesion and cardiac structure\u003c/h3\u003e\n\u003cp\u003eMorphological and functional data obtained at larval stages were further monitored and validated in 1-year-old adult -\u003cem\u003eaa\u003c/em\u003e zebrafish, to assess the long-term consequences of \u003cem\u003elgals3a\u003c/em\u003e depletion and to capture any progressive or late-onset cardiac remodelling, in line with the temporal progression of AC in humans. Notably, histological evaluations performed at 6 months of age revealed no structural abnormalities. However, analysis at one year of age revealed significantly increased ventricular dilation compared to WT controls, along with intra-myocardial adipose accumulation and ultrastructural changes, including disrupted and pale desmosomes. It is worth noting that zebrafish possess an innate ability to regenerate cardiac tissue following injury \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, primarily through cardiomyocyte proliferation and minimal extracellular matrix deposition. As a result, zebrafish hearts typically do not develop persistent fibrotic scars, even in the presence of considerable structural and functional abnormalities.\u003c/p\u003e\u003cp\u003eElectron-microscopy findings in -\u003cem\u003eaa\u003c/em\u003e mutants show that \u003cb\u003eGal-3 reduction alone is sufficient to disrupt desmosomal adhesion in the heart\u003c/b\u003e. Disruption of intercellular adhesion mediated by desmosomal proteins, such as DSG2, is a hallmark in AC pathogenesis and has also been reported in other epithelial tissues. Notably, Gal-3 associates with the transmembrane cadherin glycoprotein DSG2 via \u003cem\u003eN\u003c/em\u003e-linked glycans, resulting in a lactose-sensitive interaction that promotes DSG2 stability at the cell surface and supports epithelial intercellular adhesion \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e\u003cp\u003eOverall, our findings demonstrate for the first time that Gal-3 alone can regulate intercellular adhesion independently of genetic variants in other desmosomal-encoding genes.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eGAL-3: Role in intercellular adhesion and inflammation\u003c/h2\u003e\u003cp\u003eAltered function of intercellular junction proteins contributes not only to compromised tissue barrier but also to disruptions in tissue homeostasis observed in inflammatory disease states. Inflammatory and necroptotic/apoptotic processes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e were active at both larval and adult stages in this stable -\u003cem\u003eaa\u003c/em\u003e model, demonstrating that reduction of Gal-3 alone can initiate inflammatory cascades resembling the \u0026ldquo;hot phases\u0026rdquo; observed in human AC. L-Plastin was employed as a general marker of inflammatory cells; however it does not discriminate among immune cell subtypes such as neutrophils, macrophages, or lymphocytes. Therefore, while our immunostaining confirms an overall increase in inflammatory cells within cardiac tissue, it cannot specifically attribute this infiltration to macrophages alone.\u003c/p\u003e\u003cp\u003eSupporting our previous study hypothesis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the LGALS3 \u0026minus;/\u0026minus; mouse model \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e demonstrates that Gal-3 reduction is associated with inhibition of macrophage polarization, leading to increased expression of proinflammatory genes. Specifically, both our models and the LGALS3 -/- mouse show reduced TGFβ expression, confirming decreased activation of the TGFβ signalling pathway alongside elevated expression of proinflammatory molecules, which ultimately promote accumulation of proinflammatory macrophages. Indeed, Gal-3 knockdown in human macrophages \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e increased the expression of genes commonly associated with macrophage motility, accumulation, and repolarization, including MMP12, which plays a central role in enhancing the invasive capacity of Gal-3-deficient CD68\u0026thinsp;+\u0026thinsp;macrophage subpopulations; CCL2, which promotes further macrophage recruitment and other proinflammatory molecules such as TNFα, PTSG2 (cyclooxygenase-2), and IL-6 \u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSimilarly, RNA sequencing at adult stages revealed increased expression of genes belonging to the MMP family (e.g., MMP25 \u003csup\u003e44\u003c/sup\u003e), the CCL family (e.g., CCL38, an important macrophage-recruiting chemokine \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e), and the interleukin family (e.g., IL16, which is significantly upregulated in M1-polarized macrophages and modulates macrophage polarization by regulating IL-6 expression \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e). These findings suggest that Gal-3 deficiency fosters a proinflammatory microenvironment, with macrophages playing a central, though not exclusive, role in the observed cardiac pathology.