Skeletal muscle is a highly organized, multi-scale tissue (Williams and Holt, 2018; Holt, 2020). Sarcomeres, sub-cellular structures containing contractile protein filaments, are considered to be the functional units of muscle. Thin filaments, containing a helical actin polymer (Holmes et al., 1990; Holmes, 2009) and a regulatory troponin–tropomyosin complex (Hanson and Lowy, 1963), project from the Z-disks at the ends of the sarcomere and overlap with the central thick filaments, which contain a myosin polymer. These contractile protein filaments are held in place by a sarcomeric cytoskeleton (Horowits et al., 1986; Gautel and Djinović-Carugo, 2016), including the large protein titin (Horowits et al., 1986; Maruyama, 1976; Wang et al., 1979), and create a highly ordered lattice structure when viewed in three dimensions (Hodge et al., 1954; Millman, 1998; Shimomura et al., 2016). Sarcomeres are arranged in series and in parallel in muscle fibers, and are enveloped by the sarcoplasmic reticulum (SR), an internal calcium (Ca2+) store made up of a network of interconnected tubules. Muscle fibers are organized into entire muscles with orientations ranging from parallel to perpendicular to the line of action of the muscle (Gans, 1982; Kier and Smith, 1985; Askew and Marsh, 2001; Kargo and Rome, 2002; Taylor-Burt et al., 2018). Muscle fibers and entire muscles are surrounded by connective tissues (Purslow and Trotter, 1994; Scott and Loeb, 1995; Azizi and Roberts, 2009; Huijing, 2009; Sleboda et al., 2020) and connected to the skeleton by tendons (Alexander et al., 1982; Gronenberg et al., 1997; Roberts and Azizi, 2011).

The classic framework for understanding muscle contraction includes the excitation–contraction coupling (ECC) and crossbridge and sliding-filament theories. Together, these theories describe the process of force generation in response to activation by the nervous system. According to ECC theory, action potentials in motorneurons activate muscles and cause Ca2+ release from the SR. This Ca2+ binds to troponin, moving tropomyosin and permitting myosin heads to bind to actin and form crossbridges (Ebashi and Endo, 1968). According to the crossbridge and sliding-filament theories, these bound myosin heads then undergo a conformational change that acts to slide the actin filament past the myosin filament, generating force and potentially doing work. Upon deactivation, Ca2+ is returned to the SR and the muscle relaxes. Single activation pulses give rise to short twitch contractions, whereas high frequency activation pulses do not leave sufficient time for Ca2+ to be removed and result in tetanic contractions at fusion frequency. The formation of crossbridges and the sliding of filaments are thought to give rise to the isometric force–length and isotonic force–velocity relationships observed in maximally stimulated muscles in vitro (Hill, 1938; Huxley, 1957; Gordon et al., 1966). According to the force–length relationship, isometric force is maximal at an intermediate sarcomere length corresponding to optimum actin–myosin overlap (Fig. 1). Force declines at long lengths owing to reduced overlap, and at shorter lengths owing to excessive overlap potentially interfering with crossbridge binding (Gordon et al., 1966; Walker and Schrodt, 1974; Herzog et al., 1992a). According to the isotonic force–velocity relationship, the force a muscle can produce declines monotonically with increasing shortening velocity (Fig. 2), thus limiting power (Rome et al., 1988; Schiaffino and Reggiani, 2011). This decline is thought to result from reduced crossbridge binding probability at faster shortening speeds and opposing crossbridge forces resulting from insufficiently rapid crossbridge detachment (Huxley, 1957; Alcazar et al., 2019).

Fig. 1.

Comparison of skeletal muscle force–length curves across vertebrates and invertebrates, and across muscles with a variety of functions, highlighting variation in curve width. (A) Vertebrates; (B) invertebrates. All digitized data points were normalized to peak force and optimum fiber or sarcomere length, and fit with a polynomial function. The chameleon tongue and blowfly larva muscles have been characterized as supercontracting muscles, and the cuttlefish body wall muscle is obliquely skeletal. All other muscles are thought to be typical skeletal muscles. Not all invertebrate sarcomere lengths are reported but the crab abdominal and squid tentacle muscles have sarcomere lengths of 10.8 μm (Chapple, 1983) and 1.5 μm (Kier and Curtin, 2002; Shimomura et al., 2016), respectively.

