Changes in pH of bovine muscle during postmortem aging. The pH of bovine muscle with different pH decline rates during postmortem aging is shown in Fig. 1. The pH of the fast pH decline group decreased significantly at 24 h while the pH of the slow group decreased significantly at 48 h compared with 0.5 h (P<0.05). However, the pH in the fast group was lower than the slow group by 4.06%, 5.11%, 8.34%, 4.43%, and 2.67% at 0.5, 2, 6, 12, and 24 h (P<0.05), respectively. These findings suggest a significant difference in pH decline rates between the two groups within 24 h postmortem.
After slaughter and bleeding, glycogen metabolism shifts from aerobic to anaerobic and the metabolite lactate accumulates, resulting in a decline in muscle pH 24,25. Apaoblaza et al.26 demonstrated that the ultimate pH of carcasses was correlated with glycogen content and a sufficient amount of glycogen can supply the necessary fuel for the pH decrease. Previous studies have shown that the breakdown of ATP produced by postmortem muscle glycolysis led to the accumulation of protons (H+), which was also caused a decrease in pH27. The rate of glycolysis is determined by the activity of glycolytic enzymes. Pyruvate kinase, enolase 2, phosphoglucomutase 1, fructose diphosphate aldolase, and enolase 3 are all phosphorylation-regulated enzymes that may be implicated in postmortem muscle glycolysis28. In addition, several other pre- and post-mortem factors might affect postmortem muscle glycolysis, such as genetics, pre-slaughter stress, carcass temperature, and S-nitrosylation of glycolytic enzymes, etc.26,29,30. In the present study, the fast group of muscles may have consumed glycogen more quickly, resulting in a low pH in the early postmortem period.
Effect of pH decline rate on protein denaturation and mitochondrial ROS accumulation in postmortem bovine muscle. The decrease in pH is generally thought to induce further protein denaturation in muscle. Therefore, we evaluated the influence of pH decline rate on muscle protein denaturation temperature and ROS levels. A total of three heat absorption peaks were observed in the muscle tissue by DSC scanning, representing the denaturation temperature of the myosin head, sarcoplasmic protein, and actin31. The denaturation temperature of a protein increases when its structure is slightly altered. As shown in Table 1, both myosin and sarcoplasmic protein denaturation temperatures increased significantly in both groups during postmortem aging (P<0.05), however myosin denaturation temperature in the fast group was 2.60% higher than in the slow group at 12 h (P<0.05). Sarcoplasmic protein denaturation temperature was 1.79% and 1.39% higher than in the slow group at 6 and 12 h, respectively (P<0.05). However, actin did not show any differences in denaturation temperature during the postmortem aging in the two groups. In addition, the fluorescence images of ROS in two groups are shown in Fig. 2A. The average fluorescence values of ROS in muscle cells, reflecting the ROS levels, were significantly higher in the fast group than the slow group at 6–24 h postmortem (P<0.05; Fig. 2B), with a most significant difference at 12 h (P<0.01). The above results suggested that fast pH decline promoted myosin and sarcoplasmic protein denaturation and ROS accumulation in the postmortem muscle.
Insufficient ATP production and supply in the postmortem muscle in conjunction with the reduction of pH disrupt the normal redox system, leading to the accumulation of proapoptotic free radicals such as ROS13,14. There is a direct correlation between increased ROS levels and lower muscle pH32. Previous studies have shown that the antioxidant enzymes such as SOD, CAT, and GSH-Px showed decreased activity at the low pH in postmortem bovine muscle33. Sun et al.34 showed that an ultimate pH of 5.3–5.6 during postmortem muscle aging did not indicate that muscle glycogen was depleted, but rather that the inactivation of enzymes in the glycolytic pathway caused the reaction to halt when the pH fell below a certain threshold. These findings suggest that lower muscle pH induces a decrease in enzyme activity in the cytoplasm. The enzymes in the cytoplasm generally play an essential role in a neutral environment. Lower pH caused the active core group of the enzyme to deviate from its optimal dissociation state, reducing its capacity to bind to the substrate and catalytic activity25,35. In the present study, the fast pH decline group had greater ROS levels than the slow group, which could be owing to changes in the structure and activity of ROS scavenging enzymes by rapidly declining muscle pH. Taken together, it was clear that postmortem fast pH decline contributed to the accumulation of muscle ROS by inducing sarcoplasmic protein denaturation.
Table 1
Changes in the denaturation peak temperature of Myosin (TMyosin), Myoplasmic protein (TMyoplasmic protein) and Actin (TActin) in bovine muscles of different glycolytic rates at 0.5, 6, 12, 24, 72, 120, and 168 h postmortem.
