Michael Kristensen

Lactate and force production in skeletal muscle

Lactic acid accumulation is generally believed to be involved in muscle fatigue. However, one study reported that in rat soleus muscle (in vitro), with force depressed by high external K+ concentrations a subsequent incubation with lactic acid restores force and thereby protects against fatigue. However, incubation with 20 mm lactic acid reduces the pH gradient across the sarcolemma, whereas the gradient is increased during muscle activity. Furthermore, unlike active muscle the Na+–K+ pump is not activated. We therefore hypothesized that lactic acid does not protect against fatigue in active muscle. Three incubation solutions were used: 20 mm Na‐lactate (which acidifies internal pH), 12 mm Na‐lactate +8 mm lactic acid (which mimics the pH changes during muscle activity), and 20 mm lactic acid (which acidifies external pH more than internal pH). All three solutions improved force in K+‐depressed rat soleus muscle. The pH regulation associated with lactate incubation accelerated the Na+–K+ pump. To study whether the protective effect of lactate/lactic acid is a general mechanism, we stimulated muscles to fatigue with and without pre‐incubation. None of the incubation solutions improved force development in repetitively stimulated muscle (Na‐lactate had a negative effect). It is concluded that although lactate/lactic acid incubation regains force in K+‐depressed resting muscle, a similar incubation has no or a negative effect on force development in active muscle. It is suggested that the difference between the two situations is that lactate/lactic acid removes the negative consequences of an unusual large depolarization in the K+‐treated passive muscle, whereas the depolarization is less pronounced in active muscle.

Exercise-induced regulation of phospholemman (FXYD1) in rat skeletal muscle: implications for Na+/K+-ATPase activity

Background:u2002 Na+/K+‐ATPase activity is upregulated during muscle exercise to maintain ionic homeostasis. One mechanism may involve movement of α‐subunits to the outer membrane (translocation).

Potassium‐transporting proteins in skeletal muscle: cellular location and fibre‐type differences

Potassium (K+) displacement in skeletal muscle may be an important factor in the development of muscle fatigue during intense exercise. It has been shown in vitro that an increase in the extracellular K+ concentration ([K+]e) to values higher than approx. 10u2003mm significantly reduce force development in unfatigued skeletal muscle. Several in vivo studies have shown that [K+]e increases progressively with increasing work intensity, reaching values higher than 10u2003mm. This increase in [K+]e is expected to be even higher in the transverse (T)‐tubules than the concentration reached in the interstitium. Besides the voltage‐sensitive K+ (Kv) channels that generate the action potential (AP) it is suggested that the big‐conductance Ca2+‐dependent K+ (KCa1.1) channel contributes significantly to the K+ release into the T‐tubules. Also the ATP‐dependent K+ (KATP) channel participates, but is suggested primarily to participate in K+ release to the interstitium. Because there is restricted diffusion of K+ to the interstitium, K+ released to the T‐tubules during AP propagation will be removed primarily by reuptake mediated by transport proteins located in the T‐tubule membrane. The most important protein that mediates K+ reuptake in the T‐tubules is the Na+,K+‐ATPase α2 dimers, but a significant contribution of the strong inward rectifier K+ (Kir2.1) channel is also suggested. The Na+, K+, 2Cl− 1 (NKCC1) cotransporter also participates in K+ reuptake but probably mainly from the interstitium. The relative content of the different K+‐transporting proteins differs in oxidative and glycolytic muscles, and might explain the different [K+]e tolerance observed.

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na+–K+ pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na+–K+ pump α2 subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the α2 subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na+–K+ pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the α1 subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the α2, the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the α subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.

Preserved arterial flow secures hepatic oxygenation during haemorrhage in the pig

1 This study examined the extent of liver perfusion and its oxygenation during progressive haemorrhage. We examined hepatic arterial flow and hepatic oxygenation following the reduced portal flow during haemorrhage in 18 pigs. The hepatic surface oxygenation was assessed by near‐infrared spectroscopy and the hepatic metabolism of oxygen, lactate and catecholamines determined the adequacy of the hepatic flow. 2 Stepwise haemorrhage until circulatory collapse resulted in proportional reductions in cardiac output and in arterial, central venous and pulmonary wedge pressures. While heart rate increased, pulmonary arterial pressure remained stable. In addition, renal blood flow decreased, renal vascular resistance increased and there was elevated noradrenaline spill‐over. Further, renal surface oxygenation was lowered from the onset of haemorrhage. 3 Similarly, the portal blood flow was reduced in response to haemorrhage, and, as for the renal flow, the reduced splanchnic blood flow was associated with an elevated noradrenaline spill‐over. In contrast, hepatic arterial blood flow was only slightly reduced by haemorrhage, and surface oxygenation did not change. The hepatic oxygen uptake was maintained until the blood loss represented more than 30 % of the estimated blood volume. At 30 % reduced blood volume, hepatic catecholamine uptake was reduced, and the lactate uptake approached zero. 4 Subsequent reduction of cardiac output and portal blood flow elicited a selective dilatation of the hepatic arterial vascular bed. Due to this dilatation liver blood flow and hepatic cell oxygenation and metabolism were preserved prior to circulatory collapse.

Na + ,K + -ATPase Na + Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na+/K+-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na+,K+-ATPase activity, and the Na+ affinity of Na+,K+-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na+ affinity was higher (Km lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na+,K+-ATPase isoform analysis implied that heterodimers containing the β1 isoform have a higher Na+ affinity than heterodimers containing the β2 isoform. Immunoprecipitation experiments demonstrated that dimers with α1 are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α2 isoform with ouabain suggested that heterodimers containing the α1 isoform have a higher Na+ affinity than heterodimers containing the α2 isoform. The estimated Km values for Na+ are 4.0, 5.5, 7.5 and 13xa0mM for α1β1, α2β1, α1β2 and α2β2, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na+,K+-ATPase at physiological Na+ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.

Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1α.

Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1α are underlying factors in long-term regulation of Na,K-ATPase isoform (α,β and PLM) abundance and Na+ affinity. Repeated treatment of mice with the AMPK activator AICAR decreased total PLM protein content but increased PLM phosphorylation, whereas the number of α- and β-subunits remained unchanged. The Km for Na+ stimulation of Na,K-ATPase was reduced (higher affinity) after AICAR treatment. PLM abundance was increased in AMPK kinase-dead mice compared with control mice, but PLM phosphorylation and Na,K-ATPase Na+ affinity remained unchanged. Na,K-ATPase activity and subunit distribution were also measured in mice with different degrees of PGC-1α expression. Protein abundances of α1 and α2 were reduced in PGC-1α +/− and −/− mice, and the β1/β2 ratio was increased with PGC-1α overexpression (TG mice). PLM protein abundance was decreased in TG mice, but phosphorylation status was unchanged. Na,K-ATPase Vmax was decreased in PCG-1α TG and KO mice. Experimentally in vitro induced phosphorylation of PLM increased Na,K-ATPase Na+ affinity, confirming that PLM phosphorylation is important for Na,K-ATPase function. In conclusion, both AMPK and PGC-1α regulate PLM abundance, AMPK regulates PLM phosphorylation and PGC-1α expression influences Na,K-ATPase α1 and α2 content and β1/β2 isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism.