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Investigating the Impact of Fatty Acids on FTO Activity and RNA Methylation in Hypoxic Cardiomyocytes
Secondary Supervisor(s): Dr Hannah Bridgewater (Warwick)
University of Registration: Coventry University
BBSRC Research Themes: Integrated Understanding of Health (Diet and Health)
Project Outline
When heart cells (cardiomyocytes) are overloaded with fatty acids, they struggle to adapt to low oxygen levels (hypoxia). This difficulty is influenced by several factors, including the regulation of specific enzymes called alpha-ketoglutarate dependent oxygenases (aKGDOs). One such enzyme is the fat mass and obesity-associated (FTO) protein [1,2].
Variations in the FTO gene have been linked to conditions like obesity and type 2 diabetes, but the reasons behind this connection are not well understood [3]. Recently, FTO has been identified as an enzyme that removes methyl groups from RNA, specifically from a modification called N6-methyladenosine (m6A). The role of m6A modifications in RNA is still debated, but they likely affect RNA stability, translation, decay, and splicing. The pattern of m6A modifications on RNA appears to be more important than the amount of methylation.
This underscores the importance of studying RNA methylation patterns (using RNA-Me-Seq) to understand changes related to FTO activity. Preliminary research in Dr. Dodd’s lab has shown that fatty acids disrupt the normal function of aKGDOs in hypoxia, which may lead to increased death of heart cells. We hypothesize that fatty acids change FTO activity, leading to altered RNA methylation patterns and increased heart cell death under low oxygen conditions.
Aims
The aim of this work is to determine the action of fatty acids on FTO activity and the change in the RNA-Me-Seq profile in hypoxic fatty acid treated cardiomyocytes.
Objectives
Objective 1 – Assess the effects of various long-chain saturated and unsaturated fatty acids on FTO activity and quantity in cardiomyocytes.
Objective 2 – To measure global m6A modifications in cardiomyocytes treated with different fatty acid compositions.
Objective 3 – To preform RNAseq and m6A detection on fatty acid treated cardiomyocytes.
Methods
This project will employ advanced molecular and cellular biology techniques developed in Dr. Dodd’s and Dr. Bridgewater’s labs [1,4]. In brief, HL-1 cardiomyocytes will be cultured in hypoxia (2%) with different ratios of saturated, monounsaturated, and polyunsaturated fatty acids, including, palmitic, oleic, stearic, eicosapentaenoic, docosahexaenoic, and linolenic acid (all bound to bovine serum albumin). Around 10 different ratios and compositions will be tested. FTO activity and quantity, along with global m6A levels will be determined using ELISAs and western blotting for all conditions. Together this will provide a pattern of which fatty acids have the greatest effect on m6A and FTO levels/activity in hypoxic cardiomyocytes. This optimised composition will subsequently be used to generate cells for RNASeq and RNA-Me-Seq with Dr Bridgewater. We expect to identify specific fatty acids that significantly alter FTO activity and RNA methylation. Overall, this result could inform dietary recommendations and therapeutic interventions for conditions relating to hyperlipidaemia including cardiovascular diseases.
References
1 – Dodd, M. S., Sousa Fialho, M. D. L., Montes Aparicio, C. N. et al. Fatty Acids Prevent Hypoxia-Inducible Factor-1α Signaling Through Decreased Succinate in Diabetes.JACC. Basic to translational science,3(4), 485–498. (2018)
2- Jia, G., Fu, Y., Zhao, X.et al.N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.Nat Chem Biol7, 885–887 (2011)
3 - Scott, L. J., Mohlke, K. L., Bonnycastle, L. L., et al. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants.Science (New York, N.Y.),316(5829), 1341–1345. (2007).
4 - Feehan, K.T., Bridgewater, H.E., Stenkiewicz-Witeska, J.et al.Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.Nat Commun15, 4326 (2024).
