DEFINING THE ROLE OF MITOCHONDRIA IN FRESH MEAT QUALITY DEVELOPMENT.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1011814
• Grant No.: 2017-67017-26470
• Cumulative Award Amt.: $454,741.00
• Proposal No.: 2016-09120
• Project Start Date: Apr 1, 2017
• Project End Date: Mar 31, 2021
• Grant Year: 2017
• Program Code: [A1361]- Improving Food Quality

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Animal and Poultry Sciences

Non Technical Summary
The long-range goal of our program is to improve the quality and consistency of fresh meat produced in the US. Quality is largely predicated on the biochemistry occurring in the muscle during its conversion to meat. While acidification of muscle via the biochemistry occuringpostmortem is indisputable, we do not know howoxygen andmitochondriaaffectthe process. This impact iswhy energy in the muscle is onlysomewhat predictive of ultimate pH and fresh meat quality at 24 hr. the overall objective of this proposal is to define precisely the role mitochondria play in modulating metabolism in dying skeletal muscle and explore the ability of tissue oxygenation to predict meat quality. The central hypothesis for this research is that the variations in the rate and extent of postmortem metabolism and meat quality may be more thoroughly explained, and predicted, by tissue oxygenation levels, thereby the abundance, function, and(or) dysfunction of mitochondria. The rationale for the proposed research is that once we have established and modeled more thoroughly the mechanism(s) responsible for controlling postmortem metabolism in muscle then we will be better positioned to: 1) predict quality development; 2) optimize the process; 3) segregate carcasses on quality 'on-line'; and(or) 4) provide breeders and genetic companies tools to select for, or against quality determinants, thereby increasing the quality and consistency of fresh meat. We have assembled a highly skilled, productive, interdisciplinary team with the experience to conduct the studies outlined herein.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	3440	1000	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
3440 - Meat, dairy cattle;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

meat

metabolism

mitochondria

muscle

Goals / Objectives
the overall objective of this proposal is to precisely define the role mitochondria play in modulating metabolism in dying skeletal muscle and to explore the ability of tissue oxygenation to predict meat quality. The central hypothesis for this research is that the variations in the rate and extent of postmortem metabolism and meat quality may be more thoroughly explained, and predicted, by tissue oxygenation levels, thereby the abundance, function, and(or) dysfunction of mitochondria. We have based this hypothesis on mounting evidence from our laboratory and across the globe. We will accomplish the overall objective of this application with the following specific AIMS:Explain the biochemical basis for increased glycolytic flux in muscle containing elevated mitochondria. The working hypothesis for this AIM is that mitochondria play a significant role on glycolysis by altering the oxidation-reduction potential of the environment. Determine mitochondrial function under simulated and actual postmortem conditions.The working hypothesis for this AIM is that reduced or dysfunctional mitochondria lead to increased anaerobic glycolysis. Develop a kinetic model of postmortem energy metabolism that includes contribution of mitochondria.The working hypothesis for this AIM is mitochondria contribute significantly to the conversion of muscle to meat and models including their contribution will better predict meat quality development.

Project Methods
Mitochondria from porcine longissimus muscle will be used in the in vitro system to determine how they increase glycolytic flux. Because of its repeatability, the modified Scopes system will serve as our bioassay. First, we will test whether mitochondria need to be functioning. This will be accomplished by mechanically disrupting mitochondria. If disrupted mitochondria produce the same effect, we will then test whether the effect resides in the matrix (soluble) or is associated with the membranes. This will be accomplished by centrifuging mechanically disrupted mitochondria. We will also determine which specific component of the mitochondria is responsible.We will use two fast-glycolyzing pig models and a prominent problem in the pork industry to achieve our AIM.Halothane (HAL)-positive pigs - While we understand full-well that these genetics have been eliminated from most production operations, rapid pH declines and PSE-like pork is still evident in the industry (see attached letters). We know these pigs experience an aggressive pH decline early postmortem, mediated mostly by calcium dysregulation and this should be exploited, especially given our collective hypotheses. Further, we know that HAL-positive pigs have similar quantities of mitochondria compared to WT pigs, which is attractive for the purpose of this study. Moreover, HAL negative pigs will also be generated as part of this study. Normal pigs and mutated HAL pigs (20 ea) will be created, genotyped and reared according to industry standards. At about 120 kg, pigs will be harvested at the Virginia Tech Meat Science Center.Whole pork carcass electrical stimulation - We have shown that electrical stimulation (ES) of pork carcass results in a rapid pH declineagain most likely through increased calcium release, though not completely documented as such. Pork carcasses will be subjected to ES protocols known to elicit PSE pork. Carcasses from HAL-negative pigs will be pulsed with 500V, 26 sec on and 2 sec off within 3 min of exsanguination. Electrodes will be placed on the shoulder and the rail will be used as the ground. This procedure results in greater glycolytic flux early postmortem and PSE pork development.Muscle oxygenation, pH and metabolites will be measured in beef carcasses postmortem. Samples will be collected and mitochondria will be extracted and subjected to functional assays as outlinedusing an in vitro system. Metabolite analysis will also be evaluated on the same samples. Based on collected data and established literature for glycolysis, we will develop a kinetic model structure (a set of differential equations with to-be-determined parameters) involving the following Outputs (pH, oxygenation level and glycogen concentration and lactate) and Inputs (mitochondria concentration, initial glycogen concentration, initial oxygenation level and temperature). The first four inputs are measurable initial conditions or states of the muscle at harvest, and temperature can be controlled during the postmortem process. The optimized model will then be used to simulate and analyze the effects of mitochondria and other measurable variables on pH and oxygenation level.

