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. 2025 Feb 4;26:102255. doi: 10.1016/j.fochx.2025.102255

NO modulation of AMP-activated protein kinase: A key player in regulating beef tenderness during post-mortem maturation

Zhuo Wang a, Qiao Li b, Jibing Ma c, Aixia Li a, Guoyuan Ma a, Qunli Yu a,⁎, Ling Han a, Cheng Chen a
PMCID: PMC11889946  PMID: 40060960

Abstract

This study investigated the role of NO in regulating AMPK activity and beef tenderness during post-mortem maturation of bovine gluteus quadriceps. The study included treatments with L-NAME (NOS inhibitor), ODQ (sGC inhibitor), Compound C (AMPK inhibitor), and a saline control. Results showed a decline in NOS activity, NO, and cGMP levels over time (P < 0.05). The L-NAME group had lower NOS activity and NO levels compared to the control (P < 0.05). AMPK activity peaked at 6 h and then decreased, with the control group showing higher AMPK activity than the L-NAME and ODQ groups (P < 0.05). The shear force in the L-NAME group was higher than the control but lower than the Compound C group at 48 h (P < 0.05). Overall, NO was found to activate AMPK, leading to reduced shear force and improved beef tenderness, providing a basis for enhancing beef quality through endogenous factors.

Keywords: NO, AMPK, Beef quality, Microstructure, Postmortem maturation

Highlights

  • •

    NO activates AMPK, reducing shear force and improving beef tenderness postmortem.

  • •

    L-NAME treatment lowered NO levels, reducing AMPK activity and impacting tenderness.

  • •

    AMPK peaks at 6 h; control group shows highest levels.

  • •

    Regulating NO could be a strategy to enhance beef tenderness and quality.

1. Introduction

Nitric oxide (NO) is produced in skeletal muscle and acts as a signaling molecule in various physiological processes. Post-slaughter, muscle cells can generate NO catalyzed by nitric oxide synthase (NOS) under conditions of ischemia and hypoxia. NO, as a gaseous signaling molecule, is prevalent in muscle tissues and regulates numerous physiological activities. The concentration and activity of NOS differ among species, and varying NO concentrations influence the biochemical transition from muscle to meat, impacting meat quality (Liu et al., 2018). Early studies have detected NOS in muscles at 4 h and 8 h post-slaughter, noting that muscle type and maturation time affect NOS activity, which decreases over time (Brannan & Decker, 2002). Zhang et al. (Zhang et al., 2013) found that NO levels influence lipid and protein oxidation, affecting chicken breast meat quality on days 1 and 4 post-slaughter. The current study indicates that NO is present in muscle cells during early postmortem maturation and influences meat quality. AMP-activated protein kinase (AMPK) is found in nearly all eukaryotes and functions as a cellular energy sensor and converter (Langendorf & Kemp, 2015). NO and NOS are associated with AMPK activation, with NO serving as an AMPK activator (Zhang et al., 2019). There is a positive feedback loop between NO and AMPK in muscle, as NO donors effectively activate AMPK, and increased AMPK activity can elevate NO levels (Bairwa et al., 2016). However, most research on NO's regulation of AMPK has focused on living cells in animals, leaving the relationship between NO and AMPK in postmortem muscle unclear.

Regulating NO levels in muscle cells using NOS inhibitors and NO donors is a common method to study NO's effects on meat quality. Although evidence suggests NO regulates post-slaughter meat quality, its precise mechanism remains elusive due to its variable impact on tenderness. Cook et al. (Cook et al., 1998) reported that NO donors reduce shear force in beef, enhancing tenderness three days post-slaughter, while Cottrell et al. (Cottrell et al., 2004) results showed no significant difference in tenderness or pH using different NO donors and inhibitors. Research by Hu et al. (Hu et al., 2016) demonstrated that dietary glutamine or glucose reduces AMPK activity in broilers post-slaughter, decreasing drip loss. Huang et al. (Huang et al., 2018) found that shackling and wing beating increased AMPKα (Thr172) phosphorylation, creatine kinase, and lactate dehydrogenase activities, accelerating glycolysis, lactic acid accumulation, pH reduction, and ultimately decreasing water retention in chicken muscles. Thus, AMPK significantly regulates glycolysis and meat quality post-slaughter.

This study investigates how NO mediates AMPK to influence beef tenderness during postmortem maturation. We measured NO content, cyclic guanosine 3′,5′-monophosphate (cGMP) levels, AMPK activity, and protein expression to explore NO's regulatory mechanism on AMPK post-slaughter. Additionally, we used AMPK inhibitors to examine the NO-AMPK pathway's impact on beef tenderness. By exploring endogenous regulatory factors and targeting them for intervention, this research aims to provide a theoretical foundation for improving beef tenderness.

2. Materials and methods

2.1. Experiment samples

Meat samples were collected from Gansu Kangmei Modern Agriculture and Animal Husbandry Industry Group Co., LTD. Six healthy Simmental crossbred bulls, each weighing approximately 600 ± 30 kg and aged between 3 and 4 years, were selected. The slaughtering process was conducted according to the “Operating Procedures of Cattle Slaughter” of the National Standards of PR China including animal welfare and conditions. Within 45 min after slaughter and bloodletting, the gluteal quadriceps muscles were excised, and surface fat and connective tissue were removed for further use.

2.2. Experiment design

Two experiments were designed in this study. The first experiment investigated the effect of NO on AMPK during beef maturation after slaughter, and the second experiment investigated the effect of NO-AMPK pathway on beef tenderness.

The first experiment was soluble guanylate cyclase (sGC), a key enzyme in the NOS pathway, and soluble guanylate cyclase. The meat samples were cut into about 60 g pieces and randomly divided into three groups. The meat sample was mixed with the treatment solution 1:1 (g: mL), and punctured with a 20 G needle for uniform penetration of the treatment solution. The three treatment groups were: 1) NOS inhibition group, 20 mM L-NAME; 2) sGC inhibition group, 100 uM 1H - (Brannan & Decker, 2002; Langendorf & Kemp, 2015; Liu et al., 2018) Oxadiazolo [4,3-a] quinoxaline – 1 - one (ODQ); 3) Control group, 0.86 % NaCl. L-NAME inhibited NOS activity and decreased the production of NO in muscle. ODQ, as an inhibitor of sGC, can effectively reduce the production of cGMP.

