Nitric oxide and skeletal muscle contractile function

Abstract

Nitric oxide (NO) is complex modulator of skeletal muscle contractile function, capable of increasing or decreasing force and power output depending on multiple factors. This review explores the effects and potential mechanisms for modulation of skeletal muscle contractile function by NO, from pharmacological agents in isolated muscle preparations to dietary nitrate supplementation in humans and animals. Pharmacological manipulation in vitro suggests that NO signaling diminishes submaximal isometric force, whereas dietary manipulation in vivo suggest that NO enhances submaximal force. The bases for these different responses are unknown but could be reflect dose-dependent effects. Maximal isometric force is unaffected by physiologically relevant levels of NO, which do not induce overt protein oxidation. Pharmacological and dietary manipulation of NO signaling enhances the maximal rate of isometric force development, unloaded shortening velocity, and peak power. We hypothesize that these effects are mediated by post-translational modifications of myofibrillar proteins that modulate thick filament regulation of contraction (e.g., mechanosensing and strain-dependence of cross-bridge kinetics). NO effects on contractile function appear to have some level of fiber type and sex-specificity. The mechanisms behind NO-mediated changes in skeletal muscle function need to be explored through proteomics analysis and advanced biophysical assays to advance the development of small molecules and open intriguing therapeutic and ergogenic possibilities for aging, disease, and athletic performance.

1). Introduction

The study of muscle contractile properties is challenging because they can change acutely with a single (e.g., post-tetanic potentiation) or repetitive contractions (e.g., fatigue). These changes seem to be due in part to the effects of nitric oxide (NO), which can cause both acute gain and loss-of-function. The loss of function with levels of NO that cause protein oxidation has been reviewed extensively [1–3]. This mini-review focuses on the modulation of skeletal muscle isometric and isotonic contractile function by NO within conditions that do not cause overt protein oxidation or other modifications that impair function.

The discovery of NO synthase expression in skeletal muscle cells in the mid-1990’s [4] led to a surge of publications focused on the role of NO on skeletal muscle contractile function [1, 2, 5–10] (Figure 1). A detailed account of the main findings of studies focused on NO and/or dietary nitrate and contractile function can be found in previous reviews [3, 11–13]. Here, we present a summarized view of the initial studies focused on NO with an update from studies completed in the last 10–15 years, a brief update on the effects of dietary nitrate, and address novel emerging concepts as potential mechanisms to explain modulation of contractile function by NO.

Figure 1. Number of publications per year addressing the effects of NO on skeletal muscle contractile function.

The discovery of nitric oxide synthase in skeletal muscle and demonstration of NO modulation of contraction in 1994 [4] (arrow) led to a surge of interest and publications focused on the role of NO on skeletal muscle contractile function. This continued with the findings of beneficial effects of dietary nitrates on skeletal muscle. Search query: nitric oxide skeletal muscle contractile function.

NO in skeletal muscle, interventions, and translation of findings

There are two main sources of NO in skeletal muscle fibers: 1) nitric oxide synthases (NOS) that synthesize NO from L-arginine [3, 4], and 2) nitrate that is reduced to nitrite and NO [14, 15]. A well-defined pathway downstream of NO production involves activation of guanylate cyclase and cGMP signaling. These systems have been targeted to modulate NO levels and signaling, and to assess the impact on skeletal muscle contractile function in animals and humans [4–10, 16]. NO appears to exert cellular effects at nanomolar concentrations [3]. Interventions with NO donors and mimetics of NO signaling, even at low concentrations, demonstrate the potential of NO to modulate contractile function, but do not define the impact of physiological levels of NO and its downstream signaling. It is worth noting that skeletal muscle contraction increases myocyte NO production [17], which will affect function during repetitive contractions. The effects of NO on muscle fatigue and performance are reviewed elsewhere in this special issue.

