Length-dependent Activation Research Articles

Super-relaxed or super-stressed: Modeling length-dependent activation in cardiac muscle

Super-relaxed or super-stressed: Modeling length-dependent activation in cardiac muscle

Levosimendan's Effects on Length-Dependent Activation in Murine Fast-Twitch Skeletal Muscle.

Levosimendan's calcium sensitizing effects in heart muscle cells are well established; yet, its potential impact on skeletal muscle cells has not been evidently determined. Despite controversial results, levosimendan is still expected to interact with skeletal muscle through off-target sites (further than troponin C). Adding to this debate, we investigated levosimendan's acute impact on fast-twitch skeletal muscle biomechanics in a length-dependent activation study by submersing single muscle fibres in a levosimendan-supplemented solution. We employed our MyoRobot technology to investigate the calcium sensitivity of skinned single muscle fibres alongside their stress-strain response in the presence or absence of levosimendan (100 µM). While control data are in agreement with the theory of length-dependent activation, levosimendan appears to shift the onset of the 'descending limb' of active force generation to longer sarcomere lengths without notably improving myofibrillar calcium sensitivity. Passive stretches in the presence of levosimendan yielded over twice the amount of enlarged restoration stress and Young's modulus in comparison to control single fibres. Both effects have not been described before and may point towards potential off-target sites of levosimendan.

Cardiac length-dependent activation driven by force-dependent thick-filament dynamics

The length-dependent activation (LDA) of maximum force and calcium sensitivity are established features of cardiac muscle contraction but the dominant underlying mechanisms remain to be fully clarified. Alongside the well-documented regulation of contraction via the thin filaments, experiments have identified an additional force-dependent thick-filament activation, whereby myosin heads parked in a so-called off state become available to generate force. This process produces a feedback effect that may potentially drive LDA. Using biomechanical modeling of a human left-ventricular myocyte, this study investigates the extent to which the off-state dynamics could, by itself, plausibly account for LDA, depending on the specific mathematical formulation of the feedback. We hypothesized four different models of the off-state regulatory feedback based on (A) total force, (B) active force, (C) sarcomere strain, and (D) passive force. We tested if these models could reproduce the isometric steady-state and dynamic LDA features predicted by an earlier published model of a human left-ventricle myocyte featuring purely phenomenological length dependences. The results suggest that only total-force feedback (A) is capable of reproducing the expected behaviors, but that passive tension could provide a length-dependent signal on which to initiate the feedback. Furthermore, by attributing LDA to off-state dynamics, our proposed model also qualitatively reproduces experimentally observed effects of the off-state-stabilizing drug mavacamten. Taken together, these results support off-state dynamics as a plausible primary mechanism underlying LDA.

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.

Assessment and modelling of the activation-dependent shift in optimal length of the human soleus muscle in vivo.

Previous in vitro and in situ studies have reported a shift in optimal muscle fibre length for force generation (L0) towards longer length at decreasing activation levels (also referred to as length-dependent activation), yet the relevance for in vivo human muscle contractions with a variable activation pattern remains largely unclear. By a combination of dynamometry, ultrasound and electromyography (EMG), we experimentally obtained muscle force-fascicle length curves of the human soleus at 100%, 60% and 30% EMGmax levels from 15 participants aiming to investigate activation-dependent shifts in L0 in vivo. The results showed a significant increase in L0 of 6.5±6.0% from 100% to 60% EMGmax and of 9.1±7.2% from 100% to 30% EMGmax (both P<0.001), respectively, providing evidence of a moderate in vivo activation dependence of the soleus force-length relationship. Based on the experimental results, an approximation model of an activation-dependent force-length relationship was defined for each individual separately and for the collective data of all participants, both with sufficiently high accuracy (R2 of 0.899±0.056 and R2=0.858). This individual approximation approach and the general approximation model outcome are freely accessible and may be used to integrate activation-dependent shifts in L0 in experimental and musculoskeletal modelling studies to improve muscle force predictions. KEY POINTS: The phenomenon of the activation-dependent shift in optimal muscle fibre length for force generation (length-dependent activation) is poorly understood for human muscle in vivo dynamic contractions. We experimentally observed a moderate shift in optimal fascicle length towards longer length at decreasing electromyographic activity levels for the human soleus muscle in vivo. Based on the experimental results, we developed a freely accessible approximation model that allows the consideration of activation-dependent shifts in optimal length in future experimental and musculoskeletal modelling studies to improve muscle force predictions.

