Dottorato in Fisiopatologia Medica

Contatti

Supervisore

Franco Veglio

Curriculum vitae

Curriculum Vitae

Attività di ricerca

YEAR-END REPORT OF THE PhD RESEARCH ACTIVITY, 2^nd YEAR

 

PhD Student: Marco Mergiotti

Tutor: Prof.ssa Alessandra Ghigo

Coordinator: Prof. Franco Veglio

PhD School: Fisiopatologia Medica, 37th cycle

Project: Interrogating Early Molecular Events of Doxorubicin-induced Cardiotoxicity at Single-cell Level to Identify New Therapeutic Approaches

Narrative summary

Our research group, under the supervision of Prof. Alessandra Ghigo, focuses on the role of PI3K signaling in modulating metabolism and senescence in cardiac cells upon treatment with doxorubicin (DOX), one of the mostly used chemotherapies in clinics. Additionally, in the last few months, our group has intensified the collaboration with Prof. Michela Noseda from the Imperial College of London, in analyzing single-nucleus RNA-sequencing (snRNA-seq) data obtained from DOX-treated animals. We mainly focused on the analysis of gene signatures of early stages after treatment (3 days after the one single injection of low-dose DOX), when the cardiac dysfunction is not yet detectable clinically and thus likely to be reversible. Since cardiomyocytes are the major cell type damaged by DOX, mainly because of their inability to regenerate, we first focused on this cell type among all populations identified by snRNA-seq (endothelial cells, fibroblasts, mural cells, myeloid cells, lymphoid cells, lymphatic endothelial cells, neurons).

Background and rationale

Doxorubicin (DOX) is a highly effective chemotherapeutic agent widely used for treating various types of malignancies. Unfortunately, its clinical use is hampered by cardiotoxic side effects which are responsible for increased mortality among cancer survivors compared to the general population. Preclinical and clinical studies have highlighted potential mechanisms of anthracycline-induced cardiotoxicity (AIC), but the core molecular basis remains largely unknown. More importantly, only a few studies have focused on the characterization of the molecular events that distinguish the early reversible phase of AIC from an advanced and irreversible state of the disease.

Aim: We aim to provide an unbiased and in-depth profile of early transcriptional changes induced by DOX at single cell resolution, to identify new druggable targets for the development of cardioprotective agents to prevent AIC.

Methods: Based on our previously established murine model of AIC (Li et al, Circulation, 2019), BALB/c mice were injected with saline (Vehicle) or DOX (3 weekly injections of 4 mg/kg), and hearts were collected at either 3 days (acute cardiotoxicity) or 6 weeks after the first injection (chronic cardiotoxicity). Nuclei were isolated from frozen hearts for single-nuclei transcriptomic (snRNAseq; 10x Genomics, Chromium Single Cell 3' v3.1). Raw data were cleaned up for ambient transcripts (CellBender). Seurat pipeline was used for analysis including quality control, batch correction, dimensionality reduction, unbiased clustering and differential gene expression analysis. Scanpy was used for data visualisation. Differential abundance of each cell state was examined by Milo. SCORPIUS was applied for trajectory analysis. Zebrafish model of and hiPSC-derived cardiomycytes (hiPSC-derived CM) were used for validation of genes potentially involved in AIC.