The second experiment inhibited NOS and AMPK, respectively. The meat samples were cut into about 60 g pieces and randomly divided into three groups. The meat sample was mixed with the treatment solution 1:1 (g: mL), and punctured with a 20 G needle for uniform penetration of the treatment solution. The three treatment groups were: 1) NOS inhibition group, 20 mM L-NAME; 2) AMPK inhibition group, 40 uM Compound C; 3) Control group, 0.86 % NaCl. Among them, L-NAME mainly acts on NOS and reduces the production of NO in the body. Compound C can effectively inhibit AMPK activity as an AMPK inhibitor.

The dosages of L-NAME, ODQ, and Compound C were selected based on preliminary experiments and a review of the relevant literature. The concentration of 20 mM L-NAME was chosen as it has been shown to significantly inhibit NOS activity in muscle tissues without inducing cytotoxicity. Similarly, 100 μM ODQ was selected based on its efficacy in inhibiting sGC and reducing cGMP levels in prior studies (Ma et al., 2022). For Compound C, a concentration of 40 μM was adopted, as it effectively blocks AMPK activity in muscle cells without compromising cell viability. These concentrations were further validated in preliminary tests to ensure they produced measurable effects under the experimental conditions.

All the samples were soaked at 4 °C for 12 h and then taken out, and the surface water was absorbed with filter paper, and then matured at 4 °C. The meat samples were taken out for determination at the post-mortem maturation 0, 6, 12, 24, 48, 72 and 120 h, respectively. For the indicators that are inconvenient to determine immediately, they are stored in the −80 °C ultra-low temperature refrigerator for use. Each indicator was measured at least 3 times.

2.3. Detection of NO regulation on AMPK

2.3.1. NOS enzyme activity and NO content

Take about 1.0 g of meat sample, put it into 50 mL centrifuge tube, add cooling homogenate medium (0.86 % normal saline) at 1:9 (g: mL), and homogenize 10,000 r/min ice bath homogenate for 30 s, homogenize for several times. Then centrifuge at 4 °C at 4000 r/min for 10–15 min, discard the precipitation and take the supernatant for determination. The determination of NOS activity and NO content in meat samples was consistent with the pre-treatment, and the specific operation steps and calculation formulas were carried out according to the instructions of Nanjing Jiancheng Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.3.2. AMPK activity

According to the method of Underwood et al (Underwood et al., 2008),. about 0.6 g of meat sample was taken into a 10 mL centrifuge tube, and 1:5 (g: mL) was added to the frozen homogenate and 3000 r/min ice bath homogenate. Then centrifuge at 4 °C, 12000 r/min for 5 min, and take the supernatant for determination.

2.4. Detection of key factors in the NO-AMPK signaling pathway

2.4.1. cGMP, PKG, PLC, IP3 content

Take about 1.0 g of meat sample and add homogenate PBS (0.01 M, pH 7.4) ice bath homogenate at 1:9 (g: mL). Then centrifuge at 4 °C, 5000 ×g for 20 min, collect the supernatant, and carry out the specific operation steps and calculation according to the instructions of Nanjing Jiancheng Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.4.2. Cytoplasmic Ca2+ assay

Refer to the method of Yablokov et al (Yablokov et al., 2013),. and modify it slightly. The meat samples were added with a 9-fold volume homogenate medium (0.1 mol/L Tris-HCl, 1 mmol/L KCl, 1 mmol/L EDTA-2Na, 0.25 mol/L sucrose, pH 7.4), centrifuged at 4 °C and 2000 r/min for 10 min, then the precipitation was discarded and the supernatant was taken. Continue centrifuging at 4 °C, 18000 r/min for 15 min, and the obtained supernatant is cytoplasm. Then, the plasma was digested by microwave and the concentration of Ca2+ was determined by flame atomic absorption method.

2.4.3. CaMKKβ activity

Take about 1.0 g of meat sample, put it into a 50 mL centrifuge tube, and add PBS ice bath homogenate at 1:9 (g: mL). Then centrifuge at 4 °C, 5000 ×g for 20 min and collect the supernatant. The operation procedure and calculation are carried out according to the manual of Nanjing Jiancheng Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.5. Determination of beef protein by differential scanning calorimetry (DSC)

According to the method of Yu et al (Yu et al., 2024),. with slight modifications. Weigh approximately 2 mg of meat sample and place it in a covered aluminum crucible. The equilibrium time is set for 2 min, and the temperature is raised from 20 °C to 100 °C at a rate of 5 °C/min. The denaturation enthalpy (ΔH) and peak temperature (Tg, /°C) are calculated from the thermograms using Pyris-12 software (Perkin-Elmer Instruments, USA).

2.6. Effects of the NO-AMPK signaling pathway on beef tenderness and microstructure

2.6.1. Shear force

According to the method of Weng et al (Weng et al., 2022) Place the meat samples in a water bath at 80 °C, and when the center temperature reaches 70 °C, remove the samples and cool them to a center temperature of 0 °C to 4 °C. Using a circular corer with a diameter of 1.27 cm, drill into the meat samples parallel to the muscle fibers, ensuring that the core length is not less than 2.5 cm. The sampling position should be at least 5 mm away from the edge of the sample, and the distance between the edges of two cores should also be at least 5 mm. Discard any cores with obvious defects, and ensure that the number of samples is no fewer than three. Measure the shear force immediately after sampling using the Warner-Bratzler Shear test.

2.6.2. MFI

The method of Shen et al. (Shen et al., 2006) was slightly modified. Ice bath homogenates with 10 times the volume of MFI buffer (100 mM KCl, 20 mM K3PO4, 1 mM EDTA, 1 mM MgCl2, 1 mM NaN3, pH 7.1) were added to the meat samples. Centrifuge 5000 g for 20 min and discard supernatant. The precipitate was homogenized with 10 times buffer and centrifuged again to remove the supernatant. After the homogenization of 10 times buffer for precipitation is obtained, the gauze is filtered, and the filtrate is the myofibrillar protein solution. The protein concentration was determined, diluted to 0.5 mg/mL, and then the absorbance value was determined at 540 nm. The result was multiplied by 200 to give the MFI value.

2.6.3. Scanning Electron microscopy

The method of Bresolin et al. (Bresolin et al., 2022) was slightly modified. Meat samples slightly larger than 5 mm × 5 mm × 5 mm were taken along the direction of the muscle fibers and fixed in glutaraldehyde solution for 3 days. The samples were dehydrated using an ethanol gradient, with each step lasting approximately 10 min. After vacuum drying, the samples were gold-sputtered, and the microstructure was observed using an accelerating voltage of 20 kV and a magnification of 300.

