Xu, Xiaolei

BACKGROUND: Anthracyclines are a widely used and effective class of chemotherapy. However, a major limitation for their use is cardiotoxicity, manifesting as systolic dysfunction, congestive heart failure, and arrhythmias. The objective of this study was to identify genes and genetic risk variants for doxorubicin-induced arrhythmia. METHODS: We performed genome-wide association studies and meta-analysis across 2 independent nonoverlapping, genetically homogenous data sets from the Molecular Epidemiology Resource (cases N=77, controls N=1184) and Tapestry (cases N=13, controls N=172). Doxorubicin-related arrhythmia was the primary cardiac outcome, followed by investigation of a broader phenotype of cardiac events. Effects of doxorubicin on top-associated genes were assessed by quantitative polymerase chain reaction on RNA extracted from human cardiomyocytes following treatment with doxorubicin and by echocardiography in a zebrafish model of doxorubicin-induced cardiomyopathy. RESULTS: In the meta-analysis, rs12151014 at the KCTD15 locus was associated with doxorubicin-related arrhythmia at the genome-wide significance (minor allele frequency in Molecular Epidemiology Resource cases 0.195 versus 0.069 in controls; Tapestry cases 0.192 versus 0.079 in controls) P =9.01×10 −9 ; odds ratio, 3.61. MMP 16 rs1094199 was associated with the broader phenotype of arrhythmia and heart failure (minor allele frequency in Molecular Epidemiology Resource cases 0.079 versus 0.019 in controls; Tapestry cases 0.038 versus 0.015) P =1.98×10 − 8 ; odds ratio=6.15. In human cardiomyocytes, MMP16 and KCTD15 gene expression was significantly downregulated by doxorubicin, and in a zebrafish MMP16 knockout was protective of doxorubicin-induced decline in left ventricular ejection fraction. CONCLUSIONS: Genetic variants at KCTD15 and MMP16 are associated with doxorubicin-related arrhythmia and cardiac events. MMP16 is a potential cardioprotective therapeutic target.

Abstract Alzheimer’s Disease (AD) 1 is a progressive neurodegenerative disorder with limited therapeutic options, driving interest in drug repurposing to accelerate treatment discovery. Drug repurposing has emerged as a promising strategy to accelerate therapeutic discovery by repositioning existing drugs for new clinical indications. Recent computational repurposing approaches, including knowledge graph reasoning, transcriptomic signature analysis, and integrative literature mining, have demonstrated strong predictive capabilities 2 . However, these methods often yield divergent drug rankings, which makes it difficult to decide which candidates to advance for experimental follow-up and results in substantial gaps between computational predictions and feasible in vivo validation 2 .To bridge this computational-experimental gap, we proposed an advanced prioritization framework leveraging large language models (LLMs). Our method systematically evaluated three state-of-the-art (SOTA) and representative computational methods (TxGNN 3 , Composition-based Graph Convolutional Network (CompGCN) 4 , and a regularized logistic regression (RLR) 5 , to analyze both their predictive performance and pharmaceutical class distributions. By integrating the strengths and divergences of these models, we generated a unified, streamlined list of 90 candidate drugs for further prioritization. We then utilized an LLM-based agent to perform evidence synthesis from biomedical literature abstracts for each candidate. This process mimics expert manual curation but significantly reduces human effort and time by efficiently distilling vast textual data into actionable insights. Applying consistent and transparent selection criteria, we obtained a refined and prioritized list of drug candidates suitable for subsequent in vivo experimental validation. The robustness and clinical relevance of our framework were validated using real-world data from Alzheimer’s patient cohorts, clinical trial registries, and expert pharmacological reviews. This comprehensive validation confirmed that our LLM-driven approach enhances efficiency, consistency, scalability, and generalizability. By integrating computational predictions with scalable evidence synthesis and multifaceted validation, our framework facilitated rapid and informed prioritization of repurposed drugs. Our framework can potentially accelerate the translational pathway toward viable AD therapeutics. Moreover, the versatility of our framework can also be applied to drug repurposing efforts for other diseases beyond AD.