Fig. 1.

Comparison of skeletal muscle force–length curves across vertebrates and invertebrates, and across muscles with a variety of functions, highlighting variation in curve width. (A) Vertebrates; (B) invertebrates. All digitized data points were normalized to peak force and optimum fiber or sarcomere length, and fit with a polynomial function. The chameleon tongue and blowfly larva muscles have been characterized as supercontracting muscles, and the cuttlefish body wall muscle is obliquely skeletal. All other muscles are thought to be typical skeletal muscles. Not all invertebrate sarcomere lengths are reported but the crab abdominal and squid tentacle muscles have sarcomere lengths of 10.8 μm (Chapple, 1983) and 1.5 μm (Kier and Curtin, 2002; Shimomura et al., 2016), respectively.

Fig. 2.

Comparison of skeletal muscle force–velocity curves across vertebrates and invertebrates, and across muscles with a variety of functions, highlighting variation in Vmax and curvature. (A) Vertebrates; (B) invertebrates. All digitized data points were fit with a hyperbolic–linear equation (Marsh and Bennett, 1986). Not all invertebrate sarcomere lengths are reported, but the squid arm, squid tentacle, katydid thoracic and cockroach limb muscles have sarcomere length of 12.5 μm, 1.5 μm (Kier and Curtin, 2002; Shimomura et al., 2016), 3.1 μm (Josephson, 1984) and 3.5 μm (Fourtner, 1976), respectively.

Fig. 2.

Comparison of skeletal muscle force–velocity curves across vertebrates and invertebrates, and across muscles with a variety of functions, highlighting variation in Vmax and curvature. (A) Vertebrates; (B) invertebrates. All digitized data points were fit with a hyperbolic–linear equation (Marsh and Bennett, 1986). Not all invertebrate sarcomere lengths are reported, but the squid arm, squid tentacle, katydid thoracic and cockroach limb muscles have sarcomere length of 12.5 μm, 1.5 μm (Kier and Curtin, 2002; Shimomura et al., 2016), 3.1 μm (Josephson, 1984) and 3.5 μm (Fourtner, 1976), respectively.

ECC and the crossbridge and sliding-filament theories have dominated our understanding and comparative study of skeletal muscle since the 1950s. However, these theories do not adequately describe muscle performance. Deviations from the typical force–length and force–velocity relationships with changing muscle activation level (Rack and Westbury, 1969; Brown et al., 1999; Holt et al., 2014; Holt and Azizi, 2014, 2016), high muscle forces during lengthening (Abbott and Aubert, 1952; Nishikawa, 2016), and the dependence of muscle activation and force on contractile history (Abbott and Aubert, 1952; Sandercock and Heckman, 1997; Askew and Marsh, 1998; Josephson and Stokes, 1999a; Fukutani and Herzog, 2019) are routinely observed. Mechanisms such as changes to actin–myosin lattice spacing (Williams et al., 2013; Tune et al., 2020; Rockenfeller et al., 2022), cooperative crossbridge binding (in which the binding of one crossbridge increases the likelihood of additional binding; Daniel et al., 1998; Tanner et al., 2007) and changes to titin upon muscle activation (Kellermayer and Granzier, 1996; Herzog et al., 2012; Nishikawa et al., 2012) have been proposed to explain these deviations.

Journal of Experimental Biology has a long and rich history of the study of muscle physiology that is comparative (i.e. studying functionally diverse muscles across a range of taxa) and realistic (i.e. studying muscles under conditions somewhat relevant to in vivo muscle function). Here, we explore the role that this type of study has had in the development of classic theories, and highlight the diversity possible within, and deviations from, such theories. We suggest potential new directions for the study of comparative muscle physiology, including the routine characterization of a wider range of muscle properties and the use of phylogenetic comparative methods.

