Exercise Training Decreases Nitrite Concentration in the Heart and Locomotory Muscles of Rats Without Changing the Muscle Nitrate Content

Abstract

Background

Skeletal muscles are postulated to be a potent regulator of systemic nitric oxide homeostasis. In this study, we aimed to evaluate the impact of physical training on the heart and skeletal muscle nitric oxide bioavailability (judged on the basis of intramuscular nitrite and nitrate) in rats.

Methods and Results

Rats were trained on a treadmill for 8 weeks, performing mainly endurance running sessions with some sprinting runs. Muscle nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) concentrations were measured using a high‐performance liquid chromatography–based method, while amino acids, pyruvate, lactate, and reduced and oxidized glutathione were determined using a liquid chromatography coupled with tandem mass spectrometry technique. The content of muscle nitrite reductases (electron transport chain proteins, myoglobin, and xanthine oxidase) was assessed by western immunoblotting. We found that 8 weeks of endurance training decreased basal NO₂ ⁻ in the locomotory muscles and in the heart, without changes in the basal NO₃ ⁻. In the slow‐twitch oxidative soleus muscle, the decrease in NO₂ ⁻ was already present after the first week of training, and the content of nitrite reductases remained unchanged throughout the entire period of training, except for the electron transport chain protein content, which increased no sooner than after 8 weeks of training.

Conclusions

Muscle NO₂ ⁻ level, opposed to NO₃ ⁻, decreases in the time course of training. This effect is rapid and already visible in the slow‐oxidative soleus after the first week of training. The underlying mechanisms of training‐induced muscle NO₂ ⁻ decrease may involve an increase in the oxidative stress, as well as metabolite changes related to an increased muscle anaerobic glycolytic activity contributing to (1) direct chemical reduction of NO₂ ⁻ or (2) activation of muscle nitrite reductases.

Nonstandard Abbreviations and Acronyms

1‐Tre

trained on a treadmill for 1 week

2‐Tre

trained on a treadmill for 2 weeks

4‐Tre

trained on a treadmill for 4 weeks

8‐Tre

trained on a treadmill for 8 weeks

8cT

8 weeks of continuous forced endurance training on a treadmill

ETC

electron transport chain

GSH

reduced glutathione

GSSG

oxidized glutathione

LN₂

liquid nitrogen

MGF

fast‐twitch part of the medial gastrocnemius muscle

MGS

slow‐twitch part of the medial gastrocnemius muscle

NO

nitric oxide

NO₂ ⁻

nitrite

NO₃ ⁻

nitrate

NOS

nitric oxide synthase

NO_x

nitrite and nitrate concentrations

Sed_A

untrained sedentary control group A

Sed_B

untrained sedentary control group B

TA

tibialis anterior muscle

XO

xanthine oxidase

Research Perspective.

What Is New?

• We have found that exercise training enhances nitrite bioactivation (reduction to nitric oxide) in the heart and in the locomotory muscles and that this time course of training is rapid and is positively correlated with the distance of run covered by the animals on a treadmill.

• We have discovered that nitrite bioactivation in skeletal muscles appears, as in the heart, to be dependent on changes in intramuscular pH.

What Question Should Be Addressed Next?

• Nitrite appears to play a role in the striated muscle buffering capacity, which might explain the positive effects of nitrite/nitrate supplementation reported in the literature, especially in short‐term, high‐power output exercise performance in humans; however, this issue needs further studies.

• The physiological significance of nitrite bioactivation (reduction to nitric oxide) in skeletal muscle during exercise training needs further research, especially, the impact of nitrite bioactivation on muscle vasodilation, force generation, and mitochondrial biogenesis in relation to exercise performance.

Nitric oxide (NO) is a universal messenger that plays a crucial role in cardiovascular system protection since it acts as a key vasodilator and inhibitor of platelet aggregation and smooth muscle cell proliferation. ¹ The main classical pathway of NO synthesis is the NO synthase (NOS)‐dependent pathway, in which NO is generated from l‐arginine in the presence of oxygen. ² NO as a short‐lived molecule (~1 second) that becomes oxidized to nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻), which are not only end products of NO oxidation but constitute substrates for NO generation in the nonclassical, NOS‐independent pathway (NO₃ ⁻– NO₂ ⁻–NO pathway). ² , ³

Skeletal muscles account for about 30% to 40% of human body mass ⁴ and contain a remarkable amount of NO₃ ⁻ and NO₂ ⁻ (NO_x pool). ⁵ Therefore, they are postulated to be a potent regulator of systemic NO homeostasis. ⁶ , ⁷ Consequently, it is suggested that dietary NO₃ ⁻/NO₂ ⁻ supplementation that enhances plasma and tissue NO_x pool, is beneficial for normal cardiovascular and metabolic systems functioning, especially when NO bioavailability is limited or dysfunctional, for example, during aging and in cardiovascular disease. ⁷ , ⁸ The underlying ergogenic effects of NO₃ ⁻/NO₂ ⁻ supplementation are considered to be related to an increase in NO_x pool and in consequence in NO production via the NO₃ ⁻–NO₂ ⁻–NO pathway. ⁶

In our recent study, we demonstrated, for the first time, that in the locomotory muscles the lowest content of NO₂ ⁻ is present in the slow‐twitch oxidative soleus. Conversely, the fast‐twitch glycolytic muscle, such as a white part of the medial gastrocnemius (MGF), possesses the highest NO₂ ⁻ content in the locomotory muscle group. ⁹ Furthermore, we have reported that higher NO₂ ⁻ content in the fast muscles is accompanied by greater NO synthase activity (reflected by citrulline‐to‐arginine ratio). Based on these results, ⁹ we have concluded that fast‐twitch glycolytic muscle fibers, having higher NO₂ ⁻ content than slow‐twitch oxidative fibers, are actually predisposed to directly use NO₂ ⁻ as a substrate to generate NO via alternative (nonclassical) pathway of NO production, as suggested previously. ⁶ This feature of the fast‐twitch skeletal muscle fibers seems to be especially important during exercise performed at high exercise intensities, when the fast‐twitch muscle fibers are recruited and involved in power generation. That results in a deeper muscle metabolic instability/perturbation (compared with low/moderate intensity exercise) including faster and greater accumulation of metabolites related to fatigue such as hydrogen ions (H⁺), inorganic phosphate (P_i), ADP, AMP, and the like. ¹⁰ , ¹¹ The changes in the acid–base balance during high‐intensity exercise (eg, see Zoladz et al ¹⁰ and Majerczak et al ¹² ), as a consequence of activation of anaerobic glycolysis, especially in the fast muscle fibers may through a decrease in the intramuscular pH, accelerate NO₂ ⁻ reduction and NO generation to a greater extent than in the slow oxidative muscles. In accordance with this, it has been recently suggested that NO₃ ⁻ supplementation might be more beneficial for sprint/power athletes ¹³ than for endurance athletes, since in their case maximal power generation of the fast‐twitch muscle fibers is crucial for successful performance. ¹⁴ , ¹⁵

In the present study, we aimed to evaluate the impact of physical training on the NO bioavailability (assessed by NO₃ ⁻ and NO₂ ⁻ concentration at basal state) in the heart and in the skeletal muscles of rats, that is, in the slow‐twitch soleus and fast‐twitch MGF. We chose the soleus and MGF since they differ significantly, not only in the basal NO₂ ⁻ content, ⁹ but especially in the pattern of daily muscle activation. ¹⁶ , ¹⁷ , ¹⁸ Soleus as a postural, antigravitational muscle composed exclusively of slow motor units ¹⁶ reveals long‐lasting tonic activity (≈5–8 hours of total daily activity), whereas fast fatigable (the majority of MGF's motor units) and fast resistant motor units in MGF ¹⁷ , ¹⁸ have been shown to be active about 30 to 180 seconds and 20 to 90 minutes per day, respectively. ¹⁹ Accordingly, we assumed that the soleus muscle in our training program seemed to be active during each exercise bout from the lowest to the highest workloads. In the case of the MGF, composed mainly of fast fatigable‐type motor units, this muscle seemed to be involved during episodic exercise bouts of higher intensities.

