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.