The main approaches available for human studies are pharmacological inhibition of NOS, dietary supplementation with L-arginine (L-citrulline as alternative) and inorganic nitrate/nitrite [18, 19], and pharmacological inhibition of cGMP degradation [16]. Some of the findings in isolated muscle preparations and animal models are challenging to translate to humans in any field because of limited availability of interventions, the increasing physiological complexity, and greater individual variability. This holds true for studies of NO effects on skeletal muscle contractile function. Nitrate supplementation and inhibition of cGMP degradation elicit generally similar effects in rodents and humans, but there are high and low responders due to diet, variability in nitrate and nitrite processing [20], or differences in baseline NO signaling. Additionally, humans and rodent nitrate processing are not identical in nature, mainly differing by conversion of nitrate to nitrite via commensal bacteria in humans [21]. However, nitrate supplementation results in similar distribution of nitrate/nitrite ions in humans and rodents [21] and similar nitrate/nitrite transporters and reducers are present in skeletal muscle from humans and mice [22–24].

NO signaling and contractile properties.

Twitch and Submaximal Tetanic Force.

The consensus of earlier reviewers was that NO signaling has minimal to no effect on twitch contraction but diminishes force during submaximal (unfused) tetanic contractions [3, 11, 12]. Pharmacological inhibition of NOS in mouse limb muscle [10] or rat diaphragm ex vivo did not change peak twitch force [4, 7]. Later studies emerged showing that NOS inhibition increased peak twitch force by ~20% in rat diaphragm bundles (pharmacological, [25]) and mouse EDL (pharmacological and nNOS knockout, [26]). However, a consistent observation was that NOS inhibition ex vivo enhanced submaximal tetanic contractions – an effect that was abolished by addition of NO donors, a cGMP analog, or inhibition of phosphodiesterases that breakdown cGMP [8, 9]. In a similar fashion, studies have shown that acute exposure of mouse FDB fibers to sodium nitrite ex vivo decreased twitch and submaximal tetanic force [7, 27]. Pharmacological inhibition of neuronal NOS increased twitch force-time integral of in situ contractions of limb muscle from young [28] but not old rats [29]. Altogether, these previous studies have led to the suggestion that NO and its downstream signaling acts to inhibit peak twitch and submaximal tetanic force in a manner akin to that seen in smooth muscle (Figure 2A). This “clear” picture and interpretation was established originally in the late 1990’s and lasted for 10–15 years. In 2012, however, Hernandez et al [30] showed that dietary nitrate supplementation did not change peak twitch but increased submaximal tetanic force in the EDL and FDB muscles. Subsequent studies in human limb muscles shared these observations in males and females [31–33], with males also showing increased peak twitch force after dietary nitrate supplementation [31, 32]. Similarly, dietary nitrate supplementation increased peak twitch force in diaphragm of old male mice [34]. The general effects of dietary nitrate are summarized in Figure 2B. Based on the conversion of nitrate to nitrite and NO, the effects of dietary nitrate were assigned to NO signaling (assuming that nitrate and nitrite are biologically ‘inert’ aside from serving as pool of NO storage). It is worth noting that earlier studies had shown that replacing sodium chloride with sodium nitrate in experimental solutions of isolated fibers increased twitch force in amphibian muscles [35–37], presumably by changes in ion fluxes and enhanced depolarization unrelated to NO signaling. However, mammalian muscle incubated with sodium nitrate ex vivo showed normal contractile properties (Figure 3). Finally, a study published in 2017 showed that limb muscle of mice lacking guanylate cyclase had normal twitch and submaximal tetanic forces [38]. The latter challenges the notion that constitutive NO signaling via guanylate cyclase and cGMP acts to depress twitch and submaximal tetanic force, but also does not support the notion that constitutive NO signaling (via cGMP) enhances submaximal contractions. We will address the potential mechanisms underlying NO modulation of contractile function below. In short, however, the reason for the apparently contrasting effects of pharmacological, genetic, and dietary manipulations on twitch and submaximal tetanic contractile function are unclear.

Figure 2. Schematic illustration of NO/cGMP (A) and dietary nitrate (B) effects on isometric force-frequency relationship.

NO and cGMP (A, blue dashed line) reduce submaximal force production, shown by a rightward shift of the force-frequency relationship. Dietary nitrate supplementation (B, blue dashed line) increases submaximal force production and results in a leftward shift in the force-frequency relationship. Neither intervention alters maximal force production.

