Single Muscle Fibre Contractile Function With Ageing
by Benjamin Bunting BA(Hons) PGCert
Written by Ben Bunting: BA(Hons), PGCert. Sport & Exercise Nutrition. British Army Physical Training Instructor (MFT).
Single muscle fibres have been shown to undergo structural changes with ageing. These include decreased fibre size, decreases in maximum unloaded shortening velocity, increased fibre stiffness and increased susceptibility to contractile injury (Ochala et al., 2007).
In contrast, recent studies have reported that single fibres can be preserved in older adults. These results might be a result of a compensatory response to the widespread motoneuron and fiber death that occurs with old age.
The muscles of your body are made up of different types of muscle fibers. These fibers are responsible for movement, postural control and exercise output. They are made up of a combination of slow-twitch and fast-twitch fibers.
Slow-twitch (ST) fibres focus on endurance, small movements and postural control. They are more fatigue resistant than fast-twitch (FT) fibres and rely on more mitochondria and myoglobin.
Fast-twitch (FT) fibres are more focused on power, larger movements and short durations of exercise. They have less blood supply and are more anaerobic.
They are also more susceptible to fatigue quickly.
The structure of skeletal muscle involves repeating units called sarcomeres that contain 2 contractile proteins called myosin and actin – thick filaments and thin filaments, respectively. The interaction of these proteins with calcium releases an electrical impulse that triggers muscle contraction.
These signals are converted to ATP (a cellular energy source for muscle contraction) by specialized enzymes called myosin ATPases.
Several classification techniques differentiate muscle fibers based on different myosin structures (isoforms) or physiologic capabilities.
In addition to physiologic differences, muscle fibers can be classified by their orientation (whether they align end to end or side by side). The alignment of the muscle fibres influences the overall function of the muscle.
Myofilament is a key regulatory protein in muscle contraction with thick-thin filament interactions producing force and motion. It is a central mechanism for disease-related impairments of muscle function and has emerged as a promising therapeutic target.
Myosin and Actin, Two Major Components of Myofilament.
In muscle cells, there are three protein filaments called myofilaments that are essential for muscle contraction. The myofilaments are myosin (the contractile protein), actin (the elastic protein), and titin (an elastin-like protein).
Aspects of Myofilament Important for Muscle Contraction
In skeletal and cardiac muscle, thin and thick filaments form a sarcomere, which is the structure of a muscle cell. Histological sections of muscle reveal thick and thin filaments that overlap in a zone known as the A band. These regions are dense, and they contain many myosin heads that can form cross bridges with each other about five times per second. The cross bridges attach the myosin head to a troponin that is exposed on the actin binding site, and this activation produces a muscle contraction.
Myofilament Adaptations Explained
Myofilaments are contractile units composed of proteins that produce the end result of muscle contraction. A sarcomere is a bundle of muscles, or a "muscle bundle" that includes the Z line (myosin), M line (creatine kinase, myomesin, and M protein), thick filament, and thin filament (G-actin, nebulin, troponin, and tropomyosin).
In order to generate force, each sarcomere must undergo an initiation process whereby calcium binds to the calcium-binding subunit of the troponin complex, TnC1,2,3,4. The Ca2+ binding enables activation of the entire thin filament, which includes 300-400 globular actins, 40-60 troponin and tropomyosin molecules, and 1 nebulin molecule.
However, this cellular level mechanism is not sufficient to trigger contraction. The myofilament must interact with a variety of higher order signaling pathways that regulate the muscle fibers' overall activation status1,6,8,10,11,12, and ultimately generate force4.
The interplay between the myofilaments and other systems that regulate whole muscle function has been difficult to measure at the molecular and cellular level. Thus, it is important to study myofilament biology at the sarcomere, the unit that regulates the strength of muscle contraction, in a systematic and reliable way.
Our new biosensor is the first to incorporate myofilament specific sarcomere ligands, and has been engineered to interrogate sarcomere activation dynamics in intact skeletal muscle fibers. This novel approach uses distance-dependent fluorescence resonance energy transfer (FRET) to elicit a dynamic transient that is capable of reporting the onset, duration, and amplitude of the overall sarcomere twitch contraction.
Myofilament Adaptations and Whole Muscle Phenotypes
Skeletal muscle disuse is associated with reduced single fiber power output and diminished whole muscle function, in addition to increased mobility impairment (Narici and De Boer, 2011). The relationship between myofilament adaptations and whole muscle phenotypes has been studied at the molecular and cellular level. However, it is challenging to directly extrapolate changes in myofilament function at the cellular and molecular level to changes in whole muscle torque due to interceding effects of higher order regulatory systems such as neural, excitation-contraction coupling and connective tissue properties.
