Influenza Infection has Fiber Type-Specific Effects on Cellular and Molecular Skeletal Muscle Function in Aged Mice

Abstract

Skeletal muscle myopathies represent a common non-pulmonary manifestation of influenza infection, leading to reduced physical function and hospitalization in older adults. However, underlying mechanisms remain poorly understood. Our study examined the effects of influenza virus A pulmonary infection on contractile function at the cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) levels in soleus and extensor digitorum longus muscles of aged (20 months) C57BL/6 male mice that were healthy or flu-infected for 7 (7-days post-infection; 7-DPI) or 12 days (12-DPI). Cross-sectional area (CSA) of myosin heavy chain (MHC) IIA and IIB fibers was reduced at 12-DPI relative to 7-DPI and healthy. Maximal isometric force in MHC IIA fibers was also reduced at 12-DPI relative to 7-DPI and healthy, resulting in no change in specific force (maximal isometric force divided by CSA). In contrast, MHC IIB fibers produced greater isometric force and specific force at 7-DPI compared to 12-DPI or healthy. The increased specific force in MHC IIB fibers was likely due to greater myofilament lattice stiffness and/or an increased number or stiffness of strongly bound myosin-actin cross-bridges. At the molecular level, cross-bridge kinetics were slower in MHC IIA fibers with infection, while changes in MHC IIB fibers were largely absent. In both fiber types, greater myofilament lattice stiffness was positively related to specific force. This study provides novel evidence that cellular and molecular contractile function is impacted by influenza infection in a fiber type-specific manner, suggesting potential molecular mechanisms to help explain the impact of flu-induced myopathies.

The decline in physical function is a well-known hallmark of the aging process (1). Maintaining physical function into later adulthood is critical, as declines in function are associated with loss of independence, nursing home admission, and mortality (2). Any disease or perturbation that further exacerbates the age-related decline in physical function poses a serious concern to the health and well-being of older adults.

One common episodic health event that has the often unrecognized capacity to augment the rate and magnitude of age-related decline in physical function for older adults is the influenza (flu) virus. Flu is estimated to cause 9.2 to 36.5 million illnesses annually, resulting in an elevated risk for hospitalization and mortality (3). Flu infection can be particularly devastating for older adults, as 90% of flu-related deaths occur in those aged ≥ 65 years (4). The effects of flu infection on the immune and respiratory systems are well-established; however, less attention has been given to flu-induced myopathies, which are broadly defined as diseases of muscle weakness. In high-risk older adults, such as those with underlying illness or those who are frail, the flu can precipitate a downward trajectory in functional health. For example, nursing home residents with flu-like illness exhibited significant declines in independence (bathing, dressing, and mobility) in the 4 months following infection (5). However, in another study, even 54% of healthy older adults were unable to perform activities of daily living as a result of flu infection (6). While studies are needed that characterize the effects of flu on objective measures of physical function, existing evidence suggests the presence of myopathies in both healthy and high-risk older adults, albeit the time course and likelihood of catastrophic disability may vary based on health status. However, to date, the underlying physiological mechanisms leading to flu-induced myopathies remain understudied and poorly understood.

Preclinical animal models have been used to uncover some of the mechanisms possibly leading to skeletal muscle dysfunction with flu. Zebrafish infected with flu showed sarcolemma damage and reduced extracellular matrix adhesion in skeletal muscle fibers (7). In mice, flu resulted in reduced mobility and altered gait kinetics, which were more pronounced in aged mice compared to young (8). These mobility impairments were associated with upregulation of inflammatory and atrophy gene expression and downregulation of myogenic factors (8). While these studies provide some mechanistic insights into flu-induced myopathies, how the flu might impact underlying contractile properties is still unclear. Global indexes of physical performance and mobility decline with the flu in animal models (8) and humans (9), and flu-induced myopathies at the whole-body level (eg, altered gait) may be rooted in muscle dysfunction at lower anatomical levels (impaired cellular and/or molecular properties). At the most fundamental level, skeletal muscle contraction is achieved through the interaction of the primary contractile proteins, myosin and actin. We have previously shown that myosin-actin cross-bridge kinetics are reduced in single muscle fibers of older adults, especially among women, and these molecular deficits are associated with reduced whole muscle power (10). Similarly, flu-related impairments in mobility and function are more pronounced among aged mice compared to young (8). Thus, flu infection in older populations may contribute to myopathy through detrimental effects on myosin-actin interactions.

