Protein arginylation of cytoskeletal proteins in the muscle: modifications modifying function

Abstract

The cytoskeleton drives many essential processes in normal physiology, and its impairments underlie many diseases, including skeletal myopathies, cancer, and heart failure, that broadly affect developed countries worldwide. Cytoskeleton regulation is a field of investigation of rapidly emerging global importance and a new venue for the development of potential therapies. This review overviews our present understanding of the posttranslational regulation of the muscle cytoskeleton through arginylation, a tRNA-dependent addition of arginine to proteins mediated by arginyltransferase 1. We focus largely on arginylation-dependent regulation of striated muscles, shown to play critical roles in facilitating muscle integrity, contractility, regulation, and strength.

INTRODUCTION

The cytoskeleton drives many essential processes in normal physiology, including developmental morphogenesis, tissue integrity, muscle contraction, and functioning of virtually every organ system. Consequently, impairments in the cytoskeleton structure and the intricate molecular interactions involved in their dynamics and function can lead to catastrophic consequences and have been shown to underlie diseases such as cancer and heart failure, the two leading causes of death in developed countries worldwide. Although many proteins involved in cytoskeleton function have been identified and extensively studied over the last few decades, we still know very little about the mechanisms governing cytoskeleton regulation and its impairment in diseases. Among these mechanisms, a central role belongs to posttranslational modifications, covalent addition of chemical groups to proteins that may significantly affect their function.

One form of posttranslational modification that targets the cytoskeleton is arginylation (24, 25), a recently emerging regulatory mechanism that has implications in many physiological systems (24, 25, 33). This article provides an overview of the recently discovered role of posttranslational arginylation in the regulation of the cytoskeleton, with the focus on striated muscle regulation, of fundamental importance in many aspects of life.

PROTEIN ARGINYLATION

Posttranslational addition of amino acids to proteins was first discovered in 1963, when a group of researchers observed ribosome-independent incorporation of Leu and Phe into test proteins in an Escherichia coli-based in vitro system (18). A similar phenomenon was observed in a eukaryotic system: in liver extracts, proteins were seen incorporating Arg in the absence of ribosomes (19). Given the complexity of this system, it took years to identify the enzymes responsible for this incorporation—leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase) in bacteria (51, 52) and Arg-tRNA protein transferase (arginyltransferase 1, or Ate1) in eukaryotes (1)—and it took decades to gain any insights into the biological role of these modifications.

Ate1 is present in all eukaryotes from yeast to human and is encoded by a single gene in animals and fungi and two genes in plants. In higher eukaryotes and mammals, Ate1 gene encodes several homologous isoforms (four, in most species), generated by alternative splicing of two pairs of exons (41). Although these isoforms show some differences in tissue specificity and the repertoire of substrates, their functional distinction has not been well characterized.

The biological activity of Ate1 consists of transferring Arg from the charged Arg-tRNA to protein or peptide substrates. This modification targets primarily the acidic amino acid residues [Asp, Glu, and trioxidated Cys (8, 13, 53)], although NH₂-terminal arginylation of other residues in vivo has also been observed (59, 61). Arginylation was originally believed to solely target the protein’s NH₂ terminus, resulting in a peptide bond linkage of Arg to the protein or peptide amino group exposed at the NH₂ terminus, but recently it was found that Ate1 can also arginylate the side chains of Asp and Glu at midchain sites in proteins via an isopeptide bond (58).

Early studies implicated arginylation as a step in the ubiquitin/proteasome-dependent N-end rule pathway of protein degradation (54, 56). Using test substrates derived from bacterial β-galactosidase (β-Gal), it was found that addition of NH₂-terminal Arg leads to dramatically decreased metabolic stability of some test proteins and that this Arg-dependent destabilization can result from the presence of enzymatically active Ate1 in the case of β-Gal derivatives containing NH₂-terminal Asp or Glu. In mammalian systems, a similar effect of Ate1 on protein destabilization was also seen in the case of NH₂-terminal Cys, via a targeting mechanism that was later linked to Cys oxidation. Despite extensive follow-up studies, very few in vivo substrates that are targeted for degradation through arginylation have been found to date, and all of these [including mammalian regulator of G protein signaling 4 (RGS4), RGS5, and RGS16 (5, 31) and plant group VII ethylene response factors (ERF-VIIs; 60)] are Cys dependent.

