To maintain ATP levels postmortem muscle
To maintain ATP levels, postmortem muscle mobilizes its glycogen reserves to yield BMS 777607 6-phsphate that proceeds through glycolysis. Glycogen degradation was affected by the interaction between treatment and time (P<0.0001; Fig. 4A). Significantly lower glycogen (P=0.001) was observed in samples containing mitochondria without Na-azide at 120min compared to the rest of the treatments. At 1440min, samples containing only mitochondria had lower glycogen (P=0.05) than that of control, while samples containing Na-azide and mitochondria+Na-azide were intermediate. There was also a significant interaction of treatment×time for mean glucose 6-phosphate concentration of the in vitro system (P<0.0001; Fig. 4B). At 120min, glucose 6-phosphate was greater in samples containing mitochondria regardless of Na-azide (P<0.0001) compared to those without mitochondria. However, at 1440min the opposite was true, where samples containing mitochondria possessed lower glucose 6-phosphate levels (P<0.0001). During the transformation of muscle to meat, ATP is hydrolyzed by muscle ATPases to ADP and eventually to IMP by adenylate kinase and AMP deaminase (AMPD)-catalyzed reactions. Once formed, IMP accumulates in the muscle and no longer contributes to ATP synthesis (Scheffler, Park, & Gerrard, 2011). At 120min samples containing mitochondria without Na-azide had the lowest ATP concentration (P=0.03; Fig. 5A) compared to the rest of the treatments, while 1440min ATP was below detection. Samples containing only mitochondria accumulated more IMP at 120min (P=0.02; Fig. 5B) compared to the other treatments. Combined, lower ATP and greater IMP in system containing mitochondria indicate that ATPase activity was enhanced. However, when combined with Na-azide, mitochondria-induced ATP hydrolysis was diminished, which shows the efficacy of Na-azide treatment in the in vitro system. While Na-azide can inhibit other ATPases (Bowler et al., 2006, Vasilyeva and Forgac, 1998), myofibrillar ATPase (myosin ATPase) does not appear to be impacted, as evidenced by the lack of difference in ATP and pH between control and Na-azide treatments. Scopes (1974) showed that the rate of postmortem metabolism is dictated by the rate of ATP hydrolysis. The author also indicated that mechanisms controlling the flux through glycolysis must be determined by the rate of ATP hydrolysis. On the other hand, our lab has recently shown that postmortem PFK-1 activity rapidly decreases as muscle pH declines until complete inactivation is reached at pH5.5 (England et al., 2014). Hastened glycolysis appears to increase the potential for extended pH decline, as it allows for more substrate to pass PFK-1 before it becomes inactivated. This notion is supported by our findings where enhanced ATP hydrolysis was coupled with greater glycogen degradation, lactate accumulation, and pH decline, suggesting that glycolysis was hastened to meet ATP demand. It is important to note that while mitochondria-enhanced ATP hydrolysis was completely abolished by Na-azide (Fig. 5A), the inhibitor did not completely reverse the effect of mitochondria on pH decline (Fig. 2). This suggests that additional mitochondrial protein(s) may account for part of the observed effect. Jong and Davis (1983) reported that mitochondria can increase the NAD+/NADH ratio in the cytosol. Indeed, cytochrome c-mediated reduction of metmyoglobin coupled with oxidation of NADH to NAD+ has been previously suggested (Tang, Faustman, Mancini, Seyfert, & Hunt, 2005). The increase in NAD+ concentration may provide more substrate for GAPDH, allowing for greater glycolytic flux. Regardless, these results confirm that mitochondria F1-ATPase can significantly contribute to postmortem glycolysis. Consistent with our previous findings (Matarneh et al., 2017), the effect of mitochondria on glycolytic flux required 120min to be observed, suggesting that the effect may be cumulative over time. Another alternative explanation is that ATPase activity in the in vitro system was not limiting during the first 120min, but as the pH declines it may become limiting, then additional ATPase activity by mitochondrial F1-ATPase would enhance ATP hydrolysis and flux through glycolysis. Because myosin is the most abundant protein in skeletal muscle, myosin ATPase is responsible for the majority of postmortem ATP hydrolysis (Hamm, Dalrymple, & Honikel, 1973). Similar to PFK-1, however, the activity of myosin ATPase is pH dependent (Bowker, Grant, Swartz, & Gerrard, 2004), where the activity substantially drops as pH declines from 6.5 to 5.5, at which complete inactivation is achieved. On the other hand, mitochondrial F1- ATPase retains about 50% relative activity between pH6 and 5 (Feinstein & Moudrianakis, 1984). To address this issue, myofibrillar and mitochondrial proteins were added to the in vitro system after 240min from the initiation of the experiment. We expected that if myosin ATPase is limiting, then adding myofibrillar protein (as a source for myosin ATPase) to the in vitro system would produce the same effect as mitochondria. On the other hand, however, if the myofibrillar protein fails to produce similar effects, then mitochondria-induced enhancement in glycolytic flux would be a fuction of greater ATPase activity at a lower pH. Samples containing mitochondria had lower pH at 1440 (P<0.0001; Fig. 6) than those from control and myofibril samples, while no difference between control and myofibrillar treatment was observed. Lactate followed the same trend as pH, where samples with mitochondria had greater lactate concentration (P<0.0001; Fig. 7) than those containing myofibrillar protein. The lack of difference between control and myofibril treatments argues that ATPase activity rather than abundance was limiting at lower pH. Further, the addition of mitochondria after 240min produced a similar effect to those added at 0min, suggesting that mitochondrial ATPase activity plays an important role at lower pH (pH<6.2). Based on these results, we suggest that mitochondria can extend postmortem metabolism through maintaining greater ATPase activity at lower pH which, in turn, enhances flux through glycolysis.