Indicators | Glycolytic rate | Postmortem aging time |
0.5 h | 6 h | 12 h | 24 h | 72 h | 120 h | 168 h |
TMyosin (℃) | Fast | 51.85±1.23B | 51.81±1.78B | 52.91±1.54AB* | 52.93±1.51AB | 53.18±1.39AB | 53.68±1.45A | 53.67±1.39A |
Slow | 51.73±1.34b | 51.63±1.36b | 51.53±1.43b | 52.89±1.29ab | 53.62±1.56a | 53.81±1.48a | 53.45±1.35a |
TMyoplasmic protein (℃) | Fast | 61.38±1.44B | 62.64±1.73AB* | 63.21±1.33A* | 63.64±1.39A | 63.51±1.26A | 63.38±1.08A | 63.33±1.36A |
Slow | 61.46±1.36c | 61.54±1.47c | 62.35±1.36b | 63.39±1.28a | 63.24±1.04a | 63.61±1.27a | 63.40±1.63a |
TActin (℃) | Fast | 76.43±0.75A | 76.61±0.58A | 76.31±0.57A | 77.53±0.61A | 77.47±0.65A | 76.88±0.59A | 76.59±0.78A |
Slow | 76.51±0.66a | 77.03±0.65a | 76.53±0.65a | 77.56±0.72a | 77.18±0.98a | 77.67±0.54a | 76.42±0.68a |
A-B: Each letter denotes a statistically significant difference in the fast group at different postmortem times (P<0.05). a-c: Each letter denotes a statistically significant difference in the slow group at different postmortem times (P<0.05). * indicates the significance compared the same time points between two groups (P<0.05). All measurements were expressed as the mean ± SE. |
Effect of pH decline rate on mitochondrial dysfunction and cytochrome c redox state. A mitochondrial dysfunction is a precursor to cell apoptosis. Therefore, the effect of pH decline rate on mitochondrial membrane permeability and cytochrome c redox state were assessed. Fig. 3A showed that the mitochondrial membrane permeability significantly increased during postmortem aging in both groups (P<0.05) and significantly higher in the fast group by 14.05%, 22.39%,18.34% and 25.28% than in the slow group at 6, 12, 24 and 72 h, respectively (P<0.05). The cytochrome c reduction levels are shown in Fig. 3B. Our results showed that cytochrome c reduction levels decreased significantly postmortem in both two groups (P<0.05). Furthermore, the cytochrome c reduction in the fast group was significantly lower than the slow group by 16.71%, 23.39%, 17.05%, and 26.61% at 6, 12, 24, and 120 h, respectively (P<0.05), indicating that fast pH decline induced mitochondrial membrane dysfunction and cytochrome c oxidation during the postmortem aging.
ROS, produced in mitochondria, was an additional indicator for mitochondrial apoptosis36. The weakening of antioxidant enzymes and other defense systems in postmortem muscle inevitably led to the production of ROS37. Excessive accumulation of ROS induced swelling and destruction of mitochondria membrane and ultimately mediated mitochondrial apoptosis14,38. ROS could attack the mitochondrial membrane polyunsaturated fatty acids to produce lipid peroxides such as malondialdehyde, causing mitochondrial swelling and increasing mitochondrial membrane permeability13,39. Moreover, ROS induced cytoplasmic Ca2+ influx into mitochondria, leading to mitochondrial Ca2+ overload, which further caused an increase in mitochondrial membrane pore opening and permeability40. Subsequently, the release of cytochrome c from damaged mitochondria into the cytoplasm was a universal event for mitochondrial apoptosis41, but its catalytic activity was largely dependent on its oxidative state14. Suto et al.42 also showed that the oxidized cytochrome c was more effective in initiating mitochondrial apoptosis. In the present study, fast pH decline facilitated cytochrome c oxidation. Previous studies have showed that the oxidation of cytochrome c was associated with mitochondrial oxidants, such as H2O237,43. Together with the above findings on the role of pH decline rate in ROS, it suggested that fast pH decline indirectly induced mitochondrial dysfunction and cytochrome c oxidation by promoting the accumulation of mitochondrial ROS.
Effect of pH decline rate on caspase-3, apoptosis and tenderness in bovine muscle. To further exam the downstream events of cytochrome c oxidation and mitochondrial dysfunction, the effect of pH decline rate on caspase-3 activity, apoptotic nucleus, MFI, and shear force were evaluated during postmortem aging. As depicted in Fig. 4, in both two groups, caspase-3 activity tended to increase initially and later decrease (P<0.05), with caspase-3 activity reaching the peak at 24 h. Besides, the caspase-3 activity in the fast group was significantly higher than the slow group at 12–168 h (P<0.05), except the 72 h time point. TUNEL assay of the apoptotic nucleus is shown in Fig. 5A. The normal nucleus was labeled with overall blue fluorescence (DAPI), while the apoptotic nucleus was labeled with green fluorescence (TUNEL positive). The proportion of apoptotic nuclei of the total nuclei was quantified and shown in Fig. 5B, with a significant increase in the total number of apoptotic nuclei in both groups (P<0.05). In the fast group, the total apoptotic nuclei were significantly higher than those in the slow group at 24–168 h (P<0.05).