Significant insights into the mechanisms and role of arginylation were opened recently with the development of mass spectrometry-based methods of identification of posttranslational modifications. These methods revealed a large number of intracellular arginylation targets that potentially do not follow the N-end rule pathway and may not always be Asp/Glu/Cys dependent (59, 61). Although some of this noncanonical activity can be mediated directly by Ate1 isoforms, the possibility of other enzymes mediating arginylation in vivo remains open. More recently, arginylation was shown to directly regulate the overall switch in protein flux between proteasomal degradation and autophagy (16, 17). Overall, accumulating data suggest that arginylation is a potent global mechanism of protein regulation that can affect protein stability, protein-protein interactions, activity, and functions (23, 48).

ARGINYLATION OF CYTOSKELETON PROTEINS

In 2007, the first mass spectrometry-based study of global intracellular arginylation identified 43 proteins arginylated in vivo in mouse cells and tissues (61). Notably, over one-fourth of these proteins are key components of the cytoskeleton, including actin and tubulin, the central components of actin and microtubule cytoskeleton organization in the cell.

A breakthrough in the understanding of arginylation-dependent cytoskeleton regulation was achieved with the discovery that nonmuscle β-actin, a ubiquitously expressed, highly abundant intracellular protein, can be arginylated at its NH₂ terminus (22). Arginylation was found on the third residue in the β-actin sequence, following its NH₂-terminal preprocessing, which at present is only partially characterized. Importantly, it was found that β-actin NH₂-terminal arginylation regulates actin cytoskeleton and cell motility in mouse embryonic fibroblasts (22). This finding was the first demonstration of a nondegradation pathway of protein regulation by arginylation. The exact underlying mechanisms of the effect of NH₂-terminal arginylation on actin function are still under investigation.

The discovery of midchain arginylation (58) revealed enrichment of this posttranslational modification in some of the more stationary intracellular structures, including cardiac and skeletal muscle cells. These muscle proteins include myosin, actin, and several other highly abundant and important myofibril components (4). Most of the striated muscle proteins targeted for arginylation are directly related to muscle development, maintenance of myofibril structure, contractile activity and its regulation, and production of active and passive forces (4, 26, 34). These proteins include the myosin heavy chains (MHCs), myosin light chains, actin, α-actinin, tropomyosin (Tm), troponin T (TnT), myosin-binding protein C (MyBP-C), and titin, differentially arginylated in cardiac and skeletal muscles (Table 1). Notably, the arginylated residues in these proteins are located within regions of high sequence conservation that are identical between multiple species. This further supports the idea that regulation of these proteins by arginylation is functionally important and likely evolutionarily conserved.

Table 1.

