«Research Institute for the Biology of Farm Animals, Dummerstorf, Germany ULRICH KÜCHENMEISTER and GERDA KUHN Regulation of intracellular Ca2+ ...»
Arch. Tierz., Dummerstorf 46 (2003) 5, 445-454
Research Institute for the Biology of Farm Animals, Dummerstorf, Germany
ULRICH KÜCHENMEISTER and GERDA KUHN
Regulation of intracellular Ca2+ concentration and meat quality in
Dedicated to Prof. Dr. Dr. h. c. Klaus Ender on the occasion of his 60th birthday
The meat quality of pigs is essentially dependent on the rate and intensity of energy metabolism after slaughter.
The major cellular processes in muscle cells are regulated by the Ca2+ concentration in the cytoplasm, stimulating different energy consuming ATPases. The most essential regulator of the Ca2+ concentration is the sarcoplasmic reticulum (SR) with its membranes, the SR Ca2+ ATPase (Ca2+ pump), and the calcium release channel (CRC). Defects of one or more of these elements will be of influence on the metabolism and ultimately on the meat quality. This is widely investigated in pigs with a mutated CRC. However, pigs without a mutation in the CRC also show a wide variability in their meat quality, dependent on other factors e.g. stress or season of the year. The variability in meat quality in these “normal” pigs is at least partly a result of differences in SR Ca2+ transport and the resulting metabolism.
Key Words: pig, calcium, sarcoplasmic reticulum, meat quality Zusammenfassung Titel der Arbeit: Regulation der intrazellulären Ca2+ Konzentration und die Fleischqualität beim Schwein Die Fleischqualität von Schweinen hängt wesentlich von der Geschwindigkeit des Energieumsatzes nach dem Schlachten ab. Die zellulären Prozesse werden vorrangig durch die Ca2+ Konzentration im Zytoplasma geregelt wobei verschiedene energieverbrauchende ATPasen aktiviert werden. Der hauptsächliche Regulator für die intrazelluläre Ca2+ Konzentration ist das sarkoplasmatische Retikulum (SR) mit dessen Membranen, Kalziumkänalen (CRC) und SR Ca2+ ATPasen (Ca2+ Pumpen). Defekte an einem oder mehreren dieser Elemente führen zur Beeinflussung des Energiemetabolismus und schließlich der Fleischqualität. Nachweise erfolgten umfassend für Schweine mit mutiertem CRC. Allerdings weisen auch Schweine mit nichtmutiertem CRC eine hohe Variabilität in der Fleischbeschaffenheit auf, beeinflusst durch andere Faktoren wie z.B. nach Einwirkung von Stressoren oder die Jahreszeit. Auch bei diesen Tieren ergibt sich ein Zusammenhang zwischen Ca2+ Transport, Metabolismus und Fleischqualität.
Schlüsselwörter: Schwein, Calcium, sarkoplasmatisches Retikulum, Fleischqualität Introduction The pig breeding systems aiming for an increase of the lean meat percentage have been very successful in the preceding decades. However, parallel with an increased meat content there was an increasing part of carcasses showing inferior meat quality and also an increase in losses bydeath during transportation or other kinds of stresses.
A major reason for this relationship has been shown to be a mutated calcium release channel (CRC) of the sarcoplasmic reticulum (SR) (MCLENNAN and PHILLIPS, 1992). It turned out, that this mutation causes an insufficient regulation of the intracellular Ca2+ concentration, activating metabolic processes not only in the live pig muscle, but also after slaughtering the animals. Currently all major pig producing KÜCHENMEISTER; KUHN: Regulation of intracellular Ca2+ concentration and meat quality in pigs nations are taking measures to eliminate this mutation. This will be combined (at least temporarily) with a decreasing lean meat content, however, there are also animal welfare demands to be taken into account because mutated pigs are prone for health problems (MARTENS, 1998). With the elimination of this mutation the question arises: what are the causes for the wide variability of meat quality which occurs even in carcasses of normal pigs. A lot of work has been done to investigate the influence of different kinds of stress on meat quality. The results are not unequivocal, and now there are papers dealing with more basic approaches to this problem (ALLISON et al., 2002; HOPKINS and THOMPSON, 2002; SCHÄFER et al., 2002 ). The impact of the SR Ca2+ transport on meat quality in normal pigs has been the subject of only a small number of investigations. Some basics and some results are summarized in this paper.
Regulation of intracellular Ca2+ concentration, muscle activation, and energy metabolism The most prominent energy consumption in the muscle derives from contraction work.
Contractions are initiated by cell membrane depolarisations (action potential). By a not yet fully understood mechanism, the depolarisation of the transverse tubuli of the membrane is followed by a calcium release out of the sarcoplasmic reticulum (SR) through the calcium release channel (CRC). The increasing intracellular Ca2+ concentration activates the actin-myosin complex to contract, consuming energy by hydrolysing ATP. At the same time the increased intracellular calcium level initiates the Ca2+ ATPase (Ca2+ pump) of the SR to sequester Ca2+ back into the SR, also hydrolysing ATP.