Key Points ift140 -deficient zebrafish exhibited phenotypes observed in mammals, including skeletal defects and kidney cysts. Beyond ciliogenesis, ift140 also regulated cell polarity and intracellular microtubule stability, potentially contributing to cystogenesis. Microhomology-mediated end joining–induced mosaic ift140 crispants enabled evaluation of cystogenesis in adult fish and F0-based screening for modifiers of kidney cysts. Background Genetic modifiers are believed to play an important role in the onset and severity of polycystic kidney disease, but identifying these modifiers has been challenging due to the lack of effective methodologies. Methods We generated zebrafish mutants of IFT140 , a skeletal ciliopathy gene and newly identified autosomal dominant polycystic kidney disease gene, to examine skeletal development and kidney cyst formation in larval and juvenile mutants. In addition, we used ift140 crispants, generated through efficient microhomology-mediated end joining–based genome editing, to compare phenotypes with mutants and conduct a pilot genetic modifier screen. Results ift140 mutants developed kidney cysts and bone defects similar to those seen in mammalian models. ift140 crispants recapitulated mutant phenotypes while bypassing the early lethality of the mutants, enabling the analysis of kidney cyst formation in adult fish. In addition to cilia defects, we identified nonciliary phenotypes, including disrupted cell polarity and aberrant cytoplasmic microtubule stabilization in kidney epithelial cells, as potential contributors to ift140 -associated cystogenesis. The ability to detect ift140 -associated kidney cysts with ease allowed us to develop an F0-based genetic screen to identify potential protective modifiers. A pilot screen of 16 genes previously implicated in dysregulated signaling pathways in autosomal dominant polycystic kidney disease revealed both known and novel modifiers, including mtor and ulk1a . We further found that inhibition of mtor and ulk1a reversed both cilia-related and non–cilia-related abnormalities in the kidney. Conclusions By establishing a zebrafish model of ift140 -associated cystic kidney disease, we recapitulated ift140 ’s ciliary role and uncovered a nonciliary function in kidney cystogenesis. Importantly, we demonstrated the feasibility of using ift140 mosaic crispants to evaluate cystogenesis in adult fish and to perform F0-based screening for identifying genetic modifiers of kidney cysts.

ABSTRACT While BAG3 has been identified as a causative gene for dilated cardiomyopathy, the major pathological events in BAG3‐ related cardiomyopathy that could be targeted for therapeutic benefit remain to be discovered. Here, we aim to uncover novel pathological events through genetic studies in a zebrafish bag3 cardiomyopathy model. Given the known cardioprotective effects of mtor inhibition and the fact that transcription factor EB (tfeb ) encodes a direct downstream phosphorylation target of mTOR signaling, we generated a cardiomyocyte‐specific transgenic line overexpressing tfeb ( Tg[cmlc2:tfeb] ). This overexpression was sufficient to restore defective proteostasis and rescue cardiac dysfunction in the bag3 cardiomyopathy model. Importantly, we detected accelerated cardiac senescence in the bag3 cardiomyopathy model, which can be mitigated by Tg(cmlc2:tfeb) . We compared cardiac transcriptomes between the Tg(cmlc2:tfeb) transgenic fish and the mtor xu015/+ mutant and found that inhibition of the fatty acid binding protein a ( fabp7a ) gene exerts therapeutic effects. Consistent with this genetic evidence, we detected elevated fabp7a expression in the bag3 cardiomyopathy model, whereas cardiomyocyte‐specific overexpression of fabp7a induced dysregulated proteostasis, accelerated cardiac senescence, and cardiac dysfunction. To elucidate the functions of Fabp7a in normative cardiac aging, we turned to the African Turquoise Killifish. We noted elevated Fabp7a expression in the hearts of aged killifish, and pharmacological inhibition of Fabp7a mitigated the cardiac aging process. Together, this study uncovered accelerated cardiac senescence as a key pathological event in bag3 cardiomyopathy and reveals that manipulating the mTOR‐Tfeb‐Fabp7a axis can mitigate this pathology and confer cardioprotective effects.

Abstract Genetic modifiers are believed to play an important role in the onset and severity of polycystic kidney disease (PKD), but identifying these modifiers has been challenging due to the lack of effective methodologies. In this study, we investigated zebrafish mutants of IFT140 , a newly identified ADPKD gene, and observed phenotypes similar to those seen in mammalian models, including kidney cysts and bone defects. Using efficient microhomology-mediated end joining (MMEJ)-based genome editing technology, we demonstrated that ift140 CRISPRants recapitulate mutant phenotypes while bypassing the early lethality of the mutants to allow for renal cyst analysis in adult fish. In addition to cilia defects, non-cilia phenotypes, such as disrupted cell polarity and aberrant microtubule stabilization in kidney epithelial cells, may also contribute to ift140 -associated cystogenesis. Importantly, the ability to detect ift140 -associated renal cysts with ease allowed us to develop an F0-based genetic screen to identify potential protective modifiers. A pilot screen of sixteen genes previously linked to dysregulated signaling pathways in ADPKD revealed both known and novel modifiers, including mtor and ulk1a . Inhibition of mtor and ulk1a reversed both cilia-related and non-cilia-related abnormalities. In summary, our study underscores the potential of using zebrafish as a model for the efficient discovery of genetic modifiers of kidney cysts.

Allelic heterogeneity (AH) has been noted in truncational TTN –associated ( TTNtv -associated) dilated cardiomyopathy (DCM); i.e., mutations affecting A-band–encoding exons are pathogenic, but those affecting Z-disc–encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure were more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were present in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the full titin -encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also noted only in ttntv-A but not in ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss of function is sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.

