The role of microtubule-associated protein 1S (MAP1S) in regulating pathological cardiac hypertrophy

Abstract

Cardiac hypertrophy is an important process that may lead to the development of heart failure. Controlling pathological cardiac hypertrophy is important because it can improve the long-term prognosis of heart failure. The aim of this project is to investigate the role of Microtubule-associated protein 1S (MAP1S) in regulating pathological hypertrophy in mice. In non-cardiac cells MAP1S has been shown to regulate autophagy. It is one of the key proteins that regulate autophagy during the development of tumorigenesis to eliminate damaged organelles and proteins that cause oxidative stress and genome instability. Autophagy is a catabolic process and is considered as a survival process because it recycles damaged organelles and misfolded proteins. Autophagy is involved in various diseases including heart diseases; however, the exact role of autophagy in regulating cardiac hypertrophy is not fully understood. MAP1S has previously been identified as an interacting partner of the major autophagy regulator LC3; however, its role in the heart is unknown. In this study I investigated the role of MAP1S in regulating autophagy during cardiac hypertrophy. To study the role of MAP1S in cardiac hypertrophy I studied mice with global genetic deletion of the Map1s gene (MAP1S−/−). To induce pathological hypertrophy, MAP1S−/− mice were subjected to transverse aortic constriction (TAC) for two weeks. Analysis of heart weight/tibia length ratio showed a significant reduction of hypertrophy in MAP1S−/− mice compared with the WT group. Furthermore, expression of hypertrophic markers ANP and BNP, detected by qPCR, showed higher expression in WT-TAC mice compared to MAP1S−/− TAC mice. However, no significant difference in the cardiac function parameters fractional shortening (FS) and ejection fraction (EF) was observed in MAP1S−/− compared to WT mice after two weeks TAC. To analyse cellular hypertrophy, histological sections of cardiac tissues were also analysed. Consistently, the cardiomyocyte cell surface area was significantly smaller in MAP1S−/− TAC mice compared to the WT-TAC group. Also, the level of interstitial fibrosis detected using Masson's trichrome staining analysis was reduced in MAP1S−/− mice following TAC compared to WT-TAC, but expression levels of fibrosis markers such as COL1A1 and COL3A1 did not show any significant difference between MAP1S−/− TAC mice compared to the WT-TAC group. To investigate the level of autophagy, the expression of autophagy markers was analysed. The expression of the autophagosome formation step markers LC3-I and LC3-II were significantly reduced in MAP1S−/− TAC mice compared to WT-TAC group; however, the level of other autophagy induction markers such as Beclin-1 and P62/SQSTM1 did not show any changes. To further analyse the role of MAP1S in regulating the formation of autophagosome, in vitro studies was performed using cardiomyoblast cell line (H9C2) with siRNA-mediated knock down of the MAP1S gene. To detect the autophagosome formation, I used adenovirus to express GFP-labelled LC3 in the H9C2 cells and induced autophagy by treating cells with rapamycin and chloroquine. No significant change in the autophagosome formation was observed in MAP1S knock down cells compared to control cells. However, by using MitoTracker to visualize mitochondria, I found that the MAP1S knock down cells showed more co-localization of mitochondria with the autophagosomes, suggesting increase level of damaged mitochondria and the occurrence of mitophagy. Then, to analyse whether this phenotype also occurs in vivo in the TAC model, transmission electron microscopy was used to analyse the mitochondria structure in the heart of MAP1S-/- and WT controls. In consistent with the in vitro data, MAP1S−/− mice showed a higher number of abnormal mitochondria compared to WT controls. Finally, to investigate whether MAP1S regulates other signalling pathways during pathological hypertrophy PathScan phospho-kinase array was used to detect phosphorylation of eighteen different signalling molecules in MAP1S−/− and WT hearts after sham or TAC treatment. Although, the kinase array data showed slightly different signals of p-STAT3 and p-BAD between knockout and WT, further confirmation by Western blot did not show any significant difference in phosphorylation levels of these proteins. In conclusion, the ablation of MAP1S reduced the development of pathological cardiac hypertrophy in mice, possibly via the regulation of autophagy in cardiomyocytes.

Date of Award	3 Oct 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Xin Wang (Supervisor) & Delvac Oceandy (Supervisor)