Zebrafish models for ARVC8 analysis and drug discovery

INTRODUCTION: Desmoplakin is one the most abundant desmosomal proteins in cardiac and epithelial tissues. In humans, dominat mutations in the desmoplakin gene (DSP) cause Arrhythmogenic Right Ventricular Cardio​myopathy 8 (ARVC8), a dominant cardiomyopathy, frequently involved in juvenile sudden death. Current ARVC models are based on cell lines and transgenic mice. In this context, it has been shown that suppression of DSP expression leads to a reduction in canonical Wnt signaling, suggesting that this pathway could be a molecular target for ARVC therapeutic intervention. In order to address this issue, the present study aims to evaluate the pathogenic mechanisms of DSP mutations in vivo, using zebrafish (Danio rerio) as an innovative model for this disease. In zebrafish, the desmoplakin gene is present with two isoforms, dspa and dspb, both orthologous to the single DSP in humans. PURPOSE: The purpose of this study is the generation and the phenotypic characterization of transient ARVC8 zebrafish models using a morpholino-mediated knock-down strategy. In addition, by taking advantage of zebrafish pathway reporter lines, we aim to verify if Wnt signaling and/or other molecular cascades might be involved in ARVC8 pathogenesis. The final goal is the assessment of our ARVC8 model as a suitable tool for molecularly-targeted drug discovery. METHODS: To evaluate the expression of dspa and dspb during zebrafish embryonic development and adulthood, we used whole-mount in situ hybridization (WISH) and semi quantitative RT_PCR. Knockdown of zebrafish dspa and dspb genes was obtained by a morpholino (MO)-based antisense strategy. Specifically, we injected anti-dspa and anti-dspb MO oligos in both wild types and pathway-specific lines reporting the activity of Wnt, Bmp, TGFbeta, FGF, Shh, Notch, CREB, Hippo and Hypoxia signaling. RESULTS: We found that both dspa and dspb are expressed during zebrafish embryonic development, while the molecular analysis of cDNAs from different adult tissues demonstrates that both dspa and dspb are highly expressed in heart and skin, with dspa more strongly detectable compared to dspb. MO-mediated knock-down of both dspa and dspb leads to delayed development, microcephaly, pericardial edema and, particularly in dspb knock-down embryos, decreased heart rate. TEM analysis of cardiac and skin tissues under dspa+dspb simultaneous knock-down shows reduced and disorganized desmososmes. As far as concerns the analysis of previously mentioned signaling pathways, we observed a specific reduction of Wnt signaling responsiveness in the cardiac region of both dspa and dspb knock- down embryos (Fig. 1). CONCLUSION: Our results show that transient knock-down of zebrafish desmoplakin genes is able to phenocopy some ARVC8 features, such as cardiac and cutaneous desmosomal defects, heart rate alteration and Wnt signaling reduction, pointing to zebrafish as a suitable ARVC8 model for in vivo screening of molecularly-targeted drugs

Identification of Giovanni Battista Morgagni remains following historical, anthropological and molecular studies

Morgagni died on December 5, 1771, 89 years old, and was buried in Saint Maxim Church in Padua, where his wife and five of his 15 children, four daughters, and one son were already buried. In 1868 and 1900, the tomb was opened to identify Morgagni. Among the remains of several adult individuals, two skulls considered of very old persons were identified and replaced in an earthenware jar inside the sepulcher. In 2011, we opened the tomb and found the remains described during the first two identifications, but additionally, we found the skulls fragments of three very young individuals which could have been Morgagni\u2019s children. An anthropological analysis confirmed that one of the skulls inside the earthenware jar belonged to the oldest individuals (\u201csenilis\u201d) between those found in the tomb. A genetic analysis proved a kinship between this skull and the fragments of young individuals (onemale and two females), supporting the hypothesis that they were Morgagni and his children. In conclusion, thanks to the interaction between historical studies, anthropological research, and molecular analysis that reinforce each other, we can assume that the skull is Giovanni Battista Morgagni\u2019s and that the series of skull fragments are from his children who were buried together with their parents