In the present study, we hypothesized that 8 weeks of continuous forced endurance training (8cT) would enhance NO₃ ⁻ and NO₂ ⁻ concentrations in the heart and in the skeletal muscles of rats through NOS‐dependent pathway (assessed in our study indirectly through citrulline‐to‐arginine ratio). It has been previously shown that 4 weeks of voluntary running in mice increased NO₂ ⁻ and nitrosothiol concentrations in the heart and in the gastrocnemius muscle. ²⁰ The enhancement of the NO bioavailability after physical training has also been found in human locomotory muscles. ²¹ Furthermore, we hypothesized that an increase in the training duration (1, 2, 4 and 8 weeks) with an increasing workload will gradually enhance (mainly through NOS‐dependent pathway) the intramuscular NO₃ ⁻ and NO₂ ⁻ content in the slow‐twitch oxidative soleus, a locomotory muscle that is active in all movements.

Methods

Ethical Approval

All experimental procedures were in compliance with the guidelines of the European Community Council Directive 2010/63/UE of September 22, 2010 on the protection of animals used for scientific purposes. Experimental protocols were approved by the Local Ethics Committee on Animal Experimentation. Experimental protocol A was approved by the II Local Ethics Committee on Animal Experimentation in Krakow (Permit Number: 197/2018). Experimental protocol B was approved by the Local Ethics Committee on Animal Experimentation in Poznan (Permit Number: 3/2013).

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animals

A total of 103 male Wistar rats (Animal House of Poznan University of Medical Sciences, Poznan, Poland) comprised the experimental groups (trained or untrained sedentary groups) and participated in experiments A (n=30 rats, ≈3.5 months old) and B (n=73 rats, ≈5 months old). The individual rat was considered the experimental unit within this study. Six rats from experiment A and 3 rats from experiment B that were unable to perform the training protocol were excluded from the initial groups. Therefore, the final size of the groups in experiment A and B were n=24 and n=70, respectively (Figure 1). The animals were kept in standard laboratory cages in a room with a 12‐hour/12‐hour light/dark cycle and controlled temperature (22±2 °C) and humidity (55±10%). All rats had unrestricted access to standard rat food and tap water.

Figure 1. Experiment A (8 weeks of continuous forced endurance training) and experiment B (1, 2, 4, and 8 weeks of training) in rats.

Experiment A consisted of sedentary rats (Sed_A) and rats trained for 8 weeks (8cT). Experiment B consisted of sedentary rats (Sed_B) and rats trained for 1, 2, 4, and 8 weeks (1‐Tre, 2‐Tre, 4‐Tre, and 8‐Tre, respectively). HPLC indicates high‐performance liquid chromatography; LC‐MS/MS, liquid chromatography coupled with tandem mass spectrometry; NO_x, nitrite and nitrate concentrations; and WB, western immunoblotting.

In experiment A, rats (n=24, 428±30 g) were randomly assigned to 2 groups: 1 group was involved in the 8‐week continuous endurance training program on a treadmill (8cT group, n=12), whereas the second group consisted of sedentary untrained rats (Sed_A, n=12). In experiment B, rats (n=70, 421±57 g) were randomly assigned to 5 experimental groups: 1 untrained sedentary control group (Sed_B, n=14) and 4 groups of rats that trained on a treadmill for 1 week (1‐Tre group, n=14), 2 weeks (2‐Tre group, n=14), 4 weeks (4‐Tre group, n=14), and 8 weeks (8‐Tre group, n=14) (Figure 1). Experimental blinding and randomization strategies were employed when possible. The principal investigator was the only person aware of the group allocation. The sample size between 12 and 14 in each group for a particular measurement was evaluated on the basis of the results obtained from our previous studies.

Endurance Training

Endurance training (in experiments A and B) was performed at 22±2 °C, 5 times per week (Monday through Friday), on a standard treadmill for small rodents (Exer‐6 M Treadmill, Columbus Instruments, Columbus, OH). The endurance training in experiments A and B were performed according to the protocol adapted from Dudley et al ²² and implemented by our group. ²³ , ²⁴ It was mainly composed of an endurance bout of continuous running with 6 sprints during each training session. During the first week, to habituate rats to the treadmill, the intensity of the training was low and consisted of (1) 5 to 10 minutes of slow running at 5 to 10 m·min⁻¹ with 5‐ to 10‐second intervals of progressively increased treadmill speed, up to 30 to 40 m·min⁻¹, followed by a decrease in speed, which was repeated 10 to 20 times; (2) 60 minutes of rest; and (3) repetition of the first step of the procedure. In the subsequent weeks, training was conducted according to the following protocol: second week: 40‐minute continuous run at 30 m·min⁻¹; third week: 60‐minute continuous run at 30 m·min⁻¹; fourth week: 60‐minute continuous run at 30 m·min⁻¹, with acceleration every 10 minutes to 35 m·min⁻¹ with 30‐second intervals (6 accelerations at 10, 20, 30, 40, 50, and 60 minutes); fifth and sixth weeks: 60‐minute continuous run at 30 m·min⁻¹, with acceleration every 10 minutes to 40 m·min⁻¹ with 30‐second intervals (6 accelerations at 10, 20, 30, 40, 50, and 60 minutes). During the seventh and eighth weeks, rats involved in experimental protocol B (8‐Tre group) performed an 80‐minute continuous run at 30 m·min⁻¹, with acceleration every 13 minutes, up to 40 m·min⁻¹ for 30‐second intervals (6 accelerations at 13, 26, 39, 52, 65, and 78 minutes). In the case of rats involved in experiment A, due to their lower exercise capacity during the 7th and 8th week of training, they performed a 65‐ to 80‐minute continuous run (according to their physical capacity) at 25 m·min⁻¹, with acceleration every 13 minutes to 30 m·min⁻¹ for 30‐second intervals (6 or 5 accelerations, according to the individual rat's physical capacity at 13, 26, 39, 52, 65 and 78 minutes). Therefore, the total distance covered by rats and total time spent on a treadmill after 8 weeks of training in experiment A (8cT group) was lower than in experiment B (8‐Tre group) (Figure 1).

Muscle Tissue Extraction

In experiment A, the impact of 8cT on the metabolite concentrations and protein expressions was analyzed in the heart ventricles and in the locomotory muscles (soleus and MGF) with varied muscle fiber type composition. ²⁵ After completing the last training session in the training program (≈24 hours after the last exercise bout), rats involved in the experiment A were decapitated. All efforts were made to minimize suffering. Immediately after collecting blood, heart ventricles and locomotory muscles were dissected and frozen in the liquid nitrogen (LN₂). Tissue extraction in all animals was conducted by the same person in the same sequence as follows: (1) heart ventricles (≈2–3 minutes after decapitation), (2) medial gastrocnemius with dissection on the red, oxidative part (MGS) and white, glycolytic part (MGF) of the medial gastrocnemius, (3) tibialis anterior (TA), and finally (4) the soleus muscle. Locomotory muscles were dissected between 5 and a maximum of 10 minutes after decapitation. Care was taken to dissect the samples in the above‐mentioned order at the same time after decapitation.

In experiment B, at the end of each training period (after 1, 2, 4, and 8 weeks), ≈24 to 72 hours after completing the last training session (except group 1‐Tre, in which the muscle excision was performed a few hours after the last exercise bout) rats were anesthetized with pentobarbital sodium (initialintraperitoneal dose of 60 mg·kg⁻¹, supplemented with additional doses of 10 mg·kg⁻¹·h⁻¹ after 2 hours). The depth of anesthesia was controlled by monitoring the pinna and withdrawal reflexes. Before the electrophysiological experiments that were performed on the left leg (for details see Kryściak ²⁴ ), the locomotory muscles from the right leg (ie, the medial gastrocnemius divided during excision on the MGS and MGF as well as soleus muscle) were dissected and frozen immediately in the LN₂. After finishing the electrophysiological experiments, rats were euthanized by anesthetic overdose. MGS and MGF were used for protein expression analysis presented in our previous paper. ²⁴ The soleus muscle was stored in the LN₂ and used for the purpose of this study (Figure 1).

