Biochemical and physiological mechanisms of creatine kinase release from avian skeletal muscle during acute stress

Marked changes in the plasma activities of the intracellular enzyme creatine kinase (CK) have been observed in broiler chickens subjected to a range of stressful stimuli. Using a novel anion exchange chromatography technique to separate the various CK isoenzymes, it was demonstrated that the skeletal muscle form of CK (MM-CK) was predominant in plasma (96.8%) and that its activity increased 65.4% (p<0.05) in response to an episode of acute heat stress (2 h, 32°C/80% relative humidity). An investigation of mechanisms that might mediate the release of CK from this tissue was undertaken using a novel validated isolated in vitro chicken skeletal muscle preparation (m. tensor patigialis) and incubation system. CK loss from the muscle preparation under optimised control incubation conditions (150 min at 41.5°C, pH 7.4) was negligible, constituting less than 0.3% of total muscle CK content. Incubation at 45°C had no effect on the rate of CK loss above that observed under control conditions. Incubation under conditions of reduced O₂ availability (anoxia) was without effect on CK loss from the preparation. Limiting ATP availability by impairing muscle mitochondrial oxidative metabolism induced a 4-fold increase (p<0.05) in the rate of loss of CK. Promoting external ionic calcium (Ca²⁺) entry into the muscle cells using 4 Br-A23187 calcium ionophore (25µM) caused a 60-fold increase (p<0.001) in the release rate of CK. Incubation with the ionophore induced a significant increase in Ca²⁺accumulation (79.7%; p<0.05) as measured by the uptake of radio-labelled ⁴⁵calcium and was associated with a 8.6-fold greater total loss of CK. The results suggest that the entry of external Ca²⁺ into muscles represents an important initial event in the development of muscle membrane damage and subsequent CK loss. It is proposed that CK loss occurs as a consequence of Ca²⁺ mediated alterations in muscle cell membrane permeability, possibly caused by Ca²⁺-activated phospholipase A₂ (PLA2). In support of this suggestion, incubation with 25 µM Ro-31-4493/001 (an inhibitor of PLA₂ activity) significantly decreased CK loss by 44.6% (p<0.05) in muscle incubated with 4BrA23187 (5 µM). The role of elevated external sodium (Na⁺) entry into skeletal muscle was investigated as a potential mechanism for mediating trans-membrane changes in Ca²⁺ flux in vivo. Concentration-dependent increases in ⁴⁵calcium uptake and total CK release were observed in muscle incubated with monensin Na⁺ ionophore. The largest responses occurring at monensin concentrations of 100 |nmol or greater, corresponding to a 49.0% increase (p<0.05) in ⁴⁵calcium uptake and a 2.4-fold increase (p<0.001) in total CK release. The incubation of muscles in medium containing an elevated external Na⁺ concentration, and the prevention of Na⁺ extrusion significantly augmented increases in ⁴⁵calcium uptake and CK loss in Na⁺ loaded muscle cells, possibly through the action of Na⁺/Ca²⁺ exchange. Direct inhibition of the Na⁺/Ca²⁺ exchanger using amiloride (1 mM) had no effect on muscle ⁴⁵calcium uptake or CK efflux where active Na⁺ extrusion via Na⁺/K⁺ exchange had been prevented. However, reducing external Ca²⁺ availability for Na⁺/Ca²⁺ exchange in monensin treated muscles significantly reduced ⁴⁵calcium uptake (37.8%, p<0.05) but paradoxically produced a 84.5% increase (p<0.001) in total CK release, thus implicating the possible involvement of Ca²⁺ from some intracellular source, such as the sarcoplasmic reticulum (SR). Mobilisation and redistribution of Ca²⁺ from intracellular stores produced a significant increase in CK release (2.7-fold, p<0.001) in the absence of external Ca²⁺ entry. Inhibition of the release of Ca²⁺ from the SR through ryanodine-sensitive Ca²⁺ channels using dantrolene (25 mM) significantly reduced the release of CK (32.2%, p<0.05) in monensin treated muscles.

In complementary in vivo studies, birds treated with dantrolene (2.5 mg/kg) demonstrated no increase in plasma CK activity following exposure to acute heat stress. This contrasted with a 70.8% increase in plasma CK activity exhibited in vehicle treated controls exposed to the same conditions. It is therefore proposed that acute heat stress induces Ca²⁺ release from the SR in chicken skeletal muscle through the ryanodine-sensitive Ca²⁺ channels which in combination with the entry of external Ca²⁺ possibly through Na⁺/Ca²⁺ exchange increases PLA₂ mediated alterations in sarcolemmal permeability which facilitates the intracellular loss of CK.