Structure of mavacamten-free human cardiac thick filaments within the sarcomere by cryoelectron tomography

Heart muscle has the unique property that it can never rest; all cardiomyocytes contract with each heartbeat which requires a complex control mechanism to regulate cardiac output to physiological requirements. Changes in calcium concentration regulate the thin filament activation. A separate but linked mechanism regulates the thick filament activation, which frees sufficient myosin heads to bind the thin filament, thereby producing the required force. Thick filaments contain additional nonmyosin proteins, myosin-binding protein C and titin, the latter being the protein that transmits applied tension to the thick filament. How these three proteins interact to control thick filament activation is poorly understood. Here, we show using 3-D image reconstruction of frozen-hydrated human cardiac muscle myofibrils lacking exogenous drugs that the thick filament is structured to provide three levels of myosin activation corresponding to the three crowns of myosin heads in each 429Å repeat. In one crown, the myosin heads are almost completely activated and disordered. In another crown, many myosin heads are inactive, ordered into a structure called the interacting heads motif. At the third crown, the myosin heads are ordered into the interacting heads motif, but the stability of that motif is affected by myosin-binding protein C. We think that this hierarchy of control explains many of the effects of length-dependent activation as well as stretch activation in cardiac muscle control.

Enhancing sarcomere function and length-dependent activation in engineered human myocardium via triiodothyronine and dexamethasone treatment

Enhancing sarcomere function and length-dependent activation in engineered human myocardium via triiodothyronine and dexamethasone treatment

Length dependent reversible off-on activation of photo-switchable relay anion transporters.

A homologous series of azobenzene-derived photo-switchable ion relay transporters is reported. We reveal that both the length and geometry of the relay strongly affect transport rate, allowing the relative activity of the E and Z isomers to be reversed and hence the wavelengths of light used for on and off switching to be exchanged.

Regulating Striated Muscle Contraction: Through Thick and Thin.

Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat.

Structure of the native myosin filament in the relaxed cardiac sarcomere

The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

Mechanistic study of mechanical stretch induced increase in Ca2+ sensitivity of troponin C.

The Frank-Starling Law of the heart is a direct consequence of myofilament length dependent activation (LDA) whereby increased sarcomere length (SL) leads to both increased myocardial Ca2+ sensitivity and maximum force. The molecular mechanisms underlying LDA remain elusive. Since troponin complex binds Ca2+ and regulates myosin-actin interaction during muscle contraction, it has been considered the key element involved LDA. This study aims to explore the molecular mechanism underlying sarcomere LDA and to investigate the functional role of each troponin protein in the LDA regulation. A mutant troponin-I, in which the SP-region was replaced with a G-linker (cTnI-ΔSP), was constructed to study the molecular interaction between troponin-I, troponin-C, and myosin during LDA regulation. The cTnI-ΔSP was used to prevent the functional effect from crossbridge (XB) on the SL-induced Ca2+ sensitivity changes of troponin complex. The SL-induced Ca2+ sensitivity change was monitored by in situ FRET which represents the Ca2+-induced cTnC structural change. Our results show that there are no significant changes of the SL-induced pCa50 shift of troponin in the absence of strong XB, suggesting that besides the XB feedback, the LDA of thin filament may have an alternative pathway involving troponin itself. To further understand the mechanism underlying LDA, recombinant cTnT and cTnI mutants carrying an enzyme cutting site at one end of the IT arm region, respectively, were used in our study. By selectively breaking T1 and T2 of cTnT or separating the C-domain of cTnI from the N-domain, we were able to examine the mechanistic pathway of SL-stretch on the Ca2+-induced structural change of cTnC. Our results provide novel information on the molecular mechanism of how troponin-Ca2+ activation senses myocardial sarcomere stretches to promote myosin-actin interactions at the diastolic level of Ca2+.

Muscle length alters activation kinetics in fast-twitch edl muscle and relaxation kinetics in slow-twitch soleus muscle.

