Evolutionary isolation of ryanodine receptor isoform 1 for muscle-based thermogenesis in mammals.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

Ca2+ leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle.

Mammals rely on nonshivering thermogenesis (NST) from skeletal muscle so that cold temperatures can be tolerated. NST results from activity of the sarcoplasmic reticulum (SR) Ca2+ pump in skeletal muscle, but the mechanisms that regulate this activity are unknown. Here, we develop a single-fiber assay to investigate the role of Ca2+ leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca2+ pump in resting muscle. By inhibiting a subpopulation of RyR1s in a single-fiber preparation via targeted delivery of ryanodine through transverse tubules, we achieve in-preparation isolation of RyR1 Ca2+ leak. This maneuver provided a critical increase in signal-to-noise of the SR-temperature-sensitive dye ER thermoyellow fluorescence signal from the fiber to allow detection of SR temperature changes as either RyR1 or SR Ca2+ pump activity was altered. We found that RyR1 Ca2+ leak raises cytosolic [Ca2+] in the local vicinity of the SR Ca2+ pump to amplify thermogenesis. Furthermore, gene-dose-dependent increases in RyR1 leak in RYR1 mutant mice result in progressive rises in leak-dependent heat, consistent with raised local [Ca2+] at the SR Ca2+ pump via RyR1 Ca2+ leak. We also show that basal RyR Ca2+ leak and the heat generated by the SR Ca2+ pump in the absence of RyR Ca2+ leak is greater in fibers from mice than from toads. The distinct function of RyRs and SR Ca2+ pump in endothermic mammals compared to ectothermic amphibians provides insights into the mechanisms by which mammalian skeletal muscle achieves thermogenesis at rest.

Ryanodine receptor activity and store-operated Ca2+ entry: Critical regulators of Ca2+ content and function in skeletal muscle.

Store-operated Ca2+ entry (SOCE) is critical to cell function. In skeletal muscle, SOCE has evolved alongside excitation-contraction coupling (EC coupling); as a result, it displays unique properties compared to SOCE in other cells. The plasma membrane of skeletal muscle is mostly internalized as the tubular system, with the tubules meeting the sarcoplasmic reticulum (SR) terminal cisternae, forming junctions where the proteins that regulate EC coupling and SOCE are positioned. In this review, we describe the properties and roles of SOCE based on direct measurements of Ca2+ influx during SR Ca2+ release and leak. SOCE is activated immediately and locally as the [Ca2+ ] of the junctional SR terminal cisternae ([Ca2+ ]jSR ) depletes. [Ca2+ ]jSR changes rapidly and steeply with increasing activity of the SR ryanodine receptor isoform 1 (RyR1). The high fidelity of [Ca2+ ]jSR with RyR1 activity probably depends on the SR Ca2+ -buffer calsequestrin that is located immediately behind RyR1 inside the SR. This arrangement provides in-phase activation and deactivation of SOCE with a large dynamic range, allowing precise grading of SOCE flux. The in-phase activation of SOCE as the SR partially depletes traps Ca2+ in the cytoplasm, preventing net Ca2+ loss. Mild presentation of RyR1 leak can occur under physiological conditions, providing fibre Ca2+ redistribution without changing fibre Ca2+ content. This condition preserves normal contractile function at the same time as increasing basal metabolic rate. However, higher RyR1 leak drives excess cytoplasmic and mitochondrial Ca2+ load, setting a deleterious intracellular environment that compromises the function of the skeletal muscle.

Tiny changes in cytoplasmic [Ca2+] cause large changes in mitochondrial Ca2+: what are the triggers and functional implications?

Ca2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca2+ is reported to modulate the rate of ATP resynthesis, regulate the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1α) following exercise, and drive the generation of reactive oxygen species (ROS). Due to the latter, mitochondrial Ca2+ overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation. Recently, we described the relationship between [Ca2+]mito and resting [Ca2+]cyto and other mitochondrial Ca2+-handling properties of skeletal muscle. An important next step is to understand the triggers for Ca2+ redistribution between intracellular compartments, which determine the mitochondrial Ca2+ load. These triggers in both physiological and pathophysiological scenarios can be traced to the coupled activity of the ryanodine receptor 1 (RyR1) and store-operated Ca2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca2+ measurements relevant to mitochondrial Ca2+-handling, the steady-state relationship between cytoplasmic and mitochondrial Ca2+, and the potential implications for Ca2+ handling by muscle mitochondria and cellular function.

Occurrence and characterisation of Eustrongylides species in Australian native birds and fish.