Figure 3. Ex vivo exposure to NaNO₃ does not alter skeletal muscle isometric and isotonic contractile function.

Isolated mouse diaphragm bundles were exposed to increasing concentrations of NaNO₃ (control, open circles; NaNO₃, filled circles). (A) maximal isometric specific force. (B) maximal rate of force development during isometric contraction. (C) peak power estimated by a single isotonic release contraction against ~33% of maximal specific force. Inset shows concentrations of NaNO₃, with exposure lasting 15 min per concentration. Data are compared to control diaphragm bundles from the same animal exposed to standard buffer (no NaNO₃) with measurements taken at similar time intervals (open circles). Data are shown as percentage of pre-treatment baseline values (initial) for each diaphragm bundle. N = 6 per group.

Maximal Tetanic Force.

Nitric oxide has no effect on maximal force but modulates the rate of force development during fused tetanic contractions [10]. These findings were among the original observations related to NO effects on muscle contractile function (see review [11]). Specifically, pharmacological NOS inhibitors or modulation of NO signaling via cGMP ex vivo also had no effect on maximal tetanic force of rat diaphragm [4, 6, 8, 9], but the maximal rate of force development was not reported in those studies. NOS inhibition had no effect on maximal tetanic force ex vivo, but it slowed the rate of force development during a maximal (fused) tetanic contraction of mouse EDL at 20° C [10]. The main development since the initial studies came from reports showing that dietary nitrate supplementation accelerates the rate of tetanic force development without changing maximal force in EDL of young [30] and diaphragm of old mice [34]. In humans, dietary nitrate had no effect on maximal tetanic force [31, 32]. We are not aware of studies in humans examining the direct role of NO on the rate of force development during electrically stimulated fused tetanic contractions. However, representative data suggest that dietary nitrate accelerates the rate of tetanic force development in human quadriceps (c.f. Fig. 1C in [31]). In contrast, four days of dietary nitrate dose (8.8 mmol·d⁻¹ plus 17.6 mmol on the day of testing), which may be relatively high for young adults, did not change maximal rate of isometric force development [39]. Importantly, recent pilot studies established dose-dependent effects of dietary nitrate on muscle contractile properties in older adults [40]. Overall, constitutive NO and enhanced levels reached with low-to-medium doses of dietary nitrate seem to optimize muscle activation (rate of force development) during a fused tetanus (Figure 4).

Figure 4. Schematic illustration of force (A) and rate of contraction (B) tracings during fused isometric tetanus.

Constitutive NO signaling [10] or dietary nitrate supplementation [30, 34] (dashed blue line) accelerates the rate of contraction during a fused tetanus compared to control condition (solid red line). Panel B data show the first derivative of the force tracing shown in Panel A.

Isotonic/Isokinetic contractile properties.

The most consistent observation in animals and humans is that NO and cGMP signaling positively modulates isotonic contractile properties (Figure 5). The initial discovery of the NO role on isotonic contractile function was made by Prof. Michael Reid’s group [5], who observed that acute pharmacologic inhibition of NOS in rat diaphragm bundles ex vivo slowed shortening velocity during loaded contractions and, consequently, diminished peak power [5, 6]. A NO donor, alone, did not affect isotonic properties but rescued shortening velocity and peak power in muscles exposed to NOS inhibition. Subsequent studies in mouse EDL muscle shared generally similar findings to those in rat diaphragm, but also showed that a cGMP analog increased unloaded shortening velocity [10]. Human studies examining the role of NO and cGMP signaling on muscle isotonic/isokinetic properties emerged a decade later [16, 18]. Chronic administration of sildenafil, which enhances cGMP levels and NO signaling, increased peak power of knee-extensors by ~20% on average in older adults [16]. Prof. Andrew Coggan’s group then showed that acute dietary nitrate supplementation increased maximal shortening velocity and peak power in knee extensor muscles of young individuals, older healthy adults, and patients with heart failure [18, 40–42]. Along similar lines, a recent study by our group showed that two weeks of dietary nitrate supplementation restored diaphragm peak power of old mice [34]. Maximal shortening velocity also increased by 30% on average, albeit not reaching the threshold for statistical significance [34]. It is worth noting that much earlier studies had established that direct exposure of amphibian muscles to sodium nitrate ex vivo altered the curvature of the force-velocity relationship in a manner consistent with enhanced peak power and maximal shortening velocity [43, 44]. Nonetheless, the effects were attributed to electrolytes and muscle activation, not nitric oxide. As mentioned above, the contractile properties of mammalian muscle were unaltered by exposure to sodium nitrate ex vivo (Figure 3C). These findings reinforce the notion established by human studies suggesting that faster shortening velocity and heightened peak power with dietary nitrate requires whole-body mechanisms and involve NO signaling [18].

