Biology subjects

Irving, T. C.

Publications and source records attributed to Irving, T. C..

9 recordsLinked to original sources

bioRxiv2026

Porcine Left Atrial and Ventricular Thick Filaments Exhibit Distinct Resting Structures and Calcium-dependent Responses

The heart maintains systemic perfusion through the coordinated function of its four chambers: the left and right atria and ventricles. Each chamber has distinct structural, functional, and molecular properties tailored to its role in circulation, which may result in chamber-specific differences in myofilament structure and regulation between atria and ventricles. To test this hypothesis, we employed muscle mechanics and X-ray diffraction to investigate functional and structural differences in porcine left atrial (LA) and left ventricular (LV) tissue. Here, we report the first X-ray diffraction study of atrial tissue, demonstrating that under resting conditions, myosin filaments in LA adopted a more ON-like, structurally distinct configuration compared with those in LV. Under contracting conditions, LV generated greater force and exhibited higher sinusoidal stiffness than LA across multiple calcium concentrations. LA showed faster kTR than in LV, with no calcium-dependence, in contrast to the calcium-dependence of kTR seen in LV. Structurally, the distinct myosin head configuration seen in the relaxed LA persisted during contraction. Furthermore, using the troponin inhibitor MYK-7660 to inhibit active contraction, we showed that, unlike LV, LA showed no direct calcium-dependent thick filament activation, reconciling discrepancies between fast rat and slow porcine ventricular myocardium regarding calciums role in thick filament regulation. Altogether, our study reveals that LA myosin filaments adopt a molecular architecture and regulatory mechanism distinct from their LV counterparts, suggesting that myosin filament structure and regulation have evolved differently to meet the unique functional demands of each cardiac chamber. Moreover, atrial disease is often associated with cardiomyopathy-related genetic variants, highlighting the atrial myocardium as an important therapeutic target and understanding atrial-specific regulatory mechanisms provides new insights into therapeutic strategies for atrial diseases.

physiology↗

bioRxiv2023

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

The Frank-Starling law states that the hearts 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 titins role in the Frank-Starling mechanism.

physiology↗

bioRxiv2023

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.

physiology↗

bioRxiv2023

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.

physiology↗

bioRxiv2023

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure with Pulmonary Hypertension

RationaleRight ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in heart failure patients with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. ObjectiveTo determine components of myocyte contractile depression in HFrEF-PH, identify those reflected by clinical RV indices, and elucidate their underlying biophysical mechanisms. Methods and ResultsResting, calcium- and load-dependent mechanics were measured in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 HFrEF-PH patients undergoing cardiac transplantation and 9 organ-donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded two HFrEF-PH subgroups that in turn mapped to patients with depressed (RVd) or compensated (RVc) clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in RVd, while surprisingly, many other major myocyte contractile measures including peak power, maximum unloaded shortening velocity, and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick-filament defects, myofibrillar structure was assessed by X-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in RVd but not RVc, as compared to controls. This corresponded to reduced myosin ATP turnover in RVd myocytes, indicating less myosin in a cross-bridge ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Lastly, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human HF. ConclusionsWhile there are multiple RV myocyte contractile deficits In HFrEF-PH, clinical indices primarily detect reduced isometric calcium-stimulated force related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

biophysics↗

bioRxiv2023

Danicamtiv increases myosin recruitment and alters the chemomechanical cross bridge cycle in cardiac muscle

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue. Significance StatementDirectly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.

physiology↗

bioRxiv2020

TWO CLASSES OF MYOSIN INHIBITORS, BLEBBISTATIN AND MAVACAMTEN, STABILIZE β-CARDIAC MYOSIN IN DIFFERENT STRUCTURAL AND FUNCTIONAL STATES

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a newly-discovered low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional and the super-relaxed states are widely assumed to correspond respectively to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin. Disruption of the SRX population in different heart diseases, such as hypertrophic cardiomyopathy, results in unwarranted muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examine the structure-function relationships of two myosin ATPase inhibitors, mavacamten, and blebbistatin, and found that binding of mavacamten to myosin at a site different than blebbistatin populates myosin into the SRX state. Blebbistatin, and para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps non-functional, ultra-relaxed state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations that diverge significantly from the hypothetical open and closed states and furthermore, mavacamten treatment causes a greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and blebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing SRX states.

biochemistry↗

bioRxiv2020

In vivo x-ray diffraction and simultaneous EMG reveal the time course of myofilament lattice dilation and filament stretch

Muscles function within an organism depends on the feedback between molecular to meter-scale processes. While the motions of muscles contractile machinery are well described in isolated preparations, only a handful of experiments have documented the kinematics of the lattice occurring when multi-scale interactions are fully intact. We used time-resolved x-ray diffraction to record the kinematics of the myofilament lattice within a normal operating context: the tethered flight of Manduca sexta. Since the primary flight muscles of Manduca sexta are synchronous, we used these results to reveal the timing of in vivo cross-bridge recruitment, which occurred 24 (s.d. 26) ms following activation. In addition, the thick filaments stretched an average of 0.75 (s.d. 0.32)% and thin filaments stretched 1.11 (s.d. 0.65)%. In contrast to other in vivo preparations, lattice spacing changed an average of 2.72 (s.d. 1.47)%. Lattice dilation of this magnitude significantly impacts shortening velocity and force generation, and filament stretching tunes force generation. While kinematics were consistent within individual trials, there was extensive variation between trials. Using a mechanism-free machine learning model we searched for patterns within and across trials. While lattice kinematics were predictable within trials, the model could not create predictions across trials. This indicates that the variability we see across trials may be explained by latent variables occurring in this naturally functioning system. The diverse kinematic combinations we documented mirror muscles adaptability and may facilitate its robust function in unpredictable conditions.

biophysics↗

bioRxiv2018

Mavacamten stabilizes a folded-back sequestered super-relaxed state of β-cardiac myosin

SummaryMutations in {beta}-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity and ATPase activityof myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative modelpositsthat mutations in myosin affect the stability ofa sequestered, super-relaxed state (SRX) of the proteinwith very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here, using a combination of biochemical and structural approaches, we show that purified myosin enters aSRX thatcorresponds to a folded-back conformation, which in muscle fibersresults insequestration of heads around the thick filament backbone. Mutations that cause HCM destabilize this state, while the small molecule mavacamtenpromotes it. These findings provide a biochemical and structural link between the genetics and physiology ofcardiomyopathywith implications for therapeutic strategies.

biochemistry↗