Antarctic teleosts with and without hemoglobin behaviorally mitigate deleterious effects of acute environmental warming.

Recent studies forecast that many ectothermic animals, especially aquatic stenotherms, may not be able to thrive or even survive predicted climate change. These projections, however, generally do not call much attention to the role of behavior, an essential thermoregulatory mechanism of many ectotherms. Here we characterize species-specific locomotor and respiratory responses to acute ambient warming in two highly stenothermic Antarctic Notothenioid fishes, one of which (Chaenocephalus aceratus) lacks hemoglobin and appears to be less tolerant to thermal stress as compared to the other (Notothenia coriiceps), which expresses hemoglobin. At the onset of ambient warming, both species perform distinct locomotor maneuvers that appear to include avoidance reactions. In response to unavoidable progressive hyperthermia, fishes demonstrate a range of species-specific maneuvers, all of which appear to provide some mitigation of the deleterious effects of obligatory thermoconformation and to compensate for increasing metabolic demand by enhancing the efficacy of branchial respiration. As temperature continues to rise, Chaenocephalus aceratus supplements these behaviors with intensive pectoral fin fanning which may facilitate cutaneous respiration through its scaleless integument, and Notothenia coriiceps manifests respiratory-locomotor coupling during repetitive startle-like maneuvers which may further augment gill ventilation. The latter behaviors, found only in Notothenia coriiceps, have highly stereotyped appearance resembling Fixed Action Pattern sequences. Altogether, this behavioral flexibility could contribute to the reduction of the detrimental effects of acute thermal stress within a limited thermal range. In an ecologically relevant setting, this may enable efficient thermoregulation of fishes by habitat selection, thus facilitating their resilience in persistent environmental change.

Ca(2+) permeation and/or binding to CaV1.1 fine-tunes skeletal muscle Ca(2+) signaling to sustain muscle function.

BACKGROUND: Ca(2+) influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca(2+) permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed. METHODS: We generated a mouse with a Ca(2+) binding and/or permeation defect in the voltage-dependent Ca(2+) channel, CaV1.1, and used Ca(2+) imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca(2+) imaging techniques to define pathways modulated by Ca(2+) binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles. RESULTS: Using mice with a pore mutation in CaV1.1 required for Ca(2+) binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca(2+) stores during sustained activity. Decreases in these Ca(2+)-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca(2+) binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers. CONCLUSIONS: While not essential for excitation-contraction coupling, Ca(2+) binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function.

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 µg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.

AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation.

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca(2+) leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca(2+)-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca(2+) concentrations. If unchecked, the temperature-driven increases in resting Ca(2+) concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.

KCa1.1 potassium channels regulate key proinflammatory and invasive properties of fibroblast-like synoviocytes in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.

Methods to study CFTR protein in vitro.

CFTR is a cyclic AMP and nucleotide-related chloride-selective channel with a low unitary conductance. Many of the physiological roles of CFTR are effectively studied in intact cells and tissues. However, there are also several clear advantages to the application of cell-free technologies to the study of the biochemical and biophysical properties of CFTR. When expressed in heterologous cells, CFTR is processed relatively poorly, depending, however, on the cell-type analysed. In some cells, only 20-25% of the protein which is initially synthesized exits the endoplasmic reticulum to insert into the cell membrane [Cell 83 (1995) 121; EMBO J. 13 (1994) 6076]. Further, many of the disease-causing mutants of CFTR result in even lower processing efficiencies. Therefore, several procedures have been developed to study regulated CFTR channel function expressed in microsomal membranes and following its purification and reconstitution. These experimental approaches and their application are discussed here.

The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl- channel.

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.