Figure 5. Illustration of positive modulation of skeletal muscle isotonic contractile properties by NO and dietary nitrate.

A) Specific force-shortening velocity relationship. B) Specific force-power relationship. Power calculated as specific force (N/cm²) x shortening velocity (Lo/s). Constitutive NO signaling, NO donors and cGMP, or dietary nitrate supplementation increase shortening velocity against low to moderate submaximal loads compared with NOS blockade or control treatment (solid red line). A faster shortening velocity for each force results in higher peak power. Schematic based on references discussed in the text.

Fiber type-specific effects.

The original discovery of nitric oxide synthase (NOS) in skeletal muscle observed greater NOS content and activity in fast twitch muscles [4]. Similarly, the effects of nitrate supplementation in rodents have occurred in fast twitch muscles [30, 34]. The rationale for this specificity is based on the lower microvascular oxygen pressure (PO₂) associated with fast-twitch muscle fibers that favors nitrite conversion to NO via reduction by deoxygenated heme- or molybdenum-based proteins or via disproportionation [45]. At lower PO₂, more deoxygenated heme-based proteins (e.g. hemoglobin, myoglobin) are available for reduction to proceed. Additionally, the disproportionation reaction preferentially occurs under acidic conditions (pKA ~3.3) and it increases dramatically during ischemia where pH can plummet to ~5.5 [46, 47]. As previously shown, oxygenation is critical when investigating effects of NO on skeletal muscle [26, 27] and pH likely is as well. Fiber type-specific effects may make findings in rodent skeletal muscle, with a more homogenous distribution of muscle fibers, harder to detect in humans that typically have a heterogeneous distribution of muscle fibers. This merits fiber-type specific studies at the single fiber level. It is important to note that dietary nitrate elicits generally similar effects in healthy mice hearts [48] and fast-twitch skeletal muscles (rats and mice), but did not change contractile properties in mouse soleus muscle (~50% type I and 50% type II fibers), making it unclear whether ‘fiber type’, or specifically myosin heavy chain isoform, per se is an important determinant of NO effects on contractile function.

Physiological mechanisms and molecular signaling

The physiological mechanisms and molecular signaling mediating the modulation of contraction by nitric oxide have not been defined in detail. Most mechanistic studies have focused on detrimental effects mediated by S-nitrosylation or protein oxidation. The existing evidence suggests that beneficial effects of NO on contractile function involve steps of excitation-contraction coupling, myofilament function, or both.

Neuromuscular transmission.

NO signaling is required for neuromuscular junction formation and clustering of acetylcholine receptors [49, 50]. NO donors and cGMP analogues induce vesicle release (Drosophila, [51]), enhance transmitter release (rat [52]), and inhibit acetylcholinesterase activity (rat, [53]) at the neuromuscular junction. NO effects in the neuromuscular junction might contribute to modulation of contractile function seen with voluntary or nerve stimulated contractions in humans [18, 31, 32, 41, 42]. However, studies showing NO modulation of contractile function in rodents bypassed neuromuscular transmission with field (direct) stimulation ex vivo [5, 6, 10, 34].

Excitation-contraction coupling.