A major barrier to studying the effects of influenza infection on skeletal muscle function at lower anatomical levels (eg, cellular and molecular) in humans is the need to perform a percutaneous needle biopsy, which is an invasive technique and may not be appropriate for older adults in the presence of other flu-related health complications. In contrast, preclinical animal models represent a viable alternative experimental approach to addressing the effects of flu infection on skeletal muscle function at the cellular and molecular levels. The aim of this study was to characterize the effects of influenza A virus infection on cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) contractile function in aged mice from two hind limb muscles with different myosin heavy chain (MHC) isoform compositions, the soleus and extensor digitorum longus (EDL).

Method

Mice and Viral Infection

C57BL/6 male mice (n = 14) were obtained from Jackson Laboratories. To prevent exposure to accidental antigens, bacterium and/or virus, all mice were housed in a specific pathogen-free climate-controlled environment with a 12:12 light:dark cycle and fed standard rodent chow and water ad libitum. Experiments began when mice were 20 months old, as mice at this age are equivalent in senescence to humans aged ~66 y, based on previous research showing that one human year is equal to nine mouse days (11). All mice were cared for in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were approved by the University of Connecticut Medical School Institutional Animal Care and Use Committee, protocol number 100705. Per IACUC requirements, recumbent mice and mice that lose >30% body weight are considered moribund and should be euthanized; however, no mice met these criteria in this experiment. All mice underwent gross pathological examination (inspection for tumors in body cavity, necrosed tissue on internal organs, and splenomegaly) at time of sacrifice; however, no animals exhibited signs of gross pathology. All mice were anesthetized with isoflurane and control mice (n = 5) received a sham injection (PBS) while the remaining animals (n = 10) were intranasally inoculated with 40 μl of 400 EID₅₀ of influenza virus A/Puerto Rico/8/1934 (PR8). One inoculation was not successful, resulting in nine infected mice. These animals were weighed daily to monitor infection progression and were sacrificed at 7- (n = 4) and 12-days (n = 5) post-infection (DPI) via CO₂ asphyxiation.

Muscle Fiber Preparation

Soleus and EDL muscles were removed at the time of sacrifice. Muscle tissue was immediately placed into cold (4°C) dissecting solution (20 mM N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES), 5 mM ethylene glycol-bis(2-amino-ethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 5 mM MgATP, 1 mM free Mg²⁺, 1 mM dithiothreitol (DTT) and 0.25 mM Pi) with an ionic strength of 175 mEq, pH 7.0, and at pCa 8.0 (pCa = −log₁₀([Ca²⁺])) for isolation of single fiber bundles for mechanical measurements. Muscle bundles of approximately 75 fibers were dissected and tied to glass rods and placed in a low Ca²⁺ solution that begins the removal of the muscle fibers’ external membrane (12) (skinning solution: 170 mM potassium propionate, 10 mM imidazole, 5 mM EGTA, 2.5 mM MgCl₂, 2.5 mM Na₂H₂ATP, protease inhibitor (Roche) with an ionic strength of 175 mEq, pH 7.0) for 24 hours at 4°C. An osmotic pressure gradient, induced by the addition of glycerol, was used to further permeabilize the muscle fibers’ external membrane. This was accomplished by transferring the fiber bundles to storage solution (identical to skinning solution, but with 1 mM sodium azide and without protease inhibitor) and step-wise adding increasing amounts of glycerol to the solution: 10% v/v glycerol for 2 hours, 25% v/v glycerol for 2 hours, and 50% v/v glycerol for 2 hours. Thereafter, bundles were stored in 50% v/v glycerol that included the protease inhibitor at −20°C (glycerol protects the fibers from freezing damage) until single fibers were isolated for mechanical measurements, which occurred within 4 weeks of the dissection.