Results: Echocardiography revealed DOX-induced systolic dysfunction at 6 weeks, confirming the establishment of AIC (Data not shown). SnRNAseq data from 12 hearts allowed to identify 8 major cell types including cardiomyocytes (CM), endothelial (EC) and mural cells, fibroblasts, myeloid cells, B and T lymphocytes and neuronal-like cells. To unravel changes in CM, we performed unbiased subclustering which defined 6 different subpopulations, including a CM_Stressed population, characterized by enrichment of typical cardiac stress markers, such as natriuretic peptide hormone (Nppb) and myosin heavy chain 7 (Myh7), typically overexpressed in diseased mouse hearts. Cell composition analysis and Milo differential abundance analysis showed enrichment of the CM-Stressed population at both 3 days and 6 weeks, suggesting that DOX drives a transcriptional change toward stress status in CM both at early and late stages of cardiotoxicity. To examine how DOX specifically alters gene expression within each cell state, we performed trajectory analysis scFates and uncovered a progression path from CM_Energetic, CM_Basal, CM_Intermediate to CM_Stressed. Moreover, differential gene expression analysis revealed Golgi associated kinase 1b (Gask1b) as perfectly following the progression path towards stressed cell state and upregulated in DOX-treated groups in different CM subclusters. To understand if Gask1b gene could be a new potential determinant of DOX-induced cardiotoxicity, we performed Gask1b gene silencing in cardiac myosin ligh chain-GFP (cmlc:GFP) expressing zebrafish. Preliminary screening in a zebrafish model of AIC showed that Gask1b gene silencing partially reduced DOX-induced decline of fractional shortening. In further agreement with this data, when we infected hiPSC-derived CMs with shGask1b-lentiviral particles, cell mortality induced by DOX was partially prevented, identifying Gask1b as a new potential player in DOX cardiotoxicity. 

Conclusions: We generated a single-nucleus dataset of DOX-treated mouse hearts and identified transcriptional changes driven by DOX in CM at early and late stages of the disease. Furthermore, we identified Gask1b gene, whose role in cardiac pathophysiology was previously unappreciated, as a new potential marker and determinant of DOX cardiotoxicity.

Future Perspective: From a future perspective, further investigations would be needed to fully elucidate the role of Gask1b in AIC development. Moreover, we are deeply exploring the role of other cardiac cell populations, such as immune cells and fibroblast which are known to be associated to AIC pathophysiology.

2. Publications and other activities:

Publications:

1) A. Murabito_, M. Mergiotti, M. Li, P. Peretto,  A. Raimondi, A. Loffreda, V. Capurro, C. Tacchetti, N. Pedemonte, E. Hirsch, A. Ghigo. “A PI3Kg Mimetic Peptide promotes F508del-CFTR stabilization at plasma membrane through a PKD1-dependent mechanism”. Manuscript under preparation.  

Conference, seminars, posters and given talks:

1. Given talk at MBC “Guido Tarone” weekly seminar entitled: “A PI3Kg Mimetic Peptide promotes CFTR localization at the Plasma Membrane through a PKD1-dependent mechanism”, 30^th January 2023;

 

2. Selected abstract and given talk at 18^th European Society of Cystic Fibrosis (ECFS) Basic Science Conference 2023 entitled: “A PI3Kγ Mimetic Peptide Promotes F508del-CFTR Localization at Plasma Membrane through a PKD1-dependent Mechanism”, 29^th-1^st April 2023, Dubrovnik, Croatia;

 

3. Selected abstract for poster presentation at “Tarone Day” entitled: “A PI3Kγ Mimetic Peptide Promotes F508del-CFTR Localization at Plasma Membrane through a PKD1-dependent Mechanism”, 26^th May 2023, MBC “Guido Tarone”, Torino, Italy;

 

4. Selected abstract for poster presentation at “The PI3K-AKT-mTOR-PTEN pathway: a new era in basic research and clinical translation” entitled: “Targeting the Anchoring Function of PI3Kγ with a Mimetic Peptide as a Promising Therapeutic Strategy for Cystic Fibrosis”, 13^th-15^th September 2023, Castelldefels (Barcelona), Spain;

 

5. Selected abstract for given talk at Società Italiana di Ricerche Cardiovascolari (SIRC) congress entitled: “Interrogating Early Molecular Events of Doxorubicin-induced Cardiotoxicity at Single-cell Level to Identify New Therapeutic Approaches”, 18^th-20^th October 2023, Imola, Italy.

 

Awards:

1. Best oral presentation and travel grant awards at 18^th European Society of Cystic Fibrosis (ECFS) Basic Science Conference 2023, 29^th-1^st April 2023, Dubrovnik, Croatia;

 

2. Travel grant award at SIRC congress, 18^th-20^th, Imola, Italy.