Muscle Sample Preparation for NO Metabolite and Targeted Metabolomic Analysis

Muscle samples obtained after completing experiments A and B were homogenized in an ice‐cooled phosphate buffer using Precellys Evolution (Bertin, Montigny‐le‐Bretonneux, France) or UltraTurax T10 (IKA, Staufen, Germany). The samples were cooled down on ice during the homogenizing process as well as during the whole preparation procedure. After the homogenization, samples were centrifuged (10 000g, 10 minutes, 4 °C) and the supernatant was prepared further following the protocols described below and developed specifically for the quantification of NO metabolites as well as liquid chromatography coupled with tandem mass spectrometry–based targeted metabolomic analysis. The composition of extraction buffer was developed to reliably quantify all mentioned endogenous molecules and protein concentration as published previously. ⁹

NO Metabolite Quantification

The levels of NO metabolites including NO₂ ⁻ and NO₃ ⁻, in plasma and in muscle homogenates were determined by the application of the liquid chromatography–based method with a postcolumn derivatization using an ENO‐20 NO_x analyzer (Eicom Corp., San Diego, CA). Before the analysis, samples were precipitated using methanol (1:1; v/v), centrifuged (10 000g, 10 minutes, 4 °C), and subjected to the analysis using the method conditions and parameters described previously. ⁹ The levels of NO₂ ⁻ and NO₃ ⁻ measured in muscle samples were normalized to milligrams of total protein.

Targeted Metabolomic Analysis

The concentrations of the selected amino acids including l‐arginine and l‐citrulline, as well as reduced glutathione (GSH) and oxidized glutathione (GSSG), lactate, and pyruvate were determined in plasma and muscle samples using liquid chromatography coupled with tandem mass spectrometry targeted metabolomic methods as described previously with minor changes. ²⁶ The concentrations of all selected metabolites were measured simultaneously in muscle samples. A chromatographic separation of analytes was performed with the aid of UFLC Nexera (Shimadzu, Kyoto, Japan) using Acquity UPLC BEH C18 (1.7 μm 3.0×150 mm; Waters, Milford, MA) as an analytical column. The composition of mobile phases and gradient elution programs used for amino acid quantification as well as GSH, GSSG, lactate, and pyruvate determination was described in detail in previous work. ²⁶

The concentration of studied metabolites measured in muscle samples was normalized to milligrams of the total protein. The specific ratio of l‐citrulline to l‐arginine was calculated to describe NOS activity. ⁹ Since NOS utilizes l‐arginine (as a substrate) for NO biosynthesis and l‐citrulline is generated as a by‐product, ²⁷ the changes in the l‐citrulline and l‐arginine balance, expressed by their ratio, reflect NOS activity. ²⁸

In addition, we used pyruvate and lactate concentration in the soleus muscles (experimental protocols A and B) to calculate intramuscular pH on the basis of the equation presented by Sahlin et al,²⁹ that is, pH=−0.00532×[pyruvate+lactate] + 7.06.

All standards and chemicals were purchased from Sigma‐Aldrich or Witko (Lodz, Poland), with the exception of nicotinamide‐d₄ (D‐3457), which was purchased from CDN Isotopes (Pointe‐Claire, Quebec, Canada).

Protein Extraction and Western Immunoblotting Analysis

Protein analysis in the muscle samples was performed as described previously. ⁹ , ³⁰ In short, striated muscle–derived protein extracts were separated using 4% to 20% gradient gels (Mini‐PROTEAN TGX gels; BioRad Laboratories, Hercules, CA). Equal amounts of total protein were loaded on gels. To eliminate differences between the gels resulting from the unequal transfer, the internal standard (ie, rat muscle sample) was applied on each gel. The proteins were then transferred onto the nitrocellulose membrane (Amersham Hybond, GE Healthcare, Pittsburgh, PA) at a constant voltage (35 V) in transfer buffer at 4 °C. Following the transfer, the detection of protein bands on the western blotting membranes was performed using Ponceau S staining (0.1% w/v in 5% acetic acid; Merck KGaA, Darmstadt, Germany) to ensure equal loading and transfer of proteins. After Ponceau S staining, membranes were incubated with the primary antibodies (Abcam, Cambridge, UK), specific to subunits of the electron transport chain (ETC) protein subunits (ab110413), myoglobin (ab77232), and xanthine oxidase (XO, ab109235). After the primary antibody incubation, membranes were washed and incubated in the secondary antibodies conjugated with horseradish peroxidase. Protein bands were visualized by an enhanced chemiluminescence method, and data were imaged using GeneGnome 5 Syngene (GenSys 1.2.7.0, Syngene Bio Imaging, Cambridge, UK). Gene Tools Syngene analysis software was used for densitometric analysis. The optical density values obtained for proteins detected in the heart and in the skeletal muscle samples were normalized to the internal standard and then to the protein content using Ponceau S staining. ⁹ , ³⁰

The protein data content was presented using an arbitrary unit. We used the sum of the subunits of ETC complexes (ETC proteins) (ie, the sum of the subunits of complexes II, III, IV, and V) as a marker of mitochondrial content in the analyzed muscles (heart ventricles, soleus, MGF). ⁹

Statistical Analysis

The results obtained in this study are presented as means, SDs, and 95% CIs.

The impact of 8cT (experiment A, Figure 1) on the analyzed variables in the heart, soleus, and MGF (Figures 2 and 3) was evaluated using the Wilcoxon–Mann–Whitney test. The obtained P values were corrected for simultaneous testing using the Holm–Bonferroni method. In addition, taking into account our previous data showing no impact of the endurance training on the systemic NO bioavailability in humans (eg, see Majerczak et al ³¹ ), we evaluated NO₂ ⁻ and NO₃ ⁻ concentrations as well as NOS activity in the plasma of rats (Sed_A versus 8cT group) using the Wilcoxon–Mann–Whitney test (Figure S1).

Figure 2. The impact of 8 weeks of continuous forced endurance training (8cT) on nitric oxide (NO) bioavailability in the heart and in the locomotory muscles of rats.

Nitrite (NO₂ ⁻) concentration (n=12‐6) (A), nitrate (NO₃ ⁻) concentration (n=12‐12) (B), and NO synthase activity (reflected by l‐citrulline‐to‐l‐arginine ratio) (n=12–12) in the heart of the sedentary rats (Sed_A) and trained rats (8cT). NO₂ ⁻ concentration (n=12‐12‐12‐12) (D), NO₃ ⁻ concentration (n=12‐10‐11‐10), and NO synthase activity (n=12‐12‐12‐12) in the locomotory muscles of the Sed_A and 8cT groups. Boxes and whiskers represent, the 95% CIs for means and SDs, respectively. The Wilcoxon–Mann–Whitney test P values were corrected for simultaneous testing using the Holm–Bonferroni method. a.u. indicates arbitrary units; Heart, heart ventricles; MGF, fast part of medial gastrocnemius muscle; ns, nonsignificant; and Sol, soleus muscle.

Figure 3. The impact of 8 weeks of continuous forced endurance training on the content of nitrite reductases in the heart and in the locomotory muscles of rats.

The content of electron transport chain (ETC) proteins (n=12‐12) (A) and myoglobin (n=12‐12) (B) in the heart of sedentary rats (Sed_A) and trained rats (8cT). The ETC proteins (n=12‐12‐11‐12) (C), myoglobin (n=12‐12‐11‐12) (D), and xanthine oxidase (XO) (n=12‐12‐12‐12) (E) in the locomotory muscles of the Sed_A and 8cT groups. Boxes and whiskers represent, the 95% CIs for means and SDs, respectively. The Wilcoxon–Mann–Whitney test P values were corrected for simultaneous testing using the Holm–Bonferroni method. a.u. indicates arbitrary units; Heart, heart ventricles; MGF, fast part of medial gastrocnemius muscle; ns, nonsignificant; and Sol, soleus muscle.