\u003c/p\u003e\u003cp\u003eOur comprehensive experimental data and literature review highlight the role of \u003cem\u003elgals3a\u003c/em\u003e as an antagonist of inflammation and proinflammatory macrophage polarization. Macrophages are dynamic immune responders to diverse environmental signals, and \u003cb\u003eGal-3 deficiency induces a clear shift toward proinflammatory phenotype.\u003c/b\u003e This shift parallels findings reported in AC models across species, including humans and mice, while acknowledging inherent interspecies differences \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eGAL-3: Role in intercellular adhesion and signalling regulation\u003c/h2\u003e\u003cp\u003eGal-3 exhibits pleiotropic biologic functions. Extracellularly, it interacts with cell-surface and extracellular matrix glycoproteins and glycolipids; intracellularly, it binds to cytoplasmic and nuclear proteins to modulate signalling pathways \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eReduced expression of \u003cem\u003elgals3\u003c/em\u003e led to suppression of Wnt signalling, as evidenced by decreased levels of phospho-GSK3β at serine 9, resulting in increased GSK3β activity and subsequent β-catenin degradation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. To confirm this mechanism, pharmacological inhibition of GSK3β was achieved using SB216763 (SB), an ATP-competitive inhibitor, which induced β-catenin accumulation, a key downstream effector that activates canonical Wnt signalling. Previous studies have shown that aberrant GSK3β localization and activity contribute to AC progression, and that SB treatment can reverse disease phenotypes in both zebrafish and mouse models of AC. Specifically, SB was found to prevent myocyte injury and cardiac dysfunction \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The Wnt/β-catenin signalling pathway, which is physiologically active during epicardial and valve formation \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, was significantly downregulated at both larval and adult stages. This downregulation may contribute to epicardial and valvular defetcts, including ventricular abnormalities, bradycardia, and arrhythmia.\u003c/p\u003e\u003cp\u003ePharmacological modulation of the Wnt/β-catenin pathway in our model yielded promising results, restoring several cardiac functional parameters and reducing inflammation at the larval stage.\u003c/p\u003e\u003cp\u003eHowever, in adults, ventricular dilation was only partially rescued, and desmosomal structure remained compromised, accompanied by adipocytes accumulation and a hypertrophic myocardial response, potentially driven by prolonged Wnt/β-catenin activation. These findings suggest that, while early-stage intervention with Wnt/β-catenin modulators can mitigate cardiac deterioration, their efficacy declines in advanced disease stages due to persistent desmosomal instability. Thus, although the partial recovery of ventricular dilation is encouraging, Wnt/β-catenin activation alone may be insufficient to stabilize desmosomes, indicating that combined or complementary therapeutic strategies may be necessary.\u003c/p\u003e\u003cp\u003eThe JAK/STAT3 axis serves as a convergent node for multiple extracellular and intracellular signals, including cytokines (e.g., IL-6, IL-11), growth factors, and Toll-like receptor (TLR) signaling. The sustained proinflammatory state observed in our model may act as an activator of JAK/STAT3 signaling, which was found to be upregulated. However, pharmacological inhibition of the JAK/STAT3 pathway yielded only partial rescue at the larval stage, improving ventricular contractility and reducing arrhythmic events, but failing to achieve substantial functional recovery and ultimately resulting in heart failure. These findings suggest that targeting this pathway alone may be insufficient to