Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads.

Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-CC8C10), but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-CC1C7 to myofilament force production and regulation.

Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by ~24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

Titin-based force regulates cardiac myofilament structures mediating length-dependent activation.

The Frank-Starling law states that the heart's stroke volume increases with greater preload due to increased venous return, allowing the heart to adapt to varying circulatory demands. Molecularly, increasing preload increases sarcomere length (SL), which alters sarcomere structures that are correlated to increased calcium sensitivity upon activation. The titin protein, spanning the half-sarcomere, acts as a spring in the I-band, applying a SL-dependent force suggested to pull against and alter myofilaments in a way that supports the Frank-Starling effect. To evaluate this, we employed the titin cleavage (TC) model, where a tobacco-etch virus protease recognition site is inserted into distal I-band titin and allows for rapid, specific cleavage of titin in an otherwise-healthy sarcomere. Here, we evaluated the atomic-level structures of amyopathic cardiac myofilaments following 50% titin cleavage under passive stretch conditions using small-angle X-ray diffraction, which measures these structures under near-physiological (functional) conditions. We report that titin-based forces in permeabilized papillary muscle regulate both thick and thin myofilament structures clearly supporting titin's role in the Frank-Starling mechanism.

Myosin-binding protein C forms C-links and stabilizes OFF states of myosin.

Contraction force in muscle is produced by the interaction of myosin motors in the thick filaments and actin in the thin filaments and is fine-tuned by other proteins such as myosin-binding protein C (MyBP-C). One form of control is through the regulation of myosin heads between an ON and OFF state in passive sarcomeres, which leads to their ability or inability to interact with the thin filaments during contraction, respectively. MyBP-C is a flexible and long protein that is tightly bound to the thick filament at its C-terminal end but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7). Under considerable debate is whether the MyBP-CC1C7 domains directly regulate myosin head ON/OFF states, and/or link thin filaments ("C-links"). Here, we used a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. After cleavage, the thin filaments were significantly shorter, a result consistent with direct interactions of MyBP-C with thin filaments thus confirming C-links. Ca2+ sensitivity was reduced at shorter sarcomere lengths, and crossbridge kinetics were increased across sarcomere lengths at submaximal activation levels, demonstrating a role in crossbridge kinetics. Structural signatures of the thick filaments suggest that cleavage also shifted myosin heads towards the ON state - a marker that typically indicates increased Ca2+ sensitivity but that may account for increased crossbridge kinetics at submaximal Ca2+ and/or a change in the force transmission pathway. Taken together, we conclude that MyBP-CC1C7 domains play an important role in contractile performance which helps explain why mutations in these domains often lead to debilitating diseases.

The distinctive mechanical and structural signatures of residual force enhancement in myofibers.

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titin's function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.

Titin force in muscle cells alters lattice order, thick and thin filament protein formation.

Skeletal muscle force production is increased at longer compared to shorter muscle lengths because of length-dependent priming of thick filament proteins in the contractile unit before contraction. Using small-angle X-ray diffraction in combination with a mouse model that specifically cleaves the stretch-sensitive titin protein, we found that titin cleavage diminished the length-dependent priming of the thick filament. Strikingly, a titin-sensitive, length-dependent priming was also present in thin filaments, which seems only possible via bridge proteins between thick and thin filaments in resting muscle, potentially myosin-binding protein C. We further show that these bridges can be forcibly ruptured via high-speed stretches. Our results advance a paradigm shift to the fundamental regulation of length-dependent priming, with titin as the key driver.