Sites arginylated in cardiac and skeletal myofibrils

Gene Symbol	Muscle Type	Accession No.	Name	Arginylated Site(s)
Acta1	Skeletal	NP_033736.1	Actin-α, skeletal muscle	E74
Actn3	Skeletal	NP_038484.1	α-Actinin-3	D456, D462, D465
Ckm	Skeletal	NP_031736.1	Creatine kinase M-type	D326, D335
Capzb	Skeletal	NP_001032850.1	F-actin-capping protein subunit β isoform a	E22
Myh2	Skeletal	NP_001034634.2	Myosin heavy chain IIa	E1169
Myh4	Skeletal	NP_034985.2	Myosin heavy chain 4	E887, E1005, E1012, E1166, E1500
Myh7	Skeletal	NP_542766.1
Mybpc2	Skeletal	NP_666301.2	Myosin-binding protein C, fast-type isoform 2	E162
Tnnt3	Skeletal	NP_001157138.1	Troponin T	D63, D72
Usp21	Skeletal	NP_038947.2	Ubiquitin carboxyl-terminal hydrolase 21	D439
Actn2	Cardiac	NP_150371.4	α-Actinin-2	L510
Fbn1	Cardiac	NP_032019.2	Fibrillin 1	E640‡, Q671, L1266†, E1794†
Lamc1	Cardiac	NP_034813.2	Laminin γ1	E803†
Myh6	Cardiac	NP_034986.1	Myosin heavy chain 6	L747*, K999*, L1001*‡, V1027*, L1486*, Q1534,  L1578†, N1647
Myl3	Cardiac	NP_034989.1	Myosin light chain 3	A20, T81‡, M117
Ttn	Cardiac	NP_082280.2	Titin N2B	L7960, V15013, C24818†
Tpm1	Cardiac	NP_077745.2	Tropomyosin α-1	L50, K77
Tnnt2	Cardiac	NP_001123650.1	Cardiac troponin T2	A184

^*Also found arginylated in myosin heavy polypeptide 7 (Myh7; NP_542766.1; L745, K997, L999, V1025, L1484, and L1576);

^†Found as monomethylated arginine;

^‡Found as dimethylated arginine.

MECHANISMS OF CONTRACTION AND ARGINYLATION OF SARCOMERE PROTEINS

The mechanisms of muscle contraction have been gradually elucidated through data collected from structural (44, 45), biochemical (36), and mechanical (14, 15) studies. It is well accepted that muscle contraction is driven by cyclical interactions of the molecular motor myosin II with actin filaments in the sarcomere, a mechanical process coupled to the energy released from the breakdown of ATP. After the initial attachment between myosin and actin, phosphate is released, starting the so-called “power stroke,” responsible for force generation and actin sliding. ADP is subsequently released, establishing the rigor conformation, after which the binding of a new molecule of ATP disrupts the strong interaction between myosin and actin. The MHC in the myosin molecule is the main structure responsible for linking the chemical energy released from ATP and the mechanical work produced during myosin-actin interactions. The myosin light chains function as lever arms for the changes in myosin configurations that happen during the myosin-actin cycle.

At the sarcomere level, muscle contraction is further regulated by tropomyosin (Tm) and troponin (Tn). Biochemical and structural studies show that myosin’s interaction with actin thin filament is graded as three possible states: blocked, closed, and open (e.g., 32, 37, 57). In the absence of Ca²⁺, Tm prevents myosin from binding to actin by occupying the blocked state. Upon Ca²⁺ binding to Tn, the equilibrium position of Tm shifts toward the closed state, exposing actin sites that allow weak binding of myosin. Then myosin transits from the weak to the strong binding state, which shifts the equilibrium position of Tm further toward the open state. This shift permits cooperative binding of additional myosin heads by exposing neighboring actin-binding sites.

Besides the active force in response to muscle activation, sarcomeres also produce passive forces when they are stretched into long sarcomeres, a mechanism that stabilizes the muscle myofibrils and increases their stiffness, essential for the long-term maintenance of muscle integrity. These passive forces are produced by titin, a springlike molecule that will also maintain the A band in the center of the sarcomere upon muscle activation (7, 9).

Clearly, the myosin-actin interaction, the cross-bridge cycle, and its regulation are directly linked to the activity of proteins that are targeted by arginylation (Fig. 1). MHC presents the greatest number of target residues within the contractile proteins, including specific sites associated with the rod and tail domains of myosin responsible for self-association and the integrity of actomyosin assemblies. The actin filament (F-actin) is a polymer of actin globules (G-actin) assembled with different accessory proteins and is arginylated in skeletal muscle of adult mice and in the developing heart (4, 42; Table 1). One of these sites is accessible only before actin is incorporated into the polymer and may have important roles for actin polymerization (42).