Preparation for Mechanical Measurements

On the day of the experiment, a muscle bundle was removed from storage and placed in dissecting solution with 1% v/v Triton X-100 for 20 minutes at 4°C. Triton X-100 removes more of the muscle fiber membrane (sarcolemma), and importantly, breaks up and removes internal membranes such as the sarcoplasmic reticulum (13). This prevents any internal calcium storage or release and enables the control of Ca²⁺ concentration within the fiber. Afterward, individual fibers (~1 mm in length) were isolated from muscle bundles using a pair of fine forceps, and aluminum T-clips were placed on both ends while in dissecting solution. Individual fibers were further demembranated (dissection solution with 1% v/v Triton X-100) for 30 minutes at 4°C to ensure complete removal of sarcolemma and sarcoplasmic reticulum. Side and top fiber diameters were measured in dissecting solution at the mid-point of the fiber to determine its height-to-width ratio. The fiber was mounted on the muscle mechanical apparatus (see “Experimental Apparatus” below) by sliding the holes in the T-clips onto hooks attached to a piezoelectric motor and a strain gauge. The hooks and fiber were initially submerged in relaxing solution, which was the same as dissecting solution but with 15 mM creatine phosphate and 300 units/mL of creatine phosphokinase, at 15°C. Sarcomere length was set to 2.65 µm. Cross-sectional area (CSA) was determined by measuring fiber width on the apparatus’ compound microscope and estimating height by multiplying width by the height-to-width ratio measured during fiber preparation and presuming the fiber CSA is elliptical.

Experimental Apparatus

A custom-built muscle mechanics apparatus was used, as previously described (14). Briefly, an aluminum bath plate of 13 wells (~100 μl) was made to hold experimental solutions and a single large chamber (~450 μl) for mounting the fiber onto hooks attached to an Akers force gauge (AE-801, SensorOne, Sausalito, CA) and piezo actuator linear motor (P-841.10, Physik Instrumente, Auburn, MA). The plate slides horizontally (x-axis) within a plastic trough, past the fixed motor and force gauge, allowing the fiber to be exchanged between chambers. During solution changes, the time that the fiber was in the air was minimal (~1 second). The bathing solutions were maintained at a constant temperature by circulating cooling solution through channels milled into the chamber walls. The bathing solution assembly was mounted to an inverted microscope (Zeiss Invertiscope) with a video camera (BFLY-U3-23S6m-C, Point Grey Research Inc., Richmond, British Columbia, CA) and custom video analysis software (ImageJ) that enabled precise measurements of fiber dimensions and sarcomere length.

Single-Fiber Mechanical Measurements

All solutions used for these experiments were calculated using the equations and stability constants according to Godt and Lindley (15). Relaxing solution was the same as dissecting solution, but with 15 mM creatine phosphate and 300 μL/mL of creatine phosphokinase. Pre-activating solution was the same as relaxing solution, except at an EGTA concentration of 0.5 mM. Activating solution was the same as relaxing solution, but at pCa 4.5.

Myofilament properties and myosin-actin cross-bridge mechanics and kinetics were derived using sinusoidal analysis, as previously described (14), and were performed at 15°C under maximal Ca²⁺-activated conditions. Sinusoidal analysis can be related to specific steps in the cross-bridge cycle using the following equation:

This analysis yields three characteristic processes, A, B, and C, which relate to various mechanical (A, B, C, and k) and kinetic (2πb and 2πc) properties of the cross-bridge cycle, as described in detail (14). Briefly, the A-process (characterized by parameters A and k) describes a linear relationship between the viscous and elastic moduli that has no kinetic or enzymatic dependence (16). Under Ca²⁺-activated conditions, where myosin-actin cross-bridges are formed, the A-process represents the underlying stiffness of the lattice structure (myofilament lattice stiffness) and the attached myosin heads in series (16). The parameter A indicates the magnitude of a viscoelastic modulus and k describes the degree to which measured viscoelastic mechanics represent purely elastic (k = 0) versus purely viscous (k = 1) mechanical responses. The parameter A can be separated into an elastic (A-elastic) and viscous (A-viscous) component to characterize myofilament structural elements. The B- and C-process magnitudes (B and C) are proportional to the number of myosin heads strongly bound to actin and the cross-bridge stiffness (17). As the B and C process magnitudes produce qualitatively near-identical results, we show only the B values. The frequency portion of the B-process (2πb) has been interpreted as the apparent (observed) rate of myosin force production or, in other words, the rate of myosin transition between the weakly and strongly bound states (18). The reciprocal of the frequency portion of the C-process, or (2πc)⁻¹, represents the average myosin attachment time to actin, t_on (17).