reverse disease progression, although it may serve as a valuable adjunct to other therapeutic strategies aimed at improving cardiac outcomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStudy limitations\u003c/h2\u003e\u003cp\u003eThis study has certain limitations that should be acknowledged. The assessment of Gal-3 expression in zebrafish larvae relied on RT-PCR at the cardiac and whole-body levels, leaving room for more anatomically detailed insights that techniques like whole-mount \u003cem\u003ein situ\u003c/em\u003e hybridization could offer. Additionally, the RNA-seq data were derived from whole hearts containing a heterogeneous mixture of cardiomyocytes, fibroblasts, endothelial, and immune cells. Consequently, we cannot attribute observed transcriptional changes to specific cell types at this stage. Nonetheless, this tissue-level analysis offers valuable insight into the global transcriptional landscape impacted by the \u003cem\u003elgals3a\u003c/em\u003e depletion. Furthermore, this study did not explore whether simultaneously modulating Wnt/β-catenin and JAK/STAT3 signalling pathways could produce synergistic effects on disease phenotypes, which could represent an intriguing avenue for future research.\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, this study presents a novel and stable zebrafish model of lgals3a deficiency, providing strong evidence that Gal-3 is essential for preserving cardiac desmosome integrity. Through integrated morphological, functional, molecular, and pharmacological analyses, we demonstrate that Gal-3 depletion alone disrupts intercellular adhesion independently of other desmosomal gene variants and drives hallmark features of arrhythmogenic cardiomyopathy (AC), including inflammatory activation, Wnt/β-catenin signaling suppression, and progressive ventricular dysfunction. Emphasizing the importance of therapeutic timing, early pharmacological activation of Wnt signaling partially mitigated these defects. Collectively, our findings identify lgals3a as a critical regulator of cardiac structure and function in zebrafish and reinforce its translational value for elucidating AC pathogenesis and guiding treatment strategies.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSex as a biological variable\u003c/h2\u003e\u003cp\u003eZebrafish of both sexes were used. Sex was not considered as a biological variable in this study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eZebrafish maintenance\u003c/h2\u003e\u003cp\u003e\u003cem\u003eDanio rerio\u003c/em\u003e (zebrafish) were kept in a temperature-controlled environment (28.5\u0026deg;C), with a 12:12 light-dark cycle, staged and maintained following standard procedures. Wild-type (WT) lines used in this work, including those involved in the generation of the stable \u003cem\u003elgals3\u003c/em\u003e mutant lines, were the Tuebingen, Giotto, and Umbria strains. The following transgenic lines were used: Wnt/β-catenin reporter line \u003cem\u003eTg(7xTCF-Xla.Siam:EGFP)\u003c/em\u003e\u003csup\u003e\u003cem\u003eia4\u003c/em\u003e 53\u003c/sup\u003e and myocardial transgenic line \u003cem\u003eTg(tg:EGFP-myl7:EGFP)\u003c/em\u003e\u003csup\u003e\u003cem\u003eia300\u003c/em\u003e 32\u003c/sup\u003e. These reporter lines, with and without mutated genetic backgrounds, were outcrossed with WT fish to obtain a heterozygous fluorescent signal in all analysed embryos.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGeneration and genetic analysis of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3a\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3b\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant lines\u003c/span\u003e\u003c/p\u003e\u003cp\u003eTo specifically target an optimal CRISPR sequence in exon 3 of zebrafish \u003cem\u003elgals3\u003c/em\u003e genes (\u003cem\u003elgals3a\u003c/em\u003e and \u003cem\u003elgals3b\u003c/em\u003e), single guide RNAs (sgRNAs) were designed, using the CHOPCHOP software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chopchop.cbu.uib.no/\u003c/span\u003e\u003cspan address=\"https://chopchop.cbu.uib.no/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), generated according to Gagnon and colleagues \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and transcribed \u003cem\u003ein vitro\u003c/em\u003e using the MEGAshortscript T7 kit (AM1354, Life Technologies, Milan, Italy) (Supplementary Table\u0026nbsp;1-A). One-cell