Mammalian skeletal muscles typically comprise mixtures of slow and fast-twitch fiber types. By studying muscles that homogenously express fast-twitch (EDL) and slow-twitch soleus (SOL) fibers, we can compare the activation and relaxation kinetics in fast and slow muscle fibers in intact and permeabilized muscle preparations at different sarcomere lengths (SL). Intact EDL muscle at optimal length (L0) had significantly longer times (p<0.05) to 50% (½ time) activation at 38.02 ± 2.26 ms (t1/2ACT), compared to 95% L0 and 90% L0 (33.65±1.86 and 31.31±1.61 respectfully). There were, however, no significant differences in activation ½ times of intact SOL muscle as muscle length increases. The relaxation ½ times are indistinguishable for intact EDL muscle at different muscle lengths, while the relaxation ½ times for intact SOL muscle increased as the muscle lengthen increased. To deconvolute the contribution of calcium transients and intrinsic myofilament kinetics to the length dependent activation and relaxation kinetics seen in intact muscle, we next investigate the kinetics of skinned myofibrils at different sarcomere lengths. Rate constant values for initial myofibril activation for EDL preparations showed significant (p<0.05) length dependence, decreasing from 7.25±0.47 s−1 to 5.97±0.44 s−1, 4.99±0.477 s−1, and 3.98±0.41 s−1 at 2.2, 2.5, 2.75, and 2.90 µm respectively). In contrast, SOL myofibrils had minimal length dependence, with no significant changes in activation rate constants. Fast relaxation rates for soleus muscle increased from 11.51±0.91 s−1 to 13.26±0.86 s−1, 15.13±0.99 s−1, and 16.34±1.02 s−1 (at 2.2, 2.5, 2.75, and 2.90 μm respectively) while EDL preparations showed no change. The results suggest that activation kinetics in EDL muscle and relaxation kinetics in SOL are length dependent and are intrinsic properties of the myofilaments.

Increased length-dependent activation of human engineered heart tissue after chronic α1A-adrenergic agonist treatment: testing a novel heart failure therapy.

Chronic stimulation of cardiac α1A-adrenergic receptors (α1A-ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. Human engineered heart tissues (EHTs) provide a means of quantifying the effects of chronic α1A-AR stimulation on human cardiomyocyte physiology. EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. With a paired experimental design, EHTs were cultured for 3 wk, mechanically tested, cultured again for 2 wk with α1A-AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24 h. Separate control experiments determined the effects of EHT age (3-5 wk) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared with vehicle treatment (n = 7/group, P = 0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased by 35% relative to baseline testing (n = 7/group, P = 0.022), suggesting EHT maturation. Control experiments suggested that increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed that the α1A-AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways known to be activated by α1A-ARs, including the MAP kinase signaling pathway. In conclusion, increased LDA in human EHT after chronic A61603 treatment raises the possibility that chronic stimulation of the α1A-AR might be beneficial for increasing LDA in human myocardium and might be beneficial for treating human heart failure by restoring LDA.NEW & NOTEWORTHY Chronic stimulation of α1A-adrenergic receptors (α1A-ARs) is known to mediate therapeutic effects in animal heart failure models. To investigate the effects of chronic α1A-AR stimulation in human cardiomyocytes, we tested engineered heart tissue (EHT) created with iPSC-derived cardiomyocytes. RNA-seq analysis confirmed human EHT expressed α1A-ARs. Chronic (2 wk) α1A-AR stimulation with A61603 (10 nM) increased length-dependent activation (LDA) of contraction. Chronic α1A-AR stimulation might be beneficial for treating human heart failure by restoring LDA.

Sarcomere length affects Ca2+ sensitivity of contraction in ischemic but not non-ischemic myocardium.

In healthy hearts, myofilaments become more sensitive to Ca2+ as the myocardium is stretched. This effect is known as length-dependent activation and is an important cellular-level component of the Frank-Starling mechanism. Few studies have measured length-dependent activation in the myocardium from failing human hearts. We investigated whether ischemic and non-ischemic heart failure results in different length-dependent activation responses at physiological temperature (37°C). Myocardial strips from the left ventricular free wall were chemically permeabilized and Ca2+-activated at sarcomere lengths (SLs) of 1.9 and 2.3 µm. Data were acquired from 12 hearts that were explanted from patients receiving cardiac transplants; 6 had ischemic heart failure and 6 had non-ischemic heart failure. Another 6 hearts were obtained from organ donors. Maximal Ca2+-activated force increased at longer SL for all groups. Ca2+ sensitivity increased with SL in samples from donors (P < 0.001) and patients with ischemic heart failure (P = 0.003) but did not change with SL in samples from patients with non-ischemic heart failure. Compared with donors, troponin I phosphorylation decreased in ischemic samples and even more so in non-ischemic samples; cardiac myosin binding protein-C (cMyBP-C) phosphorylation also decreased with heart failure. These findings support the idea that troponin I and cMyBP-C phosphorylation promote length-dependent activation and show that length-dependent activation of contraction is blunted, yet extant, in the myocardium from patients with ischemic heart failure and further reduced in the myocardium from patients with non-ischemic heart failure. Patients who have a non-ischemic disease may exhibit a diminished contractile response to increased ventricular filling.