Fig. 1.

Structural model of skeletal (top) and cardiac (bottom) components of the actomyosin complex modified by arginylation. Sites that are arginylated in actin and myosin molecules are shown with the red letter “R.” Note that arginylation occurs at key interaction sites in the molecules, which can significantly affect myosin-actin interaction. ELC, myosin essential light chain; HEAD, myosin motor head; MBPC, myosin-binding protein C; RLC, myosin regulatory light chain; TnT, troponin T.

Tropomyosin (Tm) is a rod-shaped protein that interacts with the actin filament. One Tm molecule binds seven actin subunits in a row and interacts with two Tm molecules through head-to-tail contact. Troponin T (TnT) is one of the components of the Tn complex, responsible for its affinity to Tm and for its fixation on the actin filament (40). Altogether, the troponin complexes and the tropomyosins form a long regulatory strand that runs along the grooves of the whole actin filament. Each regulatory unit is composed of one Tn complex, one Tm, and seven actin monomers. Sites targeted for arginylation are found on Tm and TnT in the cardiac muscle and on two different isoforms of TnT in the skeletal muscle (Table 1), suggesting that arginylation likely regulates properties and functions of the tropomyosin/troponin complex.

The protein α-actinin is a highly conserved actin-binding protein that cross-links antiparallel actin filaments from adjacent sarcomeres on the Z-disks. α-Actinin has several areas for protein-protein binding with important implications for Z-disk assembly and sarcomere function (10). In cardiac muscle, the site for arginylation on α-actinin is located in a short linker region that may be important for dimerization. In skeletal muscle, the arginylated residues are located within the second spectrin-like repeat, which is part of the α-actinin rod, an important area for protein-protein binding and one of the essential areas for titin anchoring. A weakening of the titin-myosin and/or titin-α-actinin binding may impair titin elongation during sarcomere stretch and, consequently, the passive forces.

Myosin-binding protein C protein C (MyBP-C) is an important regulatory protein found in the thick filament of striated muscles. It interacts with myosin, actin, and titin. Studies have linked MyBP-C to actomyosin formation and possibly strengthening of the thick filament backbone (35, 47). MyBP-C is composed mainly of immunoglobulin and fibronectin domains, with a proline/alanine-rich region and a MyBP-C-specific motif (M-domain). The arginylation site (E162) found in the skeletal muscle MyBPC is located in the M-domain (4), which is important for myosin-actin interactions.

Finally, titin is the main protein responsible for passive force development in muscle fibers. Titin is arginylated at several sites in both cardiac and skeletal muscles, located in the A band region of the molecule (34). On one side, titin closely interacts with the myosin filament and anchors in the M-line (12). This interaction starts early during muscle development and has important structural functions. The NH₂ terminus of titin binds to the entire length of the Z-line. In this area, titin molecules from adjacent sarcomeres overlap through titin-telethonin and titin-α-actinin binding (6). Although the skeletal muscle titin isoform presents more sites for arginylation than cardiac titin, one site found in the heart is located in the titin serine-threonine kinase domain (C24818). During myofibrillogenesis this kinase domain is important for sarcomere formation, driving a signaling pathway that senses mechanical load and regulates transcriptional activity in the cells (30).

ROLE OF ARGINYLATION IN MUSCLE PHYSIOLOGY

On the basis of the mechanisms of muscle contraction and regulation and location of the arginylated sites in the contractile and structural muscle proteins it would be expected that arginylation affects muscle assembly, integrity, and contractile activity. Notably, some of the added arginines in the myofibrils are further modified by Arg methylation, which has also been shown to target posttranslationally added Arg in the nucleus (49). This methylation has been previously proposed to target Arg in critically important positions in a protein to prevent these sites from being destroyed by proteolysis or chemical modifications (2). Thus, it is likely that arginylation on the myofibril proteins should be stable and long-lived, suggesting that it is important for muscle myofibril integrity and maintenance.