stage embryos were injected with 2 nL of a solution containing 280 ng/\u0026micro;L of Cas9 protein (M0646, New England Biolabs, Milan, Italy) and 100 ng/\u0026micro;L of sgRNA; phenol red was used as an injection marker. Injected F0 embryos were raised to adulthood and screened, by F1 genotyping, for germline transmission of the mutation. Heterozygous mutants, harbouring the mutation of choice, were out-crossed 4 times and then incrossed to obtain homozygous mutants (F5 generation), stabilize the line and avoid CRISPR/Cas9-induced off-target effects. The WT fish used as controls in the experiments were siblings, originally obtained by outcrossing the mutants. All zebrafish lines generated in this study are available on request.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDNA extraction from zebrafish embryos and adults\u003c/h2\u003e\u003cp\u003eThe HotSHOT protocol \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e was used to prepare genomic DNA from whole embryos at 24\u0026ndash;48 hours post fertilization (hpf). Each embryo was placed in a tube with 0.16 mg/mL Tricaine anesthetizing solution in fish water. After 2 min, Tricaine was discarded and the tube was filled with 50 \u0026micro;L of DNA lysis buffer (50 \u0026micro;M NaOH). The tubes were heated at 95\u0026deg;C for 20 min and then cooled at 4\u0026deg;C for 5 min; 5 \u0026micro;L of 1 M Tris HCl (pH 7.5) were added to neutralize the DNA sample. Genomic DNA from zebrafish adults was prepared after caudal fin biopsy (fin clipping).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGenotyping of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003elgals3a and lgals3b\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emutant lines\u003c/span\u003e\u003c/p\u003e\u003cp\u003eHomozygous and heterozygous deletions in \u003cem\u003elgals3a and lgals3b\u003c/em\u003e were identified by specific PCR, followed by agarose gel electrophoresis (3.5% w/v) and then by direct sequencing (Supplementary Table\u0026nbsp;1-B).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eRNA isolation and quantitative Real-Time Reverse Transcription PCR (qPCR)\u003c/h2\u003e\u003cp\u003eFor gene expression analysis, total RNA was extracted using TRIzol reagent (15596026, Thermo Fisher Scientific, Milan, Italy) from pools of 30 zebrafish embryos at 3 days post-fertilization (dpf) or from hearts of 12-month-old zebrafish. One \u0026micro;g of total RNA was used for cDNA synthesis with M-MLV Reverse Transcriptase RNase H- (06-21-010000, Solis BioDyne, Tartu, Estonia), according to the manufacturer\u0026rsquo;s protocol. Quantitative PCR (qPCR) was performed in triplicate on batches of 3-dpf mutant larvae, and on pools of 1-year-old isolated mutant hearts, using the 5X HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne) and Light Cycler 480 II (Roche, Basel, Switzerland), following the manufacturer\u0026rsquo;s protocol. All primers were designed using the Primer3 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://primer3.ut.ee\u003c/span\u003e\u003cspan address=\"http://primer3.ut.ee\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with an optimal annealing temperature of 60\u0026deg;C (Supplementary Table\u0026nbsp;1-C).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of cardiac morphology and activity\u003c/h2\u003e\u003cp\u003eWhole hearts and cardiac chambers' morphological variations were measured using a bright field microscope (Leica M165FC) equipped with a digital camera (Leica DFC7000T, Leica Microsystems, Milan, Italy), connected to a computer with Leica Software (LAS V4.8) for image acquisition and processing. Embryos anesthetized with 0.16 mg/mL Tricaine solution in fish water were placed on microscope slides with 2% methylcellulose in PBS 1X, oriented laterally. The adult zebrafish were euthanized with an overdose of Tricaine solution (0.30 mg/mL) in fish water to allow for dissection and organ extraction. The morphological abnormalities were quantified with ImageJ software. Cardiac activities including ventricular ejection fraction, relative contractility, heartbeats, and larval ventricular chamber area were measured using the pyHeart4Fish imaging software \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This software was rigorously tested and calibrated using pharmacological compounds with well-characterized effects on heart rate and rhythm, providing a reliable framework for arrhythmia