TLR3 forms a laterally aligned multimeric complex along double-stranded RNA for efficient signal transduction

Toll-like receptor 3 (TLR3) is a member of the TLR family, which plays an important role in the innate immune system and is responsible for recognizing viral double-stranded RNA (dsRNA). Previous biochemical and structural studies have revealed that a minimum length of approximately 40–50 base pairs of dsRNA is necessary for TLR3 binding and dimerization. However, efficient TLR3 activation requires longer dsRNA and the molecular mechanism underlying its dsRNA length-dependent activation remains unknown. Here, we report cryo-electron microscopy analyses of TLR3 complexed with longer dsRNA. TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction.

Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A

PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to β-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca2+ sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C invitro, in a phosphorylation- and Ca2+-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca2+ sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca2+ sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.

Cardiac efficiency and Starling's Law of the Heart.

The formulation by Starling of The Law of the Heart states that 'the [mechanical] energy of contraction, however measured, is a function of the length of the muscle fibre'. Starling later also stated that 'the oxygen consumption of the isolated heart … is determined by its diastolic volume, and therefore by the initial length of its muscular fibres'. This phrasing has motivated us to extend Starling's Law of the Heart to include consideration of the efficiency of contraction. In this study, we assessed both mechanical efficiency and crossbridge efficiency by studying the heat output of isolated rat ventricular trabeculae performing force-length work-loops over ranges of preload and afterload. The combination of preload and afterload allowed us, using our modelling frameworks for the end-systolic zone and the heat-force zone, to simulate cases by recreating physiologically feasible loading conditions. We found that across all cases examined, both work output and change of enthalpy increased with initial muscle length; hence it can only be that the former increases more than the latter to yield increased mechanical efficiency. In contrast, crossbridge efficiency increased with initial muscle length in cases where the extent of muscle shortening varied greatly with preload. We conclude that the efficiency of cardiac contraction increases with increasing initial muscle length and preload. An implication of our conclusion is that the length-dependent activation mechanism underlying the cellular basis of Starling's Law of the Heart is an energetically favourable process that increases the efficiency of cardiac contraction. KEY POINTS: Ernest Starling in 1914 formulated the Law of the Heart to describe the mechanical property of cardiac muscle whereby force of contraction increases with muscle length. He subsequently, in 1927, showed that the oxygen consumption of the heart is also a function of the length of the muscle fibre, but left the field unclear as to whether cardiac efficiency follows the same dependence. A century later, the field has gained an improved understanding of the factors, including the distinct effects of preload and afterload, that affect cardiac efficiency. This understanding presents an opportunity for us to investigate the elusive length-dependence of cardiac efficiency. We found that, by simulating physiologically feasible loading conditions using a mechano-energetics framework, cardiac efficiency increased with initial muscle length. A broader physiological importance of our findings is that the underlying cellular basis of Starling's Law of the Heart is an energetically favourable process that yields increased efficiency.

Eccentric muscle contraction potentiates titin stiffness-related contractile properties in rat fast-twitch muscles.

This study was conducted to examine the effects of an acute bout of eccentric muscle contraction (ECC) on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ to undergo 200 repeated ECCs. Immediately after the cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. Small heat shock protein αB-crystallin in the muscle fraction enriched for myofibrillar proteins was increased by ECC. ECC resulted in an increase in the titin-based passive force. Protein kinase A-treatment decreased the passive force only in ECC-subjected but not in rested fibers. ECC decreased the maximum Ca2+-activated force at a sarcomere length (SL) of 2.4 μm and had no effect on myofibrillar-Ca2+ sensitivity at 2.6-μm SL. In both rested and ECC-subjected fibers, these two variables were higher at 3.0-μm SL than at 2.4- or 2.6-μm SL. The differences in the two variables between the short and long SLs were greater in ECC-subjected than in rested fibers. These results indicate that an acute bout of ECC potentiates titin-based passive force, maximum active force at long SLs, and length-dependent activation and suggest that this potentiation may resist muscle fatigue in the muscles of the exercising body.NEW & NOTEWORTHY It remains unclear whether eccentric contraction of skeletal muscle affects titin stiffness-related contractile properties. Here, we provide evidence that an acute bout of eccentric contraction can potentiate titin-based passive force, maximum active force at long sarcomere lengths, and length-dependent activation. This potentiation may resist muscle fatigue in the muscles of the exercising body.

