Thick Filament Proteins Research Articles

Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy

Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.

The structural and functional effects of myosin regulatory light chain phosphorylation are amplified by increases in sarcomere length and [Ca2+

Precise regulation of sarcomeric contraction is essential for normal cardiac function. The heart must generate sufficient force to pump blood throughout the body, but either inadequate or excessive force can lead to dysregulation and disease. Myosin regulatory light chain (RLC) is a thick-filament protein that binds to the neck of the myosin heavy chain. Post-translational phosphorylation of RLC (RLC-P) by myosin light chain kinase is known to influence acto-myosin interactions, thereby increasing force production and Ca2+-sensitivity of contraction. Here, we investigated the role of RLC-P on cardiac structure and function as sarcomere length and [Ca2+] were altered. We found that at low, non-activating levels of Ca2+, RLC-P contributed to myosin head disorder, though there were no effects on isometric stress production and viscoelastic stiffness. With increases in sarcomere length and Ca2+-activation, the structural changes due to RLC-P become greater, which translates into greater force production, greater viscoelastic stiffness, slowed myosin detachment rates and altered nucleotide handling. Altogether, these data suggest that RLC-P may alter thick-filament structure by releasing ordered, off-state myosin. These more disordered myosin heads are available to bind actin, which could result in greater force production as Ca2+ levels increase. However, prolonged cross-bridge attachment duration due to slower ADP release could delay relaxation long enough to enable cross-bridge rebinding. Together, this work further elucidates the effects of RLC-P in regulating muscle function, thereby promoting a better understanding of thick-filament regulatory contributions to cardiac function in health and disease. KEY POINTS: Myosin regulatory light chain (RLC) is a thick-filament protein in the cardiac sarcomere that can be phosphorylated (RLC-P), and changes in RLC-P are associated with cardiac dysfunction and disease. This study assesses how RLC-P alters cardiac muscle structure and function at different sarcomere lengths and calcium concentrations. At low, non-activating levels of Ca2+, RLC-P contributed to myofilament disorder, though there were no effects on isometric stress production and viscoelastic stiffness. With increases in sarcomere length and Ca2+-activation, the structural changes due to RLC-P become greater, which translates into greater force production, greater viscoelastic stiffness, slower myosin detachment rate and altered cross-bridge nucleotide handling rates. This work elucidates the role of RLC-P in regulating muscle function and facilitates understanding of thick-filament regulatory protein contributions to cardiac function in health and disease.

Spontaneous Alignment of Myotubes Through Myogenic Progenitor Cell Migration.

In large-volume muscle injuries, widespread damage to muscle fibers and the surrounding connective tissue prevents myogenic progenitor cells (MPCs) from initiating repair. There is a clinical need to rapidly fabricate large muscle tissue constructs for integration at the site of large volume muscle injuries. Most strategies for myotube alignment require microfabricated structures or prolonged orientation times. We utilize the MPC's natural propensity to close gaps across an injury site to guide alignment on collagen I. When MPCs are exposed to an open boundary free of cells, they migrate unidirectionally into the cell-free region and align perpendicular to the original boundary direction. We study the utility of this phenomenon with biotin-streptavidin adhesion to position the cells on the substrate, and then demonstrate the robustness of this strategy with unmodified cells, creating a promising tool for MPC patterning without interrupting their natural function. We preposition MPCs in straight-line patterns separated with small gaps. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps, similar to how they migrate and align with a single open boundary. There is a directional component to the MPC migration perpendicular (90°) to the original biotin-streptavidin surface patterns. The expression of myosin heavy chain, the motor protein of muscle thick filaments, is confirmed through immunocytochemistry in myotubes generated from MPCs in our patterning process, acting as a marker of skeletal muscle differentiation. The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary patterns, with MPC alignment based on natural regenerative behavior rather than complex fabrication techniques.

Mechanism of post-tetanic depression of slow muscle fibres.

A brief tetanic stimulation has a very different effect on the subsequent isometric twitch force of fast and slow skeletal muscles. Fast muscle responds with an enhanced twitch force which doubles that of the pre-tetanic value, whereas slow muscle depresses the post-tetanic twitch by about 20%. Twitch potentiation of fast muscle has long been known to be due to myosin light chain 2 phosphorylation. It is proposed that post-tetanic twitch depression in slow muscle is due to the dephosphorylation of the slow isoform of the thick filament protein, myosin-binding protein-C, by Ca2+/calmodulin-activated phosphatase calcineurin, whilst its phosphorylation underlies the force enhancement due to β-adrenergic stimulation in slow and fast muscle.