Comparative study is the heart of understanding the broad diversity of skeletal muscle physiology and mechanics across organisms and how they affect their movement. Such work may compare muscles within a single organism to understand variation across muscles performing different functions (e.g. Altringham et al., 1993; Kier and Curtin, 2002; Ahn et al., 2006; Azizi, 2014; Fuxjager et al., 2016; Tune et al., 2020). However, it is more commonly understood to mean the comparison across organisms that vary in factors such as ecology, behavior and size, often focusing on extremes (e.g. Josephson and Young, 1987; Josephson et al., 2001; Taylor, 2000; Tu and Daniel, 2004; More et al., 2010; Miles et al., 2018; Nelson et al., 2018; Sleboda et al., 2020; reviewed in Green et al., 2018; Clark et al., 2023). Such variation greatly affects the demands on muscles, and so potentially the underlying physiology, with the resulting physiological variation presumably reflecting adaptation (Gould and Lewontin, 1979). However, trade-offs often limit simultaneous optimization of different tasks (Garland et al., 2022). Therefore, organisms with different functional demands often show different underlying physiology, whether to maximize force production, power or contraction frequency, for example. Here, we review the comparative study of skeletal muscle physiology, focusing on the observed variation in muscle mechanical performance and physiology in the context of the ECC, crossbridge and sliding-filament theories of contraction. We highlight the relationship between muscle physiology and performance, and, where possible, examine effects of ecology, behavior and size.

Comparative physiology has demonstrated extensive variation in ECC. Such study has largely focused on twitch times and fusion frequencies, as they define the upper limit of repetitive movements. Some of the earliest comparative work showed that although locust flight muscles only develop brief twitches in vivo, they can contract tetanically, similar to frog limb muscles, if stimulated at a higher frequency (Weis-Fogh, 1956). Variation in ECC kinetics has been shown across muscle types, spatially across the body, and with body size. Tetanic fusion occurs at 20 and 50 Hz in slow and fast muscles in the sculpin (fish; Altringham and Johnston, 1988), activation and relaxation kinetics slow nearly 2-fold rostro-caudally along the body of many species of fish (Altringham et al., 1993; Rome et al., 1993; Davies et al., 1995; Swank et al., 1997; James et al., 1998; D'Aout et al., 2001), and increased twitch times with increased body size have been observed in fish (James et al., 1998) and iguanas (Johnson et al., 1993). Such variation in muscle performance has organismal-level functional consequences, such as permitting the higher stride and tailbeat frequencies used at smaller body sizes (Kram and Taylor, 1990).

Much of our study of ECC has focused on the exceedingly high contraction frequencies required for sound production (Josephson, 1973; Josephson and Young, 1985; Rome et al., 1996; Schaeffer et al., 1996.; Elemans et al., 2008, 2011; Nelson et al., 2018; Schuppe et al., 2018). Such study has yielded insights into the functional importance of contraction rate, and the physiological mechanisms responsible. Passerine bird species that use high-frequency wing claps in courtship displays have wing-muscle kinetics that are twice as fast as species without such displays (Fuxjager et al., 2016), and variation in kinetics is suggested to have contributed to speciation in manakins (birds; Miles et al., 2018). Rattlesnake tail-shaker muscles operate at frequencies as high as 90 Hz owing to an increase in SR volume (Schaeffer et al., 1996; Conley and Lindstedt, 2002), and SR volume negatively correlates with twitch times across cicada (hemipteran insect) species (Josephson and Young, 1987). The Atlantic toadfish can call intermittently at 200 Hz owing to large amounts of the Ca2+ binding protein parvalbumin (Heizmann et al., 1982; Tikunov and Rome, 2009), a troponin isoform with a low Ca2+ affinity (Rome et al., 1996; Rome, 2006), and fast crossbridge kinetics (Rome et al., 1996, 1999). In contrast, the Pacific midshipman fish uses low amplitude Ca2+ transients to call continuously at frequencies of 100 Hz (Harwood et al., 2011; Nelson et al., 2018).

