ryanodine and Fever

Heat acclimation (AC) improves cardiac mechanical and metabolic performance. Using cardiomyocytes and isolated hearts from 30-day and 2-day acclimated rats (AC and AC-2d, 34 degrees C), we characterized cellular contractile mechanisms under normothermic (37 degrees C) and hyperthermic (39-42 degrees C) conditions. To determine contractile responses, Ca2+ transients (Ca2+ T), sarcoplasmic reticulum (SR) Ca2+ pool size (fura-2/indo-1 fluorescence), force generation [amplitude systolic motion (ASM)], L-type Ca2+ channels [dihydropyridine receptor (DHPR)], ryanodine receptors (RyRs), and total (PLBt) and phosphorylated phospholamban [serine phosphorylated (PLBs) and theonine phosphorylated (PLBtr)] proteins and transcripts were measured (Western blot, RT-PCR). Cardiac mechanical performance was measured using a Langendorff system. We demonstrated that AC and AC-2d increased Ca2+ T amplitude (148% and 147%, respectively) and twitch force (180% and 130%, respectively) and desensitized myofilaments, as indicated by a rightward shift in the ASM-Ca2+ relationships, despite no change in SR Ca2+ pool size. Hence, generation of higher Ca2+ T underlies greater force development in AC and AC-2d myocytes. In isolated hearts, ryanodine administration eliminated differences between AC and control (C) hearts, implying an important role for RyRs in that acclimation phase. Increased expression of DHPR and RyRs, and decreased PLBs/PLBt in AC hearts only, suggest that different pathways increase force generation in the AC-2d vs. AC myocytes. At basal beating rates, hyperthermia (39-41 degrees C) enhanced pressure generation in AC hearts. C hearts failed to restitute pressure beyond 39 degrees C. Increased beating frequency produced negative inotropic response. In C cardiomyocytes, hyperthermia elevated basal cytosolic Ca2+ and tension, Ca2+ T, and ASM. AC myocytes enhanced Ca2+ T but showed myofilament desensitization, suggesting its involvement in cardiac protection against hyperthermia. Collectively, both Ca2+ turnover and myofilament responsiveness are important adaptive acclimatory targets during normothermic and hyperthermic conditions.

Topics: Acclimatization; Actin Cytoskeleton; Animals; Calcium; Calcium Channels, L-Type; Calcium Signaling; Calcium-Binding Proteins; Disease Models, Animal; Fever; Heart Rate; Heat Stress Disorders; Hot Temperature; Kinetics; Male; Myocardial Contraction; Myocytes, Cardiac; Phosphorylation; Rats; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Ventricular Pressure

Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), caffeine and ATP (adenosine 5'-[beta,gamma-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.

Topics: Adenosine Triphosphate; Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Caffeine; Calcium; Calcium Channels; Cell Line; Cells, Cultured; Central Nervous System Stimulants; Cresols; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fever; Glycine; Immunoblotting; Kinetics; Malignant Hyperthermia; Molecular Sequence Data; Muscle, Skeletal; Mutation; Oligonucleotides; Plasmids; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spectrometry, Fluorescence; Transfection

Topics: Alkaloids; Animals; Biological Factors; Fever; Humans; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel