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This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 μM CPI-613, 1.5 U/ml Avidin, 400 μM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 μM CPI-613 or control, were employed to track the fates of pyruvate using [ 13 C 6 ]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 μM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study examined the potential influence of mitochondrial calcium sequestering ability on calpain-1 autolysis and proteolysis in vitro. We first tested whether mitochondria can sequester calcium in an in vitro setting. Isolated bovine mitochondria (0, 0.5, or 2 mg/mL) were incubated in a buffer containing varying calcium levels (0, 50, or 100 μM). An inverse relationship between mitochondrial content and measured free calcium was observed (P < 0.05), confirming that mitochondria can sequester calcium within the concentration range tested. In the first in vitro experiment, intact mitochondria (0, 0.5, or 2 mg/mL) were incorporated into an in vitro model simulating postmortem muscle conditions, and calpain-1 autolysis and proteolysis were evaluated over a 168-h period. Adding intact mitochondria to the in vitro model decreased calpain-1 autolysis and proteolysis during the first 4 h of incubation (P < 0.05), likely through reducing calcium availability. However, accentuated calpain-1 autolysis and proteolysis were observed at 24 h. To further explore these effects, mitochondrial integrity was evaluated at varying pH and calcium levels. Mitochondrial integrity decreased as pH declined (P < 0.05), especially in the presence of calcium. Based on these results, we conducted a second in vitro experiment involving disrupted mitochondria. Unlike intact mitochondria, which exerted a suppressive effect on calpain-1 autolysis and proteolysis early on, disrupted mitochondria increased both parameters at most time points (P < 0.05). Overall, it appears that intact mitochondria initially cause a delay in calpain-1 autolysis and proteolysis, but as their integrity diminishes, both processes are enhanced., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The objective of this study was to investigate if ultrasonication of bovine longissimus thoracis et lumborum (LTL) steaks increases calpain-1 and caspase-3 activities, and if so, to explore the underlying mechanisms that trigger their activation. Post-rigor bovine LTL steaks were subjected to ultrasonication at 40 kHz and 12 W/cm 2 for 40 min and subsequently aged for 14 d at 4 °C. Ultrasonication improved beef tenderness (P < 0.05) without negatively impacting pH, color, or cook loss (P > 0.05). Improved tenderness in the ultrasonicated steaks was associated with greater degradation of titin, desmin, troponin-T, and calpastatin and increased calpain-1 autolysis and caspase-3 activity (P < 0.05). In addition, ultrasonicated steaks had greater levels of cytosolic calcium and reactive oxygen species and lower mitochondrial oxygen consumption rate (P < 0.05). These data indicate that improved beef tenderness following ultrasonication is, in part, a function of increased calpain-1 and caspase-3 activities, potentially by elevating cytosolic calcium and inducing mitochondrial dysfunction, respectively., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The purpose of this study was to develop an in situ model for dark cutting beef. Iodoacetic acid (IAA) was injected at different concentrations (0, 0.625, 1.25, 2.5, 3.75, 5, or 10 μmol/g of muscle) into pre-rigor bovine longissimus thoracis et lumborum (LTL) muscle samples, and pH and color were evaluated over a 48 h period. Injection of IAA blunted muscle pH decline and lowered lightness (L*), redness (a*), and yellowness (b*) values (P ≤ 0.05) in a concentration dependent fashion. In a follow-up study, LTL muscle samples were injected with 5 μmol IAA/g of muscle to test whether IAA maintains its effect over a 336 h post-mortem storage period. In addition to inhibiting pH decline and decreasing color values, IAA increased LTL muscle water holding capacity (WHC) and firmness (P ≤ 0.05) throughout the 336 h post-mortem storage period. Collectively, these data suggest that pre-rigor injection of IAA generates beef with dark cutting-like characteristics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Our objective was to investigate possible differences in muscle fiber characteristics of beef longissimus lumborum (LL) steaks varying in tenderness (very tender vs. intermediate tender). Therefore, the relative abundance of myosin heavy chain (MHC) isoforms and activity/abundance of several glycolytic and oxidative enzymes were compared between the two steak groups. Greater (P < 0.05) content of MHC type IIa (MHC-IIa) and activities of phosphofructokinase (PFK) and glycogen phosphorylase (GP) were observed in the very tender steaks. Conversely, intermediate tender steaks had greater (P < 0.05) contents of MHC type I (MHC-I) and succinate dehydrogenase (SDH) and greater citrate synthase (CS) activity. Increased tenderness in the very tender steaks was associated with greater (P < 0.05) proteolysis as evaluated by desmin and troponin-T degradation. Further, mitochondrial calcium uniporter (MCU) was lower (P < 0.05) in the very tender steaks than steaks of intermediate tenderness. Collectively, shifting muscle characteristics toward a more glycolytic type appears to positively impact postmortem proteolysis and tenderization., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The purpose of this study was to examine the role of mitochondria in postmortem calcium homeostasis and its effect on proteolysis and tenderness. We hypothesized that mitochondria buffer cytosolic calcium levels and delay the activation of calpain-1 and subsequently the development of meat tenderness. To test this hypothesis, pre-rigor bovine longissimus thoracis et lumborum muscle samples were injected with DS16570511 to inhibit mitochondrial calcium uptake. Free calcium, tenderness, texture profile analysis (TPA), calpain-1 activity, and proteolysis were evaluated over a 336 h aging period. Inhibition of mitochondrial calcium uptake increased (P < .0001) cytosolic calcium concentration and calpain-1 autolysis and activity at 24 h compared to control steaks. Further, tenderness and TPA at 168 and 336 h, calpastatin degradation at 24 h, and proteolysis at 168 h were all enhanced (P < .05) in the treated steaks. Collectively, these data indicate that inhibition of mitochondrial calcium uptake can enhance postmortem proteolysis and tenderization through an early activation of calpain-1., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)