The development of the first knockout (KO) mice lacking the Ate1 enzyme (28) was decisive for the understanding of arginylation in striated muscles. These first full KO mice (Ate1^−/−) presented several developmental defects and early lethality, including severe impairments in heart structure and functioning (42). These findings represented an early indication of the key role that arginylation plays in striated muscle. Subsequent to the Ate1^−/− model, investigators developed cardiac-specific KO mice, with Ate1 deletion driven by α-myosin heavy chain promoter (α-MHC-Ate1), and skeletal muscle-specific KO mice, driven by creatine kinase M-type promoter (Ckmm-Ate1; 2). Interestingly, although the different mouse strains presented different phenotypes, they shared a similar feature: they all lead to significant muscle weakness and impairments in muscle contractility. Since then, studies looking into the potential effects of arginylation have been developed with skeletal and cardiac muscles using different levels of analysis, ranging from whole muscles to isolated myofibrils and single molecules.

MUSCLE DEVELOPMENT, STRUCTURE, AND OVERALL STRENGTH

The first studies looking into the effects of arginylation on cardiac muscles were performed with complete Ate1 KO mice (42). These mice exhibited embryonic lethality, accompanied by defects in cardiovascular development and angiogenesis. Most significantly, Ate1^−/− embryonic hearts presented ventricular and atrial septum defects, as well as thinner atrial and ventricular walls (28, 42), indicating that arginylation is essential for heart development. Cardiomyocytes isolated from these mice had irregular beating pattern and a disconnection between cardiac beating and Ca²⁺ waves, suggesting impairments in the regulation of contractions. Electron micrographs showed delayed myofibril formation, with fewer and more disorganized myofibrils than would have been expected for their developmental stage. Ate1^−/− myofibrils also presented diffused sarcomeric Z-lines and defects in the intercalated disks (42). All these effects at the structural level suggested a weakened heart muscle and large-scale negative effects on the functionality and contractility of the heart.

Since the results in these initial studies were suggestive of dramatic changes in the contractility of the heart, a subsequent study used cardiac-specific Ate1 KO mice driven by α-MHC promoter that activates after cardiomyocyte differentiation and thus confines the changes to the fully developed heart muscle (26). These α-MHC-Ate1 mice showed no defects in heart septation or myocardial thickness, suggesting that these defects may initiate before heart formation. Most importantly, these mice survived to adulthood, enabling the study of the direct effects of Ate1 on heart contractility. Notably, although these mice showed no obvious phenotypic changes at young age, they developed progressive dilated cardiomyopathy, resulting in increasing rates of lethality after 3 mo of age (26). These defects were accompanied by ultrastructural abnormalities in the heart muscle, which were clearly revealed by electron microscopy (Fig. 2). Structural abnormalities in the cardiac muscle in these mice were first observed just after birth and developed progressively with age. Echocardiographic measurements revealed indirectly, for the first time, impairment in cardiac contractility in the aged α-MHC-Ate1 mice, i.e., a reduction in the shortening of α-MHC-Ate1 hearts, a phenomenon commonly associated with dilated cardiomyopathy.

Fig. 2.

Electron micrographs of cross sections of skeletal and cardiac myofibrils from matched pairs of wild-type (WT) and arginyltransferase 1 (Ate1) conditional knockout (CKO) mice, derived from mice with knockout of Ate1 in the skeletal and cardiac muscle, respectively. In skeletal muscle, Ate1 knockout does not lead to structural abnormalities, whereas cardiac myofibrils in the absence of Ate1 appear disorganized. Red arrows indicate ultrastructural abnormalities. Scale bar, 500 nm.