MHC Isoform Composition

Following sinusoidal analysis measurements, single fibers were placed in 30 μL loading buffer, heated for 2 minutes at 65°C and stored at −80°C until determination of MHC isoform composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify fiber type, as described (14), with minor modifications to achieve better separation between the four MHC isoforms expressed in mouse skeletal muscle. Specifically, the resolving gel was comprised of 8% acrylamide/bis-30% glycerol (w/v). Additionally, gels were run at 70 V for 1 hour followed by 275 V for 26 hours.

Statistical Analysis

Data expressed as mean ± SE. For each fiber type, a one-way analysis of variance (ANOVA) with condition (control, 7-DPI, or 12-DPI) as the between-subjects factor was conducted to examine the main effect of flu status on all primary outcomes. If a main effect was noted, post hoc contrasts (Fisher’s least significant differences) were examined for pairwise comparisons. We conducted Pearson correlations to examine the relationships between single fiber specific force and molecular contractile properties. Statistical significance was set at p < .05 and analyses were conducted using SPSS for Windows version 26.0 (IBM, Armonk, NY).

Results

Physical Characteristics

Body weight was not different between groups (control, 7- and 12-DPI) at baseline (Table 1). Weight loss is a marker of influenza infection (8,19), and mice at 7- and 12-DPI lost weight compared to healthy mice. Mice lost 5.3 g (~16% of body mass) at 7-DPI and 11.3 g (~29%) at 12-DPI following inoculation compared to their pre-infection baseline body weight.

Force, CSA, and Specific Force

Mouse skeletal muscle expresses four pure MHC isoforms (MHC I, IIA, IIX, and IIB) and MHC is the primary determinant of fiber functional characteristics (force, shortening velocity, and power) (20). Moreover, fiber type composition varies in different skeletal muscles, and MHC IIA and IIB fibers are the predominant myosin isoforms expressed in soleus and EDL muscle of C57BL/6 mice (21). Accordingly, we present data for single fiber contractile function in MHC IIA (n = 73) and IIB (n = 68) fibers from 14 aged mice as the number of MHC I (n = 18) and IIX (n = 9) fibers was insufficient to permit statistical analysis. All MHC IIA fibers were expressed in the soleus muscle and all but one of the MHC IIB fibers (~99%) were expressed in EDL muscle. MHC IIA fibers had decreased maximum Ca²⁺-activated force at 12-DPI, while MHC IIB fibers had increased maximum force at 7 DPI (Figure 1A). Conversely, CSA in both MHC IIA and IIB fibers was reduced in mice at 12-DPI relative to the control and 7-DPI (Figure 1B). The effects of flu on maximum specific force (maximum Ca²⁺-activated force divided by CSA) were fiber type-specific (Figure 1C). In MHC IIA fibers, the decline in single fiber maximum force at 12-DPI was accompanied by a similar reduction in CSA, yielding no change in specific force. However, in MHC IIB fibers, although CSA declined in a similar manner to IIA fibers, maximum force increased at 7-DPI and remained similar to control at 12-DPI, resulting in specific force being greater at 7-DPI (114.2 mN/mm²) relative to control (79.7 mN/mm²) and 12-DPI (95.9 mN/mm²). Thus, flu affected force production in a fiber type-specific manner, characterized by no change in MHC IIA fibers, but an increase in specific force of MHC IIB fibers after 7 days of the flu.

Myosin-Actin Cross-Bridge Kinetics

Similar to observations at the cellular level, influenza infection had fiber type-specific effects on molecular function. Namely, flu infection slowed myosin-actin cross-bridge kinetics in MHC IIA fibers (Figure 2A and B). Myosin attachment time, or the duration of time myosin remains strongly bound to actin, was significantly longer at 12-DPI compared to 7-DPI and healthy in MHC IIA fibers; however, there was no change in MHC IIB fibers (Figure 2A). In addition, the rate of force production, or the rate of myosin transition between the weakly and strongly bound states, was reduced at 7- and 12-DPI compared to healthy MHC IIA fibers (Figure 2B). Interestingly, MHC IIB fibers were mostly unaffected, with differences only noted between 7- and 12-DPI, but not compared to healthy fibers.

