Eric M. England

pH inactivation of phosphofructokinase arrests postmortem glycolysis.

Fresh meat quality development is influenced by pH decline that results from muscle glycolyzing energy substrates postmortem. The exact reason why glycolysis stops in the presence of residual glycogen remains unclear. We hypothesized that a critical glycolytic enzyme loses activity near the ultimate pH of meat. Porcine longissimus muscle samples were subjected to an in vitro system that mimics postmortem anaerobic metabolism at buffered pH values (7.0, 6.5, 6.0, 5.5 or 5.0). At pH7.0, 6.5, and 6.0, glycogenolysis and glycolysis proceeded normally while pH5.5 stopped lactate formation. Additional experimentation indicated that phosphofructokinase lost activity at pH5.5 while all other glycolytic enzymes remained active. A similar inactivation of phosphofructokinase was observed when using chicken and beef muscle. Elevated temperature hastened pH decline and phosphofructokinase activity loss. Thus, pH inactivates phosphofructokinase and arrests postmortem glycolysis, which may explain the similar ultimate pH across meat of different species.

Exploring the unknowns involved in the transformation of muscle to meat

Meat quality development, or the transformation of muscle to meat, involves a myriad of biochemical pathways that are largely well-studied in living muscle tissue. However, these pathways are less predictable when homeostatic ranges are violated. In addition, there is far less known about how various management or environmental stimuli impact these pathways, either by substrate load or altered cellular environment. Likewise, it is largely accepted that oxygen plays little to no role in the conversion of muscle to meat, as anaerobic metabolism predominates in the muscle tissue. Even so, the oxygen tension within the tissues does not fall precipitously at exsanguination. Therefore, transition to an anaerobic environment may impact energy metabolism postmortem. Antemortem handling, on the other hand, clearly impacts meat quality development, yet the exact mechanisms remain a mystery. In this paper, we will attempt to review those factors known to affect postmortem energy metabolism in muscle and explore those areas where additional work may be fruitful.

Excess glycogen does not resolve high ultimate pH of oxidative muscle

Skeletal muscle glycogen content can impact the extent of postmortem pH decline. Compared to glycolytic muscles, oxidative muscles contain lower glycogen levels antemortem which may contribute to the higher ultimate pH. In an effort to explore further the participation of glycogen in postmortem metabolism, we postulated that increasing the availability of glycogen would drive additional pH decline in oxidative muscles to equivalent pH values similar to the ultimate pH of glycolytic muscles. Glycolysis and pH declines were compared in porcine longissimus lumborum (glycolytic) and masseter (oxidative) muscles using an in vitro system in the presence of excess glycogen. The ultimate pH of the system containing longissimus lumborum reached a value similar to that observed in intact muscle. The pH decline of the system containing masseter samples stopped prematurely resulting in a higher ultimate pH which was similar to that of intact masseter muscle. To investigate further, we titrated powdered longissimus lumborum and masseter samples in the reaction buffer. As the percentage of glycolytic sample increased, the ultimate pH decreased. These data show that oxidative muscle produces meat with a high ultimate pH regardless of glycogen content and suggest that inherent muscle factors associated with glycolytic muscle control the extent of pH decline in pig muscles.

Net lactate accumulation and low buffering capacity explain low ultimate pH in the longissimus lumborum of AMPKγ3R200Q mutant pigs

Postmortem lactate accumulation in skeletal muscle is linearly associated with the extent of pH decline. Yet, pigs harboring the AMPKγ3(R200Q) mutation produce meat with similar lactate levels to that of wild-type pigs but have a lower ultimate pH. We hypothesized that lower initial lactate levels and (or) lower buffering capacity in muscle of these pigs may help explain this discrepancy. Longissimus lumborum muscle samples were harvested at 0 and 1440 min postmortem from AMPKγ3(R200Q) and wild-type pigs. As expected, AMPKγ3(R200Q) muscle exhibited a lower ultimate pH but similar lactate levels to that of wild-type pigs at 1440 min postmortem. However, the total net lactate produced postmortem was greater in the AMPKγ3(R200Q) muscle due to lower initial lactate levels at 0 min postmortem. Buffering capacity measured over the pH range of 5.5-7.0 was also lower in AMPKγ3(R200Q) muscle. Greater net lactate accumulation postmortem (i.e., glycolytic flux) coupled with a lower buffering capacity explains the lower ultimate pH of meat from AMPKγ3(R200Q) pigs.

