Exploring the role of LEM domain proteins in cardiac conduction and inherited cardiomyopathy

The nuclear envelope and associated Linker between Nucleoskeleton and Cytoskeleton (LINC) complex proteins are essential for proper cardiac and skeletal muscle development 1-3 (Fig 1). This is highlighted by the numerous mutations that lead to cardiac and skeletal myopathies in humans and mice that are collectively known as laminopathies. Interestingly, laminopathies predominantly affect striated muscle tissue which are under constant mechanical load.

Pathological mechanisms underlying the laminopathies are poorly understood, but seem to involve multiple, often overlapping factors: altered or weakened structural integrity at the nucleus, which appears to be particularly important in contractile cells, leading to aberrant morphologies, ruptures and DNA damage; altered genome organisation, adversely affecting gene expression; and altered chemical and biomechanical signalling, affecting a host of cellular functions 4-6.

Importantly, emerging data suggest that mutations in proteins within the LEM domain-containing family, which include Lem2, Emerin and Man1 cause both inherited cardiomyopathy as well as congenital heart disorders. Specifically, loss of NE protein MAN1 is associated with cardiac arrhythmias and congenital heart defects in patients. To investigate the underlying mechanisms that lead to cardiac dysfunction, we have generated a novel mouse model in which deletion of Man1 in cardiac progenitor cells leads to cardiac dysfunction and atrioventricular block.

Our pilot data suggest this is likely due to excessive fibrosis in the annulus fibrosus, through which the cardiac conduction system (CCS) passes to connect the atria to ventricles (Fig 2). Man1 has previously been shown to regulate TGFB/BMP signalling by regulating receptor-SMAD localisation and phosphorylation at the NE. Therefore, loss of Man1 may lead to deregulated TGFB/BMP signalling at the NE, driving excessive fibrosis and dysfunction.

However, the precise cellular and molecular mechanisms played by Man1 in the heart remain obscure. This proposal aims to reveal Man1's roles in the heart and which cell type(s) it is essential for proper heart development, along with the molecular sequelae that are consequent to cardiac dysfunction.

The overall goal of this project is to characterise the role of Man1 in primary epicardial cells and cardiomyocytes in vitro and conditional knockout mice in vivo. To achieve this objective, we propose the following Aims: Aim 1: Examine cardiac function in Man1 cardiomyocyte-specific KO mice Aim 2) Elucidate Man1's role in NE organisation and fragility in cardiomyocytes

Aim 3: Generation and characterisation of epicardium-specific knockout mice of LEM domain proteins using floxed mice

Aim 4: siRNA-mediated knockdown in primary murine epicardium undergoing epithelial-to-mesenchymal transition (EMT) and human embryonic stem cells and characterise the functional effects of removing Man1 on cell proliferation, death, migration, and differentiation.