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.

The ineluctable constraints of thermodynamics in the aetiology of obesity.

We exploit the detail-independence feature of thermodynamics to examine issues related to the development of obesity. We adopt a 'global' approach consistent with focus on the first law of thermodynamics - namely that the metabolic energy provided by dietary foodstuffs has only three possible fates: the performance of work (be it microscopic or macroscopic), the generation of heat, or storage - primarily in the form of adipose tissue. Quantification of the energy expended, in the form of fat metabolised, during selected endurance events, reveals the inherent limitation of over-reliance on exercise as a primary agent of weight loss. This result prompts examination of various (non-exercise based) possibilities of increasing the rate of heat loss. Since these, too, give little cause for optimism, we are obliged to conclude that obesity can be prevented, or weight loss achieved, only if exercise is supplemented by reduction of food intake.

The basis of differences in thermodynamic efficiency among skeletal muscles.

Muscles convert chemical free energy into mechanical work. The energy conversion occurs in 2 steps. First, free energy obtained from oxidation of metabolic substrates (ΔGS ) is transferred to ATP and, second, free energy from ATP hydrolysis (ΔGATP ) is converted into work by myosin cross-bridges. The fraction of ΔGS transferred to ATP is called mitochondrial efficiency (ηM ) and the fraction of ΔGATP converted into work is called cross-bridge efficiency (ηCB ). Overall cross-bridge efficiency varies among muscles from ~20% and 35% and the analysis presented in the current studies shows that this variation is largely due to differences in ηCB whereas ηM is similar (~80%) in all the muscles assessed. There is an inverse, linear relationship between maximum normalised power output and ηCB ; that is, more efficient muscles tend to be less powerful than less efficient muscles. It is proposed that differences in cross-bridge efficiency reflect the extent to which cross-bridges traverse the force-length relationship for attached cross-bridges. In this framework, cross-bridges from tortoise muscle (ηCB  = 45%) produce close to the maximum possible work a cross-bridge can perform in a single attachment cycle.

A mathematical model to quantify RYR Ca2+ leak and associated heat production in resting human skeletal muscle fibers.

Cycling of Ca2+ between the sarcoplasmic reticulum (SR) and myoplasm is an important component of skeletal muscle resting metabolism. As part of this cycle, Ca2+ leaks from the SR into the myoplasm and is pumped back into the SR using ATP, which leads to the consumption of O2 and generation of heat. Ca2+ may leak through release channels or ryanodine receptors (RYRs). RYR Ca2+ leak can be monitored in a skinned fiber preparation in which leaked Ca2+ is pumped into the t-system and measured with a fluorescent dye. However, accurate quantification faces a number of hurdles. To overcome them, we developed a mathematical model of Ca2+ movement in these preparations. The model incorporated Ca2+ pumps that move Ca2+ from the myoplasm to the SR and from the junctional space (JS) to the t-system, Ca2+ buffering by EGTA in the JS and myoplasm and by buffers in the SR, and Ca2+ leaks from the SR into the JS and myoplasm and from the t-system into the myoplasm. The model accurately simulated Ca2+ uptake into the t-system, the relationship between myoplasmic [Ca2+] and steady-state t-system [Ca2+], and the effect of blocking RYR Ca2+ leak on t-system Ca2+ uptake. The magnitude of the leak through the RYRs would contribute ∼5% of the resting heat production of human muscle. In normal resting fibers, RYR Ca2+ leak makes a small contribution to resting metabolism. RYR-focused pathologies have the potential to increase RYR Ca2+ leak and the RYR leak component of resting metabolism.

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.

Doublet stimulation increases Ca2+ binding to troponin C to ensure rapid force development in skeletal muscle.

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca2+ during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca2+ release, binding of Ca2+ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca2+ spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca2+ between the first three Ca2+ spikes of the transient, maintaining a higher overall free Ca2+ level during first 20-30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca2+ release rates during doublet stimulation and showed that Ca2+-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca2+ binding to the main intracellular Ca2+ buffers of troponin C (TnC), parvalbumin, and the SR Ca2+ pump during Ca2+ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca2+-binding site on TnC (TnC2), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in faster saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and more rapid force development.

The efficiency of muscle contraction.

When a muscle contracts and shortens against a load, it performs work. The performance of work is fuelled by the expenditure of metabolic energy, more properly quantified as enthalpy (i.e., heat plus work). The ratio of work performed to enthalpy produced provides one measure of efficiency. However, if the primary interest is in the efficiency of the actomyosin cross-bridges, then the metabolic overheads associated with basal metabolism and excitation-contraction coupling, together with those of subsequent metabolic recovery process, must be subtracted from the total heat and work observed. By comparing the cross-bridge work component of the remainder to the Gibbs free energy of hydrolysis of ATP, a measure of thermodynamic efficiency is achieved. We describe and quantify this partitioning process, providing estimates of the efficiencies of selected steps, while discussing the errors that can arise in the process of quantification. The dependence of efficiency on animal species, fibre-type, temperature, and contractile velocity is considered. The effect of contractile velocity on energetics is further examined using a two-state, Huxley-style, mathematical model of cross-bridge cycling that incorporates filament compliance. Simulations suggest only a modest effect of filament compliance on peak efficiency, but progressively larger gains (vis-à-vis the rigid filament case) as contractile velocity approaches Vmax. This effect is attributed primarily to a reduction in the component of energy loss arising from detachment of cross-bridge heads at non-zero strain.

Thermodynamic analysis questions claims of improved cardiac efficiency by dietary fish oil.

Studies in the literature describe the ability of dietary supplementation by omega-3 fish oil to increase the pumping efficiency of the left ventricle. Here we attempt to reconcile such studies with our own null results. We undertake a quantitative analysis of the improvement that could be expected theoretically, subject to physiological constraints, by posing the following question: By how much could efficiency be expected to increase if inefficiencies could be eliminated? Our approach utilizes thermodynamic analyses to investigate the contributions, both singly and collectively, of the major components of cardiac energetics to total cardiac efficiency. We conclude that it is unlikely that fish oils could achieve the required diminution of inefficiencies without greatly compromising cardiac performance.