Nitric Oxide 122–123 (2022) 54–61

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Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox

Nitric oxide and skeletal muscle contractile function

Ravi Kumar a, Andrew R. Coggan b, **, Leonardo F. Ferreira a, c, *
a
Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL, USA
b
Department of Kinesiology, Indiana University Purdue University, Indianapolis, IN, USA
c
Department of Physiology, Amsterdam UMC Locatie VUmc, Amsterdam, the Netherlands

A B S T R A C T

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 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 1.1. NO in skeletal muscle, interventions, and translation of findings

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] (Fig. 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.
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 con­
tractile 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 repeti­
tive 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 pharmaco­
logical 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

* Corresponding author. Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL, USA.
** Corresponding author.
E-mail addresses: acoggan@iupui.edu (A.R. Coggan), ferreira@hhp.ufl.edu (L.F. Ferreira).

https://doi.org/10.1016/j.niox.2022.04.001
Received 30 September 2021; Received in revised form 23 March 2022; Accepted 5 April 2022
Available online 8 April 2022
1089-8603/© 2022 Published by Elsevier Inc.
R. Kumar et al.
Fig. 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" AND "skeletal muscle" AND "contractile function".
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 ef­
fects 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 pro­
cessing are not identical in nature, mainly differing by conversion of
nitrate to nitrite via commensal bacteria in humans [21]. However, ni­
trate 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].
1.2. NO signaling and contractile properties
1.2.1. Twitch and submaximal tetanic force
The consensus of earlier reviews was that NO signaling has minimal
to no effect on twitch contraction but diminishes force during submax­
imal (unfused) tetanic contractions [3,11,12]. Pharmacological inhibi­
tion 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 bun­
dles (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 inhi­
bition 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 (Fig. 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 sup­
plementation 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],
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Nitric Oxide 122–123 (2022) 54–61
Fig. 2. Schematic illustration of NO/cGMP (A) and dietary nitrate (B) ef­
fects 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.
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 Fig. 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 biologi­
cally ‘inert’ aside from serving as pool of NO storage). It is worth noting
that earlier studies had shown that replacing sodium chloride with so­
dium 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. Howev­
er, mammalian muscle incubated with sodium nitrate ex vivo showed
normal contractile properties (Fig. 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 pharma­
cological, genetic, and dietary manipulations on twitch and submaximal
tetanic contractile function are unclear.
1.2.2. Maximal tetanic force
Nitric oxide has no effect on maximal force but modulates the rate of
force development during fused tetanic contractions [10]. These find­
ings were among the original observations related to NO effects on
muscle contractile function (see review [11]). Specifically, pharmaco­
logical 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 devel­
opment in human quadriceps (c.f. Fig. 1C in Ref. [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
R. Kumar et al.
Fig. 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 mice (1 diaphragm bundle per condition).
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 (Fig. 4).
1.2.3. Isotonic/isokinetic contractile properties
The most consistent observation in animals and humans is that NO
and cGMP signaling positively modulates isotonic contractile properties
(Fig. 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,
Fig. 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.
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Nitric Oxide 122–123 (2022) 54–61
Fig. 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 in­
crease 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 ref­
erences discussed in the text.
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 en­
hances 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 con­
tractile properties of mammalian muscle were unaltered by exposure to
sodium nitrate ex vivo (Fig. 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].
R. Kumar et al.
1.2.4. 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 ef­
fects may make findings in rodent skeletal muscle, with a more ho­
mogenous 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.
1.3. 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.
1.3.1. 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) stim­
ulation ex vivo [5,6,10,34].
1.3.2. 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-nitro­
sylation [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 supple­
mentation [30–32,34], but may not explain changes in rate of contrac­
tion (see Myofilament function). Dietary nitrate effects were also
accompanied by increased abundance of calsequestrin and dihy­
dropyridine 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
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Nitric Oxide 122–123 (2022) 54–61
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 Vmax and
peak power must arise at least in part from effects of NO signaling on
myofilament proteins.
1.3.3. Myofilament function
The classical view of muscle activation and subsequent force devel­
opment 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 in­
teractions 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 pro­
cess 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 intracel­
lular 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 acti­
vation 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
(Fig. 6) and presumably nitric oxide.
The details on the process of thick filament activation during iso­
metric contraction and regulation of cross-bridge function and short­
ening 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, mecha­
nosensing, 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 (Fig. 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 short­
ening 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
R. Kumar et al.
Fig. 6. Relationship between maximal rate of isometric force develop­
ment and peak power. Data shown are contractile properties of diaphragm
bundles from old mice receiving water (red circles) or dietary nitrate supple­
mentation (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 after­
loaded 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.
cross-bridge kinetics. However, we cannot discard an increase in
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Nitric Oxide 122–123 (2022) 54–61
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.
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 deter­
mined 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 short­
ening 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 sub­
maximal 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 sub­
maximal load (Fig. 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 ap­
proaches 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 per­
formed 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 short­
ening rises [72,74], lowering the energetic demand of contraction for
matched power outputs. These mechanisms could contribute to the
Fig. 7. Illustration of sarcomere (top) and thin
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 ve­
locity, 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 sec­
ondary events downstream of currently known path­
ways. Figure prepared and published with permission
from BioRender.com.
R. Kumar et al.
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. Fig. 7 illustrates myofi­
brillar 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 reg­
ulatory 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 sensi­
tivity [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 supplemen­
tation [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 arginyla­
tion, 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 contrac­
tile 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 physi­
ology 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.
1.4. Summary
NO can modulate skeletal muscle contractile function in a dose-
dependent manner, opening intriguing therapeutic and ergogenic pos­
sibilities 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
59
Nitric Oxide 122–123 (2022) 54–61
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 posi­
tively impact isometric and isotonic properties.
Author disclosure statement
The authors declare no conflict of interest.
Acknowledgements
The authors work on the topic of this review has been funded by the
American Heart Association (20PRE35200047 to R.A. Kumar) and the
National Institutes of Health (R01-HL130318 and R03-AG040400 to L.F.
Ferreira; R21-AG053606 and R34-HL138253 to A.R. Coggan).
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