Examining the Role of Lmod2 and its Functional Domains in Striated Muscle

Abstract

Striated muscles are highly organized structures that convert chemical energy into mechanical work. they consist of both cardiac and skeletal muscles and are referred to as “striated” due to the repeating bands of functional units observed along the muscle fibers called sarcomeres. A sarcomere is the most basic contractile unit of muscle, consisting of myosin-thick filaments, actin-thin filaments and a plethora of structural and regulatory proteins. In response to increases in cytosolic calcium levels, thick filaments bind the thin filaments and pull them towards the center of the sarcomere, resulting in muscle contraction. Actin filament regulation and length is crucial for efficient contraction and is regulated by a myriad of thin filament binding proteins like the tropomodulin family of proteins. The tropomodulin family of proteins consists of tropomodulins (Tmods) and leiomodins (Lmods). Although they share similar domain structures (they share a tropomyosin binding site (TMBS) and two actin binding sites(ABS)), key differences provide them with opposing functions (Tmod has an extra TMBS while Lmod has an extra ABS). Tmods are known to regulate actin filament length by preventing thin filament polymerization and depolymerization while Lmods allow controlled actin polymerization by acting as a leaky cap at the thin filament pointed ends. The function of Lmod2 as a leaky cap was recently proposed and it is thought to be mediated via ABS1. In this dissertation, the contribution of ABS1 to Lmod2’s leaky cap activity was studied, first by determining the specific residues within ABS1 that interact with actin and second by creating a quadruple mutant predicted to decrease binding of ABS1 to actin. We found that Lmod2’s ABS1 binds actin both via a N-terminal disordered region and a C-terminal amphipathic helix, and introduction of the quadruple mutations abolished binding of the C-terminal helix to actin. This resulted in the inability of Lmod2 to control pointed end elongation as we observed extraordinarily long thin filaments in cells in culture expressing the quadruple mutations. We confirmed the role of Lmod2 as a leaky cap in vivo by expressing the quadruple mutant in the Lmod2 KO mouse model, which in contrast to mice expressing the wild-type construct, developed abnormally long thin filaments. Overall, we were able to identify key residues within ABS1 that interact with actin and demonstrated that ABS1 of Lmod2 is crucial to control elongation at the pointed end of thin filaments both at the cell and at the whole organism level. Due to the severe cardiac phenotype observed in the Lmod2 KO mice and in patients diagnosed with LMOD2 pathogenic variants, the study of Lmod2’s function has been mainly focused on the heart, while its role in skeletal muscle is relatively unknown. In this dissertation, the role of Lmod2 in skeletal muscle was investigated by creating a skeletal-specific Lmod2 KO mouse model. We found that absence of Lmod2 in skeletal muscle results in an overall decrease in contractility in both slow and fast twitch fibers. Since Lmod2 regulates thin filament length in slow but not fast twitch skeletal muscle, it was determined that Lmod2 regulates contractility, independent of its role in thin filament length regulation. Lmod2 loss had a more profound effect on slow-twitch muscle, where Lmod2 is more abundant, suggesting a dose dependent mechanism. In addition to a decrease in force, the slow-twitch soleus muscles experience a fiber type switch (from fast myosin heavy chain (MHC) IIA fibers, to the slower MHC I fibers) as well as increased Lmod3 levels and abnormally short actin filaments. These additional changes observed in soleus muscle were attributed to compensatory mechanisms in response to the absence of Lmod2. Overall, we demonstrated that ABS1 of Lmod2 is necessary to control thin filament elongation at the pointed end. We also found that Lmod2 plays an important role in regulating actomyosin interactions in skeletal muscle, a function which is unrelated to its role in thin filament length regulation.