The underlying mechanisms of these myofilament adaptations remain unknown, but have been linked to altered sarcomere function, and, in particular, to a decreased ratio of thick to thin filaments which is thought to contribute to the reduced contractile capacity observed with skeletal muscle atrophy. A recent review of this phenomenon demonstrates that atrophy may result in stoichiometric reductions in myofilament content and/or composition relative to fiber CSA.
To examine these myofilament adaptations in more detail, we expressed a FRET biosensor based on the calcium-binding subunit TnC (TnC-FRET) into the sarcomere of live fast twitch extensor digitorum longus (EDL) muscles under steady state conditions. Data showed stoichiometric incorporation of the biosensor into the sarcomere where it replaced 64% of endogenous fsTnC in the myofilaments.
The sarcomere-based TnC biosensor reports a Ca2+-activated myofilament activation signal, which is uncoupled from the sarcomere-dependent contractile tension, as in previous studies in intact muscle systems. The myofilament activation transient is reported through the TnC conformational change that occurs in response to initial binding of Ca2+ to TnC in a theoretical twitch contraction. This global conformational change in TnC is subsequently translated through the troponin complex and tropomyosin alignment along the actin thin filament.
These changes in TnC, troponin and tropomyosin conformation are believed to trigger changes in myofilament performance that allow force to be generated, thereby facilitating movement. This system sets the foundation for future studies examining the roles of various activating ligands in regulating myofilament performance in live skeletal muscle fibers.
Type I and IIa fibres
Single muscle fibres have become an important tool for studying skeletal muscle contractile function with ageing. These studies can provide information on a number of factors, including the role of oxidative stress in skeletal muscle dysfunction and the effects of short-term disuse.
Using a combination of electrophoretic analysis and the differential sensitivity (pCa50- pSr50, D50) of individual fibres, it has been possible to distinguish between type I and IIa fibres. These fibre types are characterized by differences in their myosin heavy chain (MHC) isoform composition, which is reflected in the ratio of MHC I and IIa CSA. This is also associated with their Ca2+ sensitivity, which is reduced in type IIa fibres when subjected to short-term disuse.
To investigate the effect of ageing and short-term disuse on single muscle fibre function, a study compared the contractile properties of MHC I and IIa fibres from young (24 +- 1 years) and old (67 +- 2 years) men with similar levels of physical activity. The results indicate that, while ageing did not appear to affect the contractile properties of type I fibres, a marked impairment in MHC IIa fibres was observed following 2 weeks of lower limb cast immobilisation.
The present data suggest that the observed impairments in single fibre contractile properties are due to a combination of ageing and short-term disuse, rather than ageing per se. Therefore, future studies should focus on the relationship between fibre type and aging in order to better understand the mechanism of skeletal muscle contractile dysfunction with ageing.
It is well known that the type of ATP synthase (SDH) used by a single muscle fibre to generate ATP is highly dependent on its fibre type. This is reflected in the SDH staining of muscle sections from different age-points, showing that type-IIX and -IIA fibres are more oxidative than type-IIB fibres.
As a consequence, the proportion of oxidative fibres decreases with ageing. This reflects the fact that oxidative fibres are more vulnerable to aging-induced processes, such as mitochondrial dysfunction. They are also more prone to breaking when subjected to strain. Moreover, they show a higher breakage rate at low and high strains compared with young fibres.
A deterioration of skeletal muscle mass, strength, and function is a common feature of ageing and has been associated with reduced quality of life and increased risk of disability, frailty, and death. Sarcopenia is a major cause of this decline in function and is commonly associated with a decrease in physical activity and exercise training.
Although many different factors contribute to the onset and progression of sarcopenia, one important factor involved is the reduction of single muscle fibre contractile function. This is a key mechanism for the sarcopenia-related functional impairment, as it primarily affects activities of daily living such as dressing and eating.
Numerous studies have revealed changes in single fibre contractile properties with ageing, which include: decreased specific force and unloaded shortening velocity in type I and IIa fibres; increased instantaneous stiffness; and increased passive elastic modulus and stress, as well as reduced resistance to the contraction induced injury. The underlying causes of this decline in single muscle fibre function are complex and require further study.
In this regard, the CSA method is an excellent tool to measure these changes. However, it has been observed that the results derived from these studies vary among different groups of participants (see e.g., D'Antona et al., 2003; Frontera et al., 2000; Power et al., 2015; and others).