2.6.4. Hematoxylin and eosin (HE) staining

The HE staining procedure was conducted following the method described by Cardiff (Cardiff et al., 2014), the meat samples were fixed in 4 % paraformaldehyde for 24 h at room temperature to preserve tissue morphology. After fixation, the samples were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections of 5 μm thickness were cut using a microtome and mounted on glass slides. The slides were then deparaffinized, rehydrated, and stained with hematoxylin for 5 min, followed by differentiation in 1 % acid alcohol and bluing in running tap water. Subsequently, the slides were counterstained with eosin for 2 min, dehydrated, cleared, and coverslipped with a mounting medium. For image analysis, high-resolution photographs of the stained slides were taken using an optical microscope at 40× magnification. Myocytes were selected for analysis based on clear cellular boundaries and the absence of artifacts. A minimum of 30 myocytes per image were analyzed, with three images taken from each sample to ensure representativeness. The analyzed images were processed using ImageJ software, with parameters standardized across all samples to maintain uniformity.

2.7. Statistical analysis

The data were analyzed using SPSS Statistic 19.0 (SPSS Inc., Chicago, IL, USA) and expressed as mean ± standard errors (SE). Before conducting the analyses, the data were checked for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene's test. A one-way analysis of variance (ANOVA) was performed to evaluate the effects of treatment type, postmortem time, and their interactions. Duncan's new multiple range test was applied post hoc to identify significant differences among group means. The treatment type and postmortem time were treated as fixed effects, while individual animals were considered as random effects to account for biological variability. Differences between means were considered statistically significant at a 5 % significance level (P < 0.05). The results were interpreted with respect to the biological relevance of observed changes, and all statistical analyses were conducted following established guidelines to ensure reliability and reproducibility of findings.

3. Results

3.1. Results of NO regulation on AMPK

3.1.1. Changes in the NOS enzyme activity and NO content during post-mortem maturation

NO has become an important regulator of skeletal muscle homeostasis, and it interacts with many physiological pathways affecting meat quality. L-NAME competed with L-arginine for the NOS catalytic site, thereby inhibiting NOS activity and reducing NO production. As shown in Fig. 1 (A), NOS activity showed a decreasing trend during postmortem maturation. The NOS activity in 0 h L-NAME group and control group was 0.32 U/mg prot and 0.56 U/mg prot, respectively, and the NOS activity in 0–72 h L-NAME group was significantly lower than that in control group (P < 0.05). At 120 h of maturity, NOS activity was not significantly different between the two groups, but the L-NAME group was 45.37 % lower than the control group. It can be seen from the results that L-NAME can be used as an inhibitor of NOS activity. As shown in Fig. 1 (B), the change of NO content after slaughter also showed a decreasing trend. NO content in 0 h L-NAME group and control group was 8.00 umol/g prot and 8.48 umol/g prot, respectively, and NO content in 6 to 72 h L-NAME group was significantly lower than that in control group (P < 0.05). At 120 h, NO content was not significantly different between the two groups, but the L-NAME group was 8.21 % lower than the control group. In conclusion, the NOS inhibitor L-NAME decreased NOS activity and decreased NO content during beef maturation after slaughter.

Fig. 1.

Fig. 1

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Changes in NOS enzyme activity, NO content and AMPK activity during post-mortem maturation. (A) NOS enzyme activity. (B) NO content. (C) AMPK activity. The small letters (a-g), the capital letters (A-G), and the small letters (u-z) in the figure respectively indicate the significant difference between the control group, the L-NAME group, and the ODQ group (P < 0.05). The * indicates the significant difference between groups, *: (P < 0.05).

3.1.2. Changes in the AMPK activity during post-mortem maturation

AMPK can regulate the energy metabolism balance of cells and affect the glycolysis process of beef after slaughter. As shown in Fig. 1 (C), AMPK activity in all three groups increased first and then decreased, reaching the maximum at 6 h. At this time, AMPK activity in L-NAME group and ODQ group decreased by 17.51 % and 28.90 % compared with control group, respectively. From 0 to 24 h, the AMPK activity in L-NAME group was higher than that in ODQ group and lower than that in control group (P < 0.05). L-NAME and ODQ decreased the activity of AMPK, and the effect of ODQ was more obvious. With the decrease of NO and cGMP, the activity of AMPK also decreased. cGMP is the downstream kinase of NO, suggesting that NO can enhance AMPK activity through cGMP.

3.2. Results of the detection of key factors in the NO-AMPK signaling pathway

3.2.1. Changes in cGMP content and PKG content during post-mortem maturation

sGC is a direct physiological target of NO, which catalyzes the conversion of guanosine 5 ‘-triphosphate (GTP) to cGMP. The combination of NO with sGC greatly improved the synthesis of cGMP. In order to determine whether cGMP is involved in the molecular mechanism of NO downstream during the maturation of beef after slaughter, the cGMP content of meat samples in three groups was first determined. ODQ, as a cGMP inhibitor, has been widely used to study the biological function of cGMP, so in this study, ODQ was used as a cGMP inhibitor to regulate the content of cGMP. As shown in Fig. 2 (A), cGMP content showed a decreasing trend from 0 to 120 h. At 6 h, the cGMP content in L-NAME group, ODQ group and control group was 55.21 nmol/L, 47.19 nmol/L and 61.37 nmol/L, respectively, and the cGMP content in L-name group was significantly lower than that in control group at 6 to 48 h (P < 0.05). The ODQ group was significantly lower than the control group at 6 to 72 h (P < 0.05), and the ODQ group was significantly lower than the L-NAME group at 6 to 24 h (P < 0.05). At 120 h postmortem maturation, cGMP content in L-NAME group and ODQ group was 9.32 % and 19.20 % lower than that in control group, respectively, but the difference was not significant (P > 0.05). These results indicate that NO promotes cGMP production.

Fig. 2.

Fig. 2

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Effects on the activity of key enzymes in the NO-AMPK signaling pathway. (A) cGMP content. (B) PKG content. (C) PLC content. (D) IP3 content. (E) CaMKKβ activity. (F) Ca2+ content. The small letters (a-g), the capital letters (A-G), and the small letters (u-z) in the figure respectively indicate the significant difference between the control group, the L-NAME group, and the ODQ group (P < 0.05). The * indicates the significant difference between groups, *: (P < 0.05).