detection and classification in zebrafish larvae. \u0026ldquo;Arrhythmia events\u0026rdquo; were defined as irregularities in cardiac rhythm, including beat-to-beat interval variability, bradyarrhythmia, and transient pauses, observed during high-speed video acquisition of larval zebrafish cardiac function. Embryos anesthetized with 0.16 mg/mL Tricaine solution in fish water were placed on agarose stamps made for zebrafish larvae (Stampwell, Idylle, Twin Helix, Milan, Italy), oriented ventrally to expose the heart. Parameters were derived from distinct and independent pools of larvae, collected and analyzed separately at 3 dpf and 6 dpf, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eAcridine Orange/Ethidium Bromide staining\u003c/h2\u003e\u003cp\u003eAfter manually removing the chorion \u003cem\u003ein vivo\u003c/em\u003e with needle tools, the 3-dpf embryos were incubated for 30 minutes at RT in \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAcridine Orange/Ethidium Bromide (\u003c/span\u003eAO/EB) staining solution: 15 \u0026micro;g/mL (5 \u0026micro;g/mL x dpf) of AO (A6014, Sigma-Aldrich, Milan, Italy) and 15 \u0026micro;g/mL (5 \u0026micro;g/mL x dpf) of EB (J62282.AB, Thermo Fisher Scientific, Milan, Italy) in fish water. Three 5-minute washes in fish water at RT were performed to remove the excess staining, with the last wash containing 0.16 mg/mL Tricaine to anesthetize the embryos and allow for easier acquisition. Embryos were then immediately placed on microscope slides with 2% methylcellulose and oriented laterally to acquire the signals as described in the \"Fluorescent expression analysis\" section in the supplementary materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eLocomotion assay\u003c/h2\u003e\u003cp\u003eBehavioural experiments were performed using the DanioVision tracking system (Noldus Information Technology, Wageningen, The Netherlands). Zebrafish larvae at 5 dpf were placed in 48-well plates, with one larva per well in 1 mL of fish water. After a 20-min acclimation period, movements of larvae were recorded over three cycles of 10 min light and 10 min dark, as previously described \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003ePharmacological modulation of the Wnt/β-catenin and JAK/STAT3 signalling pathways\u003c/h2\u003e\u003cp\u003eZebrafish mutant embryos were treated for 48 h with 40 \u0026micro;M SB216763 (SB) to activate Wnt/β-catenin signalling (S3442, Sigma-Aldrich, Milan, Italy) \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. To inhibit JAK/STAT3 overexpression, a 50 \u0026micro;M concentration of AG490 (T3434, Sigma-Aldrich, Milan, Italy) was used \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. All drugs administered at the larval stages were directly dissolved in fish water. At the adult stage, the SB drug was mixed with dry food and administered daily for 1 month. The dosage was determined by comparing a single fish with mouse models treated with the same drug by other groups \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The dosage in these studies varied from 0.6 to 4.8 mg/kg; therefore, we decided to use 2.4 mg/kg, the mean value. Fish were weighed (average weight: 380 mg) and the corresponding dose was calculated, resulting in 0.912 \u0026micro;g of drug per fish. This amount was dissolved in 0.123 \u0026micro;L of DMSO and mixed with food prior to administration.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eFluorescent expression analysis\u003c/h2\u003e\u003cp\u003eEmbryos anesthetized with 0.16 mg/mL Tricaine solution in fish water were placed on microscope slides with 2% methylcellulose in PBS 1X, oriented laterally. The emitted signals from the fluorescent tissues were acquired under a fluorescence microscope (Leica M165FC, Leica Microsystems, Milan, Italy) equipped with a digital camera (Leica DFC7000T, Leica Microsystems, Milan, Italy) and connected to a computer with Leica Software (LAS V4.8) for image acquisition and processing. The pixel intensity was analysed using ImageJ software.