NO modulates EC coupling in a dose dependent manner. Exposure of isolated fibers to NO donors increased intracellular calcium (but not force) during contraction [7]. NO and NO donors activate skeletal muscle Ca²⁺ release channel/ryanodine receptor (RyR1) via S-nitrosylation [54, 55] and inhibit sarco-endoplasmic reticulum Ca²⁺ ATPase [56]. These combined effects would result in elevated intracellular calcium and an expected higher submaximal force during contraction. The faster and higher [Ca²⁺] release (and submaximal force) are seen during submaximal tetanic contraction after dietary nitrate supplementation [30–32, 34], but may not explain changes in rate of contraction (see Myofilament function). Dietary nitrate effects were also accompanied by increased abundance of calsequestrin and dihydropyridine receptor calcium channel in mouse limb muscle [30], but not human limb muscle [31] and mouse diaphragm [34]. However, constitutive NO acts as a negative modulator of isometric muscle force (see above), presumably due to diminished myofilament calcium sensitivity [7], in a manner that is puzzling to reconcile with NO effects on RyR1 and the impact of dietary nitrate on intracellular [Ca²⁺] and contractile function. Notably, the positive modulation of isotonic contraction by NO (and dietary nitrate) reported in rodent muscle ex vivo [5, 6, 10, 34] seems independent of EC coupling as experiments were carried out during maximal activation. Therefore, enhanced V_max and peak power must arise at least in part from effects of NO signaling on myofilament proteins.

Myofilament function.

The classical view of muscle activation and subsequent force development and shortening is that an action potential causes a rise in intracellular [Ca²⁺] that turns the thin filament ON and allows actin-myosin interaction. In this model, intracellular [Ca²⁺] and its interactions with thin-filament proteins dictates the ON or OFF state of skeletal muscle (and force development or shortening) while the thick filament is a bystander, always ready for contraction. Based on this model, NO modulation of the rate of isometric force development should be explained by effects on calcium-handling proteins and intracellular [Ca²⁺] [10, 30]. However, thick filament activation is a regulated process and muscle force development and shortening requires activation of both thin and thick filaments. The concept of thick filament regulation of muscle contraction has been discussed in detail previously [57] and reinforced by a recent study [58]. Herein, we advance the notion that NO (and dietary nitrate) acts on the thick filament to modulate the rate of isometric force development, maximal shortening velocity, and peak power.

The rate of isometric force development is not limited by intracellular calcium or calcium binding to troponin [57]. The kinetics of the rise in intracellular [Ca²⁺] is approximately 10× faster than force development, and calcium-binding sites on troponin become fully occupied within 1 ms after peak intracellular [Ca²⁺] during an isometric contraction [57–59]. Similarly, the movement of tropomyosin and exposure of myosin-binding sites on actin precedes thick filament activation and force development [57]. The dynamic relationship between these processes was not captured in mammalian muscle [58], but tropomyosin movement occurs in ½ the time of the fastest structural change in the thick filament in amphibian muscle [60]. Therefore, the acceleration of isometric force development by NO and dietary nitrate, although accompanied by faster and higher intracellular [Ca²⁺], is most likely mediated by effects on the thick filament. We cannot discard calcium activation of the thick filament [61], although this mechanism is a point of contention [57]. Interestingly, data from our study [34] show a correlation between the rate of isometric force development and peak power (measured during isotonic release), suggesting that similar mechanisms contribute to a faster rate of isometric force development and heightened peak power after dietary nitrate supplementation (Figure 6) and presumably nitric oxide.

Figure 6. Relationship between maximal rate of isometric force development and peak power.

Data shown are contractile properties of diaphragm bundles from old mice receiving water (red circles) or dietary nitrate supplementation (blue circles). Data are replotted from our recent study [34], with permission from John Wiley & Sons, Inc. The relationship suggests that similar mechanisms are involved in the modulation of the rate of force development during a fused tetanic isometric contraction and peak power with NO signaling. Emerging concepts and experimental conditions for assessment of peak power suggest that thick filament regulation of contraction is the underlying basis of the relationship shown here and most likely the mediator of enhanced function with dietary nitrate and NO signaling. It is important to note two aspects: 1) power measurements were performed during isotonic release instead of afterloaded contractions that would implicate an inherent relationship between maximal rate of isometric contraction and power [94]; and 2) during whole muscle ‘isometric’ (end-held) contraction there is a small degree of sarcomere shortening [58] such that a faster shortening velocity can contribute to an ‘apparent’ faster rate of force development and, therefore, the relationship shown above. However, published studies suggest NO signaling does not accelerate shortening velocity during high loads and the data above are from a maximal tetanic contraction.