Progress 04/01/17 to 03/31/21

Outputs
Target Audience:Academicians, pork producers, meat scientists, nutritionists, pig breeding companies, geneticisits. Changes/Problems:No changes or problems, though the modeling data has lagged publication. What opportunities for training and professional development has the project provided?Two PhD and two MS degree graduate students were partially educated with funding from this project. The two PHD students took positions at major land grant institutions and the two MS students work for major meat processing companies. How have the results been disseminated to communities of interest?Yes, all data have been publiished in scientific journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We showed that mitochondria shoulder (delayed glycolysis) glycolytic flux through glycolysis and that elements of mitochondria spectifically complex 5 are partialy responsible for the lowered ultimate pH of meat in Rendement Napole pig genetics. A kinetic model has been developed showing that phoshofructokinase is the main culprits of altered glycolytic flux.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Stufft, K.M., J. Elgin, B.A. Patterson, S.K. Matarneh, E.M. England, T.L. Scheffler, R.H. Preisser, H. Shi, E.W. Mills and D.E. Gerrard. 2017. Muscle characteristics only partially explain color variations in fresh hams. Meat Sci. 128:88-96.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: England, E.M., S.K. Matarneh, H. Shi, T.L. Scheffler, E.M. Oliver, E.T. Helm, and D.E. Gerrard. 2017. Chronic activation of AMP-activated protein kinase increases MCT2 and MCT4 expression in skeletal muscle of pigs. J. Anim. Sci. 95(8):3552-3562.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Matarneh, S.K., E.M. England, T.L. Scheffler, C.-N. Yen, S. Hao and D.E. Gerrard. 2017. A mitochondrial protein increases glycolytic flux in muscle postmortem. Meat Sci. 133:119-125.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Matarneh S.K., M. Beline, S. de Luz E Silva, H. Shi and D.E. Gerrard. 2017. Mitochondrial F1-ATPase extends glycolysis and pH decline in an in vitro model. Meat Sci. 137:85-91.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, H., T.L. Scheffler, D.E. Gerrard, M.R. Larsen and R. Lametsch. 2018. Quantitative proteomics and phosphoproteomics analysis revealed different regulatory mechanisms of Halothane and Rendement Napole genes in porcine muscle metabolism. J. Proteome Res. 3;17(8):2834-2849.
• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Wang, C. S.K. Matarneh, D. Gerrard and J. Tan. Modeling of Energy Metabolism and Analysis of pH Variations in Postmortem Muscle (Meat Science, submitted)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Matarneh, S.K, C-N. Yen, J. Bodmer, S.W. El-Kadi and D.E. Gerrard. 2020. Mitochondria influence glycolytic and tricarboxylic acid cycle metabolism under postmortem simulating conditions. Meat Sci.172:108316.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Matarneh, S.K., S.L. Silva and D.E. Gerrard. New Insights in Muscle Biology that Alter Meat Quality Annual Review of Animal Biosciences 2021 9:1, 355-377.

Progress 04/01/19 to 03/31/20

Outputs
Target Audience:Animal Scientists, meat scientists, swine breeding companies, pork production management, field Extension Educators and pork producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students were partically educated with project. Also, several undergraduates were exposed to the art of scientific discovery and associated methodologies. How have the results been disseminated to communities of interest?Data and information is in review for publication. What do you plan to do during the next reporting period to accomplish the goals?Continuing the modelling work, which has lagged in the process.