NO is an important signaling molecule in many systems such as metabolism. NO produces cGMP, and the biological effect of cGMP is mainly mediated by PKG. As shown in Fig. 2 (B), PKG content in postmortem L-NAME group, ODQ group and control group showed a trend of first increasing and then decreasing and reached the maximum value at 6 h (5.26μg /mL, 4.72μg /mL, 5.75μg /mL, respectively). PKG content in L-NAME group was significantly lower than that in control group at 6 to 48 h (P < 0.05), PKG content in ODQ group was significantly lower than that in control group at 6 to 72 h (P < 0.05), and PKG content in L-NAME group and ODQ group was 3.16 % and 11.01 % lower than that in control group at 120 h, respectively. PKG content in ODQ group was significantly lower than that in L-NAME group at 6 to 12 h (P < 0.05). These results indicate that NO and cGMP promote the production of PKG during the maturation of beef after slaughter.

3.2.2. Changes in PLC content and IP3 content during post-mortem maturation

Phospholipase C (PLC) is also a key signaling enzyme in cells, interacting with substances in other signaling pathways (Yadav et al., 2018). Studies have shown that cGMP activates PLCS through PKG. As shown in Fig. 2 (C), the overall PLC content showed a downward trend after slaughter. At 0 h, the PLC content in L-NAME group and ODQ group was 5.26 % and 9.73 % lower than that in control group, respectively. From 0 to 120 h, the PLC content in L-NAME group was lower than that in control group, but the difference was significant only at 6 and 24 h (P < 0.05). The ODQ group was always lower than the other two groups, and the difference between the control group and the ODQ group was significant at 6 and 24 h (P < 0.05), and only the difference between the ODQ group and the L-NAME group was significant at 6 h (P < 0.05). When the maturity time reached 120 h, the L-NAME group and ODQ group were 6.26 % and 11.73 % lower than the control group, respectively. It can be concluded that NO and cGMP can promote the production of PLC during the maturation of beef after slaughter, but the difference becomes insignificant with the extension of post-mortem maturation.

PLC is a key enzyme in the production of IP3, and the activation of PLC promotes the production of inositol triphosphate (IP3), which then participates in many metabolic processes. IP3 is produced and released into cells and binds to the inositol 1,4,5 triphosphate receptor (IP3R) to regulate Ca2+ storage and release in the endoplasmic reticulum and regulate calcium homeostasis in cells. As shown in Fig. 2 (D), with the extension of post-mortem maturation, the IP3 content showed a gradual decreasing trend. The content of IP3 in control group was 49.36μg /L at 6 h, which was reduced by 3.63 % and 6.25 % after L-NAME and ODQ treatment, respectively. The IP3 content in L-NAME group was significantly lower than that in control group at 12 to 24 h (P < 0.05), the IP3 content in ODQ group was significantly lower than that in control group at 6 to 24 h (P < 0.05), and the IP3 content in ODQ group was significantly lower than that in L-NAME group at 12 to 24 h (P < 0.05). It can be concluded that L-NAME treatment and ODQ treatment can reduce the content of IP3 during the maturation of beef after slaughter. Therefore, NO and cGMP promote the production of IP3 after slaughter.

3.2.3. Changes in CaMKKβ activity and Ca2+ content during post-mortem maturation

CaMKKβ acts as an upstream kinase of AMPK and activates AMPK by phosphorylating Thr172 of AMPKα. As shown in Fig. 2 (E), the CaMKKβ activity of beef in the three groups first increased and then decreased 0–120 h after slaughter. The activity of CaMKKβ in 12 h L-NAME group was significantly lower than that in control group and higher than that in ODQ group (P < 0.05), and the activity of CaMKKβ in 6 to 48 h ODQ group was significantly lower than that in control group (P < 0.05), indicating that compared with L-NAME treatment, ODQ treatment reduced camkβ activity to a greater extent. NO and cGMP increased the activity of CaMKKβ during postmortem maturation.

Ca2+ is also an important cellular messenger, as shown in Fig. 2 (F), with the extension of maturation time, Ca2+ decreases overall. Ca2+ concentration in L-NAME group was significantly lower than that in control group and higher than that in ODQ group at 12 h (P < 0.05). Ca2+ concentration in ODQ group was significantly lower than that in control group at 6 to 72 h (P < 0.05). These results indicated that NO and cGMP could increase the concentration of Ca2+ in cytoplasm.

3.3. Results of beef protein determination by DSC

In DSC thermograms, the peak area represents the enthalpy change, and the size of the peak area reflects the degree of protein denaturation. When the peak area disappears, it indicates that the protein has completely denatured. Fig. 3 shows that all three groups exhibit endothermic peaks in the DSC heat flow curves of beef muscle protein, with peak I being less pronounced and peak II being more prominent. The temperatures corresponding to peak II in the three groups are as follows: Compound C group > L-NAME group > control group. Both the thermal denaturation temperature and peak area are related to the degree of protein degradation. As shown in Table 1, with the extension of post-mortem aging time, the thermal denaturation temperature of the protein generally decreases. The L-NAME group has a lower denaturation temperature than the Compound C group and a higher temperature than the control group. These results indicate that there are differences in the denaturation temperatures of beef protein among the three groups. Compared to the Compound C and L-NAME groups, the control group shows a more severe degree of degradation in beef protein, further suggesting that NO promotes the denaturation of beef protein via AMPK.

Fig. 3.

Fig. 3

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Effects of the NO-AMPK signaling pathway on the differential scanning calorimetry (DSC) profiles of beef protein under different treatments. (A) L-NAME group; (B) Compound C group; (C) Control group.

Table 1.

Changes in denaturation temperatures (Tmax) and denaturation enthalpy (ΔH) of muscle proteins during postmortem aging.

Group Maturation 0 h 6 h 12 h 24 h 48 h 72 h 120 h
Peak I
L-NAME Tg /°C 67.64 ± 0.63ab 66.19 ± 0.75b 68.64 ± 0.55a 66.73 ± 0.64a 64.44 ± 0.59b 64.10 ± 0.33a 61.88 ± 0.86b
Compound C Tg /°C 67.99 ± 0.40a 68.2 ± 60.39a 68.03 ± 0.66a 66.32 ± 0.60a 65.76 ± 0.41a 65.26 ± 0.17a 64.07 ± 0.25a
Control Tg /°C 66.79 ± 0.54b 66.28 ± 0.26b 64.42 ± 0.37b 63.39 ± 0.30b 60.72 ± 0.51c 60.39 ± 1.00b 60.75 ± 0.46b
L-NAME ΔH/Jg-1 0.33 ± 0.01a 0.31 ± 0.01a 0.32 ± 0.00a 0.30 ± 0.01a 0.30 ± 0.01b 0.27 ± 0.00a 0.26 ± 0.00a
Compound C ΔH/Jg-1 0.34 ± 0.01a 0.32 ± 0.01a 0.32 ± 0.01a 0.32 ± 0.01a 0.31 ± 0.01a 0.28 ± 0.01a 0.27 ± 0.01a
Control ΔH/Jg-1 0.33 ± 0.02a 0.32 ± 0.02a 0.31 ± 0.01a 0.30 ± 0.01a 0.28 ± 0.00c 0.25 ± 0.01b 0.26 ± 0.02a