The details on the process of thick filament activation during isometric contraction and regulation of cross-bridge function and shortening during loaded contractions are still evolving. In resting skeletal muscle, most myosin heads are in the OFF configuration (super-relaxed state). Post-translational modifications of sarcomeric proteins, mechanosensing, and inter-filament signaling switch the thick filament to the ON configuration that facilitates cross-bridge attachment and the weak-to-strong binding transition [57, 61, 62].

Shortening velocity is determined by cross-bridge kinetics. Against very low loads, muscle shortening appears to be supported by a small number of cross-bridges that are constitutively ON [57, 62, 63]. Dietary nitrate, NO, and cGMP acceleration of shortening velocity at low loads could result from a higher number of cross-bridges constitutively ON, a faster cross-bridge kinetics (rate of detachment = rate-limiting step), or both (Figure 7). The transition from OFF to ON state heightens myosin ATPase activity and skeletal muscle metabolic rate [64]. Yet, dietary nitrate supplementation lowers oxygen consumption at rest and during exercise [65, 66]. The latter observations suggest that the faster shortening velocity with dietary nitrate, NO, and cGMP is not due to more cross-bridges in the ON state, but may instead be due to acceleration of cross-bridge kinetics. However, we cannot discard an increase in number of cross-bridges constitutively ON based solely on whole muscle metabolic rate data. Dietary nitrate could enhance metabolic efficiency of other ATP-dependent steps [67] and counter an increased myosin ATPase activity with the ON state.

Figure 7. Illustration of sarcomere (top) and thick filament proteins (bottom).

Bold font highlights the proposed proteins and biophysical events that might contribute to faster rate of isometric force development, faster unloaded shortening velocity, and higher peak power with enhanced NO signaling. XB, cross-bridge. Based on the widely known activation of kinases by NO (e.g., PKG), phosphorylation is the logical post-translational modification (not illustrated here). However, other modifications might be involved as primary or secondary events downstream of currently known pathways. Figure prepared and published with permission from BioRender.com

The hyperbolic force-velocity relationship dictates peak power, which is equal to force × velocity. Muscle force is the product of the number of attached cross-bridges (force-generating in parallel) and the force per individual cross-bridge [68, 69]. Shortening velocity is determined by cross-bridge detachment rate (ADP-release step) [62], which is strain-dependent and contributes to the hyperbolic nature of the force-velocity relationship [62, 63]. Loss of force production as shortening velocity increases results from a lower number of attached cross-bridges and less force per cross-bridge over the hyperbolic region of the force-velocity relationship (5–80%) [70]. Therefore, we consider three scenarios that could explain the increased peak power mediated by NO signaling. First, a higher force per cross-bridge at each submaximal shortening velocity. This appears unlikely, as maximal isometric force is unchanged. Second, a higher number of attached cross-bridges at submaximal shortening velocity. An increase in cross-bridge compliance raises the number of attached cross-bridges and peak power during shortening despite a small loss in force per cross-bridge [71]. However, increases in cross-bridge compliance slow maximal shortening velocity, which contrasts the effects of NO signaling on contractile function. Third, a lower strain-dependence of cross-bridge detachment such that detachment rate (and shortening velocity) increases for any given submaximal load (Figure 7). In this setting, ADP release or detachment of negatively strained cross-bridges (without ATP splitting) would be enhanced at low to moderate loads, but unchanged as the load approaches maximal force. We propose that the third scenario is the most likely explanation for the positive effect of NO on isotonic contractile properties. Facilitation of ADP release from the myosin head would minimize the number of cross-bridges carried into the negative strain or ‘drag region’ after ATP splitting. Individual cross-bridges that complete the cycle and detach prior to entering negative strain will have performed more net work while still hydrolyzing one ATP molecule [72]. Faster detachment of highly strained cross-bridges that occurs via an ATP-independent mechanism is also plausible [73, 74]. In both cases, the thermodynamic efficiency of the cross-bridge cycle and muscle shortening rises [72, 74], lowering the energetic demand of contraction for matched power outputs. These mechanisms could contribute to the diminished oxygen uptake and ATP hydrolysis measured in exercising humans after dietary nitrate supplementation [75]. However, the exact biophysical and biochemical events are currently difficult to define due to technical challenges of studying cross-bridge function during isotonic contractions.

