Staff PhD

Cellular stress and metabolic responses to ante- and post mortem stress factors elucidated in primary porcine muscle cell cultures using confocal microscopy and NMR-based metabolic profiling

Ida Krestine Straadt

Background

The handling of pigs prior to slaughter is critical for obtaining good pork quality. It is recognized that stress exposure during this process is a key factor causing huge variations in meat quality, which cause economic loss for the meat industry. Stress exposure will affect the glycogen content as well as the rate of glycolysis and hence the pH development post mortem, and it has been found that pH and temperature measured early post mortem are good indicators for the subsequent meat quality development. However, the basic mechanisms at the cellular level involved in the post mortem processes are still far from fully understood. An understanding of how the muscle cells respond to stress exposure before slaughter as well as the post mortem conditions would be extremely helpful for the overall understanding of how these conditions affect the meat quality. An increased knowledge of these basic cellular responses would contribute to optimising the handling of slaughter pigs, thereby ensuring an optimal meat quality and reduce economic loss in the pork industry.

The cellular events that are initiated in the muscle cells by slaughtering are complex. Slaughter pigs are withdrawn from feed prior to slaughtering and furthermore exposed to stressful conditions related to transport and slaughter, which will activate a release of stress hormones (adrenaline/ cortisol). Because of these conditions the muscle cells are in a state of starvation, and therefore dependent on endogenous fat oxidation and high activity in the mitochondrial respiratory chain, which is the site of oxidative stress formation, and may thus increase stress susceptibility. In addition, due to termination of the blood supply, the situation post mortem is associated with a combined energy and oxygen deprivation (anoxia/ischemia).

Using primary porcine muscle cell cultures, the present study aims at investigating the cellular responses triggered by these events. Satellite cells are isolated from pigs and cultured to form fully differentiated poly-nuclear myotubes. Thus, these cells are not subjected to any form of manipulation and is regarded the closest possible model system facilitating strictly controlled conditions and thereby enable to study these mechanisms comprehensively.

Aim

The overall aim of this project is to elucidate stress responses in primary porcine muscle cell cultures using confocal laser scanning microscopy (CLSM) techniques combined with nuclear magnetic resonance (NMR)-based metabolic profiling in order to understand cellular responses to conditions analogue to the ante- and post mortem conditions. The combination of CLSM and NMR will enable an elucidation of both cellular stress response and the subsequent metabolic response, hereby providing a new strong tool in our understanding of ante- and post mortem processes determining meat quality development.

Research Outline

The objective of this project is to elucidate the response of primary porcine myotube cultures to stressors analogue to ante- and post mortem conditions by developing and using an integrated tool consisting of CLSM and NMR-based metabolic profiling. Additionally various molecular biological methods will be used to confirm the CLSM investigations.

The objective will be achieved through the following activities:

1. Establishing large homogeneous cultures of primary porcine cells isolated from genetically closely related pigs.
2. Using CLSM and NMR-based metabolic profiling in the investigation of the response of primary porcine myotubes to various stressors simulating:
   1. H2O2 exposure as a model for extreme oxidative stress.
   2. A systemic hormone response to stress exposure i.e. adrenaline and cortisol.
   3. An increased temperature as a consequence of the stress exposure prior to slaughter.
   4. Oxygen deprivation as a response to bleeding.
   5. Glucose deprivation as a response to bleeding.
   6. A decreased temperature post mortem.
3. The presence and regulation of stress proteins will be investigated with qPCR, and semi-quantitative or quantitative methods such as Western blot and Luminex, respectively, may be used to quantify the stress proteins.

Various combinations of the stressors will be relevant in the stress simulation model. The working hypothesis is that the combined effects of anoxia and state of starvation in the muscle cells will reduce or block pyruvate oxidation by pyruvate dehydrogenase, facilitating conversion of lactate and the release of H+ from ATP hydrolysis. The release of H+ causes cellular acidification. The ATP hydrolysis activates the mitochondrial system and cellular energy metabolism. The regulation mechanisms of intracellular pH involve ionic exchange over the cell membrane, and therefore perturbations in intra-and extra-cellular ion localisation may occur. Moreover, in attempt to maintain cellular homestasis, the muscle cell will induce various specific heat shock proteins, this will especially occur when the muscle cell experience temperature perturbations as in the post mortem situation.

In summary the stressors are hypothesised to trigger:

i.Cellular acidification.

ii.Perturbations in intra-and extra-cellular ion localisation (Ca2+, Na+).

iii.Changes in mitochondrial activity.

iv.Expression of specific heat shock proteins.

v.Influence on cellular energy metabolism.

Cellular acidification:

In order to study the sub-cellular heterogeneity of intracellular H+ concentration triggered by the various stressors, the pHand a potential pH gradient in the cytosol of the primary porcine muscle cells will be measured using CLSM and pH indicator dyes. The pH can be quantitatively measured by use of dyes that are pH-sensitive within the physiological ranges.

Perturbations in intra-and extra-cellular ion localisation (Ca2+, Na+):

Intracellular calcium [Ca2+] will be determined using confocal microscopy together with two calcium-sensitive dyes [acetoxymethyl esters of fluo 3 (fluo 3-AM) and rhod 2 (rhod 2-AM)] to analyze the calcium distribution in primary porcine muscle cells as a function of the various stressors. By use of the two dyes and the argon laser beam at excitation wavelengths of 488 and 514 nm it is possible to follow the temporal changes of intracellular calcium concentration.

Changes in mitochondrial activity:

Confocal microscopy will be used to visualize mitochondrial activity. The two fluorophores Mito Tracker green-FM (MTG) and tetramethylrhodamine methylester (TMRM), which selectively label mitochondria, and JC-1 which measures mitochondrial transmembrane potential will be used. Furthermore, a possible change in the rate of apoptosis and a potential relation to mitochondria activity under stress conditions can be followed e.g. by use of the dye YO-PRO-1.

Expression of specific heat shock proteins:

By means of confocal microscopy the expression of specific heat shock proteins, such as cytosolic proteins HSP70, HSP27 and HO1, mitocondrial proteins HSP60, ClpP, Lon and the endoplasmic reticulum HSP70 homologue GRP78, will be elucidated in primary porcine muscle cells upon induction of the various stressors. During stress, changes in the localisation of stress proteins has been observed, hence, the cellular localisation of the heat shock proteins will be emphasised.

Influence on cellular energy metabolism:

The effect of the various stressors on cellular energy metabolism in primary porcine muscle cells will be investigated by NMR-based metabolic profiling. Phosphorus metabolism will be studied using 31P NMR spectroscopy, glucose metabolism will be studied using 13C NMR spectroscopy, and metabolism involving fatty acids, lactate and creatine will be studied using 1H NMR spectroscopy.