Peak II
L-NAME Tg /°C 82.94 ± 0.20a 82.20 ± 0.34a 81.38 ± 0.50b 80.87 ± 0.26a 79.62 ± 0.33b 78.65 ± 0.36b 77.71 ± 0.56b
Compound C Tg /°C 83.19 ± 0.59a 82.63 ± 0.37a 82.56 ± 0.41a 81.63 ± 0.41a 80.98 ± 0.46a 79.64 ± 0.24a 78.70 ± 0.29a
Control Tg /°C 81.60 ± 0.48b 80.44 ± 0.79b 78.55 ± 0.45c 77.73 ± 0.69b 75.62 ± 0.43c 75.26 ± 0.25c 74.82 ± 0.20c
L-NAME ΔH/Jg-1 0.85 ± 0.03a 0.83 ± 0.02a 0.74 ± 0.02ab 0.67 ± 0.02a 0.59 ± 0.02b 0.55 ± 0.02b 0.46 ± 0.02b
Compound C ΔH/Jg-1 0.88 ± 0.02a 0.84 ± 0.03a 0.76 ± 0.03a 0.70 ± 0.01a 0.64 ± 0.02a 0.60 ± 0.02a 0.50 ± 0.02a
Control ΔH/Jg-1 0.84 ± 0.02a 0.80 ± 0.04a 0.69 ± 0.05b 0.62 ± 0.04b 0.53 ± 0.03c 0.50 ± 0.02c 0.42 ± 0.02c
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Note. The lowercase letters in the upper right corner indicate the significance of differences between treatment groups over time. (P < 0.05).

3.4. Results of the detection on NO-AMPK signaling pathway on beef tenderness and microstructure

3.4.1. Changes in beef tenderness during post-mortem maturation

Tenderness is also an important index to judge the quality of meat, and the greater the shear force value of meat, the worse the tenderness. As shown in Fig. 4 (A), with the extension of post-mortem maturation, the shear force increases first and then decreases. At 24 to 48 h L-NAME group was significantly higher than control group (P < 0.05), at 12 to 120 h Compound C group was significantly higher than control group (P < 0.05), at 12, 48, 72 h Compound C groups was significantly higher than L-NAME group (P < 0.05). The above results showed that compared with L-NAME group and Compound C group, the shear force of beef in the control group was reduced, and it was further indicated that NO reduced the shear force of beef and improved the tenderness through AMPK.

Fig. 4.

Fig. 4

Fig. 4

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Effects of the NO-AMPK signaling pathway on beef tenderness and microstructure. Changes in beef quality during post-mortem maturation. (A) shear force. (B) MFI. The small letters (a-g), the capital letters (A-G), and the small letters (u-z) in the figure respectively indicate the significant difference between the control group, the L-NAME group, and Compound C group (P < 0.05). The * indicates the significant difference between groups, *: (P < 0.05). (C) Myofibril microstructure. (D) HE staining imaging results. A, B, C: respectively indicate L-NAME group, Compound C group, Control group; 0, 6, 12, 24, 48, 72, 120 respectively indicate maturation time 0, 6, 12, 24, 48, 72, 120 h.

With the extension of maturation time, the structure of cytoskeleton protein is destroyed, which leads to the destruction of myofibrillar structure and the formation of small slices, thus improving the tenderness of meat. As shown in Fig. 4 (B), MFI in the three groups showed an increasing trend from 0 to 120 h. The MFI of the L-NAME group was significantly lower than that of the control group at 12–120 h (P < 0.05), the Compound C group was significantly lower than that of the control group at 6–120 h (P < 0.05), and the Compound C group was significantly lower than that of the L-NAME group at 24–120 h (P < 0.05). At 120 h, MFI in L-NAME group, Compound C group and control group increased by 49.00, 40.67 and 57.60 compared with 0 h, respectively. In conclusion, NO can increase MFI through AMPK, accelerate the ripeness and improve the tenderness of beef after slaughter.

3.4.2. Changes in beef microstructure during post-mortem maturation

The Microstructure of Muscle Cells was shown in Fig. 4 (C), at 0 h post-slaughter, the muscle fibers in all three groups were arranged in an orderly and tight manner, with no significant gaps between them. As the post-slaughter maturation time increased, the gaps between the muscle fibers gradually widened, the tissue structure became increasingly disrupted, and the arrangement became progressively disordered. By 72 and 120 h of maturation, the gaps between the muscle fibers in all three groups had significantly increased, the degree of separation had also increased, and the arrangement had become increasingly chaotic. Comparatively, the degree of muscle fiber disruption was greatest in the control group, followed by the L-NAME group, and was least in the Compound C group. These results indicate that NO promotes the glycolysis process post-slaughter through AMPK, leading to the disruption of the muscle fiber structure.

3.4.3. Changes in beef HE staining during post-mortem maturation

The muscle fiber structure is closely related to its water-holding capacity and tenderness. With the extension of aging time, the structure of muscle fibers is damaged, leading to issues such as moisture loss and meat tenderness. As shown in Fig. 4 (D), the imaging results indicate that the cross-sectional area of muscle fibers gradually decreases, while the distance between muscle fibers increases. In the control group, the distribution of muscle fibers begins to disperse after 48 h. In contrast, the muscle fibers in the L-NAME and Compound C groups are more densely arranged, starting to disperse gradually after 72 h, with an increasing gap between fibers. Additionally, as aging time progresses, the cell edges become less smooth. By 120 h of aging, the cross-sectional area and diameter of muscle fibers are at their minimum, while the degree of fiber dispersion and the distance between fibers are at their maximum.

Table 2 indicates that with the extension of post-mortem aging time, the cross-sectional area and diameter of myocytes show a decreasing trend, while the gaps between myocytes gradually increase. The interstitial spaces in the L-NAME group are significantly smaller than those in the control group at 6, 24, and 120 h (P < 0.05). In the Compound C group, the interstitial spaces are smaller than those in the control group and the L-NAME group at 6 to 120 h and 12 to 72 h, respectively (P < 0.05). Overall, the average cross-sectional area and diameter of myocytes in the control group from 0 to 120 h are lower than those in the L-NAME and Compound C groups, while the cross-sectional area of myocytes in the L-NAME group is lower than that in the Compound C group.