This array of studies demonstrates the diversity in both contraction rates and the physiological mechanisms responsible for those rates across muscles. However, none of the above mechanisms require substantial deviation from classic ECC theory. The range of physiological changes seen suggests that there is not a rate-limiting step (Mead et al., 2017), and the mechanism used may depend on the behavior required and trade-offs incurred (Josephson, 1973; Schaeffer et al., 1996; Nelson et al., 2018). For example, the use of parvalbumin in the intermittent calling of Atlantic toadfish may not work in the continuously calling Pacific midshipman fish, as parvalbumin would saturate (Nelson et al., 2018). Moreover, given constant muscle-fiber volume, increases in SR volume seen in the rattlesnake tail-shaker muscle come at the cost of reduced contractile-protein volume and force production (Lindstedt et al., 1998), potentially explaining why increased SR volume is a common strategy in sound production, where force and power requirements might be lower than in locomotion. Hence, although exceedingly high-frequency contraction can be achieved within classic ECC theory, trade-offs may limit the use of these high frequencies to movements requiring little force or power.

Although significant variation in contraction kinetics occurs within classic ECC theory, comparative physiology has demonstrated significant deviations from this theory in muscles that produce sustained force or contract asynchronously. Sustained force has been observed in the forearm muscles of ranid frogs (Peters and Aulner, 2000; Navas and James, 2007) and the jaw muscles of southern alligator lizards (Nguyen et al., 2020), both used in prolonged mate-holding behaviors (Wells, 1977; Nguyen et al., 2020). These muscles fail to relax fully before the subsequent contraction during prolonged bouts of activity, developing high sustained forces. The mechanisms responsible, and the extent to which they deviate from classic ECC theories, are unclear. Activation and relaxation kinetics can vary widely across muscles, and fatigue prolongs relaxation (Edwards et al., 1975; Allen et al., 1989). Hence, sustained force could be explained by the slow contraction kinetics of these muscles (Peters and Aulner, 2000; Navas and James, 2007; Nguyen et al., 2020) and an early onset of fatigue. However, sustained force can develop in the absence of the declines in peak force typical of fatigue (Navas and James, 2007). Hence, we might expect that these muscles would exhibit changes in Ca2+ handling or crossbridge kinetics that go beyond previously observed variation, potentially deviating from classic ECC theories.

Asynchronous muscle is arguably the clearest and best studied example of a deviation from the classic ECC theory. It is thought to have evolved multiple times in insect flight and sound-producing muscle (Pringle, 1949; Cullen, 1974; Josephson and Young, 1981; Dudley, 2000; Iwamoto, 2011). In contrast to the classic ECC framework, in which there is a synchronous relationship between motorneuron action potentials and muscle contractions, the contraction of asynchronous muscle is decoupled from action potentials. Low-frequency neural input to asynchronous muscle maintains Ca2+ at a level that permits crossbridge cycling (Jewell and Ruegg, 1966), and force cyclically rises and falls as a result of delayed stretch activation and shortening deactivation (Machin and Pringle, 1960; Pringle, 1978; Dickinson and Tu, 1997). For example, in insect flight, the thoracic muscle is stretched during wing upstroke. This increases muscle activation after a short delay, which increases muscle force, drives muscle shortening, and generates work and power during the downstroke. A variety of potential mechanisms for delayed stretch activation have been proposed, including a stretch-activated troponin isoform (Agianian et al., 2004), connections between myosin heads and troponin that move tropomyosin upon stretch (Perz-Edwards et al., 2011), a stretch-dependent transition from weakly to strongly bound crossbridges (Iwamoto and Yagi, 2013), and cooperative crossbridge binding (Iwamoto and Yagi, 2013). A variety of coordinated changes may be required for delayed stretch activation (Cao and Jin, 2020), and the precise mechanism may vary across species if asynchronous muscle indeed has multiple independent origins.