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eEmbryos at 3 dpf or small pieces of 12-month-old zebrafish hearts were fixed in 4% PFA/PBS overnight at 4\u0026deg;C and stored at -20\u0026deg;C in 100% MetOH. Samples were rehydrated in MetOH/PBS series (75%, 50%, 25%), 5 minutes each, depigmented using 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 1% KOH in PBS 1X, and washed 2 times in PBS 1X\u0026thinsp;+\u0026thinsp;0.5% Triton X. Samples were washed in distilled water for 5 minutes and frozen in acetone at -20\u0026deg;C for 7 minutes for tissue permeabilization. After additional washes for 5 minutes in distilled water and 5 minutes in PBS 1X\u0026thinsp;+\u0026thinsp;0.5% Triton X, embryos were blocked for 30 minutes in PBDT (PBS 1X\u0026thinsp;+\u0026thinsp;1% BSA\u0026thinsp;+\u0026thinsp;1% DMSO\u0026thinsp;+\u0026thinsp;0.5% Triton X) plus 2% Goat serum at room temperature, and then incubated for 2 days at 4\u0026deg;C in PBDT\u0026thinsp;+\u0026thinsp;2% Goat serum\u0026thinsp;+\u0026thinsp;Rabbit Anti-L-Plastin antibody (ab210099, Abcam, Cambridge, UK) 1:5000. After four 15-minute washes in PBDT, embryos were incubated in PBDT\u0026thinsp;+\u0026thinsp;Goat-anti-Rabbit AP (Alkaline Phosphatase) conjugated antibodies at 1:1000, overnight at 4\u0026deg;C in the dark. Samples were washed four times for 15 minutes in PBDT and stained with 0.25 mg/mL Fast Blue BB (F3378, Sigma-Aldrich, Milan, Italy)\u0026thinsp;+\u0026thinsp;0.25 mg/mL Naphthol-AS-MX-phosphate (N5000, Sigma-Aldrich, Milan, Italy) in staining buffer (100 mM Tris-HCl pH 8.2, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20). Finally, samples were stored in 4% PFA at 4\u0026deg;C in the dark or embedded in 1.5% low melting agarose on a glass dish for acquisition with the confocal microscope (Leica SP5, Leica Microsystems, Milan, Italy), exploiting the far-red emission from Fast Blue. The images were analysed using Volocity 6.0 software (Perkin Elmer, Milan, Italy).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eTransmission Electron Microscopy\u003c/h2\u003e\u003cp\u003eSmall pieces of heart tissue (about 2\u0026ndash;3 mm\u003csup\u003e3\u003c/sup\u003e) were fixed with 2.5% glutaraldehyde (16220, EMS, Hatfield, PA, USA) plus 2% paraformaldehyde (P6148, Sigma-Aldrich, Milan, Italy) in 0.1 M sodium cacodylate buffer pH 7.4 overnight at 4\u0026deg;C. Subsequently, the samples were post-fixed with 1% PFA in 0.1 M sodium cacodylate buffer for 1 hour at 4\u0026deg;C. After three water washes, samples were dehydrated in graded EtOH series and embedded in epoxy resin (46345, Sigma-Aldrich, Milan, Italy). Ultrathin sections (60\u0026ndash;70 nm) were obtained with a Leica Ultracut EM UC7 ultramicrotome, counterstained with uranyl acetate and lead citrate, and viewed with a Tecnai G2 transmission electron microscope (FEI, Hillsboro, OR, USA) operating at 100 kV. Images were captured with a Veleta digital camera (Olympus Soft Imaging System, Olympus, Segrate, Italy). At the desmosome level, the extracellular space distance was measured three times, at five different points, along each desmosome considered in the analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eBirefringence analysis\u003c/h2\u003e\u003cp\u003eAnesthetized embryos were placed in 2% methylcellulose in PBS 1X solution and subsequently positioned on a glass slide. The sample was then inserted between two polarizing-rotating filters and analyzed under a Leica M165FC microscope (Leica Microsystems, Milan, Italy). Skeletal muscle birefringence was recorded in a bright field with a DFC7000T digital camera (Leica Microsystems, Milan, Italy), rotating the upper polarizing filter until the light refracted by the skeletal muscles was uniform and homogeneous along the entire larval body. The pixel intensity was then analyzed with ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eHistology\u003c/h2\u003e\u003cp\u003eTwelve-month-old adult zebrafish were euthanized and fixed in Bouin's solution (picric acid in milliQ water/formalin/glacial acetic acid) for a minimum of 24 h up to a maximum of 72 h at room temperature, depending on the size of the fish. To allow better fixation, the abdomen was opened along the ventral midline starting from the anal pore. The fixed samples were washed in 70% EtOH plus ammonia solution until the fish appeared with normal colour and the washing solution was clear. After dehydration in a series of graded EtOH and clearing by xylene, samples were embedded in paraffin. Paraffin sectioning (2\u0026ndash;3 \u0026micro;m), hematoxylin \u0026amp; eosin (H\u0026amp;E) and Masson's trichrome staining were conducted based on standard procedures, and the sections were photographed under a light microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing analysis\u003c/h2\u003e\u003cp\u003eZebrafish