Accuracy of scoring of the epiphyses at the knee joint (SKJ) for assessing legal adult age of 18 years

Important aspects of forensic practice are age estimation and discrimination of individuals of unknown age as adults and minors. The developing knee joint was recognized as a potential site for age examination in late adolescence. We analyzed a sample of anteroposterior x-rays of the knee joints from 446 living individuals from Umbria, Italy (234 males and 212 females), aged between 12 and 26 years. We evaluated the ossification of the distal femoral (DF), proximal tibial (PT), and proximal fibular (PF) epiphyses. We took into account possible persistence of the epiphyseal scars in the ossified epiphyses by the adopted stages of those previously introduced by Cameriere et al. (2012). We also used measurements from all three epiphyses to calculate the total score of maturation for the knee joint (SKJ). Cohen Kappa coefficients of intrarater agreement for staging the DF, PT, and PF epiphyses were 0.839, 0.894, and 0.907, while interrater agreement was 0.919, 0.791, and 0.907, respectively. The resulting receiver operating characteristic (ROC) curves of SKJ show better discriminatory power than those for DF, PT, and PF epiphyses in predicting that the participant, either male or female, was an adult or a minor. The areas under the curves for SKJ were 0.991 and 0.968 vs. 0.944, 0.962, 0.974 and 0.891, 0.910, 0.918 for males and females, respectively. The results of the 2 by 2 contingency tables showed that SKJ score of 4 in males and SKJ score of 5 in females were the most suitable cut-off value in discriminating between adults and minors. Principally, the sensitivity test for males was 0.94, with 95 % confidence interval (95 % CI) 0.90 to 0.97 and specificity was 0.96 (95 % CI 0.91 to 0.98). The proportion of correctly classified individuals was 0.95 (95 % CI 0.91 to 0.97). For females, the sensitivity test was 0.89 (95 % CI 0.84 to 0.92) and specificity was 0.92 (95 % CI 0.87 to 0.96), the proportion of correctly classified individuals was 0.90 (95 % CI 0.85 to 0.94). These results indicate that the SKJ method may give valuable supporting information in forensic procedures for discriminating individuals of legal adult age of 18 years. Further studies should address the usefulness of the SKJ method in different populations

Desmoplakin knock-downs in zebrafish recapitulate arrhythmogenic cardiomiopathy type 8 and involve Wnt/beta-catenin signaling in AC8 pathogenesis

Background: Mutations in the desmoplakin (DSP) gene have been identified in patients affected with Arrhythmogenic Cardiomyopathy type 8 (AC8), a progressive heart muscle disorder, frequently involved in juvenile sudden death due to cardiac arrhythmias. In spite of the recent discovery of genes whose mutations cause ACs, early molecular events leading to cell death and arrhythmias remain elusive. In the present study we evaluate the pathogenic mechanisms of DSP dysfunction in vivo, using zebrafish (Danio rerio) as an innovative and promising model for this life-threatening arrhythmic disorder. Purpose: The aim of the present study is the generation of transient AC8 zebrafish models, using a morpholino-mediated knock-down strategy, and their structural and functional characterization. In addition, by exploiting zebrafish pathway reporter lines, we aim to study cell signaling alterations potentially involved in AC8 pathogenesis. The final goal is the assessment of our zebrafish AC8 models as a suitable tool for pathway-directed drug screening. Methods: A morpholino (MO)-based antisense strategy was used to obtain the knockdown of zebrafish dspa and dspb genes, both orthologous to human DSP. Transient AC8 zebrafish models were morphologically characterized and, subsequently, functionally tested for alterations in Wnt, Bmp, TGFbeta, FGF, Shh, Notch, CREB, Hippo and Hypoxia signaling. Results: The knock-down of both dspa and dspb leads to delayed development, microcephaly, pericardial edema and altered heart rate. Interestingly, TEM analysis of zebrafish tissues under dspa+dspb simultaneous knock-down shows highly reduced and disorganized desmososmes, resembling \u201cpale\u201d desmosomes identified in endomyocardial biopsies from AC patients. Moreover, the analysis of signaling pathways detects a cardiac-specific reduction of Wnt signaling responsiveness in both dspa and dspb knock-down embryos, confirming previous evidences that DSP suppression leads to a reduction of canonical Wnt signaling in cellular and mouse models. Conclusions: Our transient knock-down of zebrafish Dsp genes is able to recapitulate some AC8 features, such as desmosomal defects and heart rate alteration, pointing to zebrafish as a suitable model for the in vivo screening of molecularly-targeted drugs. Moreover, confirmation of the reduction in canonical Wnt signaling due to DSP knock-down suggests that this pathway could be a general mechanism involved in the pathogenesis of desmosomal-associated AC forms, and, thus, a promising target for AC therapeutic intervention