Myofilament Properties

The A-elastic and A-viscous parameters reflect the elastic and viscous properties of the nonenzymatic, passive structural elements in the myofilaments, while the B parameter is proportional to the number of myosin heads strongly bound to actin and the cross-bridge stiffness. A-elastic, A-viscous, and B were unchanged with flu infection in MHC IIA fibers (Figure 2C–E). In contrast, in MHC IIB fibers, A-elastic and B were greater at 7-DPI relative to controls and 12-DPI while A-viscous was greater at 7-DPI relative to controls (Figure 2C–E). These results indicate that myofilament properties, as well as the number and/or stiffness of the cross-bridges, increased at 7-DPI in MHC IIB fibers. Alterations at the molecular level likely have functional consequences as increased myofilament stiffness and a greater number and/or stiffness of myosin cross-bridges should each translate to improved single fiber function (ie, greater force production).

Correlations Between Specific Force and Molecular Properties

We examined correlations between single fiber specific force and molecular contractile properties for MHC IIA and IIB fibers. In both fiber types, A-elastic, which represents myofilament lattice stiffness, was positively associated with specific force (Figure 3A and B). When fibers from all conditions (eg, healthy, 7-DPI, and 12-DPI) were pooled together, the strength of association between A-elastic and specific force was identical between myosin isoforms (MHC IIA: r = .92; MHC IIB: r = .92, both p < .01). Likewise, within each isoform, the correlations were similar between conditions (ie, healthy vs 7-DPI). We also found that a greater number and/or stiffness of strongly bound myosin-actin cross-bridges, or B, was correlated with greater specific force in both fiber types when all fibers were pooled (MHC IIA: r = .38; MHC IIB: r = .70, both p < .01); however, the relationship was stronger in MHC IIB fibers (Figure 3C and D). Interestingly, in MHC IIA fibers, number and/or stiffness of myosin cross-bridges was related to specific force in flu-infected mice (7- and 12-DPI), but not healthy. In both fiber types, the strongest correlations between B and specific force were observed in fibers from 12-DPI mice, suggesting that cross-bridge number and/or stiffness plays a greater role in force production as flu infection progresses. Thus, the higher specific force at 7-DPI in MHC IIB fibers occurs, in part, due to a shift to a higher range of values in myofilament stiffness and number and/or stiffness of strongly bound cross-bridges (A-elastic: 2.3–6.5 N/mm²; B: 7.2–29.9 N/mm²) compared to control (A-elastic: 1.1–5.5 N/mm²; B: 1.3–25.4 N/mm²) and 12-DPI (A-elastic: 1.1–5.5 N/mm²; B: 3.1–20.4 N/mm²).

Calcium Sensitivity

Previous human studies have shown reduced single fiber calcium sensitivity with aging (22,23) and chronic heart failure (14), as well as acute perturbations such as short-term disuse (24). Thus, we examined specific force under relaxed conditions (pCa 8.0) as a proxy for calcium sensitivity (Figure 4). Relaxed specific force was lower at 7-DPI compared to controls in both MHC IIA and IIB fibers, suggesting a reduced calcium sensitivity with the flu. Relaxed specific force remained lower at 12-DPI in MHC IIA fibers, but not MHC IIB.

Discussion

Flu infection can have devastating consequences for older adults, ranging from disability (5) to death (4). However, understanding how flu leads to various myopathies has received less attention. The aim of this study was to investigate cellular and molecular skeletal muscle function in aged mice infected with the flu. Our results show that the infection had fiber type-specific effects on contractile function. In particular, we saw reduced MHC IIA CSA accompanied by a commensurate decline in isometric force, resulting in unchanged specific force. MHC IIB fibers exhibited a similar change in CSA, but, in contrast, there was an increase in maximum force at 7-DPI relative to healthy animals, leading to increased specific force after 1 week of infection. Cellular force production was related to molecular properties, namely myofilament lattice stiffness in both fiber types and the number and/or stiffness of strongly bound myosin-actin cross-bridges in MHC IIB fibers. In addition, myosin-actin cross-bridge kinetics slowed with infection in MHC IIA fibers, which would likely lead to reduced contractile velocity at the cellular level. This study provides preliminary evidence that influenza infection in aged mice impacts cellular and molecular contractile properties in a fiber type-specific fashion, with the increased specific force in MHC IIB fibers representing a potential compensatory mechanism in response to infection.