The differences between these results may be attributed to the fact that the single muscle fibres were tested in isolation from other muscle preparations (i.e., fresh frozen preparations) in some of these studies. This is particularly true when testing myofilaments or genetically altered animal models (Okada et al., 2008; Miller et al., 2010; and Callahan et al., 2014a).
Moreover, studies that used short-term disuse, such as 2 weeks of cast immobilisation, have also shown alterations in single muscle fibre contractile function. In particular, these studies have found that disuse results in a decrease in the maximum Ca(2+)-activated force and specific force production by MHC IIa fibers as well as an increase in the sensitivity of MHC I fibers to Ca(2+). These effects are not reflected in the instantaneous stiffness or passive elastic modulus values.
The plasticity of single muscle fibres can be influenced by many factors, including mechanical force, hormones, neurotransmitters, chemicals and nutrition. These influences can affect contractile properties and, in turn, affect whole muscle adaptability.
Aging is a common and often debilitating process that results in muscle weakness and atrophy, which can impair function and quality of life. The deterioration in muscle function seen with aging is largely a result of changes in the molecular profile of skeletal muscle fibres. However, the role of these alterations in affecting muscle plasticity and function at the microscopic level has yet to be fully elucidated.
Different studies have investigated the impact of aging on single muscle fibre contractile performance in humans, with differing results. Some have shown decreases in MHC I and IIa fibre contractile performance (isometric tension and velocity) while others show no change or even an increase (Larsson et al. 1997; Frontera et al. 2000, 2008; Krivickas et al. 2001; D'Antona et al. 2003; Trappe et al. 2003, 2004; Korhonen et al. 2006; Ochala et al. 2007; Yu et al. 2007).
These studies have provided important information on the effects of ageing and short-term disuse on contractile function. These findings suggest that a better understanding of the myofilament adaptations to ageing, disease and disuse in men and women would assist with the development of preventive and rehabilitative interventions.
Moreover, there is evidence that short-term disuse of skeletal muscle results in the production of reactive oxygen species (ROS) and the accumulation of calcium ions in myofilaments. These changes may lead to reduced sarcopenia and muscle strength. Consequently, the ability of myosin fibres to produce high torque and power is compromised. Additionally, post-absorptive myofibrillar protein synthesis rates decline and are blunted after brief periods of muscle disuse. This decrease in muscle protein synthesis (MPS) rates is attributed to a combination of decreased systemic and local amino acid uptake, and the induction of anabolic resistance to protein intake. These mechanisms contribute to the onset of sarcopenia and muscle loss in old age. In addition, the reduction in MPS and anabolic resistance to dietary protein ingestion may also contribute to muscle atrophy in aged individuals.
There are reports of impairments in single muscle fibre contractile properties with aging, although it is unclear whether these are directly related to ageing or to other factors. One of the most interesting findings is that a decline in the maximum unloaded shortening velocity, V0, has been observed in muscle fibers from older adults and can be correlated with a decrease in muscle strength in the same subjects. Using an approach that enables the measurement of the force response following a stepwise length change of a single muscle fiber, we evaluated how contractile function is affected by ageing in skeletal muscle.
Despite the apparent decline in V0 with age, the research suggests that single muscle fibres from older adults were able to generate force as they did in young individuals, although the specific force was reduced in both groups. This is a significant finding because it provides the first evidence of the ability of human muscle to maintain contractile function, even in the absence of physical activity.
Researchers also assessed the expression of proteins involved in regulating single muscle fibre size, including genes encoding IGF-1, IGF-1EA, IGF-1EC/MGF and myostatin. Compared to the young group (YG), the expression of IGF-1 and myostatin was attenuated in the older subjects (OW); however, there was no difference in expression of IGF-1EA, MGF or GDF-11 (Fig. 3).
This finding demonstrates that the balance between protein synthesis and protein degradation is partially preserved in the older group. This suggests that atrophy may be mainly associated with neural system dysfunction rather than a reduction in cellular mass.
Another interesting finding is that the abundance of proteins in sarcoplasmic reticulum and T-tubule membranes is not significantly modified with ageing. We also detected no differences in protein abundance in the fast and slow muscle fibers.
To further investigate the influence of ageing on single muscle fibre function, researchers measured the maximum Ca 2+ activated force and the sensitivity to submaximal Ca 2+ concentrations in skinned fibers from young (YG) and old (OW) subjects. We found no differences between YG and OW in the amount of Ca 2+ required to generate force, whereas the sensitivity to submaximal Ca2+ was significantly decreased in OW muscles. The sensitivity to submaximal Ca2+ concentrations is important because it determines the extent to which submaximal Ca2+ concentrations activated muscle fibers, thus affecting contractile function.