Fig. 1: Energy consuming processes in the live muscle and after slaughter: Activated by membrane depolarisation the dihydropyridine receptor (integrated in the transverse tubule) opens the calcium release channel, releasing Ca2+ into the cytosol. The increased intracellular Ca2+ concentration activates the actin-myosin filaments to contract, thereby consuming ATP. In the process of relaxation different ATPases are activated: Na+K+ ATPase to restore the membrane potential, the sarcolemmal and the SR Ca2+ ATPases to lower the intracellular Ca2+ concentration. After slaughter these processes do not stop, the energy consumption by the different ATPases continues until ATP depletion. Whereas in the live animal the mitochondria produce ATP, after slaughter the mitochondrial ATPase can split ATP (Energie verbrauchende Prozesse in lebenden Muskel und nach dem Schlachten: Der durch die Depolarisierung aktivierte Dihydropyridinrezeptor öffnet den Kalziumkanal (calcium release channel). Die dadurch erhöhte intrazelluläre Ca2+ Konzentration führt zu einer Kontraktion der Aktin-Myosin Filamente, verbunden mit ATP Arch. Tierz. 46 (2003) 5 Verbrauch. Zur Muskelrelaxion werden verschiedene ATPasen aktiviert: Na+-K+ ATPase zur Membranpolarisierung, Sarkolemm- und SR Ca2+ ATPasen zur Absenkung der intrazellulären Ca2+ Konzentration. Nach der Schlachtung setzen sich die energieverbrauchenden Vorgänge bis zur ATP Erschöpfung fort. Die im lebenden Tier energieproduzierenden Mitochondrien wirken im Schlachtkörper als ATPase und tragen zum Energieverbrauch bei.) Figure 1 shows schematically elements of a muscle cell involved in the contractionrelaxation circle, including different ATP consuming ATPases. The largest energy consumer with about 70% is the myofibrillar ATPase (SPRIET, 1989), followed by the SR Ca2+ ATPase with about 30%. These relations depend on the muscle activity. The energy consumption by the SR Ca2+ ATPase at rest is partly used for “Ca2+ -cycling” (SIMONIDES and VAN HARDEVELD, 1988) and partly for heat production (DUMONTEIL et al., 1993).
In the live animal the ATP concentration is restored in different ways:
- in the aerobic way by oxidative phosphorylation by mitochondria
- by conversion of ADP to ATP by adenylate kinase
- ADP phosphorylation by creatine kinase from creatine phosphate
- by glycolysis.
After slaughter the oxygen delivery ceases whereas most biochemical processes in the muscle proceed post mortem. Following a short time interval the only way to regenerate ATP is by glycolysis.
Investigations of muscle Ca2+ transport of the sarcoplasmic reticulum Investigations on the SR Ca2+ transport are supported by a very comfortable characteristic of the phospholipid cell membranes. By homogenising muscle tissue the membranes are destroyed. However, the SR membranes readjust themselves to vesicles in a way that the Ca2+ pumps are so arranged that the Ca2+ will be pumped into the vesicle. About 50% of the produced SR-vesicles contain CRC, derived from the terminal cisterns of the SR, and about 50 % of the vesicles come from the longitudinal SR, not containing calcium channels (KÜCHENMEISTER et al., 1999a;
O'BRIEN and LI, 1997). Generally, two ways for determining the Ca2+ uptake and the activity of the SR Ca2+ ATPase are used. Firstly, the SR is isolated by several steps of centrifugations and the more or less pure SR is used for the measurement procedures.
This way is time consuming and includes some uncertainties in the purity of the isolates (purity to be determined by measuring marker enzymes), also dependent on the degree of protein denaturation at the time of sampling the muscle. An easier way is to use the muscle homogenates directly and inhibit unwanted enzyme activities (e.g.
mitochondrial ATPase, myofibrillar ATPase, sarcolemmal ATPases) by including biochemical inhibitors in the measuring medium. This has become the mostly used method for determining Ca2+ uptake or activity of ATPase (CHEAH et al., 1990;
KÜCHENMEISTER et al., 1999a; O'BRIEN and LI, 1997; SIMONIDES and VAN HARDEVELD, 1990). The CRC (ryanodine receptor) can biochemically be closed or opened by incubating the muscle homogenate/isolated SR for different time intervals and with different concentrations of ryanodine (FEHER and LIPFORD, 1985;
NAGASAKI and FLEISCHER, 1988; O'BRIEN and LI, 1997). To determine the integrity of the SR membrane, the activity of the Ca2+ ATPase has to be measured once with a Ca2+ ionophore (e.g. A23187), and once without an ionophore in the KÜCHENMEISTER; KUHN: Regulation of intracellular Ca2+ concentration and meat quality in pigs measuring medium. The ratio of these two activities is a measure for the integrity of the membrane (BYRD et al., 1989).
