Sarcomere Dynamics Research Articles

Sarcomere. The structural unit of the myofibrillar complex of typical cardiomyocytes. Isometric aspects of sarcomere organization

Background. The complexity of understanding the structure and function of cardiomyocytes in the myocardium is a fundamental problem in the field of heart morphology. Sarcomeres are the main structural and functional units of myofibrils. Understanding the organization and dynamics of sarcomeres in cardiomyocytes is crucial for establishing the mechanisms responsible for the process of forming the contractile system of cardiomyocytes. Objective. The main purpose of this study is a review of literary sources that complement and expand the essential findings about the construction of myofibrils and their components when studied by effective methods of microscopic techniques. Materials. Literature review databases, systematic search of primary sources related to the research topic. Methods of visual examination of the components of sarcomeres and elements of their organization. Results. Transmission electron microscopy provides high-resolution imaging of sarcomeres, which is necessary for detailed analysis of their ultrastructure, including visualization of sarcomere components such as Z-discs, M-lines, thick and thin filaments. Morphometric methods are crucial for studying the compaction and orientation of myofibrils in cardiomyocytes. Conclusion. Morphometric methods provide valuable information about the structural remodeling of myofibrils in response to physiological stimuli or pathological conditions. These techniques are needed to improve our understanding of the mechanisms underlying muscle contraction and to develop methods to study cardiac remodeling processes in response to the adaptive response.

Measuring sarcomere dynamics following immunofluorescent labelling of constitutive structural and contractile proteins.

Myofibrils are subcellular organelles made of sarcomeres. Characterizing the interaction between constitutive proteins may rationalize mechanisms of muscle contraction. Labelling of titin with immunofluorescent antibodies can reveal segmental elongation of domains while subverting shortcomings including inconsistent intensities and asymmetricity of striations (A-band, I-band) inherent to bright field or phase contrast microscopy methods. Considering antibody labelling has been associated with impairments of sarcomere dynamics, we investigated the effect of labelling α-actinin (Z-disc), myomesin (M-line), and I-band titin epitopes to determine how specific antibodies and solution condition parameters might compromise passive force and sarcomere lengths (SLs). Myofibrils were harvested and purified from psoas of New Zealand White rabbits. Labelled samples were incubated with anti-α-actinin, anti-myomesin, and AlexaFluor488 antibodies. Samples were flushed with relaxing solution, restrained in needle-nanolever systems, and observed under 100x magnification (65 nm/pixel). Myofibrils (labelled, n > 6, 150 sarcomeres; controls n = 7, 141 sarcomeres) were stretched passively from SL ranges of 2.5-2.6 μm (relaxed position; RP) to 3.1-3.25 μm (stretched position; SP). Investigating SPs, forces of 66 ± 22 and 60 ± 23 nN/μm2 were obtained in control and labelled myofibrils. No statistical significance was identified, indicating antibodies did not constrain dynamics in limited passive stretch conditions. Standard deviation of individual SLs decreased from 0.15 to 0.09 μm (39%) and 0.17 to 0.13 μm (24%) in RP1 and SP1. Decrease in SL non-uniformity was pronounced in RPs, indicating antibodies may have stiffened lattice structures to induce subtle friction. SL measurement precision was improved in fluorescent compared to phase-contrast trials by 21%. Antibodies had minimal influence at longer lengths but became increasingly constraining at shorter SLs. These results inform increasingly invasive experiments to identify how titin participates in muscle contraction.

Sarcomere dynamics simulations to uncover mechanisms in hypertrophic cardiomyopathy.

Mutations in the beta myosin heavy chain (MYH7) can cause hypercontractility and cardiac remodeling in hypertrophic cardiomyopathy (HCM), but the mechanisms of specific mutations can be difficult to predict. Many mutations change intrinsic rates of myosin activity and their force sensitivity, which can dynamically alter chemo-mechanical cycle events and resultant force generation during cardiac contraction. Our team's multiscale approach enables measurement of the kinetics and force generation in purified myosin proteins, isolated myofibrils, and single and multicellular human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) platforms. Computational modeling can integrate results across these scales and provide additional insights into the individual and combinatorial effects of different changes in myosin kinetics. We have previously used a continuum model to highlight the significance of dysregulation of the super relaxed state of myosin driving hypercontractility in hiPSC-CMs with the P710R mutation. Sensitivity analysis also supported variable myofibril density and sarcomeric organization as a source of population heterogeneity in the force generating capacity of individual hiPSC-CMs. In our ongoing studies, we are leveraging computational modeling to compare the predicted and measured effects of HCM mutations in our different experiments. We have incorporated the measured changes in unloaded myosin to predict force generation after isometric maximal calcium activation (myofibril measurements) and twitch stimulation in single hiPSC-CMs on gels of physiologic stiffness. Additional simulations with spatially explicit models enable more direct prediction of the impact of heterogeneous mixtures of myosins with different molecular profiles and on cooperativity effects associated with the transmission of tension along the length of a sarcomere. Finally, we have visualized the strain profiles of individual sarcomeres in contracting myofibrils in live cells and are correlating these measurements to our simulations. These studies will give new insights into the key biophysical drivers of HCM.