Zebrafish models for ARVC8 analysis and drug discovery

Desmoplakin is one the most abundant desmosomal proteins in cardiac and epithelial tissues. In humans, dominat mutations in the desmoplakin gene (DSP) cause Arrhythmogenic Right Ventricular Cardio\u200bmyopathy 8 (ARVC8), a dominant cardiomyopathy, frequently involved in juvenile sudden death. Current ARVC models are based on cell lines and transgenic mice. In this context, it has been shown that suppression of DSP expression leads to a reduction in canonical Wnt signaling, suggesting that this pathway could be a molecular target for ARVC therapeutic intervention. In order to address this issue, the present study aims to evaluate the pathogenic mechanisms of DSP mutations in vivo, using zebrafish (Danio rerio) as an innovative model for this disease. In zebrafish, the desmoplakin gene is present with two isoforms, dspa and dspb, both orthologous to the single DSP in humans. PURPOSE: The purpose of this study is the generation and the phenotypic characterization of transient ARVC8 zebrafish models using a morpholino-mediated knock-down strategy. In addition, by taking advantage of zebrafish pathway reporter lines, we aim to verify if Wnt signaling and/or other molecular cascades might be involved in ARVC8 pathogenesis. The final goal is the assessment of our ARVC8 model as a suitable tool for molecularly-targeted drug discovery. METHODS: To evaluate the expression of dspa and dspb during zebrafish embryonic development and adulthood, we used whole-mount in situ hybridization (WISH) and semi quantitative RT_PCR. Knockdown of zebrafish dspa and dspb genes was obtained by a morpholino (MO)-based antisense strategy. Specifically, we injected anti-dspa and anti-dspb MO oligos in both wild types and pathway-specific lines reporting the activity of Wnt, Bmp, TGFbeta, FGF, Shh, Notch, CREB, Hippo and Hypoxia signaling. RESULTS: We found that both dspa and dspb are expressed during zebrafish embryonic development, while the molecular analysis of cDNAs from different adult tissues demonstrates that both dspa and dspb are highly expressed in heart and skin, with dspa more strongly detectable compared to dspb. MO-mediated knock-down of both dspa and dspb leads to delayed development, microcephaly, pericardial edema and, particularly in dspb knock-down embryos, decreased heart rate. TEM analysis of cardiac and skin tissues under dspa+dspb simultaneous knock-down shows reduced and disorganized desmososmes. As far as concerns the analysis of previously mentioned signaling pathways, we observed a specific reduction of Wnt signaling responsiveness in the cardiac region of both dspa and dspb knock- down embryos (Fig. 1). CONCLUSION: Our results show that transient knock-down of zebrafish desmoplakin genes is able to phenocopy some ARVC8 features, such as cardiac and cutaneous desmosomal defects, heart rate alteration and Wnt signaling reduction, pointing to zebrafish as a suitable ARVC8 model for in vivo screening of molecularly-targeted drugs

Zebrafish models for Arrhythmogenic Cardiomyopathy Type 8: a starting platform for exercise stress test and drug treatment