While flu infection has prominent effects on the immune and respiratory systems, myalgia is a common symptom and skeletal muscle myopathy can occur. Preclinical animal models have been used to uncover some of the mechanisms responsible for muscle dysfunction with the flu, such as upregulation of inflammatory and atrophy gene expression (8) as well as myocyte sarcolemma damage (7). Our study builds upon the existing literature by elucidating the ways in which flu infection impacts cellular and molecular contractile properties in an older population, where impairments in skeletal muscle function are more pronounced (8). First, we observed significant muscle atrophy, indicated by a reduction in the CSA of MHC IIA and IIB fibers following 12 days of infection. In MHC IIA fibers, we found a concomitant decrease in single fiber force, which was not surprising as CSA and isometric force are closely related (25). However, in MHC IIB fibers, specific force was greater after 7 days of infection compared with the healthy mice or 12-DPI (Figure 1C). This short-term improvement in MHC IIB single fiber function may represent a compensatory mechanism in order to maintain whole muscle performance when infection is present. This is not unprecedented as there are examples of improved single fiber mechanics under conditions that are generally thought to reduce function, such as aging and chronic disease. For example, in the Health ABC study, octogenarians had 28% and 63% greater normalized power in MHC I and IIA fibers, respectively, compared with fibers from healthy 20-year-old adults (26). This finding corroborated results from a previous longitudinal study showing improved single fiber contractile function in older adults after 3 years of follow-up (27). Additionally, data from our own laboratory show that isometric specific force was greater in MHC I and IIA fibers of older adults than young, and that this age effect was driven primarily by older women (10). In the context of disease, pure (MHC IIA) and hybrid (MHC IIAX) single fibers from individuals with spinal cord injury demonstrate greater unloaded shortening velocity and absolute and/or normalized peak power than fibers from able-bodied controls (28). The observation that MHC IIB fibers produced greater specific force following 1 week of infection may be an adaptation to help counteract acute flu-induced declines in function in an effort to maintain some level of physical performance. The notion that a compensatory adaptation would be present in the EDL, which extends the digits in the foot, rather than the soleus, a primary plantar flexor that is important in posture and locomotion, is not intuitive. However, recent animal work showed that specific force of single fibers from the flexor digitorum brevis (flexes digits in the foot) is unchanged with amyotrophic lateral sclerosis, and that fibers actually exhibited increased fatigue resistance, accompanied by greater expression of myoglobin and respiratory chain proteins (29). Collectively, human and animal studies suggest that in the presence of an acute or chronic perturbation that potentially causes a loss of fibers, some (but not all) of the remaining or surviving muscle fibers may adapt in such a way that preserves function, but the underlying mechanisms require further investigation. Notably, the increased specific force with the flu occurred in MHC IIB fibers, which is a myosin isoform that humans do not express and thus does limit translation to humans. For that reason, the effects of flu on this fiber type should be interpreted cautiously. The effects of infection on other MHC isoforms not examined in this study, including pure (I and IIX) and hybrid (I/IIA and IIAX) fibers, the latter of which exist in a greater proportion in skeletal muscles of older adults (30,31), is unclear and should be examined in future studies.