Furthermore, to analyze the impact of training duration (1, 2, 4, and 8 weeks of training) on the muscle metabolites and protein content in the soleus (experimental protocol B, Figure 1), 1‐way ANOVA was used with 5 groups (Sed_B, 1‐Tre, 2‐Tre, 4‐Tre, and 8‐Tre), and then the significance of the differences was checked with respect to the Sed_B group using Dunnett's post hoc test (Figures 4 and 5). To estimate the sensitivity of the experiment, we performed power calculations. For the Wilcoxon–Mann–Whitney test between 2 independent groups, each consisting of 12 subjects, as in experiment A, and for the standard effect sizes of f=0.10, 0.25, and 0.40 (or, equivalently, Cohen's d=0.2, 0.5, and 0.8), conventionally attributed to small, median, and large effects, the powers at the significance level 0.05 equal 0.07, 0.21, and 0.45. Similarly, for the balanced, 1‐way ANOVA with 5 groups and 70 observations, as in experiment B, the powers of the corresponding F‐tests equal 0.08, 0.32, and 0.74.

Figure 4. Training workload, the markers of training intensity and total antioxidant capacity in the slow‐twitch oxidative soleus muscle after 1, 2, 4 and 8 weeks of training.

Total distance (A) and total time spent on running (B) after 1, 2, 4 and 8 weeks of training. Intramuscular lactate concentration (n=14‐12‐13‐13‐14) (C), GAPDH content (n=13‐13‐14‐14‐13) (D), and total antioxidant capacity (reflected by reduced glutathione‐to‐oxidized glutathione ratio) (n=13‐11‐14‐14‐13) (E) after 1, 2, 4 and 8 weeks of training in the soleus muscle of the sedentary rats (Sed_B) and trained rats (1‐Tre, 2‐Tre, 4‐Tre, and 8‐Tre). Boxes and whiskers represent, the 95% CIs for means and SDs, respectively. Two‐sided P value represents the statistical significance of the training period (1, 2, 4 or 8 weeks) with respect to the sedentary control group (Sed_B) (Dunnett's post hoc test). *P<0.05, **P<0.01, ***P<0.001. ^#Denotes value that tended to be higher (P=0.08). 1‐Tre indicates 1 week of training; 2‐Tre, 2 weeks of training; 4‐Tre, 4 weeks of training; and 8‐Tre, 8 weeks of training.

Figure 5. Nitric oxide bioavailability and nitrite reductases in the slow‐twitch oxidative soleus after 1, 2, 4, and rats 8 weeks of training.

Concentrations of nitrite (NO₂ ⁻; n=14‐14‐14‐14‐14; A) and nitrate (NO₃ ⁻; n=14‐14‐14‐14‐14; B) and nitric oxide synthase (NOS) activity (n=14‐11‐14‐14‐14; C), as well as the content of the sum of electron transport chain (ETC) proteins (n=13‐14‐13‐14‐14; D), myoglobin (n=14‐14‐14‐14‐12; E), and xanthine oxidase (XO; n=14‐14‐13‐14‐14; F) after 1, 2, 4, and 8 weeks of training in the soleus muscle of sedentary rats (Sed_B) and trained rats (1‐Tre, 2‐Tre, 4‐Tre and 8‐Tre). Boxes and whiskers represent the 95% CIs for means and SDs, respectively. Two‐sided P value represents the statistical significance of the training period (1, 2, 4, or 8 weeks) with respect to the control group (Dunnett's post hoc test). *P<0.05, **P<0.01, ***P<0.001. 1‐Tre indicates 1 week of training; 2‐Tre, 2 weeks of training; 4‐Tre, 4 weeks of training; and 8‐Tre, 8 weeks of training.

The data points that deviated from the group means by more than 3 SDs were treated as outliers and excluded from further analysis. In case of heart homogenates (heart ventricles–8cT group) the concentration of NO₂ ⁻ after training in 6 muscle samples was below the limit of quantification (<0.02 μmol/L); therefore, the Wilcoxon–Mann–Whitney test was performed using sample size of n=12 for the heart ventricles–Sed_A group and n=6 for the heart ventricles–8cT group.

Spearman's rank correlation analysis was performed to analyze the relation between variables (Figure 6; Tables 1 and 2).

Figure 6. Spearman correlations of nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) contents in the soleus muscle with the training load expressed as total distance (A and C) and time of training (B and D) (n=56).

The data for the sedentary, untrained group (Sed_B) is also presented as dark circles in case of NO₂ ⁻ and dark diamonds in case of NO₃ ⁻, but they were not used in the correlation analysis.

Table 1.

Spearman Correlations of NO₂ ⁻ and NO₃ ⁻ Content in the Soleus Muscle With Total Muscle Antioxidant Capacity, Anaerobic Glycolysis Markers, and Muscle Nitrite Reductase Content (n=70)

	NO2 −, pmol·mg of protein−1	NO3 −, pmol·mg of protein−1
Total antioxidant capacity, a.u.	0.56‡	−0.11
Lactate, nmol. mg of protein−1	−0.10	0.28§
GAPDH, a.u.	−0.32*	0.05
ETC proteins, a.u	−0.32*	−0.01
Myoglobin, a.u.	−0.06	−0.05
XO, a.u.	−0.02	0.20

Total antioxidant capacity of muscle reflected by a reduced glutathione‐to‐oxidized glutathione ratio. a.u. indicates arbitrary units; NO₂ ⁻, nitrite; NO₃ ⁻, nitrate; ETC, electron transport chain; and XO, xanthine oxidase content in the soleus muscle.

*P<0.05, ^† P<0.01, ^‡ P<0.001.

^§Represents a tendency (P>0.05 and <0.09).

Table 2.

Spearman Correlations of Basal NO₂ ⁻ and NO₃ ⁻ Content in the Soleus Muscle With Intramuscular pH, Markers of Anaerobic Glycolysis, and Total Antioxidant Capacity in the Sedentary Groups (Sed_A and Sed_B) of Rats (n=26)

	NO2 −, pmol mg of protein−1	[NO3 −], pmol mg of protein−1
Intramuscular pH	0.60†	0.43*
Lactate, nmol. mg of protein−1	−0.55†	−0.46*
Lactate‐to‐pyruvate ratio, a.u.	−0.54†	−0.46*
Total antioxidant capacity, a.u.	0.53†	0.38§

Total muscle antioxidant capacity of muscle reflected by a reduced glutathione‐to‐oxidized glutathione ratio. a.u. indicates arbitrary units; NO₂ ⁻, nitrite; NO₃ ⁻, nitrate concentration in the soleus muscle; Sed_A, sedentary untrained rats; and Sed_B, untrained sedentary control group.

*P<0.05, ^† P<0.01, ^‡ P<0.001. ^§Represents a clear tendency (P>0.05 and <0.06).

Considering reports on the impact of oxygen tension and pH on the tissue NO bioavailability (eg, see Li et al ³² ), we checked the impact of the muscle extraction procedure (after decapitation versus deep anesthesia) in experiments A and B (see Muscle Tissue Extraction section in Methods) on the muscle metabolite concentrations (NO₂ ⁻, NO₃ ⁻, lactate) and intramuscular pH in the soleus muscle of sedentary rats (Sed_A and Sed_B) using the Wilcoxon–Mann–Whitney test (Figure 7).

Figure 7. Nitric oxide bioavailability, intramuscular lactate concentration, and intramuscular pH in the slow‐twitch oxidative soleus muscle of the sedentary groups of rats involved in experiment A (Sed_A) and in experiment B (Sed_B).