Mitochondria influence postmortem metabolism and pH in an in vitro model.

Our objective was to determine the influence of mitochondria on metabolites and pH decline using an in vitro model of postmortem muscle metabolism. Mitochondria were isolated from porcine longissimus lumborum and added (0, 0.5, or 2.0mg) to powdered muscle in reaction media containing either a combination of inhibitors for mitochondria complexes (I, IV, and V) or diluent (without inhibitors). In the absence of inhibitors, adding mitochondria (0.5 and 2.0mg) reduced ATP loss from 30 to 120 min, but did not alter glycogen or lactate during this time. In reactions with mitochondria, inhibitors decreased ATP levels by 30 min and increased glycogen degradation by 60 min. Regardless of mitochondria content, inhibitors enhanced lactate accumulation from 15 to 240 min, and decreased pH from 15 min to 1440 min. In the in vitro model, mitochondria influence the maintenance of ATP, and inhibition of mitochondria enzyme activity contributes to accelerated metabolism and pH decline.

Altered AMP deaminase activity may extend postmortem glycolysis

Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK γ3(R200Q) mutated pigs (RN⁻) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN⁻ muscle than wild-type. RN⁻ muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN⁻ pork.

Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle.

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity

Objective Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. Methods We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. Results We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Conclusions Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders.

Contribution of the phosphagen system to postmortem muscle metabolism in AMP-activated protein kinase γ3 R200Q pig Longissimus muscle

Pigs with the AMP-activated protein kinase γ3 R200Q (AMPKγ3(R200Q)) mutation generate pork with low ultimate pH (pHu). We hypothesized that reducing muscle creatine (Cr) and phosphocreatine (PCr) may accelerate postmortem ATP consumption and prevent extended pH decline in AMPKγ3(R200Q) longissimus muscle. Wild type and AMPKγ3(R200Q) pigs were assigned to control diet or diet supplemented with the creatine analog β-guanidinopropionic acid (β-GPA, 1%) for 2 wk. β-GPA reduced muscle PCr (P = 0.006) and total Cr (P<0.0001). In general, AMPKγ3(R200Q)+β-GPA exhibited more rapid metabolism than control, AMPKγ3(R200Q), and β-GPA treatment, evidenced by more rapid loss of ATP, more rapid increase in IMP, and decreased pH during the first 90 min postmortem. Overall, pHu was similar despite elevated glycogen (AMPKγ3(R200Q)), reduced total Cr (β-GPA) or both (AMPKγ3(R200Q)+β-GPA). Thus, reducing muscle phosphagens did not affect pHu in AMPKγ3(R200Q) muscle, but it hastened ATP depletion and pH decline.

Chapter 4 – Perimortal Muscle Metabolism and its Effects on Meat Quality

Muscle tissue experiences profound changes during its conversion to meat. Indeed, these changes are necessary and requisite for meat to assume those unique properties are keenly coveted by consumers of this highly nutritious and palatable food source. However, when this process is perturbed, the resulting quality is dramatically impacted. Therefore, understanding those reactions forming the basis of postmortem metabolism is incumbent to those interested in meat animal production. Initially, the biochemistry responsible for postmortem metabolism is simply an extension of those processes responsible for supplying energy to functioning skeletal muscle, responsible for locomotion. However, at some yet-to-be defined point, muscle tissues broach the physiological threshold where they begin to die and at this juncture, biochemical reactions dysregulate according to physiological normalcy. Even with the seemingly dearth of information available on the topic, our intent is to outline the biochemical processes responsible for postmortem metabolism and the conversion of muscle to meat as currently known.