Mice with Ate1 KO in the skeletal muscle driven by muscle-specific Ckmm promoter (Ckmm-Ate1) have also been developed (26, 27). In these mice, Cre activation occurs in skeletal myocytes upon their differentiation from myoblasts, resulting in complete deletion of Ate1 in the skeletal muscle with no expected changes in any nonmuscle tissues. Differently from cardiac muscles, the Ckmm-Ate1 mice were viable, fertile, and outwardly normal with no obvious defects in skeletal muscle appearance or size (Fig. 2). Ultrastructural studies of the soleus muscle from these mice failed to show marked structural difference from their littermate controls. Therefore, arginylation is not required for proper assembly of sarcomeric proteins in skeletal muscle, unlike in the heart. The reason for the difference in muscle development may be in the different residues and proteins that are arginylated in these muscles. For instance, levels of actin arginylation are higher in the developing heart than in skeletal muscles, and additional arginylated sites in actin monomers may be important for filament alignment. Furthermore, a specific titin site targeted by arginylation found in the heart but not in skeletal muscles is located in the titin serine-threonine kinase domain (C24818). This domain is important for sarcomere formation during myofibrillogenesis, driving a signaling pathway that senses mechanical load and regulates transcriptional activity in muscle cells.

Despite the apparent lack of structural differences, Ckmm-Ate1 mice showed a significant reduction in muscle strength. In tests for grip duration, grip strength, and ability to move hand over hand to the end of a horizontal wire (the phenotypic tests commonly used to test weakness linked to muscle dystrophy) the Ckmm-Ate1 mice performed poorly compared with wild-type (WT) mice (4). Some mice also presented with severe difficulties with locomotion and symptoms of muscular dystrophy that were induced by lack of arginylation (4).

In summary, although lack of arginylation has different effects on the ultrastructure of the muscle—severe in the heart and undetectable in the skeletal muscle—both the heart and the limb muscles present significant weakness in the absence of arginylation.

EFFECTS OF ARGINYLATION ON SUBCELLULAR FORCES: MECHANISTIC INSIGHTS

To investigate the cellular and subcellular consequences of the lack of arginylation, separate studies evaluated the active and passive forces developed by isolated myofibrils and sarcomeres, preparations that exclude any factor external to the sarcomere proteins. These preparations are the smallest muscle structures that still maintain the three-dimensional lattice space intact and can be activated and relaxed rapidly and consistently (e.g., 3, 38, 43, 46, 50). Myofibrils and sarcomeres allow force measurements without the interference of the processes leading to excitation-contraction coupling. Given their small diameter (~1 μm), myofibrils allow precise measurements of the “specific force” (the force produced during contractions normalized by the myofibril cross-sectional area), which prevents potential complications arising from changes in muscle size observed in muscular atrophy. Myofibrils allow a precise measurement of sarcomere length across a preparation, which is of vital importance when comparing forces from different muscles, as there is a strong length dependence of force production in striated muscles.

Myofibrils were isolated from cardiac (26, 46) or skeletal muscles (4, 34) and activated to develop isometric contractions using a fast-perfusion system. The active and passive myofibril forces were measured using advanced atomic force microscopy (29). In different studies, myofibrils isolated from the left ventricle from α-MHC-Ate1 mice produced ~30–50% less specific force than wild-type (WT) myofibrils during full activation (26, 46). Such a large decrease in force shows unequivocally that the loss in force observed in the whole muscle is a characteristic of the sarcomeres and, thus, the contractile proteins. The decrease in force was actually observed at all sarcomere lengths investigated in one of these studies (Fig. 3), showing that arginylation affects the contractile system directly, independent of the degree of filament (myosin and actin) overlap and of the active force produced by the contractile system.

Fig. 3.

Experiments performed with isolated myofibrils from the cardiac muscles of the mice. A: forces produced by the myofibrils during activation, following a shortening-stretch protocol, and during relaxation. B: average active forces produced by myofibrils at three sarcomere lengths. The force produced by the wild-type (WT) myofibrils is considerably higher than the force produced by the knockout (KO) myofibrils. Symbols indicate means ± SE.