Impacts
What was accomplished under these goals? Mitochondria added to in vitro glycolyziing reactions lowered the enrichment of pyruvate and lactate and differentially influenced the enrichment of TCA cycle intermediates.Mitochondria isolated from different (red versuses white )musclespossessed enrichment profiles for metabolites in the TCA cycle. These results arenovel because they provide new evidence of mitochondrial functionality under conditions that simulate those found in postmortem muscle. These dataalso show thatmitochondrial enzymes alterpostmortem energy metabolism even if mitochondria were disrupted, arguing that mitochondria whether alive (functioning) or dead(non-functional) can impactthe transformation of muscle to meat, and resulting meat quality development.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: 130) Matarneh, S.K, C-N. Yen, J. Bodmer, S.W. El-Kadi and D.E. Gerrard. 2020. Mitochondria influence glycolytic and tricarboxylic acid cycle metabolism under postmortem simulating conditions. Meat Sci.172:108316

Progress 04/01/18 to 03/31/19

Outputs
Target Audience:Swine producers, hog processors, geneticists, nutritionist, extension educators, acamedicians, meat scientists and seed-stock producers. Changes/Problems:We have struggled with modelling work, partly because of the complexity of the factors impacting the biochemistry but also because many of these studies needed to be accomplished before we could model the process. What opportunities for training and professional development has the project provided?One graduate student (PhD) was partially educated with this work. Also, five undergraduates of various age were exposed to the process of science and the creation of knowledge. How have the results been disseminated to communities of interest?Publications have been published. Furthermore, capstone-basedactivities were presented locally. What do you plan to do during the next reporting period to accomplish the goals?Continue kenetic modelly work.

Impacts
What was accomplished under these goals? TheRendement Napole (RN) mutation or acid meat gene or hampshire effect affected theexpression of those proteins inmuscle related to mitochondrial function andenergy metabolism which is hallmark of this muscle, more oxidative in nature. Ahigh content of UDP-glucose pyrophosphorylase 2in RN pig muscle likely contributes to the high glycogen storage capacity.The halothane (HAL) mutation, on the other hand up-regulates the activation of proteins (phosphorylation)related to calcium signaling, muscle contraction, glycogen, glucose, and energy metabolism, and cellular stress.Increased Ca2+/calmodulin-dependent protein kinase IIin muscle of these pigs may act as a key regulator. These findings support two seperate mechanisms for creating adverse meat quality inRN and HAL pigs, both related tomuscle energy metabolism.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, H., T.L. Scheffler, D.E. Gerrard, M.R. Larsen and R. Lametsch. 2018. Quantitative proteomics and phosphoproteomics analysis revealed different regulatory mechanisms of Halothane and Rendement Napole genes in porcine muscle metabolism. J. Proteome Res. 3;17(8):2834-2849

Progress 04/01/17 to 03/31/18

Outputs
Target Audience:Pork producers, animal scientists, pork processing plants, swine breeding companies, and geneticists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several undergraduates (5) were exposed to science and the process of creating new information. Also, three graduate students were partially educated with studies outlined herein. How have the results been disseminated to communities of interest?Information has been published in scientific journals, and a number of seminars have been given at various universities during graduate student interviews. What do you plan to do during the next reporting period to accomplish the goals?Continue with AIMS 2 and 3 and continue to build the kinetic model for predicting postmortem metabolism.

Impacts
What was accomplished under these goals? Mitochondria remain functionally competent for some time postmortem. As such, even a small increment in oxidative phosphorylation peri-mortem could significantly improve maintenance of ATP levels compared to anaerobic glycolysis and extend glycolysis in dying pig muscle.In an in vitro model of postmortem metabolism in muscle, reactions without mitochondria or those containing inhibitors of oxidation phosphorylation exhibit more rapid ATP loss than reactions with mitochondria alone. We have shown thatmitochondrial fractions, specifically the F¬1 ATPase, extend postmortem glycolysis and pH decline in vitro and partially explains the low pH formed in pigs harboring the hampshire effect.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Stufft, K.M., J. Elgin, B.A. Patterson, S.K. Matarneh, E.M. England, T.L. Scheffler, R.H. Preisser, H. Shi, E.W. Mills and D.E. Gerrard. 2017. Muscle characteristics only partially explain color variations in fresh hams. Meat Sci. 128:88-96.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: England, E.M., S.K. Matarneh, H. Shi, T.L. Scheffler, E.M. Oliver, E.T. Helm, and D.E. Gerrard. 2017. Chronic activation of AMP-activated protein kinase increases MCT2 and MCT4 expression in skeletal muscle of pigs. J. Anim. Sci. 95(8):3552-3562.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Matarneh, S.K., E.M. England, T.L. Scheffler, C.-N. Yen, S. Hao and D.E. Gerrard. 2017. A mitochondrial protein increases glycolytic flux in muscle postmortem. Meat Sci. 133:119-125.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Matarneh S.K., M. Beline, S. de Luz E Silva, H. Shi and D.E. Gerrard. 2017. Mitochondrial F1-ATPase extends glycolysis and pH decline in an in vitro model. Meat Sci. 137:85-91.