Concentrations of nitrite (NO₂ ⁻) (A), nitrate (NO₃ ⁻) (B), and lactate (C) and intramuscular pH (D) in the soleus muscle of the Sed_A group (n=12) and Sed_B group (n=14). The results of the Wilcoxon–Mann–Whitney test are presented.

Additionally, since in our previous study ⁹ we had not yet analyzed the muscle distribution of XO, in the present study we show the impact of muscle type (soleus, MGS, TA, and MGF) on the XO content using 1‐way ANOVA with post hoc Tukey test (Figure S2C). Statistical analyses with ANOVA were performed after checking normality of distribution and homogeneity of variance. Some variables in the original data were transformed to the logarithmic scale (NO₃ ⁻ concentration and myoglobin content) to be able to perform valid analysis of variance.

Statistical significance was set at P=0.05, and 2‐tailed P values were calculated. Statistical analyses were performed using STATISTICA 13.1 (TIBCO Software Inc., Palo Alto, CA) and Origin version 9.9 (OriginLab Corporation, Northampton, MA). All statistical power calculations were computed using G*Power v.3.1.9.7.

Results

Total Time of Training and Distance Covered by Rats in Experiments A and B

Total distance covered by rats after 8cT (experiment A) amounted to 36.8±12.8 km, and total time spent by rats running on a treadmill amounted to 26.9±9.0 hours (Figure 1).

In experiment B, total distance covered by rats from groups 1‐Tre, 2‐Tre, 4‐Tre, and 8‐Tre amounted to 1.4±0.2, 6.5±1.6, 19.5±6.8, and 46.5±16.0 km, respectively. Accordingly, total time of running on a treadmill by rats amounted to 1.6±0.1, 5.0±0.4, 14.4±2.8, and 35.7±7.3 hours, respectively for rats from the groups 1‐Tre, 2 Tre, 4‐Tre, and 8‐Tre (Figure 1). Total distance covered by rats and total time spent on a treadmill after these 8 weeks in experiment A (8cT group) was lower than in experiment B (8‐Tre group; Figure 1) due to a lower exercise capacity of rats involved in experiment A (Figure 1).

Systemic (Plasma) NO₂ ⁻ and NO₃ ⁻ Concentrations After 8cT in Rats

Eight weeks of continuous forced endurance training had no impact (P>0.05) on the basal plasma NO₂ ⁻, NO₃ ⁻, and NOS activity (reflected by l‐citrulline‐to‐l‐arginine ratio) (Figure S1).

NO₂ ⁻ and NO₃ ⁻ Concentrations in the Heart and Locomotory Muscles After 8cT in Rats

In our previous paper, we showed that the lowest NO₂ ⁻ content is found in the heart compared with locomotory muscles, whereas in the group of locomotory muscles NO₂ ⁻ content in the slow‐twitch oxidative soleus is lower than in the fast muscles, including fast‐twitch glycolytic MGF. Contrary to that, intramuscular NO₃ ⁻ has been found to be muscle type independent. ⁹ In the present study, we show that the 8cT program resulted in a significant reduction of the basal NO₂ ⁻ concentration (by ≈75%; P=0.0009; Figure 2A) in the heart, accompanied by no significant change in NO₃ ⁻ content (Figure 2B) and NOS activity (Figure 2C). In the locomotory muscles, 8cT significantly decreased NO₂ ⁻ in the slow‐twitch soleus (by ≈54%; P=0.0005), as well as in the fast‐twitch MGF (by ≈24%; P=0.01) (Figure 2D). The decrease in NO₂ ⁻ in the locomotory muscles (soleus and MGF) after the 8‐week training program was accompanied by no changes in NO₃ ⁻ concentration (Figure 2E) and NOS activity (reflected by l‐citrulline‐to‐l‐arginine ratio; Figure 2F).

Content of Nitrite Reductases in the Heart and Locomotory Muscles After 8cT in Rats

Previously, we have shown, as others before, that the heart possesses a higher content of ETC proteins and myoglobin compared with locomotory muscles. ⁹ In the present study, we found that the applied 8cT program had no impact on the sum of the ETC proteins as well as myoglobin content in the heart (Figure 3A and 3B). In the locomotory muscle group, a training‐induced increase in the sum of ETC proteins was found in the slow‐twitch soleus (P=0.02), whereas in the fast MGF, no impact of 8cT on the sum of ETC proteins was noticed (Figure 3C). No impact of the 8cT on the myoglobin and XO content in the locomotory muscles was observed (Figure 3D and 3E).

Additionally, since in our previous paper ⁹ we did not analyze the expression of XO in the muscles with varied muscle fiber type composition (soleus, MGS, TA, and MGF) we have included such an analysis in Figure S2C. We found that in the locomotory muscles expression of XO was muscle type dependent, meaning that XO expression in the slow‐twitch soleus was significantly greater (P<0.05) compared with fast‐twitch MGS, TA, and MGF. In addition, higher XO expression was found in the MGS compared with MGF as well as between TA and MGF (Figure S2C). We found no expression of XO in the heart using our antibody (Figure S2A and S2B).

Training Workload and the Markers of Training Intensity in the 1‐, 2‐, 4‐, and 8‐Week Training Program

In the present study, we also evaluated the impact of training duration (1, 2, 4, and 8 weeks of training; experiment B, Figure 1) on the muscle markers of anaerobic glycolysis, muscle antioxidant capacity and NO bioavailability in the locomotory slow‐twitch soleus muscle of rat. The soleus muscle is one of the most homogeneous striated muscles composed mainly of oxidative, mitochondria‐rich muscle fibers (ie, ≈91% of type I and type IIA muscle fibers ²⁵ ) that are active during each movement (ie, in our experiment in each exercise bout).

Total distance and time spent running after 1, 2, 4, and 8 weeks of training (experiment B) are presented in Figure 1. The training model in the subsequent weeks (1, 2, 4, and 8 weeks of training) was based on the same methodology as in experiment A (8cT program; see Kryściak et al ²⁴ ). Distance covered by rats from groups 2‐Tre, 4‐Tre, and 8‐Tre in experiment B was ≈5, ≈14, and ≈34 times greater, respectively, than distance covered by rats from 1‐Tre group (Figure 4A). Total time spent on a treadmill by rats from groups 2‐Tre, 4‐Tre, and 8‐Tre was ≈3, ≈9, and ≈22 times longer, respectively, than the total training time of rats from 1‐Tre group (Figure 4B).

Impact of Training Duration (1, 2, 4, and 8 Weeks of Training) on Anaerobic Glycolysis Markers and Total Antioxidant Capacity in the Slow‐Twitch Oxidative Soleus Muscle

The applied training program with the increased total distance and time spent by rats on a treadmill after 1, 2, 4, and 8 weeks of training (Figure 4A and 4B) was accompanied by changes in the markers of anaerobic glycolysis. Specifically, muscle lactate concentration was found to be higher after 4 and 8 weeks of training (Figure 4C). The increase in the intramuscular lactate was associated with a significant increase in the content of the glycolysis enzyme GAPDH found after 4 and 8 weeks of training (Figure 4D). Using the equation for intramuscular pH calculation, based on the measurements of intramuscular lactate and pyruvate concentrations, as proposed by Sahlin et al, ²⁹ we have observed a significant decrease (P<0.01) in the muscle pH at the fourth and eighth weeks of training. However, the absolute differences in the calculated muscle pH in the group of 70 rats were noticed at the third place after the comma (eg, 7.0587±0.0007 versus 7.0579±0.0007) after the fourth week of training for the Sed_B and 4‐Tre groups, respectively, and 7.0578±0.0007 versus 7.0578±0.0008 after the eighth week of training for the Sed_B and 8‐Tre groups, respectively, which we considered physiologically irrelevant. Statistical significance of such small effects might result from extremely small variability of the computed pH values. This, in turn, might be caused by the fact that the computed pH values were close to (or sometimes even beyond) the limit of the range in which Sahlin's formula may be reliably applied, which might make the functional relationship too flat and thus reduce the variability of the calculated pH.