mutant (n\u0026thinsp;=\u0026thinsp;6) and WT (n\u0026thinsp;=\u0026thinsp;6) hearts were immediately minced with a sterile scalpel on ice, submerged in QIAzol (1.0 mL per ~\u0026thinsp;10 mg of tissue; Qiagen, Milan, Italy) and homogenized using 5-mm steel beads in a TissueLyser instrument (Qiagen, Milan, Italy), with 2 cycles of 2 min at 30 mHz. The homogenized samples were then incubated with 20 \u0026micro;L of Proteinase K (20 mg/mL; Roche Diagnostics GmbH, Mannheim, Germany) for 10 min at 56\u0026deg;C. Subsequently, 200 \u0026micro;L of chloroform was added to each homogenized sample, which was then centrifuged at 12,000 rcf for 15 min at 4\u0026deg;C. Nucleic acids were purified using spin columns of the RNeasy mini kit with a 20-minute on-column DNase I digestion (Qiagen, Milan, Italy). RNA concentration was determined using a Qubit fluorometer (Life Technologies, Milan, Italy), and integrity was evaluated using a 4200 Tapestation (Agilent Technologies, Milan, Italy). cDNA library preparation and sequencing were performed following standard protocols of the CORALL Total RNA-Seq V2 kit (Lexogen, Vienna, Austria). Specifically, mRNA was obtained from total RNA using the RiboCop HMR plus Globin kit (Lexogen, Vienna, Austria). Individual zebrafish libraries were pooled, and sequencing was carried out for 76 cycles on the NextSeq 550 platform (Illumina, San Diego, CA, USA). Transcriptome assembly was performed on Illumina BaseSpace sequence Hub. Differential gene expression analysis was conducted using DESeq2. Genes were considered differentially expressed if presenting a Log2 FoldChange\u0026thinsp;\u0026plusmn;\u0026thinsp;1 and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Enriched pathways were analyzed using NetworkAnalyst (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.networkanalyst.ca/\u003c/span\u003e\u003cspan address=\"https://www.networkanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Ingenuity Pathway Analysis IPA (Qiagen, Milan, Italy).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSignal quantification and statistical analysis\u003c/h3\u003e\n\u003cp\u003eSignal quantification analysis was performed using the Measurements option of Volocity 6.0 software (Perkin Elmer, Milan, Italy) and ImageJ software. Pairwise analysis was carried out by unpaired t-test. Multiple comparisons were performed by one-way ANOVA followed by Tukey\u0026rsquo;s test, while survival analysis was conducted using Log-rank (Mantel-Cox) test (GraphPad Prism V7.0 software). The equality of the variances was analyzed by F-test. In the charts, error bars display standard errors of the mean. Asterisks indicate significant differences from controls. The correspondence between asterisks and significance levels is indicated in the figure legends. Sample final sizes were obtained after collection and randomization from multiple mating events, to reduce confounding effects from fish background, and excluding unfertilized eggs or embryos displaying very early developmental defects in all conditions. The sample size was preliminarily calculated using G*Power and Sample Size Calculator analysis. Single-blind experiments were performed at least in duplicate, with at least 10 processed individuals per condition, measured with at least two technical replicates, and with at least three samples per condition acquired for imaging analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eStudy approval\u003c/h2\u003e\u003cp\u003e All experiments were performed in accordance with Italian and European Legislation (Directive 2010/63/EU), and with permission for animal experimentation from the Ethics Committee of the University of Padova (OPBA) and the Italian Ministry of Health (Authorization numbers 407/2015-PR and 1111/2024-PR).\u003c/p\u003e\u003cp\u003eData and materials availability\u003c/p\u003e\u003cp\u003eAll raw data are available upon request to the corresponding author.