Our study provides novel evidence of fiber type-specific alterations in molecular contractile properties with flu infection. Importantly, flu-related changes in myofilament properties tracked with changes in specific force. Primarily, we found that myofilament lattice stiffness was unchanged with infection in MHC IIA fibers, but was greater in IIB fibers at 7-DPI, and stiffness was strongly related to specific force in both fiber types (Figure 3A and B). We have previously shown that reduced myofilament lattice stiffness explains, in part, obesity-related decreases in single fiber specific force in older adults (32). How flu might impact myofilament lattice stiffness is not entirely clear, but one possibility is through an effect on myosin binding protein-C, a component of the thick filament backbone. In mouse cardiac muscle, phosphorylation of myosin binding protein-C is important in maintaining radial rigidity of the myofilament lattice (33). The phosphorylation state of skeletal myosin binding protein-C may exert a similar impact on myofilament lattice stiffness, thereby altering force transmission, but this notion requires further investigation. While cardiac myosin binding protein-C does not modulate force production of skeletal muscle (34), the two skeletal isoforms of this protein have recently been shown to regulate thin filament activation in a Ca²⁺-dependent manner using the in vitro motility assay (35), although reviews call for a better understanding of their role in force production (36,37). Additionally, we also found that the number and/or stiffness of strongly bound myosin cross-bridges was greater after 7 days of infection in MHC IIB fibers, and there was a strong and positive relationship between the number and/or stiffness of myosin cross-bridges and single fiber specific force (Figure 3C and D). Mathematically, single fiber isometric force is equivalent to the number of strongly bound myosin-actin cross-bridges formed and the force generated per cross-bridge (38). As the force a cross-bridge can produce is determined by its elastic stiffness and unitary displacement during the myosin power stroke (38), a change in either (a) the number of cross-bridges formed and/or (b) the stiffness of the cross-bridges (and thus force per cross-bridge) will impact Ca²⁺-activated isometric force. Thus, favorable changes in these molecular parameters offer a potential explanation for improved force production in IIB fibers at 7-DPI.

Although cellular force production in MHC IIA fibers remained unchanged with the flu, myosin-actin cross-bridge kinetics were slower (Figure 2A and B). Relative to controls, myosin rate of force production, or myosin’s transition between the weakly and strongly bound states (18), was lower in 7- and 12-DPI MHC IIA fibers (Figure 2B). Another kinetic parameter, myosin attachment time, or the duration myosin is strongly bound to actin (17), was longer in MHC IIA fibers from 12-DPI mice compared to healthy controls (Figure 2A). Notably, these kinetics were not altered in MHC IIB fibers. As myosin and actin are the primary myofilament proteins dictating contraction (38), slower cross-bridge kinetics have been implicated in skeletal muscle dysfunction resulting from a wide variety of conditions, including aging (10), obesity (32), congestive heart failure (14), and cancer (39). Why flu infection would slow myosin-actin cross-bridge kinetics is unclear, but one potential etiologic factor is oxidative stress. The pathogenic role of oxidative stress in the flu virus has recently been reviewed in detail (40). In terms of skeletal muscle function, previous work has shown that simulating oxidative modifications by exposing human MHC I fibers to the thiol-specific agent N-ethylmaleimide (NEM) increased myosin attachment time (41), an alteration reflective of slower cross-bridge kinetics. In the context of the present study, longer myosin attachment times in MHC IIA fibers may compromise single fiber or whole muscle function through a detrimental impact on contractile velocity and/or power (force × velocity). Myosin attachment time is inversely related to contractile velocity (42) and based on the observation that flu resulted in longer attachment times, we predict this would lead to slower shortening velocity in MHC IIA fibers. Older mice infected with influenza exhibit reduced mobility and altered gait kinetics (8) and older humans experience functional decrements following the flu (5,6). The cellular and molecular changes we observed likely contribute, in part, to these limitations in physical performance as single-fiber contractile properties are related to whole muscle function in older adults (10,22). Studies that measure single fiber shortening velocity and power following the flu, in animal models, but, more importantly, humans, represent an important next step in advancing our understanding of flu-induced myopathy.

