Absence of complement factor H reduces physical performance in C57BL6 mice

Abstract

Complement (C) system is a double edge sword acting as the first line of defense on the one hand and causing aggravation of disease on the other. C activation when unregulated affects different organs including muscle regeneration. However, the effect of factor H (FH), a critical regulator of the alternative C pathway in muscle remains to be studied. FH deficiency results in excessive C activation and generates proinflammatory fragments C5a and C3a as byproducts. C3a and C5a signal through their respective receptors, C5aR and C3aR. In this study, we investigated the role of FH and downstream C5a/C5aR signaling in muscle architecture and function. Using the FH knockout (fh-/-) and fh-/-/C5aR-/double knockout mice we explored the role of C, specifically the alternative C pathway in muscle dysfunction. Substantial C3 and C9 deposits occur along the walls of the fh-/- muscle fibers indicative of unrestricted C activation. Physical performance assessments of the fh-/- mice show reduced grip endurance (76 %), grip strength (14 %) and rotarod balance (36 %) compared to controls. Histological analysis revealed a shift in muscle fiber populations indicated by an increase in glycolytic MHC IIB fibers and reduction in oxidative MHC IIA fibers. Consistent with this finding, mitochondrial DNA (mtDNA) and citrate synthase (CS) expression were both reduced indicating possible reduction in mitochondrial biomass. In addition, our results showed a significant increase in TGFβ expression and altered TGFβ localization in this setting. The architecture of cytoskeletal proteins actin and vimentin in the fh-/- muscle was changed that could lead to contractile weakness and loss of skeletal muscle elasticity. The muscle pathology in fh-/- mice was reduced in fh-/-/C5aR-/- double knockout (DKO) mice, highlighting partial C5aR dependence. Our results for the first time demonstrate an important role of FH in physical performance and skeletal muscle health.

Introduction

The complement (C) system is an important component of muscle inflammation that occur in settings such as in autoimmune disease, and modified muscle use (Sewry et al., 1987; Spuler and Engel, 1998; Walport, 2001a, b; Zipfel and Skerka, 2009). C proteins (Wang et al., 2017) participate in functions such as inhibition and restoration of muscle regeneration (Naito et al., 2012; Zhang et al., 2017) and in recruitment of macrophages (Wang et al., 2017). However, the role of complement factor H (FH) in muscle health and disease remains unknown. FH (FH) is an important C regulator generated mostly by the liver (Pangburn, 1988) and also by other organs, including muscle (Alexander and Quigg, 2007; Ferreira et al., 2010). (Legoedec et al., 1995). FH is traditionally involved in innate host defense, but also participates in noncanonical roles such as immune-evasion, regulation of B-cell differentiation and platelet activation (Alexander and Quigg, 2007; Ferreira et al., 2010). In addition to FH, the role of other complement proteins such as C3, C3a and C5a in muscle health is just beginning to emerge (Puri and Quigg, 2007; Sorgenfrei et al., 1982). (Behan and Behan, 1977). C causes lysis of muscle membranes in myasthenia gravis (Ashizawa and Appel, 1985)and C3 and C9 deposition in necrotic fibers (Cornelio and Dones, 1984; Morgan et al., 1984). C5b-9 can lead to a loss in membrane integrity and necrosis of the targeted cells (Mathey et al., 1994; Schafer et al., 1986). However, the exact underlying mechanism/s by which C activation causes alteration in muscle needs to be explored.

Muscle function is closely related to the fiber population and quantity of connective tissues between fibers (Richmonds and Kaminski, 2001). Skeletal muscle is composed of a mosaic of muscle fiber types (type I, type IIA, type IIB, and type IIX), which have different amounts of myosin heavy chain (MHC) proteins, mitochondria, and capillary density, and different susceptibility to loss of strength and fatigue. Based on the MHC isoforms skeletal muscle is characterized as both oxidative slow twitch fibers with a higher mitochondrial density (type I and type IIA) (Buckingham et al., 1986; Schiaffino et al., 1989; Staron et al., 1990) and glycolytic fast twitch fibers more reliant on glycolysis to metabolize glucose (types IIb) that differentially impact muscle metabolism (Clarke et al., 1975; Gulick et al., 1997; Moncman et al., 1993). The maximum velocity of contraction (Vmax) for fibers composed of a single MHC increases in the order type I, IIA, IIX, IIB (Bottinelli et al., 1991; Galler, 1994). Intermediate hybrid fibers, containing type I and IIA, can be observed in normal muscles. Muscle is a dynamic organ in which fiber type shifts take place, either in response to exercise or electrical stimulation or during muscle wasting associated with atrophying conditions. The optimal functioning of the different fibers are based on the energy demands being met, which is maintained by fine tuning of the mitochondrial density and function (Zong et al., 2002). ATP generation in muscle occurs by both glycolysis and mitochondrial function (Rangaraju et al., 2014). Since both fiber composition and metabolic properties affect function, it is interesting to know the extent to which they co-vary. Muscle cytoarchitecture is another important aspect underlying muscle integrity and function. Cellular cytoskeleton network can induce protein conformational change (Sawada et al., 2006) leading to disruption of intermediate filaments resulting in a number of diseases (Traub and Shoeman, 1994). The actin cytoskeleton undergoes dynamic assembly and disassembly during cell migration and adhesion (Tang and Gerlach, 2017) and therefore is regulated by a variety of actin-associated protein and signaling pathways, including the vimentin network and by members of the TGFβ family (Jiu et al., 2017).

In brief, for the first time our study presents evidence that CFH deficiency in mice results in reduced muscle strength, altered muscle fiber composition and a concomitant reduction in mitochondrial DNA and citrate synthase activity compared to the wildtype animals. Our results demonstrate that C activation plays a critical role in muscle pathogenesis in an inflammatory setting and reveal its potential as a therapeutic target.