\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST STATEMENT\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThis work was supported by the Italian Ministry of University and Research (MUR) (PRIN grant 20229FE439 - CUP C53D23004670006 \"\u003cem\u003eThe mechanistic link between genetic substrate and immune reactions in inflammatory cardiomyopathies\u003c/em\u003e\"), Rome; the Registry for Cardio-cerebro-vascular Pathology, Veneto region, Venice; and ARCA (Associazione Ricerche Cardiopatie Aritmiche), Padova, Italy. GR is a Postdoc Fellow supported by MUR (PRIN grant 2022WZCXRZ). RC and MBM are Postdoc Fellows supported by the Italian Ministry of Health, PNRR Next-Generation EU grant PNRR-MR1-2022-12376614. RBC is a Postdoc Fellow supported by DeBio, UniPD (grant 2024DIBIO1SIDASSEGNI-00095). NT was supported by the Italian Telethon Foundation (grant GGP19287) and MUR (grant PNRR M4C2 CN00000041). We thank the Zebrafish Facility staff at the Department of Biology, University of Padova, Italy.\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\u003cp\u003eConceptualization, G.R., R.C., K.P., N.T; Methodology and Investigation, G.R., R.C., R.B.C., M.C., K.P., N.T.; Writing\u0026mdash;original draft preparation, All Authors; Writing\u0026mdash;review and editing, All Authors; Visualization,, G.R., R.C; supervision, K.P., N.T. and C.B.; project administration, K.P., N.T.; funding acquisition, K.P., N.T., C.B, F.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOz, F., et al.: Galectin-3 correlates with arrhythmogenic right ventricular cardiomyopathy and predicts the risk of ventricular -arrhythmias in patients with implantable defibrillators. Acta Cardiol. \u003cb\u003e72\u003c/b\u003e, 453\u0026ndash;459 (2017)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlanda, V., Bracale, U.M., Di Taranto, M.D., Fortunato, G.: Galectin-3 in Cardiovascular Diseases. IJMS. \u003cb\u003e21\u003c/b\u003e, 9232 (2020)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCason, M., et al.: Novel pathogenic role for galectin-3 in early disease stages of arrhythmogenic cardiomyopathy. 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Neurochem. \u003cb\u003e116\u003c/b\u003e, 1148\u0026ndash;1159 (2011)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7855683/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7855683/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGalectin-3 (LGALS3/Gal-3) dysregulation has emerged as a critical mediator of inflammatory processes in arrhythmogenic cardiomyopathy (AC), playing pivotal roles in modulating Wnt/β-catenin signaling and regulating macrophage polarization. AC is a rare genetic disorder, primarily driven by desmosomal gene variants, characterized by fibro-fatty replacement of the ventricular myocardium, progressive ventricular dysfunction, and heightened arrhythmic risk in the young and athletes. To investigate the role of this multifaceted lectin in AC pathogenesis, we developed and characterized a stable lgals3a knock-out zebrafish model. Gal-3 deficiency alone was sufficient to recapitulate hallmark AC features, including ventricular adipose infiltration, chamber dilation, pericardial effusion, and progressive arrhythmias, spanning from larval to adult stages. Ultrastructural analyses revealed disrupted desmosomes, directly implicating Gal-3 in intercellular adhesion independent of other desmosomal gene variants. Transcriptomic analyses demonstrated suppression of both Wnt/β-catenin and TGFβ signaling. Early-stage pharmacological activation of Wnt signaling partially rescued cardiac function, but structural defects persisted in adults, indicating irreversible desmosomal instability. Inflammatory profiling revealed significant immune cell infiltration and upregulation of macrophage-related proinflammatory genes (e.g., MMP12, CCL38, IL16), consistent with AC \u0026ldquo;hot phases.\u0026rdquo; This study establishes Gal-3 depletion as a sufficient driver of AC-like pathology and identifies Gal-3\u0026ndash;related pathways as promising targets for therapeutic intervention.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"A zebrafish stable model of Galectin-3 to elucidate its role in Arrhythmogenic Cardiomyopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 09:36:05","doi":"10.21203/rs.3.rs-7855683/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b11470c8-d0f1-4cc3-a18a-025936250892","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56781997,"name":"Health sciences/Cardiology/Cardiovascular biology/Cardiovascular genetics"},{"id":56781998,"name":"Biological sciences/Biotechnology/Expression systems"}],"tags":[],"updatedAt":"2025-11-26T09:36:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 09:36:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7855683","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7855683","identity":"rs-7855683","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}