Figure 2 depicts schematically a model of a SR vesicle with essential elements responsible for the Ca2+ transport: SR membrane, CRC, and SR Ca2+ ATPase. A disturbance of one or more of these elements results in a disturbed regulation of intracellular Ca2+ concentration.
These different possibilities leading to an increased (disturbed) intracellular Ca2+
- the efflux through the CRC is higher than the activity of the Ca2+ ATPase can cope with to sequester the Ca2+ into the SR.
- the Ca2+ ATPase is disturbed/inactivated and is unable to pump the Ca2+ back into the SR even with normal efflux.
- the phospholipid membrane is destroyed so that there is no way to hold the Ca2+ in the SR.
Fig. 2: Model of an isolated SR (sarcoplasmic reticulum) vesicle, consisting of SR membrane, calcium release channel (CRC; ryanodine-receptor RyR), SR Ca2+ ATPase (Ca2+ pump) (Modell eines isolierten SR (sarkoplasmatisches Retikulum) Vesikels, bestehend aus der SR-Membran, dem Calcium Release Channel (CRC; Ryanodin-Rezeptor RyR) und der SR Ca2+ ATPase (Ca2+ Pumpe)) Mutation of the CRC, Ca2+ transport and meat quality Especially in pigs with a high muscle meat content it turned out that there is a defect in the calcium release channel (CRC) causing inferior meat quality (MCLENNAN and PHILLIPS, 1992; REMPEL et al., 1995). By a mutation in the CRC-protein the efflux of Ca2+ is facilitated compared to a normal CRC. At rest and without additional stress the appearance and the muscle contractions of pigs with mutated CRC are inconspicuous. However, physical stress like transportation, stunning, mixing with unknown pigs etc. can cause that the Ca2+ efflux can not be compensated by the Ca2+ pump and will result in a lasting increased intracellular Ca2+ concentration and lasting muscle contractions. These effects do not only happen in the live animal but also after Arch. Tierz. 46 (2003) 5 slaughter in the carcass, often evidenced by muscle twitches up to one hour p.m. and an early rigor.
Investigations with normal (MHR = malignant hyperthermia resistant = normal CRC) and mutated (MHS = malignant hyperthermia susceptible = mutated CRC) pigs have consistently shown the development of inferior meat quality of MHS pigs (KÜCHENMEISTER et al., 1999a; REMPEL et al., 1995). These differences are related to changes in the SR Ca2+ transport. Immediately after slaughter the SR Ca2+ uptake did not differ between MHS and MHR muscles, however in the time course p.m. the decrease of uptake occurs at a much higher rate in MHS pigs (Fig. 3). Four hours p.m. the uptake rate has been shown to be decreased (in vitro) to about one third in MHS, but only by about 15% in MHR (KÜCHENMEISTER et al., 1999a). Though the CRC can biochemically be opened and closed even 4 h p.m. (KÜCHENMEISTER et al., 1999a), without manipulating the CRC of MHS muscle is (in vitro) almost fully open. Interestingly, the SR Ca2+ ATPase activity has been shown to be higher immediately after slaughter in MHS muscle, but the activity decreased much faster than in MHR muscle (Fig. 4), showing similarities with the shape of uptake decrease in MHS as well as in MHR (KÜCHENMEISTER et al., 1999a).
Fig. 3: Rate of Ca2+ uptake of longissimus homogenates of malignant hyperthermia susceptible (MHS) (––––) and malignant hyperthermia resistant (MHR) (– – –) pigs and different states of the calcium release channel (CRC): ( ), closed CRC; (▲), basic CRC (Rate der Ca2+ Aufnahme durch das Homogenat vom m. longissimus dorsi von (MHS) (––––) und (MHR) (– – –) Schweinen bei verschiedenen Zuständen des CRC: ( ), geschlossener CRC; (▲), unbeeinflusster CRC) Fig. 4: SR Ca2+ ATPase activity of longissimus homogenates of MHS and MHR pigs, determined with inclusion of Ca2+ ionophore A23187 (Aktivität der SR Ca2+ ATPase des Homogenates vom m. longissimus dorsi von MHS und MHR Schweinen, bestimmt unter Einschluss des Ionophor A23187) KÜCHENMEISTER; KUHN: Regulation of intracellular Ca2+ concentration and meat quality in pigs Besides an inactivation of the ATPase, there seems to be a change in the permeability to Ca2+ in the membrane of the SR with a greater permeability in MHS samples (KÜCHENMEISTER et al., 1999b). Investigations with heterozygous pigs indicate that the meat quality as well as the Ca2+ transport are intermediate to those of homozygous negative and homozygous positive pigs (O'BRIEN and LI, 1997;
SHOMER et al., 1995; CHEAH et al., 1995).