Table 2.

Changes in cross-sectional area and spacing of muscle cells during maturation.

Maturation 0 h 6 h 12 h 24 h 48 h 72 h 120 h
Myocyte diameter (μm)
L-NAME 70.35 ± 2.83a 64.99 ± 2.35b 60.07 ± 2.32b 59.19 ± 2.02b 52.55 ± 2.22b 50.11 ± 2.69b 48.27 ± 2.86b
Compound C 71.41 ± 1.96a 69.86 ± 2.56a 64.88 ± 2.83a 62.12 ± 2.25a 55.44 ± 2.35a 51.82 ± 2.61a 50.24 ± 2.21a
Control 70.16 ± 2.97a 62.26 ± 3.05c 58.62 ± 2.98c 58.98 ± 2.06b 50.99 ± 1.37c 49.49 ± 2.54b 46.43 ± 3.13b



Myocyte interstitial space (μm)
L-NAME 7.64 ± 1.77a 11.10 ± 1.52b 12.98 ± 1.82a 14.70 ± 1.95b 16.27 ± 2.82b 16.93 ± 1.62b 17.04 ± 1.99b
Compound C 7.41 ± 1.52a 10.67 ± 1.81b 11.02 ± 0.94b 13.64 ± 1.74c 14.10 ± 1.58c 15.10 ± 1.80c 16.80 ± 2.19b
Control 7.74 ± 1.13a 12.70 ± 1.62a 13.74 ± 1.61a 16.14 ± 2.25a 18.31 ± 2.78a 18.08 ± 2.78a 19.01 ± 1.98a



Myocyte cross-sectional area (μm2)
L-NAME 4006.07 ± 273.69b 3637.97 ± 193.65b 3275.90 ± 221.39b 3129.17 ± 168.2b 2428.17 ± 169.02b 2095.90 ± 169.55b 1943.57 ± 191.14b
Compound C 4165.6 ± 205.79a 4156.17 ± 203.23a 3530.00 ± 271.75a 3337.33 ± 242.54a 2659.83 ± 168.71a 2333.27 ± 212.49a 2133.27 ± 212.02a
Control 3918.43 ± 269.62b 3442.03 ± 186.14c 3034.70 ± 161.97c 3017.43 ± 194.80c 2150.33 ± 146.79c 2002.53 ± 171.65b 1810.10 ± 198.13b
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Note. The lowercase letters in the upper right corner indicate the significance of differences between treatment groups over time. (P < 0.05).

4. Discussion

NO is an endogenous molecule involved in a variety of physiological processes. NO regulates energy metabolism in skeletal muscle. AMPK can sense the energy state of cells and regulate metabolic pathways, including glucose uptake and glucose metabolism in skeletal muscle. Early studies have shown a relationship between NO and the expression and activity of AMPK in cells (Chen et al., 1999). There is growing evidence that NO regulates intracellular AMPK. For example, in human skeletal muscle, NO donors increase glucose transport by 2.4-fold, cGMP levels by 80-fold, and NO donors simultaneously increase the activity associated with AMP-activated protein kinase AMPK-α1 (1.7-fold) (Deshmukh et al., 2010). In addition, studies by Park (Park et al., 2002) and An et al. (An et al., 2007) showed that SNP (NO releaser) could increase the phosphorylation level of AMPK Thr-172 and the activity of AMPK in endothelial cells. Zhang et al. (Zhang et al., 2008) suggested that No-dependent AMPK activation was sGC - and calcium-dependent. sGC activation converts GTP into cGMP, and the biological effects of cGMP are mainly mediated by PKG. Ca2+ signals activate AMPK in cells via CaMKKβ (Angela et al., 2005). Camkβ can activate AMPK by phosphorylating Thr172 of AMPKα subunit (Hardie, 2008). Previous studies have shown that the effect of NO on AMPK is concentrated in live animal cells. However, the regulation of NO on AMPK during post-mortem maturation of muscle has not been reported. Therefore, this study first investigated the effect of NO on AMPK during post-mortem maturation of beef, and then explored the effect of NO-AMPK pathway on beef tenderness.

4.1. Effects of NO regulation on AMPK

AMPK is an important receptor and regulator of intracellular energy balance. AMPK activity and P-AMPK expression in the three groups increased first and then decreased, and AMPK activity and P-AMPK expression in the control group were significantly higher than those in the L-NAME group at 0 to 24 h (P < 0.05), indicating that NO increased P-AMPK expression and AMPK activity during postmortem maturation. This result is similar to that of previous studies, NO donor SNP induces phosphorylation and activation of α1-AMPK in rat muscle (Higaki et al., 2001). Lira et al. (Lira et al., 2007) reported that in both in vivo and in vitro tests in rats, AMPK-induced GLUT4 mRNA upregulation required NOS activity, and there was a positive feedback interaction between NOS and AMPK. The results showed that the activity of AMPK and the expression of p-AMPK protein in ODQ group were lower than those in L-NAME group and control group, and ODQ inhibited the production of cGMP, indicating that cGMP plays a key role in the signaling mechanism of AMPK regulated by NO. Asdrubal et al. (Aguilera-Méndez & Fernández-Mejía, 2012) showed that the increase of cGMP content in mouse liver cells would cause AMPK activation. Deshmukh et al. (Deshmukh et al., 2010) found that increased cGMP level in skeletal muscle cells was accompanied by AMPK activation to promote glucose transport, and this effect was blocked in the presence of sGC inhibitors. Studies by Asdrubal and Deshmukh support the results of this study. In addition, the biological effects of cGMP are mainly mediated by PKG, and the cGMP/PKG signaling pathway regulates AMPK activity (Ramnanan et al., 2010). Studies have shown that AMPK is activated in cells in the presence of cGMP, and the expression and activity of AMPKα1 protein are increased during PKG immunoprecipitation, indicating that PKG regulates AMPK in vivo. cGMP can activate PLC through PKG and induce the subsequent production of IP3. IP3 induces the release of Ca2+ from the sarcoplasmic reticulum, which increases intracytoplasmic Ca2+, and the increase of Ca2+ activates CaMKKβ. Therefore, NO can affect Ca2+ and CaMKKβ through cGMP/PKG/PLC/IP3, and ultimately regulate AMPK. In addition to the cGMP/PKG pathway, NO may directly or indirectly activate AMPK through other pathways. cGMP and Ca2+ downstream of sGC are one of the NO-induced activation mechanisms of AMPK. However, the possibility that NO may affect AMPK through mechanisms other than cGMP cannot be ruled out. Further studies are needed to explore the potential alternative pathways involved in NO-mediated AMPK activation. Understanding these mechanisms will provide deeper insight into the regulatory role of NO in cellular energy balance and metabolism.