The study was followed by similar experiments performed with skeletal muscle myofibrils, which also showed a substantial decrease in force, although smaller than in cardiac myofibrils, ~20% smaller in Ate1 knockout than WT (4). The smaller decrease in the force observed in skeletal muscle myofibrils compared with cardiac myofibrils may be associated with the differences observed in muscle structure; in cardiac muscles the misalignment of the contractile filaments may contribute significantly to an impairment of force.

Passive Forces

At the sarcomeric level, passive force is developed mainly by titin, a giant multifunctional protein that spans the half sarcomere, from the Z-lines to the M-lines. In the I band, titin forms springlike domains that work synchronously to adjust the tension within each sarcomere in response to its length (Fig. 4A). As the sarcomere stretches from slack to long lengths, the immunoglobulin-like domains (Ig domains) within this region start to develop force first, followed by the Glu-rich segment named proline-glutamate-valine-lysine-abundant structural motif (PEVK). The PEVK domains start to develop force at long sarcomere lengths (55). In cardiac muscles the springlike domain called N2B follows PEVK and produces force when the sarcomere reaches longer lengths (11).

Fig. 4.

A: schematic representation of a half sarcomere stretched passively. During sarcomere stretch (panel at bottom), the spring domains of titin stretch, producing passive forces. B: average passive forces produced by myofibrils after they are stretched into long sarcomeres without activation. The passive force produced by the wild-type (WT) myofibrils is higher than the force produced by the knockout (KO) myofibrils. Symbols indicate means ± SE. Ig domain, immunoglobulin-like domain; PEVK domain, proline-glutamate-valine-lysine-abundant structural motif domain.

Studies have shown that lack of arginylation affects passive force development in conditions where the cardiac and skeletal muscle myofibrils are stretched sequentially at low Ca²⁺ concentrations, i.e., no activation of the myosin-actin system (4, 26, 34, 46; Fig. 4B). In the case of skeletal muscles, this reduction was independent of titin phosphorylation (34) and thus must be associated with a direct effect of arginylation on titin properties. Since myofibrils may present a significant degree of sarcomere length nonuniformity, some of these experiments were followed by a study with individual sarcomeres (34), and the results were consistent: the passive forces in skeletal myofibrils from KO mice were smaller than the passive forces produced by WT myofibrils.

A smaller capacity of developing passive forces when stretched may lead to an increased risk of damage of the sarcomeres and myofibrils during stretches and also during contractions, as the development of passive force by titin within sarcomeres represents key elements to stabilizing the myosin filaments during activation, centralizing the A band in the sarcomeres and avoiding unbalances of forces in half sarcomeres (7, 39).

In summary, arginylation of different myofibrillar proteins affects the development of cardiac muscles, but not skeletal muscles. However, lack of arginylation affects substantially the capacity of cardiac and skeletal muscles to produce active and passive forces at the sarcomeric level.

Cross-Bridge Kinetics and Mechanisms

Seeking the mechanisms of a decreased active force in the KO models, investigators evaluated the rates of force development during activation (K_act), force redevelopment following a shortening-stretching maneuver (K_tr), and relaxation (K_rel) of activated myofibrils (4, 26, 46). These parameters are putative indicators of myosin cross-bridge kinetics, as they are closely associated with the transitions of cross bridges between different states while attached to actin filaments. K_act and K_tr are indicative of cross bridges transitioning from a weakly bound state to a strongly bound state, culminating with force generation. K_rel is indicative of cross-bridge detachment from actin upon reduction of Ca²⁺ concentration. Interestingly, K_act and K_tr did not change in cardiac KO mice, but they were lowered in skeletal KO mice (4, 26, 46). Since Ca²⁺ activation is maintained maximal during these experiments, the results suggest that in cardiac muscles there was a lower number of cross bridges attached to actin in a given time, whereas in skeletal muscles there was also a lower rate of cross bridges transitioning from the weakly bound to the strongly bound state and a lower detachment rate from actin. Altogether, the results suggest a reduction in the number cross bridges being formed due to the lack of arginylation.