Background Arrhythmogenic Cardiomyopathy (AC) is an inherited heart disease characterized by progressive substitution of the myocardium with fibro-fatty tissue, leading to electrical instability and high risk of sudden death, particularly in young subjects and athletes. In recent years, our laboratory has produced zebrafish (zf) mutant lines modelling AC type 8, an AC form linked to mutations in the junctional protein Desmoplakin (Dsp). Mutations in the DSP gene have been identified in both dominant and recessive AC cases, characterized by left-dominant and biventricular forms of the disease. Sports medicine has highlighted that they are the most dangerous forms, being less easily identifiable by ECG. Purpose Taking advantage of our zf Dsp mutant lines, we aim to fully characterize the pathological phenotype, analyze the perturbation of cell communication pathways, evaluate the role of the physical exercise, and test the efficacy of candidate drugs. Methods Among our zf lines we have identified double mutant animals, bearing both zf dspa and dspb mutations in heterozygous condition, as the best model able to recapitulate the human AC phenotype. This model underwent physical stress tests in the presence/absence of candidate drug treatment. Phenotyping included heart rhythm measurement, gene expression analysis using Real Time PCR and signaling pathway transgenes, immunehistochemistry, whole-mount in situ hybridization, standard histology and ultrastructural TEM analysis. Results Preliminary results from mutant phenotyping indicate alterations in heart rate, sudden cardiac death, structural alterations of the myocardium associated with junctional disorganization and, in parallel, dysregulation of Wnt, Hippo and TGFbeta pathways. Specifically, Dsp mutant animals can range from an 8% decrease to a 14% increase of heart rhythm compared to the physiological range (120-140 beats per minute in zf larvae). At the adult stage, about 1% of the fish mutant population dies suddenly. The histological examination shows a 50% reduction of the myocardial cell mass, in parallel with a 50% decrease of Dsp signal, detected by TEM, associated with the so-called "pale desmosome" phenotype. Signaling dysregulation includes an 80% loss of Wnt/Beta-catenin, a 300% increase of TGFbeta and a 500% increase of Hippo/YAP-TAZ signaling in the cardiac tissue. Physical stress tests and pathway-directed drug treatment have clarified that these factors can modulate the pathological phenotype, as preliminarily evidenced by the rescue of Wnt signal decrease to normal levels through SB216763 treatment of Dspdeficient individuals at rest. Conclusion Preliminary evidences corroborate the zf organism as a suitable model for AC cellular and molecular phenotyping, exploitable for the dissection of the genetic events leading to the onset and progression of th

Loss of cardiac Wnt/\u3b2-catenin signalling in Desmoplakin-deficient AC8 zebrafish models is rescuable by genetic and pharmacological intervention

Abstract AIMS: Arrhythmogenic cardiomyopathy (AC) is an inherited heart disease characterized by life-threatening ventricular arrhythmias and fibro-fatty replacement of the myocardium. More than 60% of AC patients show pathogenic mutations in genes encoding for desmosomal proteins. By focusing our attention on the AC8 form, linked to the junctional protein Desmoplakin (DSP), we present here a zebrafish model of DSP deficiency, exploited to identify early changes of cell signalling in the cardiac region. METHODS AND RESULTS: To obtain an embryonic model of DSP deficiency, we first confirmed the orthologous correspondence of zebrafish dsp genes (dspa and dspb) to the human DSP counterpart. Then, we verified their cardiac expression, at embryonic and adult stages, and subsequently we targeted them by antisense morpholino strategy, confirming specific and disruptive effects on desmosomes, like those identified in AC patients. Finally, we exploited our DSP-deficient models for an in vivo cell signalling screen, using pathway-specific reporter transgenes. Out of nine considered, three pathways (Wnt/\u3b2-catenin, TGF\u3b2/Smad3 and Hippo/YAP-TAZ) were significantly altered, with Wnt as the most dramatically affected. Interestingly, under persistent DSP deficiency, Wnt signalling is rescuable both by a genetic and a pharmacological approach. CONCLUSION: Our data point to Wnt/\u3b2-catenin as the final common pathway underlying different desmosomal AC forms and support the zebrafish as a suitable model for detecting early signalling pathways involved in the pathogenesis of DSP-associated diseases, possibly responsive to pharmacological or genetic rescue

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes

Abstract Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). We present here the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease, on which environmental factors and candidate drugs can be tested. Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, oedema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed “pale”, disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/β-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-β pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/β-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Overall, our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/β-catenin signalling cascade