4.2. Effects of key factors in the NO-AMPK signaling pathway

In this study, NO content and NOS enzyme activity in L-NAME group and control group were measured first. NO content and NOS activity in L-NAME group were significantly lower than those in control group at 6–48 h, indicating that L-NAME could inhibit NOS enzyme activity and reduce the production of NO during beef maturation. Tian et al. (Tian et al., 2022) reported that NOS activity and NO content in longiseason dorsi muscle of yaks were significantly reduced after NOS inhibitor treatment, and the results of this experiment were similar. NO/cGMP/PKG is a classical pathway for NO to transmit signal molecules in the body, and sGC is a direct physiological target of NO, which catalyzes the production of cGMP. Many studies have confirmed that ODQ is an inhibitor of sGC and can effectively inhibit cGMP production. The results showed that the cGMP content in the three groups showed a decreasing trend, and the cGMP content in 6–24 h L-NAME group was significantly higher than that in ODQ group and lower than that in control group (P < 0.05). PKG content decreased after reaching the maximum at 6 h, and PKG content in L-NAME group was significantly higher than that in ODQ group and lower than that in control group at 6 to 12 h (P < 0.05). During the maturation process, cGMP content and PKG content generally showed a downward trend, which was similar to the results of Liu et al (Liu et al., 2021),. whose research results showed that the expression levels of sGC, cGMP and PKG in the smooth muscle of cerebral arteries slowly decreased at 3, 6, 12 and 24 h after death. The study of Young et al. (Young et al., 1997) also supported this result. The glucose transport rate of rat soles was studied with NO donor (SNP) treatment, and it was found that SNP could maximize the cGMP content (about 80 times), and the sGC inhibitor LY-83583 could significantly reduce the increase of cGMP content caused by SNP. The biological effect of NO is mainly realized through PKG, which is one of the main receptors of cGMP and constitutes a positive feedback metabolic process, which plays an important role in the mechanism of NO action. L-NAME and ODQ inhibited the production of NO and cGMP, respectively. In the results of this study, the content of cGMP and PKG in the control group was higher than that in the L-NAME group, and that in the L-NAME group was higher than that in the ODQ group, indicating that NO promoted the production of cGMP during the post-slaughter maturation of beef, and then cGMP promoted the production of PKG. The NO/GMP/PKG pathway still exists during beef maturation.

PLC is also a key enzyme in signal transduction, interacting with other substances to participate in signal transmission pathways. IP3 is catalyzed by PLC, and activated PLC hydrolyzes PIP2 to generate IP3, which then participates in many metabolic processes (Koschinski & Zaccolo, 2017). cGMP can activate PLC through PKG and induce subsequent IP3 production (Moustafa et al., 2011). IP3 binds to its receptor IP3R to induce the release of Ca2+ by the endoplasmic reticulum (Yadav et al., 2018). The results of this study showed that the PLC content and IP3 content in L-NAME group were lower than those in control group, and the PLC content and IP3 content in ODQ group were lower than those in L-NAME group. The PLC content and IP3 content in three groups were significantly different at 6 h (P < 0.05), and the IP3 content was significantly different at 12–24 h (P < 0.05). These results indicate that NO and cGMP can increase the PLC content and IP3 content in muscle cells during the maturation of beef after slaughter. Moustafa et al. (Moustafa et al., 2011) found that different concentrations of NO donors (SNPS) would promote the increase of IP3 content. In the presence of sGC inhibitor ODQ, the increase was completely inhibited. Different concentrations of cGMP analogue 8-Br cGMP also increased the content of IP3. Pretreatment of cells by PLC inhibitor U73122 inhibited the induction of IP3 by cGMP analogs. KT5823, a potent and highly selective PKG inhibitor, also eliminated 8-Br-cGMP induced IP3 production, which was supported by results showing that NO stimulates IP3 formation through the sGC/cGMP signaling pathway and ultimately induces Ca2+ increase. In the study of Potter et al. (Potter, 2011) IP3 was rapidly accumulated in pancreatic alveolar cells of rats after NO treatment, and this effect could be blocked by ODQ, KT5823 (PKG inhibitor) and U-73122 (PLC inhibitor), indicating that the generation of IP3 requires a cascade reaction of cGMP, PKG and PLC. This is similar to the results of this experiment. Combined with the above results, it is speculated that NO is dependent on cGMP/PKG/PLC pathway to stimulate IP3 synthesis during beef maturation after slaughter.

CaMKKβ is the upstream kinase of AMPK. CaMKKβ enhances AMPK activity and is activated immediately when the level of free Ca2+ in cytoplasm is increased (Gao et al., 2019). The results showed that Ca2+ concentration in L-NAME group was significantly higher than that in ODQ group at 12 h, and significantly lower than that in control group (P < 0.05). The activity of CaMKKβ in the control group was significantly higher than in the L-NAME group at 12 h (P < 0.05), and the L-NAME group had significantly higher activity than the ODQ group (P < 0.05). This indicates that the concentration of cytoplasmic Ca2+ increased in the control group, leading to enhanced CaMKKβ activity compared to the L-NAME and ODQ groups. The results indicated that NO and cGMP increased Ca2+ concentration and CaMKKβ activity during beef maturation after slaughter. Gao Yongfang et al. (Gao et al., 2019) found that Ca2+ significantly increased the activities of CaMKKβ and AMPK during beef maturation, and at the same time increased the phosphorylation level of AMPK. Raney et al. (Raney & Turcotte, 2008) demonstrated that Ca2+ dependent activation activates AMPK through CaMKKβ, while caffeine-induced activation of AMPK is a result of elevated intracellular Ca2+ levels. Increased Ca2+ in cytoplasm is the primary cause of activation of CaMKKβ, which phosphorylates Thr-172 on the AMPKα subunit (Hurley et al., 2005). Chu et al. (Chu et al., 2021) directly proposed that NO could cause the release of calcium from intracellular calcium reservoir and increase the concentration of calcium ions. Zhang et al. (Zhang et al., 2008) reported that NO-induced AMPK activation was not affected by inhibition of LKB1 (AMPK kinase), on the contrary, inhibition of CaMKKβ eliminated the role of NO in cells. Based on the above studies, it is further speculated that NO activates PLC through cGMP/PKG, then stimulates IP3 synthesis, induces sarcoplasmic reticulum to release Ca2+, and increases intracytoplasmic Ca2+ to activate CaMKKβ. These findings underscore the critical role of intracellular calcium dynamics in regulating CaMKKβ and AMPK, and further emphasize the complex interaction between NO, cGMP, and Ca2+ in cellular signaling. Further research is required to fully elucidate the intricate mechanisms by which these pathways modulate cellular metabolism.