We found additionally that the total muscle antioxidant capacity (reflected by the GSH‐to‐GSSG ratio) decreased gradually from the first week of training and remained attenuated until the eighth week of training (Figure 4E).

Impact of Training Duration (1, 2, 4, and 8 Weeks of Training) on NO Bioavailability and Content of Nitrite Reductases in the Slow‐Twitch Oxidative Soleus Muscle

The applied training program (1, 2, 4, and 8 weeks of training) gradually decreased the NO₂ ⁻ content in the soleus muscle. Specifically, NO₂ ⁻ in the soleus was significantly lower by ≈24% (P=0.003), 42% (P<10⁻⁴), 54% (P<10⁻⁴), and 59% (P<10⁻⁴) after 1, 2, 4, and 8 weeks of training, respectively, compared with the sedentary control group (Figure 5A). Interestingly, the level of muscle NO₃ ⁻ as well NOS activity remained unchanged during the applied training program (Figure 5B and 5C).

A decrease in NO₂ ⁻ content in the soleus from the beginning of the training (after the first week of training; Figure 5A) was accompanied by no changes in the nitrite reductases (ie, the sum of ETC proteins, myoglobin, and XO content) until the fourth week of training. At the eighth week of training, an increase in the sum of ETC proteins was found, whereas no significant change in the muscle content of myoglobin and XO were noticed (Figures 5D through 5F).

Correlations Between Muscle NO Bioavailability and Training Workload, Markers of Anaerobic Glycolysis and Antioxidant Capacity, and Nitrite Reductases in the Slow‐Twitch Oxidative Soleus Muscle

Intramuscular NO₂ ⁻ concentration was negatively correlated with the total distance covered and total time spent on a treadmill by rats during the training period (Figure 6A and 6B). We also found a small statistically significant effect of training performance on the NO₃ ⁻ level (Figure 6C and 6D), despite no effect of training on the muscle NO₃ ⁻ content when compared with untrained sedentary status (Figure 5B).

Additionally, as presented in Table 1, intramuscular NO₂ ⁻ content was negatively correlated with muscle GAPDH content. Furthermore, a significant positive correlation between muscle NO₂ ⁻ and total muscle antioxidant capacity (reflected by a GSH‐to‐GSSG ratio) was noticed (Table 1). Interestingly, no such correlations were found between muscle NO₃ ⁻ and the above‐mentioned variables (n=70), except a weak positive correlation between muscle NO₃ ⁻ and lactate concentration (Table 1, n=70).

Impact of Muscle Extraction on Basal NO₂ ⁻ and NO₃ ⁻ Concentrations in the Slow‐Twitch Oxidative Soleus Muscle

Since we have noticed that basal levels of NO₂ ⁻ and NO₃ ⁻ in the soleus muscle of the Sed_B group were significantly higher than that of the Sed_A group (Figure 7A and 7B), we assumed that the muscle extraction had the impact on the NO_x pool in the analyzed soleus muscle (see Methods). Specifically, in experiment A, muscle extraction was performed after the animal's decapitation (ie, in the condition of a lack of tissue perfusion) and in experiment B, muscle extraction was performed under deep anesthesia in the condition of normal circulation and normal oxygen supply to the muscles. Therefore, we hypothesized that the presence of the significant difference in the basal NO₂ ⁻ and NO₃ ⁻ concentrations in the soleus of the rats from Sed_A compared with the Sed_B group might result from varied oxygen pressure (not measured in the present study) and acid–base balance conditions (reflected by the calculated intramuscular pH) in 2 different procedures of tissue extraction (experiments A and B, see Methods). Accordingly, we found that a lower NO_x pool in the soleus muscle of the Sed_A group compared with the Sed_B group was accompanied by a significantly lower pH and higher intramuscular lactate concentration (Figure 7C and 7D).

Furthermore, we found that at basal conditions in the soleus muscle of the Sed_A and Sed_B, groups, both NO₂ ⁻ and NO₃ ⁻ have been strongly positively correlated (n=26) with the calculated intramuscular pH value (Table 2). Both NO₂ ⁻ and NO₃ ⁻ were negatively correlated with the anaerobic glycolysis markers such as intramuscular lactate concentration and lactate‐to‐pyruvate ratio (Table 2). We also found a significant positive correlation between muscle NO₂ ⁻ content and total muscle antioxidant capacity (Table 2).

Discussion

The main and original finding of this study is that in the heart and in the locomotory muscles of rats with varied muscle fiber type composition (slow‐twitch oxidative soleus and fast‐twitch glycolytic MGF), 8 weeks of forced endurance training results in a significant decrease in the NO₂ ⁻ content, contrary to the muscle NO₃ ⁻ content and NOS activity (reflected by a l‐citrulline‐to‐l‐arginine ratio), that are found to be training resilient (experiment A). This is an unexpected result, since based on the previous report ²⁰ we had hypothesized that endurance training would increase intramuscular NO₂ ⁻ and NO₃ ⁻ content in the NOS‐dependent pathway.

Impact of 8cT on Plasma NO Bioavailability

The data concerning the impact of physical training on the systemic NO bioavailability are conflicting. ³¹ , ³³ In the present study, we observed no impact of the 8cT on the plasma NO₂ ⁻ and NO₃ ⁻ concentrations as well as on the NOS activity (Figure S1). This is in accordance with the results presented earlier in humans (eg, see Majerczak et al ³¹ ).

Impact of 8cT on the Heart and Locomotory Muscles NO Bioavailability

NO₃ ⁻ and NO₂ ⁻, both present in the skeletal muscle at higher concentrations than in plasma, ³⁴ constitute an important reservoir of bioactive NO, ⁵ especially in a situation of a decrease in circulating NO bioavailability, such as during aging and in cardiovascular disease. ³⁵ As a result, dietary strategies that involve NO₃ ⁻ or NO₂ ⁻ supplementation, leading to an augmentation of the systemic, skeletal muscle34, 36 and heart NO bioavailability, ³⁷ have been found to exert positive effects on cardiovascular health ³⁷ , ³⁸ , ³⁹ and exercise tolerance. ¹³ , ⁴⁰

When considering the impact of NO₂ ⁻/NO₃ ⁻ supplementation on exercise tolerance, an acute administration of NO₃ ⁻ has been shown to improve physical exercise capacity by decreasing oxygen cost of submaximal exercise (by ≈5%) ⁴⁰ and augmenting muscle force generation (by ≈7%). ¹³ More importantly, sodium nitrite has been found to improve exercise tolerance in patients with heart failure. ⁴¹ It needs to be added that, so far, it seems that older subjects or patients with cardiovascular disease benefit more from NO₂ ⁻/NO₃ ⁻ supplementation ⁴¹ , ⁴² , ⁴³ than healthy subjects, especially athletes, in which less consistent effects of NO₃ ⁻ supplementation have been observed. ⁴⁴

Physical training, apart from dietary NO₂ ⁻/NO₃ ⁻ supplementation (that might evoke tolerance formation), seems to be a promising strategy in enhancing heart and skeletal muscle NO bioavailability. ²⁰ In the present study, contrary to our expectations, we found that 8cT with increasing workload, during which rats covered distance amounting to about 36.8±12.8 km and spent ≈27 hours during running on a treadmill (experimental protocol A; Figure 1), decreased NO₂ ⁻ content both in the heart ventricles by ≈75% (Figure 2A), in the slow‐oxidative soleus by ≈54% and in the fast‐glycolytic MGF by ≈27% (Figure 2D) compared with the sedentary group.