Calcium plays a fundamental role in skeletal muscle contraction (43). The responsivity of a single muscle fiber to calcium (eg, calcium sensitivity) is a measure of cellular contractility that is compromised in the presence of aging (22,23), chronic heart failure (14), and short-term muscle disuse (24). To further probe the impact of influenza on muscle function, we measured specific force under relaxed conditions (pCa 8.0) as a proxy for calcium sensitivity, as higher force at low calcium concentrations indicates a potentially greater sensitivity. Relaxed specific force was lower at 7-DPI in MHC IIA and IIB fibers and at 12-DPI in MHC IIA fibers compared with healthy control mice (Figure 4), suggesting a reduction in calcium sensitivity with flu infection. While the underlying mechanisms are unclear, this reduction may be related to impaired function of regulatory proteins on the thin filament (eg, Troponin C) or post-translational modifications such as phosphorylation of the myosin regulatory light chain (44). Nonetheless, decreased relaxed force is a potentially relevant finding as single fiber calcium sensitivity predicts whole muscle performance in older adults (22). We note that relaxed force was used as a surrogate for calcium sensitivity, which is typically characterized using a pCa-force curve where relative force is plotted across a range of calcium concentrations. However, as we are not aware of any studies that have evaluated single fiber calcium responsivity with flu, these preliminary data suggest it may be impaired and a possible contributor to muscle dysfunction.

We observed several alterations in cellular and molecular contractile properties that may help explain the functional phenotype associated with influenza infection, but the pathophysiology remains unclear. First, both fiber types atrophied with infection, but we were unable to determine the reasons for this. Chronic disease, such as congestive heart failure, results in a loss of myosin protein content (45) and it is possible that there is a loss of contractile elements with the flu. Conversely, if atrophy was due to the loss of sarcoplasm (and not myosin or actin), this could explain the maintenance of isometric force in MHC IIB fibers despite a decrease in CSA. In addition, body composition of the animals was not measured in the present study, and therefore the percentage of weight loss due to changes in muscle and adipose tissue is not clear. Future studies that account for body composition, as well as food intake and hydration status, will be helpful in better characterizing the weight loss that occurs with flu. Additionally, mature skeletal muscle fibers do not seem to be capable of a productive flu infection. Indeed, they lack the appropriate sialic acid receptors to allow for flu virus entry into the cell. Previous work has shown that there are no viral copies of flu polymerase in gastrocnemius muscle of aged mice, suggesting no direct infection of skeletal muscle (8), although other studies indicate myoblasts and myotubes may produce viral copies (46,47). In humans, however, isolation of flu virus from the skeletal muscle of infected individuals is quite rare, agreeing with other in vivo experiments. Nevertheless, biomarkers of muscle damage have been associated with flu infection in clinics. For example, elevated serum creatine kinase has been reported in patients with the flu (48,49). Further, one study of 505 patients with influenza A pH1N1 reported elevated creatine kinase levels were associated with worse clinical outcomes, such as greater intubation risk and duration of mechanical ventilation (48), suggesting greater muscle involvement during more severe flu infections. Another etiologic factor that may contribute to muscle degradation with flu infection is inflammation. Increased expression of pro-inflammatory cytokines has been reported in skeletal muscle using preclinical animal models (7,8), and this may confer muscle damage resulting in reduced function. While inflammation is also caused by other conditions, such as respiratory syncytial virus (50), and not unique to the flu, it represents a response to severe infection and may be a primary antecedent of myopathy. Thus, additional research is needed to understand how flu infection translates to changes in skeletal muscle structure and function, and, on a broader scale, measures of physical performance such as gait speed, grip strength, and activity levels.

In summary, we examined the effects of flu infection on cellular and molecular contractile properties of hindlimb muscles from aged mice, as they exhibit prolonged functional deficits compared with young (8). We found that infection had fiber type-specific effects on cellular and molecular function, characterized by a flu-induced increase in specific force of MHC IIB fibers from the EDL muscle, and a slowing of myosin-actin cross-bridge kinetics that was most pronounced in MHC IIA fibers from the soleus muscle. Myofilament lattice stiffness was strongly related to single fiber specific force in both fiber types, but the number and/or stiffness of myosin cross-bridges only contributed to force in IIB fibers. These findings suggest that (a) cellular force production increases in MHC IIB fibers in response to infection as a potential compensatory mechanism and (b) changes in the interaction between myosin and actin may underlie acute reductions in physical performance with influenza.

Funding

This work was supported by the National Institutes of Health/National Institute on Aging grants AG021600 and AG060389 (to L.H.). This research was partially conducted while Jenna Bartley was a Glenn/AFAR Postdoctoral Fellow.

Conflict of Interest

None reported.

References