Post-translational modifications of myofibrillar proteins appear to determine the effects of NO on muscle contractile function [13, 34]. Phosphorylation of myofibrillar proteins is an established modification that generally enhances contractile function. Figure 7 illustrates myofibrillar proteins that might mediate NO effects on contractile function. Based on the canonical NO−GC−cGMP−PKG pathway, it is reasonable to suspect that phosphorylation is the signaling event responsible for faster shortening velocity and increased peak power [13]. Myosin regulatory light chain (RLC) and myosin-binding protein C (MyBP-C) are known regulators of thick filament activation and cross-bridge function [57, 76, 77]. Phosphorylation of myosin RLC increases calcium sensitivity [78, 79], maximal rate of force development [80], and supports peak power [81]. MyBP-C plays an essential role on inter-filament signaling and inhibits shortening velocity and peak power [76, 82, 83]. Phosphorylation of cardiac MyBP-C accelerates cross-bridge kinetics [84, 85] and phosphorylation sites are present in skeletal MyBP-C [77]. Titin is a mechano-sensor protein that is sensitive to phosphorylation that modulates contractile function [86, 87]. Being a target of NO signaling in cardiomyocytes [88], titin could mediate the effects of NO on skeletal muscle contraction. In a recent study, we found no changes in myofibrillar protein phosphorylation with dietary nitrate supplementation [34]. However, our technique (gel electrophoresis and ProQ staining) was likely insensitive to subtle changes in specific amino acid residues that may occur with NO signaling elicited by dietary nitrate and was not suitable to detect modifications to titin. Moreover, other myofibrillar proteins or types of post-translational modification may explain enhanced contractile function with NO signaling. For example, acetylation and arginylation are positive modulators of skeletal muscle contractile function [89–91] and NO-cGMP signaling could modulate the activity of acetyltransferases/deacetylases or arginyltransferase [92, 93].

It is possible that the positive modulation of contractile function with very low levels of NO signaling arise from subtle effects on multiple proteins and mechanisms that act synergistically to enhance contractile function (e.g. small changes in rate of cross-bridge attachment or detachment, force per cross-bridge, number of attached cross-bridges, thick filament activation, stiffness, etc.). These changes might stem from a combination of factors that includes serine/threonine/tyrosine phosphorylation, lysine acetylation, aspartic or glutamic acid arginylation, and thiol redox modifications of specific sites in different proteins. In this setting, unbiased proteomics assessment of post-translational modification of myofibrillar proteins offers the best approach moving forward to resolve the biochemical event leading to enhanced contractile function with dietary nitrate and NO/cGMP. Once the potentially relevant modifications are identified, single amino acid mutations that mimic or prevent the post-translational modification can be used to establish a cause-and-effect relationship with standard muscle physiology measures and sophisticated biophysical assays. These approaches, albeit challenging, could prove insightful for the development of small-molecules and application of polypharmacology to enhance skeletal muscle contractile function in sports, aging, and disease.

Summary

NO can modulate skeletal muscle contractile function in a dose-dependent manner, opening intriguing therapeutic and ergogenic possibilities in aging, disease, and human performance. Consuming dietary nitrates such as those found in beets and leafy greens can boost NO, raising important implications for diet interventions and nutraceuticals. Sex-specific responses may also occur and should be examined further for its translational and mechanistic relevance. The mechanisms behind NO-mediated changes in skeletal muscle function are complex and challenging to resolve with methods currently available. We propose that the primary actions of NO (and dietary nitrate) are through post-translational modifications of myofibrillar proteins that alter thick filament regulation of contraction. These mechanisms will need to be explored through proteomics analysis and advanced biophysical assays. Advances in this field are critical to further the understanding of muscle contractile function and the development of small molecules that positively impact isometric and isotonic properties.