Delayed stretch activation and shortening deactivation in asynchronous muscle seem to represent a fundamental departure from the classic ECC framework; force develops in response to mechanical, rather than chemical (Ca2+), stimuli (Hooper et al., 2008; Cao and Jin, 2020). This departure is thought to permit high contraction frequencies while avoiding the reduced-force trade-off associated with increasing SR volume. The removal of the need to cycle Ca2+ in each contraction allows for the maintenance of contractile protein volume, and therefore force and power (Josephson et al., 2000a; Josephson and Young, 1981; Syme and Josephson, 2002). Hence, asynchronous muscle contraction is thought to represent an adaptation that allows for flight in the smallest insects, whose body size requires high wingbeat frequencies (Josephson et al., 2000b). More detailed comparative studies across insect species and beyond may elucidate whether this mechanical activation represents an adaptation seen only in asynchronous muscle, or illustrate whether this phenomenon contributes to muscle performance to varying degrees across a wider range of muscles.

The first support for the sliding-filament and crossbridge theories of muscle contraction was provided by images of sarcomeres in frog muscle that showed length of the myosin-containing region remaining constant during shortening while the actin-only region shrank, suggesting the sliding of actin filaments past myosin (Huxley and Niedergerke, 1954). A subsequent study in locust flight muscle suggested that this was a generalizable mechanism of contraction (Weis-Fogh, 1956). The sliding-filament and crossbridge theories have been further supported by the apparent ubiquity of isometric force–isometric length relationships and the relationship between peak muscle force and contractile protein filament length. These theories predict that the instantaneous length of a given sarcomere, and the variation in contraction protein filament length across muscles, determines the amount of overlap and therefore the number of potential crossbridges that could be formed and force that could be generated (Gordon et al., 1966; Josephson, 1975; Taylor, 2000). Isometric force–length relationships have been described in limb, abdominal, thoracic and jaw muscles across vertebrates and invertebrates (Fig. 1; Weis-Fogh, 1956; Gordon et al., 1966; Zachar and Zacharová, 1966; Herzog et al., 1992b; Tu and Daniel, 2004; Guschlbauer et al., 2007; Gidmark et al., 2013; Bohm et al., 2019, Moo et al., 2020). And although actin and myosin filament length are relatively consistent in vertebrates (Walker and Schrodt, 1974; Herzog et al., 1992a), more than 10-fold variation has been observed across invertebrates (Hoyle, 1969; Taylor, 2000; Hooper et al., 2008; Shimomura et al., 2016). This variation in contractile protein length appears to correlate with stress (Taylor, 2000) and have functional importance. Crayfish muscles with 10.5 µm sarcomeres (Zachar and Zacharová, 1966) produce a maximum isometric stress of 65 N cm−2, whereas frog muscles with 2.6 µm sarcomeres produce only 35 N cm−2 (Hodgkin and Horowicz, 1960). Moreover, across 22 species of mantis shrimp, the resting sarcomere length of the muscle actuating the raptorial appendage is ∼25% longer in species requiring high forces for prey capture (Blanco and Patek, 2014).