Background: Anthracyclines are highly effective chemotherapeutics, but their use is limited by Anthracycline-Induced Cardiotoxicity (AIC) with notable decline in ejection fraction (EF). Recovery of cardiac function is slow and often only partial, mandating novel and more efficacious treatment approaches to AIC. Hypothesis: Based on high throughput genetic screening in zebrafish we identified autophagy activation, e.g. via Atg7, a key protein in the induction of autophagy, as a potential novel translational therapeutic approach to AIC. Methods: In a mouse model of cardiotoxicity (4 x weekly 5 mg/kg intravenous doxorubicin treatment) two approaches were tested starting one week after the last treatment: 1) AdenoAssociated Viral vector 9-mediated overexpression of Atg7 in the myocardium (AAV9-ATG7, one time injection) and 2) 3 FDA approved autophagy activating (FAA) drugs (pravastatin (40 mg/kg), spironolactone (20 mg/kg) and empagliflozin (10 mg/kg), by daily gavage). Echocardiograms were performed at baseline, before start of therapy and after 28 days before sacrifice. Results: All three FAA drugs were able to rescue AIC and Atg7 overexpression by AAV9 alone sufficed to rescue from AIC (Figure 1). All three FAA drugs were able to either restore Atg7 levels to baseline (pravastatin and empagliflozin) or 2.5 times above baseline levels (spironolactone) versus more than 60fold above baseline by an AAV9-based approach. Conclusion: Atg7 overexpression and autophagy activation suffices to rescue from AIC and can be recapitulated to different levels by pravastatin, empaglifozin and spironolactone.

Zebrafish is a well-established model organism for cardiovascular disease studies in which one of the most popular tasks is to assess cardiac functions from the heart beating echo-videos. However, current techniques are often time-consuming and error-prone, making them unsuitable for large-scale analysis. To address this problem, we designed a method to automatically evaluate the ejection fraction of zebrafish from heart echo-videos using a deep-learning model architecture. Our model achieved a validation Dice coefficient of 0.967 and an IoU score of 0.937 which attest to its high accuracy. Our test findings revealed an error rate ranging from 0.11% to 37.05%, with an average error rate of 9.83%. This method is widely applicable in any laboratory setting and can be combined with binary recordings to optimize the efficacy and consistency of large-scale video analysis. By facilitating the precise quantification and monitoring of cardiac function in zebrafish, our approach outperforms traditional methods, substantially reducing the time and effort required for data analysis. The advantages of our method make it a promising tool for cardiovascular research using zebrafish.

Background: TITIN truncational variants (TTNtv) is the predominant genetic factor for dilated cardiomyopathy (DCM). Therapeutic development for TTNtv DCM has been confounded by allelic heterogeneity (AH), i.e. while truncations in the C-terminal A-domain (TTNtv-As) are pathogenic, truncations in the N-terminal Z-disc domain (TTNtv-Zs) are largely benign. Molecular mechanisms underlying AH remain elusive, partially because of the lack of an in vivo animal model. Objective: We enquire whether zebrafish is a faithful vertebrate model for deciphering AH in TTNtv DCM and for therapeutic development. Methods and Results: Through comparing phenotypes among a series of ttntv -As and ttntv -Zs in adult zebrafish, we detected DCM-like phenotypes only in ttntv -As but not ttntv -Zs. Similar to human TTNtv DCM and corresponding rodent models, zebrafish ttntv -As manifest decreased ejection fraction, enlarged heart size, myofibril dys-organization, compromised exercise capacity, and dysregulated autophagy. Moreover, we noted exaggerated cardiac senescence at the later stage of the pathogenesis of ttntv DCM. Because AH in autophagy dysregulation was also detected in homozygous ttntv-A zebrafish embryos, we screened autophagy genes and identified ulk1 inhibition that is able to repair the dysregulated autophagy. A similar repairing effect of ulk1 inhibition on dysregulated autophagy was noted in an adult heterozygous ttntv-A model, which is sufficient to attenuate the cardiac senescence and improve cardiac function. Conclusion: Our data suggested adult zebrafish as the first vertebrate model that is able to recapitulate AH in TTNtv DCM patients. Conserved phenotypes including autophagy dysregulation are recapitulated in this lower vertebrate model, while cardiac senescence has been suggested as a novel pathological event. Genetic studies in this animal strongly suggest that repairing autophagy dysregulation is a therapeutic avenue for TTNtv DCM, and identified ulk1 as a candidate therapeutic target gene.

Arrhythmogenic cardiomyopathy (ACM) is largely an autosomal dominant genetic disorder manifesting fibrofatty infiltration and ventricular arrhythmia with predominantly right ventricular involvement. ACM is one of the major conditions associated with an increased risk of sudden cardiac death, most notably in young individuals and athletes. ACM has strong genetic determinants, and genetic variants in more than 25 genes have been identified to be associated with ACM, accounting for approximately 60% of ACM cases. Genetic studies of ACM in vertebrate animal models such as zebrafish (Danio rerio), which are highly amenable to large-scale genetic and drug screenings, offer unique opportunities to identify and functionally assess new genetic variants associated with ACM and to dissect the underlying molecular and cellular mechanisms at the whole-organism level. Here, we summarize key genes implicated in ACM. We discuss the use of zebrafish models, categorized according to gene manipulation approaches, such as gene knockdown, gene knock-out, transgenic overexpression, and CRISPR/Cas9-mediated knock-in, to study the genetic underpinning and mechanism of ACM. Information gained from genetic and pharmacogenomic studies in such animal models can not only increase our understanding of the pathophysiology of disease progression, but also guide disease diagnosis, prognosis, and the development of innovative therapeutic strategies.