4.3. Effects of the NO-AMPK signaling pathway on beef protein denaturation

The degree of protein denaturation can also be assessed through DSC analysis of its thermal properties (Niu et al., 2025). In DSC thermograms, the peak temperature (Tm) represents the temperature at which protein denaturation occurs, indicating protein structural stability, while the enthalpy (ΔH) represents the energy required to induce protein denaturation (Kaushik et al., 2016). Higher denaturation temperatures and enthalpy values indicate greater protein structural stability (Zhang et al., 2024). The results of this experiment show two peaks in the thermograms of beef protein. In the control group, peak I nearly disappears, indicating mild denaturation in the control group compared to the L-NAME group, and severe denaturation compared to the Compound C group. Aktas (Aktas et al., 2005) suggests that severe degradation of myofibrillar protein can lead to a decrease in phase transition temperature and enthalpy, thereby affecting meat quality. Additionally, L-NAME and Compound C inhibit the postmortem glycolysis process, delaying postmortem aging and pH reduction in beef, which further prevents muscle fiber structure degradation, with Compound C exerting a stronger inhibitory effect than L-NAME. Consequently, myofibrillar degradation is most severe in the control group, while proteins in the Compound C group are least susceptible to degradation. Compared to the control group, the denaturation temperature in the L-NAME group is delayed, and thermal stability increases; similarly, the denaturation temperature in the Compound C group is delayed relative to the L-NAME group, with greater thermal stability. Among the three groups, the Compound C group shows the highest Tmax and enthalpy values, while the control group has the lowest, indicating that the Compound C group has the most thermally stable protein structure, while the control group has the least stable. These findings suggest that NO, through AMPK, reduces the thermal stability of myofibrillar proteins.

4.4. Effect of NO-AMPK signaling pathway on beef tenderness

The tenderness of muscle is also affected by the diameter of muscle cells and the distance between muscle cells (Warner et al., 2017). Postmortem muscle pH affects muscle tenderness. During the post-mortem maturation, muscle fibers break into small sheets, and the lamination index of myofibrillar fibers also affects the tenderness of beef (Volpelli et al., 2005). The greater the MFI value, the higher the degree of muscle fiber degradation, the greater the degree of muscle structure destruction, the looser the tissue, the more tender the meat (Wang et al., 2024). The results of this experiment showed that the shear force of the three groups of meat samples increased first and then decreased at 0–120 h, and reached the maximum at 72 h. MFI has been increasing. The glycolysis rate of the control group was the fastest, which promoted the maturation of meat after slaughter. Therefore, the shear force of the control group was lower than that of the L-NAME group and that of the L-NAME group was lower than that of the Compound C group, while the MFI changes were opposite. These results indicated that NO could improve beef tenderness through AMPK. The changes of histological structure and microstructure of muscle fibers in this experiment also verified the changes of beef tenderness during post-mortem maturation. As can be seen in Fig. 4 (C), the microstructure of muscle fibers in the control group was damaged to a certain extent, and the gaps between muscle fibers became larger and the structure became loose. The muscle fibers of L-NAME group and Compound C group were closely arranged and the gaps were relatively small. However, with the extension of postmortem maturation time, the muscle fiber structure of the three groups was gradually destroyed, the damage degree of the control group was the highest, and the damage degree of the Compound C group was the least. With the extension of postmortem maturation time, the cross-sectional area and diameter of myocytes decreased, and the distance between myocytes increased, gradually separating and shrinking from the initial tightly arranged state. The cross-sectional area and diameter of muscle fiber cells are also related to muscle tenderness, and studies have confirmed that the larger the diameter and area of muscle fiber cells, the lower the tenderness of meat (Gondret et al., 2006). Muscle tenderness is closely related to tissue structure (myocyte diameter, myocyte interstitial space, myocyte cross-sectional area). At 48 h after slaughter, the cross section of the muscle fibers in the control group began to shrink significantly, and the intercellular arrangement was more discrete, while the muscle fibers in the L-NAME group and Compound C group began to shrink significantly from 72 h, and the tenderness was improved. At 120 h of maturity, the separation between the three groups of muscle fibers was further strengthened. Therefore, NO can improve the tenderness of beef by increasing AMPK activity during post-slaughter maturation. The observations made in this study highlight the critical role of NO in influencing muscle fiber degradation and collagen solubility, both of which contribute to improved meat tenderness. Further investigation into the molecular pathways linking NO to these structural changes could provide deeper insights into meat aging and quality control.

5. Conclusion

In summary, after beef slaughter, NO binds to sGC to produce cGMP, which activates PLC through PKG and induces IP3, and IP3 induces the release of Ca2+ by endoplasmic reticulum. When the level of free Ca2+ in cytoplasm increases, CaMKKβ is activated and AMPK activity is improved. After AMPK activation, beef shear force decreased, myofibrillar fragmentation index increased, muscle fiber destruction degree increased, and tenderness improved. Therefore, the regulation pathway of NO to AMPK can be regarded as an important regulatory target to improve the tenderness of beef in the post-mortem maturation.

Funding

This study was supported by the National Key Research and Development of China (2021YFD1600204–2), the National Natural Science Foundation of China (Grant No. 31760482), the Science and Technology Project of Gansu Province (Grant Nos: 24JRRA647), the program for Agriculture Research System of China (No. CARS-37).

Informed Consent

Informed consent was obtained from all individual participants included in the study.

CRediT authorship contribution statement

Zhuo Wang: Writing – original draft, Investigation, Data curation. Qiao Li: Writing – review & editing, Formal analysis, Data curation. Jibing Ma: Visualization, Software. Aixia Li: Writing – review & editing. Guoyuan Ma: Writing – review & editing. Qunli Yu: Supervision, Funding acquisition. Ling Han: Supervision. Cheng Chen: Supervision.

Declaration of competing interest

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.

Data availability

The authors do not have permission to share data.

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