Despite the support for the sliding-filament and crossbridge theories provided by the dependence of force on actin–myosin overlap, considerable variation seems to exist in the width of the normalized force–length curves (Fig. 1). Some caution should be taken with these comparisons owing to the variety of methods used across labs and muscles. Nevertheless, in the squid mantle, the shape of the force–length relationship varies across muscle fibers from different regions under identical conditions (Thompson et al., 2014). The shape of the force–length curve has functional implications. Broader curves allow for higher forces over larger ranges of motion and in some cases are presumed to reflect adaptation. For example, supercontracting muscles in which myosin filaments are thought to pass through the Z-disk and interact with actin in adjacent sarcomeres have been described in both invertebrates and vertebrates, and are thought to allow for the large strains required in ballistic tongue projection in chameleon feeding (Hoyle et al., 1965; Hardie, 1976; Wainwright et al., 1991; Herrel et al., 2001; Anderson and Deban, 2010). In addition, obliquely striated muscles, in which sarcomeres are at an oblique angle to the long axis of the muscle fiber and can rotate during shortening, have been suggested to allow for broader and more variable force–length relationships and may permit distention of the body wall in feeding leeches (Rosenbluth, 1965; Kier, 1985; Gerry and Ellerby, 2011; Taylor-Burt et al., 2018). Supercontracting and obliquely skeletal muscles expand our notions of the variation possible largely within crossbridge and sliding-filament theories. However, significant variation in force–length curve width is observed across muscles with more typical sarcomeres (Fig. 1). A variety of mechanisms have been proposed to explain this variation in width, including variation in troponin isoform and actin–myosin lattice structure, and phosphorylation of troponin (Gordon et al., 2000; Josephson and Stokes, 1987; Thompson et al., 2014; Tu and Daniel, 2004; Williams et al., 2013; Rockenfeller et al., 2022). Furthermore, the very narrow curves of stiff flight muscles (Josephson, 1997; Josephson et al., 2000b; Tu and Daniel, 2004) suggest that the passive properties of muscle may be related to the shape of the force–length curve (Hardie, 1976). Detailed comparative study of force–length relationships, especially across muscles with varying in vivo strains, lattice structures and passive properties, may further our understanding of the mechanisms of muscle contraction and the scope for variation and adaptation in the shape of the force–length relationship.

The shortening side of the isotonic force–velocity relationship is arguably the most characterized feature in comparative muscle physiology (Fig. 2; Hill, 1938, 1950; Medler, 2002; Alcazar et al., 2019). Shortening force–velocity relationships are typically characterized by maximum unloaded shortening velocity (Vmax) and their curvature. More than 40-fold variation in Vmax has been reported across muscles and species (Fig. 2; Altringham and Johnston, 1982; Josephson and Stokes, 1987; Chan and Dickinson, 1996; Holmes et al., 1999; Kier and Curtin, 2002; Astley, 2016; Anderson and Roberts, 2020; Rummel et al., 2022). Moreover, the shape of the relationship varies from highly curved to almost linear (Fig. 2; Woledge, 1968; Josephson, 1984; Schiaffino and Reggiani, 2011; Alcazar et al., 2019). This variation in Vmax and curvature of the force–velocity relationship has important functional consequences. The two-fold variation in ankle extensor Vmax across 14 frog species positively correlates with jump performance (Astley, 2016), and a flatter force–velocity relationship, which results in higher powers, is associated with increased calling frequency in two species of tree frogs (Marsh, 1999a).

Variation in Vmax and curvature is largely attributed to variation in myosin isoforms and contractile-protein length. Slower myosin isoforms decrease Vmax and increase curvature (Bottinelli et al., 1991; Medler, 2002; Schiaffino and Reggiani, 2011), and muscle fibers with shorter sarcomeres have a higher Vmax owing to the increased number of serial sarcomeres (Josephson, 1975; Kier and Schachat, 1992; Kier and Curtin, 2002), Thus, such variation can be explained within classic crossbridge and sliding-filament theories. However, interaction between the contractile proteins and structural elements of muscle also modulate the force–velocity relationship. The requirement of muscle to move both its own mass and external loads has been suggested to reduce Vmax, decrease curvature, and restrict the region of the force–velocity relationship over which a muscle can operate (Günther et al., 2012; Richards and Clemente, 2013; Holt et al., 2014; Ross et al., 2020; Ross and Wakeling, 2016; Richards and Eberhard, 2020). Hence, in larger muscles and animals, we might expect that observed shortening velocity would be lower than predicted from myosin kinetics. In muscles with a substantial tendon, stretch and recoil of this elastic element can decouple muscle shortening speed from myosin kinetics, leading to very rapid yet forceful shortening (Peplowski and Marsh, 1997; Azizi and Roberts, 2010; Ilton et al., 2018; Longo et al., 2019). Variation in the elastic properties of these tendons is a major determinant of jump performance across three frog species (Roberts et al., 2011; Mendoza and Azizi, 2021).

In contrast to the extensive comparative study of