The number of the MFI reflects the degree of fragmentation of myofibrils and positively correlates with the degradation of myofibrillar proteins. Table 2 showed a significant increase in MFI during aging for both groups (P<0.05), however, the fast group had a significantly higher MFI than the slow group at 12–168 h (P<0.05). Shear force was used to evaluate the tenderness of bovine muscle, and the smaller the value indicates the tenderer of the muscle. As shown in Table 2, the shear force of both fast and slow groups kept growing until 12 h (P<0.05). However, it plummeted at 24–168 h (P<0.05). In the fast group, the shear force was significantly higher than in the slow group at 6 h (P<0.05), but significantly lower than in the slow group at 12–168 h (P<0.05) and 10.49% lower than in the slow group at 168 h. The above results suggested that fast pH decline promoted caspase-3 activation, apoptosis, and bovine muscle tenderization during postmortem aging.
Caspase-3 is one of the most important effector caspases in the apoptotic pathway and plays a critical role in muscle tenderization44. The oxidized cytochrome c interacts rapidly with apoptotic protease activating factor (Apaf-1) and procaspase-9 in the presence of dATP to activate caspase-9, which further activates the downstream effector caspase-312,13,41. The results of this study indicated that the caspase-3 showed the highest activity at 24 h time point. Our finding was confirmed by previous reports45, in which they suggested that caspase-3 activity in bovine skeletal muscle gradually increased to a maximum at approximately 24 h time point. Besides, our study also showed that the caspase-3 activity in the fast group was higher than that in the slow group. Combining the above effect of pH decline rate on ROS accumulation, mitochondrial dysfunction, and cytochrome c oxidation, we concluded that the fast pH decline promoted postmortem caspase-3 activation and apoptosis in the bovine muscle.
Postmortem muscle tenderization is a complex biochemical process involving the degradation of myofibrillar proteins and is controlled by a variety of protein hydrolases, including caspase-3 and calpain-146. According to Kemp and Parr47, the formation of 28- and 30-kDa troponin-T degradation products and the degradation of desmin improved the muscle tenderness. Although these proteins were more likely to be recognized by calpain-1, caspase-3 also played an important role in this process. Huang et al.41 reported that caspase-3 also recognized and interacted with actomyosin, titin, nebulin, and tropomyosin in myofibrillar protein and this interaction can trigger further hydrolysis. As a result, caspase-3 is vital in enhancing the tenderness of bovine muscle during aging. In the present study, the bovine muscle tenderness was higher in the fast pH decline group than in the slow group at 12–168 h postmortem and matched the caspase-3 activity. Hwang and Thompson7found that bovine longissimus dorsi with lower pH before the onset of rigour mortis produced more tender meat in the later stages of aging, which backed up the current findings. Given the essential role of pH decline rate in caspase-3 activation, it is possible that fast pH decline increases bovine muscle tenderization via the mitochondrial apoptotic pathway. Our experiment also observed that the tenderness of the fast group was lower than that of the slow group at 6 h postmortem, which could be associated to calpain-1 activity. Previous research has revealed that calpain-1 activation occurred around 6 h postmortem in bovine muscle48, and that calpain-1 degradation activity on the myofibrillar protein was strongest at pH 7.549. Lomiwesa et al.50 concluded that although calpain-1 autolysis was fast at a low pH, the hydrolysis of myofibrillar proteins was diminished in pork muscle. Despite the fact that no experiments involving calpain-1 activity were conducted in this study, it can be hypothesized, based on previous studies, that the fast pH decline group may have decreased calpain-1 activity at 6 h due to the lower pH microenvironments. Therefore, various processes other than mitochondrial apoptosis may also be involved in the regulation of postmortem pH decline rate on bovine muscle tenderness during aging, and this needs to be investigated further in the future. The current study is simply a first step towards understanding the role of postmortem pH in muscle tenderization during aging and we believe our findings will encourage more research in this field.
Table 2
Changes in the myofibril fragmentation index (MFI) and shear force in bovine muscles of different glycolytic rates at 0.5, 6, 12, 24, 72, 120, and 168 h postmortem.
Indicators | Glycolytic rate | Postmortem aging time |
0.5 h | 6 h | 12 h | 24 h | 72 h | 120 h | 168 h |
MFI | Fast | 64.07±7.11F | 65.13±6.53F* | 108.67±5.52E* | 125.60±7.63D* | 142.53±6.11C* | 167.40±6.77B* | 183.47±7.04A* |
Slow | 66.07±6.03g | 79.06±6.80f | 93.53±6.11e | 108.73±7.71d | 128.72±6.74c | 149.33±7.63b | 166.87±6.93a |
Shear force (kgf) | Fast | 6.62±0.37D | 7.79±0.53B* | 8.45±0.33A* | 7.18±0.42C* | 6.07±0.40E* | 5.61±0.52EF* | 5.12±0.31F* |
Slow | 6.53±0.63d | 7.20±0.39c | 8.96±0.35a | 7.74±0.37b | 6.91±0.41cd | 6.24±0.34de | 5.72±0.35e |
A-B: Each letter denotes a statistically significant difference in the fast group at different postmortem times (P<0.05). a-c: Each letter denotes a statistically significant difference in the slow group at different postmortem times (P<0.05). * indicates the significance compared the same time points between two groups (P<0.05). All measurements were expressed as the mean ± SE.