Citations

Abstract Anthracycline-induced cardiotoxicity (AIC) remains a dose-limiting complication of cancer treatment, often leading to irreversible heart failure, yet effective cardioprotective therapies remain limited. Graph-driven drug repurposing offers a rapid pathway to identify cardioprotective therapies, but different computational models often generate divergent candidate lists that exceed experimental validation capacity. We applied a novel "Union-then-Filter" framework to prioritize therapeutics for AIC using AI-based approaches and evaluated candidates in zebrafish models. Three state-of-the-art graph learning models (TxGNN, CompGCN, and RLR) were trained on the PrimeKG to generate an initial candidate pool. A Large Language Model (LLM) agent automated evidence synthesis across 4,286 PubMed abstracts for the top 50 drugs from each model. Candidates were stratified using dual decision criteria and filtered against existing clinical trials. The pipeline narrowed 150 initial candidates to four novel candidates: Cysteine, Dasatinib, Tranilast, and Tretinoin. Prospective in vivo validation was subsequently conducted using an adult AIC (aAIC) zebrafish model. Phenotypic screening via echocardiography revealed that Cysteine, Tranilast, and Tretinoin significantly restored ejection fraction (EF) (overall one-way ANOVA: \(\:F\left(\text{4,95}\right)=47.66,\:\:p<0.0001\); Tukey's post hoc: \(\:\:\:p<0.01\) for all three treatments vs. DOX+ control) and normalized cardiac remodeling markers in doxorubicin-treated subjects (Kruskal-Wallis: \(\:{\chi\:}^{2}\left(4\right)=17.21,\:\:p=0.0018\); Dunn’s post hoc: \(\:\:\:p<0.01\) for all three comparisons vs. DOX control). This study demonstrates the translational utility of an LLM-integrated AI pipeline for drug repurposing and highlights zebrafish AIC as an efficient bridge between in silico prediction and in vivo phenotypic validation in cardio-oncology.

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 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.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma ( P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

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.

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.

Myosin heavy chain 7 (MYH7) is a major causative gene for hypertrophic cardiomyopathy, but the affected signaling pathways and therapeutics remain elusive. In this research, we identified ventricle myosin heavy chain like ( vmhcl ) as a zebrafish homolog of human MYH7 , and we generated vmhcl frameshift mutants. We noted vmhcl -based embryonic cardiac dysfunction (VEC) in the vmhcl homozygous mutants and vmhcl -based adult cardiomyopathy (VAC) phenotypes in the vmhcl heterozygous mutants. Using the VEC model, we assessed 7 known cardiomyopathy signaling pathways pharmacologically and 11 candidate genes genetically via CRISPR/Cas9 genome editing technology based on microhomology-mediated end joining (MMEJ). Both studies converged on therapeutic benefits of mTOR or mitogen-activated protein kinase (MAPK) inhibition of VEC. While mTOR inhibition rescued the enlarged nuclear size of cardiomyocytes, MAPK inhibition restored the prolonged cell shape in the VEC model. The therapeutic effects of mTOR and MAPK inhibition were later validated in the VAC model. Together, vmhcl/myh7 loss of function is sufficient to induce cardiomyopathy in zebrafish. The VEC and VAC models in zebrafish are amenable to both efficient genetic and chemical genetic tools, offering a rapid in vivo platform for discovering candidate signaling pathways of MYH7 cardiomyopathy.

Rationale: Anthracycline-induced cardiotoxicity (AIC) is a major side effect that limits the use of anthracyclines as effective chemotherapeutics. No mechanism-based therapy is available when cardiac function deteriorates. Objective: We aim to elucidate the dynamic autophagic defects in AIC and to identify a mechanism-based therapy via both genetic and pharmacological studies. Method and Results: Through phenotyping an adult AIC zebrafish model, we detected a biphasic response in autophagy: activation in the early stage and suppression in the later phase that is characterized by a decline in cardiac function. We conducted conditional genetic studies with atg7 , which encodes a rate-limiting autophagy core protein, and found that atg7 overexpression leads to therapeutic effects in the late phase but deleterious effects in the early phase of adult AIC. We then assessed the therapeutic effects of 37 Food and Drug Administration–approved autophagy activators using an embryonic AIC zebrafish model and identified spironolactone, pravastatin, and minoxidil as top-ranking drugs. We demonstrated the therapeutic efficacy of these Food and Drug Administration–approved autophagy activators in the adult AIC model and confirmed that these drugs exert therapeutic effects in the late phase but not in the early AIC phase. Finally, we demonstrated that the time-dependent therapeutic effects are conserved in a mouse AIC model and that spironolactone and rapamycin activated autophagy in an Atg7-dependent fashion. Conclusions: Our findings suggest that atg7-based autophagy activation is an effective therapeutic avenue to reversing the decline in cardiac function in AIC, highlighting the time-dependent nature of autophagy-based therapy.

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.