In Australia, nematodes belonging to the genus Eustrongylides were believed to be endemic species until the late 20th century when they were all considered to be E. excisus, invalid or inquirendae. Although these nematodes have frequently been reported in Australian fish, reptiles, and birds and cause disease or mortality among them, there has been no attempt to date to characterise them genetically. Globally, also, no one has validated or defined suitable genetic markers to distinguish between species of Eustrongylides. In this study, adult Eustrongylides from little black cormorant (Phalacrocorax sulcirostris; n = 3) and larvae from mountain galaxias (Galaxias olidus, n = 2) and a Murray cod (Maccullochella peelii, n = 1), and a Murray cod-trout cod hybrids (Maccullochella peelii x Maccullochella macquariensis, n = 1) were available for morphological examination and molecular characterisation. The adult nematodes from cormorants were identified as E. excisus. Sequences of the 18S and ITS regions were then obtained for all nematodes, which were identical among all specimens (larvae and adults) and also identical to those of E. excisus available in the GenBank. However, only one base pair difference exists between the 18S sequences of E. excisus and E. ignotus, with limited sequences available in GenBank accompanied with proper morphological data for the nematodes. With that limitation in mind, identifying our specimens as E. excisus suggests spill-over - that it is an introduced parasite species that has successfully established its life cycle among Australian native species - may have occurred. Our study is the first report of E. excisus in the little black cormorant, P. sulcirostris. Our results do not exclude the possibility of the occurrence of other species of Eustrongylides, either native or exotic, in Australia. This parasite is zoonotic and with increasing demand for fish and changing dietary preferences, such as the consumption of raw or undercooked fish, its occurrence in the flesh of the fish is concerning. This parasite is also associated with anthropogenic habitat alteration affecting the reproductive success of the infected hosts. Therefore, awareness among the relevant authorities of the presence of the parasite in Australia and its adverse impact on native animals is crucial for the success of conservation plans such as fish recovery and relocation efforts.

Parasites of Selected Freshwater Snails in the Eastern Murray Darling Basin, Australia.

Aquatic snails serve an important role in the ecosystem. They also play an essential role in the life cycle of many parasites as hosts and may pose risks to animal and human health. In Australia, the role of snails in the transmission of parasites of livestock is well studied. However, despite the country's unique biodiversity and wildlife, little is known about the role of snails in the transmission and survival of parasites in other ecosystems, including aquatic and aquaculture systems. This study aimed to determine the occurrence of parasites in freshwater snails in the eastern Murray Darling Basin. A total of 275 snails were collected from various localities, including aquaculture fishery ponds and natural creeks during the summer and autumn months in the southern hemisphere. Three different species of freshwater snails, all common to the area, were found, including Bullastra lessoni (n = 11), Isidorella hainesii (n = 157), and Haitia acuta (n = 107), of which 9.1%, 1.3%, and 4.7%, respectively, were found to be harboring various developmental stages of Trematoda. No other parasite was found in the examined snails. Parasites were identified as Choanocotyle hobbsi, Plagiorchis sp. and Petasiger sp. based on the sequences of their ITS2, 18S, and 28S ribosomal DNA region. Herein, we report a native parasite Choanocotyle hobbsi in an introduced snail, Haitia acuta, from both natural and aquaculture ponds. As there are no genetic sequences for adult specimens of Petasiger spp. and Plagiorchis spp. collected in Australia for comparison, whether the specimens collected in this study are the larval stage of one of the previously described species or are a new, undescribed species cannot yet be determined. Our results also suggest snails collected from aquaculture ponds may be infected with considerably more parasites.

Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle.

Muscle contraction depends on tightly regulated Ca2+ release. Aberrant Ca2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca2+ leak initiates a cascade of events that cause precise redistribution of Ca2+ among the SR, cytoplasm, and mitochondria through altering the Ca2+ permeability of the transverse tubular system membrane. This redistribution of Ca2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.

Detailed distribution of lipids in Greenshell™ mussel (Perna canaliculus).

Greenshell™ mussels (GSM-Perna canaliculus) are a source of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA). Farmed GSM are considered to be a sustainable source of LC-PUFA as they require no dietary inputs, gaining all of their oil by filter-feeding microorganisms from sea water. GSM oil is a high-value product, with a value as much as 1000 times that of fish oils. GSM oil has important health benefits, for example, anti-inflammatory activity. It also contains several minor lipid components that are not present in most fish oil products, and that have their own beneficial effects on human health. We have shown the lipid content of the female GSM (1.9 g/100 g ww) was significantly greater than that of the male (1.4 g/100 g ww). Compared with male GSM, female GSM contained more n-3 LC-PUFA, and stored a greater proportion of total lipid in the gonad and mantle. The higher lipid content in the female than the male GSM is most likely related to gamete production. This information will be useful to optimize extraction of oils from GSM, a local and sustainable source of n-3 LC-PUFA.