Molecular Studies

The results of the studies with myofibrils indicate that lack of arginylation causes changes in the myosin-actin interaction and cross-bridge kinetics, especially in skeletal muscles. Such results would imply that arginylation has a direct effect on the molecular interactions, which could then be targeted by modulators. Although the kinetics of myosin-actin interactions can be affected by different mechanisms, it is governed mostly by the mechanics of the molecular motor myosin II and the rates of attachment/detachment to/from actin. Our group decided to use a unique technique to investigate the effects of arginylation directly on the myosin-actin interaction. We first isolated single myosin and actin filaments from muscles dissected from KO and WT mice. We then used a system with microfabricated cantilevers to grab the filaments and manipulate them so that they would interact and produce force (20, 21). After the filaments attached, they started to slide past each other, producing force until they detached (Fig. 5). This preparation allowed us to evaluate whether the reduction of myofibril force arises directly from posttranslational modification at the myosin-actin level.

Fig. 5.

A: setup to work with individual filaments of myosin and actin. The filaments are attached to microfabricated cantilevers of known stiffness. When the filaments attach, they slide causing a deflection of the cantilevers, allowing for the measurement of force. B: raw data showing force produced by individual filaments isolated from wild-type (WT) and knockout (KO) mice. The force is higher in the WT filaments. C: average force produced by filaments extracted from WT and KO filaments and also in KO filaments after rearginylation (Re-Arg).

The filaments isolated from KO mice produced less force than the WT muscle, showing that the force is reduced because of a decreased force produced by individual myosin molecules during attachments with actin. These results suggest that arginylation is essential for a normal myosin-actin cycle. We then rescued the filaments by rearginylation with Ate1, and astonishingly, the filaments were able to restore the force to the control level (Fig. 5; 4).

MODEL TO EXPLAIN ARGINYLATION EFFECTS ON CONTRACTILITY AND PASSIVE FORCES

On the basis of the results of the studies evaluating arginylation sites in muscles, measuring the contractile and passive forces at different levels of analysis expanding from whole muscles to single molecules, and measuring the kinetics of activation and relaxation, we propose the following mechanism to explain the role of arginylation in striated muscle contraction.

In the heart, arginylation affects myofibril and sarcomere alignment, and lack of arginylation leads to myofibrils with sarcomeres and sets of muscle filaments that are disaligned, affecting force production. In the heart and in skeletal muscles, the downstream effect of arginylation of the MHC and the rod region on the myosin molecule and the arginylation of actin result in conformational changes in myosin and actin filaments that facilitate myosin-actin interactions, resulting in normal cross-bridge kinetics. In the absence of arginylation, this conformational change cannot be easily achieved, with two possible consequences: the force produced by individual cross bridges in a given myosin-actin interaction cycle is decreased, or the number of myosin cross bridges associated with actin at a given time is reduced. Both possibilities would lead to a decrease in active force. Finally, arginylation also affects the rigidity of titin molecules, and lack of arginylation leads to a decreased passive force production in both cardiac and skeletal muscles.

PHYSIOLOGICAL RELEVANCE

Arginylation is a potent regulator of striated muscle contractility, and therefore it has fundamental physiological importance. Although the role of arginylation in human muscles is still to be investigated, our studies predict that arginylation is required for maintaining normal muscle strength and that deregulation of Ate1 may accompany skeletal myopathies in human patients. Therefore, the understanding of arginylation and striated muscle contractions has important implications in human health and disease.

GRANTS

D. E. Rassier is funded by Canadian Institutes of Health Research Grant 125898 and Natural Science and Engineering Research Council of Canada Grant 2016-05317. A. Kashina is funded by NIH National Institute of General Medical Science Grant R35-GM-12250.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.E.R. and A.K. prepared figures; D.E.R. and A.K. drafted manuscript; D.E.R. and A.K. edited and revised manuscript; D.E.R. and A.K. approved final version of manuscript.