It should be noted that tissue NO₂ ⁻ (including vascular reserve of NO₂ ⁻), which is a naturally occurring anion derived from NO oxidation, represents the largest accessible direct source of NO ³² , ⁴⁵ , ⁴⁶ and independently of NO plays a role as a signaling molecule. ⁴⁶ , ⁴⁷ As presented in the study of Li et al, ³² NO generation from NO₂ ⁻ occurs primarily in tissues (liver and heart), not in blood, and its usage (termed also bioactivation of NO₂ ⁻) ⁴⁶ strictly depends on the oxygen pressure and hydrogen ion concentration.^32,48 In reference to the aforementioned unexpected result showing muscle NO₂ ⁻ attenuation after the 8cT, we also evaluated the impact of the applied training program on the content of the nitrite reductases such as a sum of mitochondrial ETC proteins, myoglobin and XO. However, the only nitrite reductase content that changed after the training was the ETC proteins, but its increase after 8 weeks of training was found only in the soleus muscle (Figure 3C), whereas a training‐induced decrease in NO₂ ⁻ content was also observed in MGF and in the heart (Figure 2A and 2D).

When considering the role of varied nitrite reductases in the muscle, one should take into account that complexes of the ETC, such as complex III, cytochrome c, and complex IV possess an ability to reduce NO₂ ⁻; however, they operate as nitrite reductase at extremely low concentration of oxygen (≈2%), as oxygen inhibits NO₂ ⁻ reduction in a competitive process. ⁴⁵ Therefore, it seems that the nitrite reductase activity of the mitochondrial ETC might be limited to an acute drop in oxygen tension such as during myocardial ischemia. ³ , ³² As suggested, XO is the main muscle nitrite reductase in the skeletal muscles in the physiological conditions, ³² , ⁴⁵ , ⁴⁹ and its ability to reduce NO₂ ⁻ increases in lower pH and Po ₂ ³² , conditions in the muscle and blood that are present during high‐intensity exercise. ¹⁰ , ¹² , ⁵⁰ , ⁵¹ In the present study, we showed no impact of 8cT on the XO content in the locomotory muscles (Figure 3E). Interestingly, we found no expression of XO in the heart despite using varied protein concentrations (Figure S2A and S2B), contrary to the locomotory muscles in which the XO expression was found to be muscle type dependent (Figure S2C). This is an unexpected result; however, the data concerning the XO activity/content in the hearts of mammals is conflicting. ⁵² , ⁵³ , ⁵⁴ In the rat heart, at basal conditions, XO activity has been shown to be low; that is, it accounts for about 6% of the sum of xanthine dehydrogenase and XO activity. However, in ischemic conditions XO activity increases up to 25% due to conversion of xanthine dehydrogenase into XO. ⁵² This low basal activity of XO in the rat heart might explain our result (Figure S2A and S2B). On the other hand, contrary to the heart, the present study shows that the XO expression in the locomotory muscle depends on the muscle type (Figure S2C). Nevertheless, as shown in our study, XO content was resilient to 8cT in the locomotory muscles, both in the slow‐twitch soleus and in the fast‐twitch MGF (Figure 3E).

Based on our results, it seems that the training‐induced attenuation of NO₂ ⁻ present in the heart and locomotory muscles, not accompanied by a change in NO₃ ⁻ and NOS activity, is not a result of the changes in the abundance of the muscle nitrite reductases. However, we cannot exclude that the decrease in NO₂ ⁻ content in the analyzed muscles (heart ventricles, soleus, MGF) after the training might be an effect of the changes in muscle metabolites released during exercise bouts in the applied training (such as H⁺ and others) that enhanced the NO₂ ⁻ reduction reaction (a simple chemical reaction) or influenced the activity of the nitrite reductase (or both) in the heart and in the locomotory muscles. Furthermore, a deeper training‐induced decrease of the NO₂ ⁻ in the soleus (by ≈54%) compared with the MGF (by ≈24%) might suggest that metabolic disturbances related to the performed training should have been more pronounced in the slow‐twitch oxidative soleus than in the fast‐twitch glycolytic MGF. This difference can be at least partly explained by the relatively lesser recruitment of the MGF muscle during the applied training than the soleus. ¹⁸ , ¹⁹ Namely, the running velocity applied during the training did not exceed 40 m·min⁻¹, which most likely did not exceed the critical speed of running. As shown by Copp et al, ⁵⁵ exceeding the critical speed by rats leads to a great recruitment of the fast‐twitch glycolytic muscles, as judged by a disproportional increase in blood flow when compared with exercise intensity below the critical speed. Interestingly, the critical running velocity of the Sprague–Dawley rats ⁵⁵ was 48.6 m·min⁻¹, which is far above the maximal running velocity applied in the training in our study (Methods section). Therefore, our study shows that the magnitude of the training‐induced decrease in the muscle NO₂ ⁻ concentration differs between varied muscles and seems to be dependent upon the level of their recruitments during the training.

Based on our results, one may conclude that in the locomotory muscles bioactivation of NO₂ ⁻ after the applied forced continuous endurance training program (experiment A) was more pronounced in the soleus muscle. Therefore, in the next part of this study (experiment B), we analyzed the impact of training duration with increasing workload on the basal NO bioavailability in the soleus muscle. The soleus muscle specimens were obtained in our previous experiment (see Kryściak et al ²⁴ ).

Impact of Training Duration With Increasing Workload on NO Bioavailability in the Slow‐Twitch Oxidative Soleus Muscle of the Rat

In this part of the study (experiment B, Figure 1), we analyzed the NO bioavailability and the content of nitrite reductases in the slow‐twitch oxidative soleus muscle after 1, 2, 4, and 8 weeks of training with an increasing workload. The total distance covered by rats when running on a treadmill increased significantly from the first to eighth week of training as the result of an increase in velocity of running and duration of exercise sessions (Methods, Figures 1 and 4A and 4B). Accordingly, we found that when exceeding 4 weeks of training, an increase in the muscle anaerobic glycolysis markers (such as lactate concentration and GAPDH content) were present (Figure 4C and 4D). Interestingly, the increase in the muscle GAPDH content, as found in this study, questions the applicability of this protein as a loading control in the study involving physical training. This is in accordance with data presented by Stuewe et al ⁵⁶ showing that endurance training increases GAPDH content and activity in the heart.

When considering the impact of the performed training on the markers of anaerobic glycolysis (measured at basal state ≈24–48 hours after the last exercise bout), one may take into account that a higher basal content of lactate after 4 and 8 weeks of training suggests a concomitant increased concentration of muscle hydrogen ions (or lower pH value). ¹² , ⁵⁰ , ⁵¹ Hence, we have calculated intramuscular pH and found a significant decrease in the intramuscular pH after 4 and 8 weeks of training; however, we found these changes in the intramuscular pH as physiologically irrelevant.

Furthermore, we found that an increase in training workload was accompanied by a significant decrease in the total antioxidant capacity as reflected by a significant gradual attenuation of the GSH‐to‐GSSG ratio in the muscle from the first to eighth week of training (Figure 4E). Similar time‐dependent changes, as in case of GSH‐to‐GSSG ratio, were observed in case of muscle NO₂ ⁻ content. Specifically, we found that the applied training program gradually decreased the NO₂ ⁻ content from the first to eighth week of training, whereas at the same time no significant changes in the muscle NO₃ ⁻ content and muscle NOS activity was present (Figure 5A through 5C). Therefore, in the independent experiment (experiment B), we showed that muscle NO₂ ⁻ content is metabolite sensitive to the training workload, whereas NO₃ ⁻ is training resilient. Interestingly, we found a similar level of decrease in the nitrite content in the soleus muscle after 8cT in experiment A (by ≈54%, 8cT group) and experiment B (by ≈59%, 8‐Tre group) (Figures 2D and 5A), regardless of a significant difference in the basal NO₂ ⁻ level (Figure 6A).

As demonstrated, NO generation from NO₂ ⁻ (bioactivation of NO₂ ⁻) in the tissues such as the liver and the heart depends on the intramuscular oxygen tension and pH. ³² In the present study, the NO₂ ⁻ decrease in the soleus muscle was found as early as after the first week of training (Figure 5A) and preceded the changes in muscle lactate and GAPDH content, which were present no earlier than after 4 weeks of training (Figure 4C and 4D). Interestingly, the attenuation of the muscle NO₂ ⁻ from the beginning of the training (Figure 5A) was accompanied by a significant decrease in the muscle antioxidant capacity present also from the first week of training (Figure 4E).

