Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans)

Abstract

A thermogenic organ is found beneath the brain of billfishes (Istiophoridae), swordfish (Xiphiidae) and the butterfly mackerel (Scombridae). The heater organ has been shown to warm the brain and eyes up to 14°C above ambient water temperature. Heater cells are derived from extraocular muscle fibers and express a modified muscle phenotype with an extensive transverse-tubule (T-tubule) network and sarcoplasmic reticulum (SR) enriched in Ca2+-ATPase (SERCA) pumps and ryanodine receptors (RyRs). Heater cells have a high mitochondria content but have lost most of the contractile myofilaments. Thermogenesis has been hypothesized to be associated with release and reuptake of Ca2+. In this study, Ca2+ fluxes in heater SR vesicles derived from blue marlin (Makaira nigricans) were measured using fura-2 fluorescence. Upon the addition of MgATP, heater SR vesicles rapidly sequestered Ca2+. Uptake of Ca2+ was thapsigargin sensitive, and maximum loading ranged between 0.8 μmol Ca2+ mg-1 protein and 1.0 μmol Ca2+ mg-1 protein. Upon the addition of 10 mmoll-1 caffeine or 350 μmoll-1 ryanodine, heater SR vesicles released only a small fraction of the loaded Ca2+. However, ryanodine could elicit a much larger Ca2+ release event when the activity of the SERCA pumps was reduced. RNase protection assays revealed that heater tissue expresses an RyR isoform that is also expressed in fish slow-twitch skeletal muscle but is distinct from the RyR expressed in fish fast-twitch skeletal muscle. The heater and slow-twitch muscle RyR isoform has unique physiological properties. In the presence of adenine nucleotides, this RyR remains open even though cytoplasmic Ca2+ is elevated, a condition that normally closes RyRs. The fast Ca2+ sequestration by the heater SR, coupled with a physiologically unique RyR, is hypothesized to promote Ca2+ cycling, ATP turnover and heat generation. A branch of the oculomotor nerve innervates heater organs, and, in this paper, we demonstrate that heater cells contain large 'endplate-like' clusters of acetylcholine receptors that appear to provide a mechanism for nervous control of thermogenesis.