Structure of the native myosin filament in the relaxed cardiac sarcomere

The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent invitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, insitu Foerster resonance energy transfer-fluorescence lifetime imaging(FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). Invitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with β-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

Thick filament dynamics: A mechanism for protein replacement within the sarcomere.

The heart contracts continually throughout one's lifetime through antiparallel sliding of actin-based thin filaments along myosin-based thick filaments, organized within muscle sarcomeres. A recent biochemical study from our lab demonstrated that the half-life of cardiac thick filament proteins is ∼10 days in adult mice, suggesting that continual protein replacement supports structural and functional fidelity. These data further suggest that newly synthesized molecules are randomly incorporated into preexisting thick filaments by a stochastic mechanism. Therefore, we hypothesize that myosin replacement involves the dynamic exchange of single molecules into thick filaments in vivo. To test this hypothesis, we generated an adeno-associated virus that replaced 27±7% of the endogenous regulatory light chain (RLC) on myosin molecules with a fluorescent GFP-RLC. We then visualized myosin within intact hearts using two-photon microscopy. At high magnification, individual sarcomeres were observed with GFP-RLC incorporated into thick filaments. Next, we measured the movement of fluorescently labeled myosin within the heart by irreversibly photobleaching limited areas of the cells and quantifying fluorescence recovery after photobleaching (FRAP). No FRAP occurred within 30 minutes, when photobleaching regions encompassed entire sarcomeres. However, rapid FRAP occurred after photobleaching small portions of single sarcomeres at rates orders of magnitude faster than protein synthesis. Recovery appeared to come from the exchange of molecules in the unbleached portion of the sarcomere and was fastest near the tips of the thick filaments, suggesting enhanced exchange where myosin is less densely packed in the filament. Collectively, these data demonstrate dynamic thick filament structure in vivo with myosin molecules freely exchanging between thick filaments within single sarcomeres. Therefore, these large macromolecular complexes appear to be designed to allow for the rapid incorporation and release of myosin molecules while maintaining the structural integrity that is essential to support contractility.

RLC phosphorylation amplifies Ca2+ sensitivity of force in myocardium from cMyBP-C knockout mice.

Hypertrophic cardiomyopathy (HCM) is the leading genetic cause of heart disease. The heart comprises several proteins that work together to properly facilitate force production and pump blood throughout the body. Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein, and mutations in cMyBP-C are frequently linked with clinical cases of HCM. Within the sarcomere, the N-terminus of cMyBP-C likely interacts with the myosin regulatory light chain (RLC); RLC is a subunit of myosin located within the myosin neck region that modulates contractile dynamics via its phosphorylation state. Phosphorylation of RLC is thought to influence myosin head position along the thick-filament backbone, making it more favorable to bind the thin filament of actin and facilitate force production. However, little is known about how these two proteins interact. We tested the effects of RLC phosphorylation on Ca2+-regulated contractility using biomechanical assays on skinned papillary muscle strips isolated from cMyBP-C KO mice and WT mice. RLC phosphorylation increased Ca2+ sensitivity of contraction (i.e., pCa50) from 5.80 ± 0.02 to 5.95 ± 0.03 in WT strips, whereas RLC phosphorylation increased Ca2+ sensitivity of contraction from 5.86 ± 0.02 to 6.15 ± 0.03 in cMyBP-C KO strips. These data suggest that the effects of RLC phosphorylation on Ca2+ sensitivity of contraction are amplified when cMyBP-C is absent from the sarcomere. This implies that cMyBP-C and RLC act in concert to regulate contractility in healthy hearts, and mutations to these proteins that lead to HCM (or a loss of phosphorylation with disease progression) may disrupt important interactions between these thick-filament regulatory proteins.

Titin force in muscle cells alters lattice order, thick and thin filament protein formation

Skeletal muscle force production is increased at longer compared to shorter muscle lengths because of length-dependent priming of thick filament proteins in the contractile unit before contraction. Using small-angle X-ray diffraction in combination with a mouse model that specifically cleaves the stretch-sensitive titin protein, we found that titin cleavage diminished the length-dependent priming of the thick filament. Strikingly, a titin-sensitive, length-dependent priming was also present in thin filaments, which seems only possible via bridge proteins between thick and thin filaments in resting muscle, potentially myosin-binding protein C. We further show that these bridges can be forcibly ruptured via high-speed stretches. Our results advance a paradigm shift to the fundamental regulation of length-dependent priming, with titin as the key driver.

Mechanisms of fish protein degradation caused by grass carp spoilage bacteria: A bottom-up exploration from the molecular level, muscle microstructure level, to related quality changes

To demonstrate the roles of bacteria in fish protein degradation and related quality changes, three major grass carp spoilage bacteria were individually inoculated into grass carp flesh, and their effects on protein molecules, muscle structures, and quality indices were evaluated through peptidomics, optical microscopy and transmission electron microscope, and texture and water distribution analyses, respectively. Results showed that Pseudomonas putida degraded obscurin, nebulin, and titin, caused disarrangement of myofilaments and fragmented myofibers, and induced great loss of free water in muscle. Shewanella putrefaciens was active in hydrolyzing collagen and degraded both thick and thin filament proteins. Mutual separation of myofibers and severe texture softening were also observed in S. putrefaciens-inoculated samples. Aeromonas rivipollensis degraded myosin heavy chain and some thin filament proteins but less affected muscle microstructure and quality indices. Therefore, this study revealed the mechanisms of bacteria-induced grass carp protein degradation and provided guidance for developing quality control strategies.

Thin filament cardiomyopathies: A review of genetics, disease mechanisms, and emerging therapeutics.

All muscle contraction occurs due to the cyclical interaction between sarcomeric thin and thick filament proteins within the myocyte. The thin filament consists of the proteins actin, tropomyosin, Troponin C, Troponin I, and Troponin T. Mutations in these proteins can result in various forms of cardiomyopathy, including hypertrophic, restrictive, and dilated phenotypes and account for as many as 30% of all cases of inherited cardiomyopathy. There is significant evidence that thin filament mutations contribute to dysregulation of Ca2+ within the sarcomere and may have a distinct pathomechanism of disease from cardiomyopathy associated with thick filament mutations. A number of distinct clinical findings appear to be correlated with thin-filament mutations: greater degrees of restrictive cardiomyopathy and relatively less left ventricular (LV) hypertrophy and LV outflow tract obstruction than that seen with thick filament mutations, increased morbidity associated with heart failure, increased arrhythmia burden and potentially higher mortality. Most therapies that improve outcomes in heart failure blunt the neurohormonal pathways involved in cardiac remodeling, while most therapies for hypertrophic cardiomyopathy involve use of negative inotropes to reduce LV hypertrophy or septal reduction therapies to reduce LV outflow tract obstruction. None of these therapies directly address the underlying sarcomeric dysfunction associated with thin-filament mutations. With mounting evidence that thin filament cardiomyopathies occur through a distinct mechanism, there is need for therapies targeting the unique, underlying mechanisms tailored for each patient depending on a given mutation.

Distinct Mechanisms for Increased Cardiac Contraction Through Selective Alteration of Either Myosin or Troponin Activity

Modulation of sarcomere contractility represents a new therapeutic opportunity for the treatment of heart failure by directly targeting the thick and thin filament proteins of the sarcomere to increase cardiac muscle contraction. This study compared the effect of 2 small molecules (M and T) that selectively alter myosin thick filament (M) or troponin thin filament (T) activity on overall cardiac muscle mechanics. This study revealed key differences related to the mechanism utilized by M and T to increase contractile force generation and suggeststhat targeting different proteins within the sarcomere may result in differentiating therapeutic profiles.

Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament.

The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158-166) and a highly flexible region (residues 167-203). Critically, this region modulates the troponin/tropomyosin complex's position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca2+ conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker's positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.

Strucutrally defining the flexible cardiac troponin T linker

Contraction of the heart is driven by cyclic interactions between the thick and thin filament proteins, mediated by Ca2+ level fluctuations. Recent advances in electron microscopy (EM) and molecular dynamics studies have provided structural understanding of the cardiac thin filament (cTF). However, for certain cTF-domains the structure and precise nature of protein-protein interactions remain unknown. One such region is the extended cardiac troponin t (cTnT) linker between TNT1 and TNT2, which remains structurally undefined due to its flexibility.

Integration of proteomic and genetic approaches to assess developmental muscle atrophy.

Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, we chose to investigate changes in protein profiles during muscle atrophy in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms as these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, we first defined protein signature changes during M. sexta intersegmental muscle (ISM) atrophy and then used genetic approaches to confirm their functional importance in the D. melanogaster dorsal internal oblique muscles (DIOMs). Our data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A-band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. We also uncovered the actin filament regulator ciboulot (Cib) as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may eventually lead to therapeutic targets.