Abstract P1062: Regional Differences In Sarcomeric Glycogen Synthase Kinase 3β Maintain Normal Chamber Level Function

The structure of the left ventricle is spatially and temporally heterogeneous. The heart’s electrical system helps synchronize these events, but mechanical events play an important role as well. However, experimental evidence of the regional mechanical processes that underpin a “normal contraction” remain largely unknown. Despite the differences in deformation between the anterior and posterior walls, sarcomere lengths during filling and ejection are the same in these regions. One explanation for this disconnect is the myocyte’s force production is different despite similar sarcomere lengths, a relationship known as Length Dependent Activation (LDA). Indeed, a modeling study suggested LDA is a critical regulator of mechanical synchrony in the left ventricle. We recently found the serine/threonine kinase GSK-3β localizes to the z-disc and can modulate LDA, and thus hypothesized it may play a role in maintaining normal cardiac synchrony. We used inducible cardiomyocyte specific GSK-3β KO mice and GSK-3βfl/fl/Cre- littermate Con treated with tamoxifen in which GSK-3β was KO for three weeks. We assessed regional differences in GSK-3β protein levels and found that Con mice had substantial regional heterogeneity: 50% more cytosolic GSK-3β in the posterior wall compared to the anterior wall; however, the myofilament-bound GSK-3β was the opposite, with 50% less GSK-3β in the posterior wall. This regional heterogeneity was lost in KO mice, in which protein levels were homogenously low. There were no differences detected by m-mode echo between Control and KO mice. However, strain analysis revealed a significant interaction (p=0.037) between genotype and region (Anterior vs. Posterior), in which regional differences in Systolic Strain Rate were ablated in the knockout. These data suggest that the regional differences in GSK-3β sarcomeric levels were important for maintaining regional functional heterogeneity. We further observed that sarcomeric GSK-3β levels (but not cytosolic) decreased in a mouse myocardial infarction model, which these findings suggest could worsen mechanical dyssynchrony post-infarct. Perhaps a possible therapy for mechanical dyssynchrony could involve regional restoration of sarcomeric GSK-3β levels to restore synchrony.

Abstract P1056: Stretch Reduces Inter-filament Spacing And Triggers Alterations In Thick-filament Structure In Healthy Left-atrial Fibers

Introduction: Left-atrial dilatation is thought to play a key pathological role in facilitating remodeling in both genetic and acquired cardiac myopathies, as well as enabling the onset and maintenance of atrial fibrillation. However, despite its likely role in disease, the structural responses of the atria to stretch remain understudied. The myofilament responses to stretch have been extensively studied in ventricular tissue, where strain-dependent recruitment of reserve, OFF-state, myosin heads from the thick-filament contribute to length dependent activation (LDA, i.e., greater forcer at longer sarcomere lengths). In these experiments, the effects of stretch in both atrial sarcomere and thick-filament structure were evaluated. Methods: Myofilament structure was evaluated via x-ray diffraction at two sarcomere lengths (SL, SHORT: 2.1 μm, and LONG: 2.3 μm) in relaxed left-atrial and left-ventricular myocardial skinned fibers from Yucatan mini-pigs. In these experiments, both equatorial and meridional reflections reflecting sarcomeric structure and ordering were evaluated. In addition, the biomechanical effects of stretch were also evaluated in both skinned porcine fibers and engineered heart tissues (EHT) from atrial and ventricular hIPSc-derived myocytes via a proprietary system (MyoPod; Propria Biosciences). Results: In both atrial skinned fibers and EHTs, stretch triggered moderate increases in force, demonstrating LDA. LA fibers, stretch decreased lattice spacing but failed to alter the I 11 /I 10 equatorial intensity ratio, suggesting negligible length-dependent recruitment of OFF-state myosin heads from the thick-filament. LA fibers, however, tended to have higher I 11 /I 10 values at short SL (vs. LV fibers) suggesting an intrinsically higher population of ON-state myosin heads. In addition, stretch triggered alterations in thick filament ordering in both LA and LV fibers, as reflected by changes in both the 3 rd and 6 th order meridional reflections (M3 and M6), suggesting effects in both myosin heads and thick-filament backbone. Conclusion: Atrial stretch results in marked sarcomere structural alterations, reducing both inter-filament spacing and thick-filament ordering that likely contribute to length dependent activation. The role of these alteration in atrial pathophysiology remains to be studied