Nevertheless, the attenuation of muscle NO₂ ⁻ content after 1, 2, 4, and 8 weeks of training that was not accompanied by changes in muscle NO₃ ⁻ suggests that muscle NO₂ ⁻ was used in the NO₂ ⁻reduction reaction (Figure 5A and 5B). Since it has been shown that NO₂ ⁻ reduction might originate from the nonenzymatic NO₂ ⁻ reduction (a simple chemical process, that accounts for ≈15%–20% of total NO produced from NO₂ ⁻), whereas a majority (≈76%) of NO generation from NO₂ ⁻ results from an enzymatic process (mostly with involvement of molybdenum enzymes such as XO), ³² , ⁴⁵ we also assessed the content of muscle nitrite reductases (Figure 5D through 5F). We found no training‐induced changes in the content of skeletal muscle nitrite reductases including myoglobin and XO (Figure 5E and 5F), whereas an increase in the ETC proteins was present no sooner than after 8 weeks of training (Figure 5D), which is in accordance with the results of experiment A (Figure 3C through 3E).

Therefore, similarly to the data discussed above (experiment A) the training‐induced NO₂ ⁻ attenuation in the locomotory soleus muscle was unrelated to the changes in the content of muscle nitrite reductases, but we cannot exclude the possibility of the impact of the muscle metabolites (mainly H⁺) released during training with increased workload (Figure 4A and 4B) on the direct (simple chemical reaction) or indirect (through an increase in XO activity) muscle NO₂ ⁻ reduction. Accordingly, in the present study, we had an opportunity to assess the impact of muscle pH on the basal NO_x pool in the soleus muscle obtained in 2 different muscle extraction procedures. In short, in experiment A muscle extraction was performed after decapitation, that is, in the condition of the lack of tissue perfusion (a longer time after cessation of circulation to muscle freezing in LN₂), and in experimental protocol B, muscle extraction was performed under deep anesthesia, that is, in the condition of normal circulation and normal oxygen supply to the muscles (a shorter time between muscle excision to muscle freezing in LN₂). We found indeed lower NO₂ ⁻ and NO₃ ⁻ content accompanied by a higher lactate concentration and lower intramuscular pH (reflecting enhanced anaerobic glycolysis in the muscles) in the soleus obtained in the conditions of lack of tissue perfusion (Sed_A) compared with soleus obtained during normal tissue perfusion (Sed_B) (Figure 7A through 7D). This is in line with data showing that the NO generation from NO₂ ⁻ in the tissues (such as the liver and the heart) depends on the intramuscular oxygen tension and the pH. ³² In addition, we found that muscle NO₂ ⁻ content in the sedentary group of rats was strongly positively correlated with calculated intramuscular pH and inversely correlated with the intramuscular lactate concentration and lactate‐to‐pyruvate ratio (Table 2). This is in accordance with the results presented earlier in the heart. ³ , ³² , ⁴⁸ Based on these results, one may conclude that an acceleration of anaerobic glycolysis (higher lactate and lactate‐to‐pyruvate ratio), even at resting conditions, but related to the lack of tissue perfusion, accompanied by an increase in hydrogen ion concentration (a decrease in pH from 7.059 to 6.850), has a strong negative impact on the muscle NO₂ ⁻ but also on the NO₃ ⁻ content in the locomotory muscle (Table 2). The background of muscle NO₂ ⁻ attenuation at a lower pH likely originated from a nonenzymatic or enzymatic (mainly XO‐dependent) process, or both. Based on the data showing a strong similarity between NO₂ ⁻ and bicarbonate, ⁵⁷ this could suggest that NO₂ ⁻ in the locomotory muscles simply functions as a proton acceptor and is not only the source of NO but participates in the muscle intracellular pH homeostasis.

By reviewing our results, we noticed that a training‐induced decrease in the muscle NO₂ ⁻ content (Figure 5A) might result from an increase in the oxidative stress, reflected by a decrease in total antioxidant capacity (Figure 4E) as well as possibly being an effect of the NO₂ ⁻ reduction process (NO₂ ⁻ as a proton acceptor) due to the enhancement of ATP resynthesis in the anaerobic glycolysis, especially at the highest training workloads (Figure 4C). Accordingly, we found a significant positive correlation between intramuscular NO₂ ⁻ and total muscle antioxidant capacity (Table 1) as well as between NO₂ ⁻ and intramuscular pH (Table 2).

In addition, the study showing the similarity between NO₂ ⁻ and bicarbonate ⁵⁷ might suggest that the NO₂ ⁻/ NO₃ ⁻ supplementation might simply enhance muscle buffer capacity and in this way may exert similar physiological effects to sodium bicarbonate supplementation. ⁵⁸ However, this issue needs further study.

Muscle NO₂ ⁻ Content and Mitochondrial Biogenesis

In experiment B, we found that muscle NO₂ ⁻ content (Figure 5A) and total antioxidant capacity (Figure 4E) decreased as early as after the first week of training and preceded the changes in the mitochondrial biogenesis markers (ETC proteins) that were observed in the soleus no earlier than after 8 weeks of training (Figure 5D). This is in accordance with the data obtained from the soleus in experiment A (Figure 3C). The specific pattern of changes in NO₂ ⁻ and muscle antioxidant capacity (reflected by GSH‐to‐GSSH ratio) dependent on the training workload might suggest that intramuscular NO₂ ⁻ and cellular redox status play an important role in the muscle adaptation to training, including one of the most important: mitochondrial biogenesis. Furthermore, the training‐induced decrease in muscle NO₂ ⁻ level that was negatively correlated with the ETC proteins (Table 1) may suggest that training‐induced bioactivation of NO₂ ⁻ (a decrease in NO₂ ⁻ not accompanied by a change in NO₃ ⁻) might be involved in the mitochondrial biogenesis through NO₂ ⁻ alone ⁴⁷ or through nitrite‐derived NO‐dependent signaling, as suggested previously. ⁵⁹ , ⁶⁰ The role of NO₂ ⁻ bioactivation for mitochondrial biogenesis after training in various muscle types needs further study.

Limitations

A limitation of this study is that in this experiment, we estimated (but did not measure) the pH of muscle and heart tissue. A future study should include direct measurement of muscle and cardiac tissue pH using an appropriate probe or nuclear magnetic resonance method. This would allow for a deeper interpretation of the role of muscle acid–base status in the mechanisms underlying tissue NO bioavailability at rest and during exercise.

Conclusions

Muscle NO₂ ⁻ level, but not NO₃ ⁻, decreases over the course of training both in the heart and in the locomotory muscles. In the slow‐twitch oxidative soleus, this effect is rapid and visible already after the first week of training. The underlying mechanisms of the training‐induced muscle NO₂ ⁻ decrease may involve an enhancement of muscle anaerobic glycolytic activity, resulting in (1) direct chemical reduction of NO₂ ⁻or (2) indirect reduction of NO₂ ⁻ (activation of muscle nitrite reductases). Muscle NO₂ ⁻ bioactivation (NO₂ ⁻ reduction to NO) may contribute to an increased NO‐dependent vasodilatory response after exercise training and a potentiation of force generation, leading to enhancement of training‐induced improvement of exercise tolerance. Furthermore, it seems that NO₂ ⁻ bioactivation might be involved in training‐induced mitochondrial biogenesis in the locomotory muscles.

Sources of Funding

This work was supported by the funds from National Science Centre 2017/27/B/NZ7/01976 awarded to Joanna Majerczak. The experiments were partially performed with the use of equipment cofinanced by the qLIFE Priority Research Area under the program Excellence Initiative–Research University at Jagiellonian University. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Disclosures

None.

Supporting information

Figures S1–S2

Reference 61