Abstract 112: The 3CL Protease Of SARS-CoV2 Induces Sarcomeric Degradation Through Cleavage Of The Giant Protein Obscurin

We hypothesized that the 3CL protease (3CLPro) is responsible for the sarcomere degradation observed in cardiomyocytes infected by SARS-COV2. Overexpression of 3CLPro, but not a catalytically inactive mutant, resulted in breakdown of sarcomeres characterized by intact Z-disk/thin filament subunits that has been recently reported (Perez-Bermejo, et al, 2021) in SARS-COV2 infection. To identify potential host protein targets of 3CLPro in an unbiased fashion we screened the human proteome using a cut-site scoring algorithm that we developed. Scoring, ie likelihood of 3CL protease cleavage, was based off experimental data (Chuck et al, 2010) previously published on the highly homologous (96%) 3CL protease from SARS-COV. This scoring was followed by refinement by secondary structure prediction to identify cut-sites that lie in unstructured regions that are thus thus more likely to be accessible to the protease. Using this method, we identified >1000 potential high-likelihood cut sites across the proteome. Further filtering by proteins with cardiomyocyte expression showed 5 high-likelihood sites within the giant sarcomeric protein, Obscurin (OBSCN), as well as many other structural and signaling proteins which we experimentally validated. Expression of 3CLPro in IPSC cardiomyocytes resulted in significantly reduced OBSCN staining without alterations in Z-disk, thin filament, or thick filament proteins by both western blot and immunocytochemistry. In addition, imaging showed loss of OBSCN at sites with intact Z-disk/thin filament subunits. Thus we propose that activity of 3CLPro is a significant contributor to sarcomere breakdown in SARS-COV2 infection via degradation of Obscurin.

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5–10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5–10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7–10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S–S bonds providing for general stability.

Hypothesis: Single Actomyosin Properties Account for Ensemble Behavior in Active Muscle Shortening and Isometric Contraction.

Muscle contraction results from cyclic interactions between myosin II motors and actin with two sets of proteins organized in overlapping thick and thin filaments, respectively, in a nearly crystalline lattice in a muscle sarcomere. However, a sarcomere contains a huge number of other proteins, some with important roles in muscle contraction. In particular, these include thin filament proteins, troponin and tropomyosin; thick filament proteins, myosin binding protein C; and the elastic protein, titin, that connects the thin and thick filaments. Furthermore, the order and 3D organization of the myofilament lattice may be important per se for contractile function. It is possible to model muscle contraction based on actin and myosin alone with properties derived in studies using single molecules and biochemical solution kinetics. It is also possible to reproduce several features of muscle contraction in experiments using only isolated actin and myosin, arguing against the importance of order and accessory proteins. Therefore, in this paper, it is hypothesized that “single molecule actomyosin properties account for the contractile properties of a half sarcomere during shortening and isometric contraction at almost saturating Ca concentrations”. In this paper, existing evidence for and against this hypothesis is reviewed and new modeling results to support the arguments are presented. Finally, further experimental tests are proposed, which if they corroborate, at least approximately, the hypothesis, should significantly benefit future effective analysis of a range of experimental studies, as well as drug discovery efforts.

Maintenance of sarcomeric integrity in adult muscle cells crucially depends on Z-disc anchored titin

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness ‘myosinopathy’ patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.

Contractility and Myofibrillar Content in Skeletal Muscle are Decreased During Post-Sepsis Recovery, But Not During the Acute Phase of Sepsis.

Convalescence in humans after severe sepsis occurs over weeks to months and is associated with prolonged functional disabilities and impaired quality-adjusted survival. While much is known regarding the acute early phase of sepsis, there is a knowledge gap pertaining to restoration of muscle mass and function after elimination of the septic nidus. We used a sepsis-recovery model-where cecal-ligation-puncture (CLP) was performed in adult male mice followed 24 h later by removal of the cecum and antibiotic treatment-to assess changes in the abundance of muscle contractile proteins and function during the acute phase of sepsis (24 h post-CLP) and during the recovery phase (day 10 post-CLP). Although body weight and food consumption decreased acutely with sepsis, both had normalized by day 10; however, extensor digitorum longus mass remained decreased 10%. During acute sepsis, there were few contractile defects or significant changes in contractile proteins. In contrast, during sepsis recovery, specific maximum isometric twitch and specific maximum tetanic force were decreased ≈50%, compared with time-matched pair-fed controls, and defects were independent of the concomitant reduction in muscle mass. Force generation in sepsis-recovery mice was decreased 30% with increasing stimulus frequency. Contractile defects during sepsis-recovery were associated with 50% to 90% reductions in thin filament (troponin T, troponin I, tropomyosin, α-sarcomeric actin), thick filament (myosin heavy and myosin light chains), Z-disc (α-actinin 3), and M-band (myomesin-2) proteins, but no change in the intermediate filaments desmin and vimentin. During sepsis recovery, myofibrillar protein synthesis did not differ from control, but synthesis of sarcoplasmic proteins was increased 60%. These data suggest intrinsic defects in muscle contractile function exist during the recovery phase of sepsis and may negatively impact convalescence.