Abstract 13073: Antisense Transcripts Regulate Titin Alternative Splicing and Sarcomere Function in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

The giant protein Titin is an essential structural component of the sarcomere and a key determinant of passive tension during diastole. Titin tension is regulated by alternative splicing within the spring-like domains in the I-band region of TTN pre-mRNA. The cardiac RNA-binding protein RBM20 has been shown to promote TTN exon skipping during splicing, but additional factors are likely involved. We hypothesized that a natural antisense transcript in the TTN locus ( TTN-AS1 ) could regulate TTN splicing in cis through recruitment of spliceosome components. Through single nuclei RNA-Seq and RNA fluorescence in situ hybridization (ISH) on human cardiac tissue and iPS-derived cardiomyocytes (iPS-CM), we showed that the expression of TTN-AS1 was restricted to cardiomyocyte nuclei. The percent spliced in (PSI) of each TTN exon was calculated based on RNA-Seq data from iPS-CM (n=3) and 26 exons (all in the I-band region) that are normally spliced out were included to a significantly higher extent (adjusted p<0.05) upon siRNA-mediated TTN-AS1 knock down. Reduced exon skipping from TTN exon 50 was confirmed with exon-junction spanning qPCR probes (p<0.05, n=3). Similar effects on splicing were seen in cells where RBM20 had been knocked down. The consequences of TTN-AS1 knock down on cardiomyocyte contractile function was assessed using sarcomere tracking. Transgenic expression of ACTN2-GFP was used to visualize z-disks in iPS-CM, sarcomeres (n=14,636) were recorded during contractions using wide-field epifluorescence microscopy and analyzed using Sarc-Graph software. The analysis revealed that in cells where TTN-AS1 had been knocked down, sarcomere length and fractional shortening, as well as contraction and relaxation time were increased (all p<0.001). Again, similar effects were seen in iPS-CM where RBM20 had been knocked down. We observed co-localization of TTN-AS1 and RBM20 protein during active TTN splicing in iPS-CM nuclei using multiplex RNA ISH and immunofluorescence and confirmed interaction between TTN-AS1 and RBM20 using RNA Immunoprecipitation and qPCR. In conclusion, we show that TTN-AS1 can affect cardiomyocyte contractile properties and sarcomere dynamics through regulation of TTN alternative splicing.

Abstract 14927: Real-Time Super-Resolution Analysis of Sarcomere Motion Reveals Context-Dependent Stochastic Heterogeneity in Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Studies of sarcomere dynamics in cardiac myofibrils are key to understand how myosin-actin contractility translates to whole heart contractions. Such analyses require high spatial (nm) and temporal (ms) resolution of multiple sarcomeres in parallel. Here, we present a new imaging and computational method to analyze sarcomere structure and function in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. For this we (1) engineered a fluorescent z-band tag (ACTN2-Citrine) by CRISPR-editing, (2) developed micropatterned soft (7:1, 10 kPa) substrate cultures to mimic in vivo conditions, (3) made use of high-speed microscopy (66 fps), and (4) designed an automated AI-based sarcomere detection and analysis tool (SarcAsM - Sarcomere Analysis Multitool). Cardiomyocytes were differentiated from the ACTN2-Citrine iPSC-line and purified using a staged growth factor and metabolic selection protocol (>95% purity with a ventricular myocyte phenotype). Our analysis revealed highly heterogeneous single sarcomere motion, i.e., shortening and lengthening patterns during the same time of the contraction cycle. Notably, the observed rich dynamics on single sarcomere-level are not apparent from whole cell analyses. Interestingly, culture on stiffer substrates (>20 kPa; mimicking cardiac fibrosis) resulted in reduced cell-level contraction, but constant sarcomere level contractility with however enhanced heterogeneity. Analysis of a large data set (>1,200 cells) in conjunction with a biophysical model showed that heterogeneous sarcomere motion is not caused by static non-uniformity (e.g., strong/weak sarcomeres), but can be primarily attributed to the stochastic and non-linear nature of sarcomere dynamics and thus occurs intrinsically during cardiomyocyte beating. Our findings show that that sarcomere heterogeneity is an important feature of cardiomyocyte contractility. The introduced tools for unbiased high-throughput analysis of sarcomere-level dynamics can be exploited to gain a better understanding on how sarcomere level contractility translates to whole cardiomyocyte contractions under simulated physiological and pathological conditions.

Altered contractility in mutation-specific hypertrophic cardiomyopathy: A mechano-energetic in silico study with pharmacological insights.

Introduction: Mavacamten (MAVA), Blebbistatin (BLEB), and Omecamtiv mecarbil (OM) are promising drugs directly targeting sarcomere dynamics, with demonstrated efficacy against hypertrophic cardiomyopathy (HCM) in (pre)clinical trials. However, the molecular mechanism affecting cardiac contractility regulation, and the diseased cell mechano-energetics are not fully understood yet. Methods: We present a new metabolite-sensitive computational model of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) electromechanics to investigate the pathology of R403Q HCM mutation and the effect of MAVA, BLEB, and OM on the cell mechano-energetics. Results: We offer a mechano-energetic HCM calibration of the model, capturing the prolonged contractile relaxation due to R403Q mutation (∼33%), without assuming any further modifications such as an additional Ca2+ flux to the thin filaments. The HCM model variant correctly predicts the negligible alteration in ATPase activity in R403Q HCM condition compared to normal hiPSC-CMs. The simulated inotropic effects of MAVA, OM, and BLEB, along with the ATPase activities in the control and HCM model variant agree with in vitro results from different labs. The proposed model recapitulates the tension-Ca2+ relationship and action potential duration change due to 1µM OM and 5µM BLEB, consistently with in vitro data. Finally, our model replicates the experimental dose-dependent effect of OM and BLEB on the normalized isometric tension. Conclusion: This work is a step toward deep-phenotyping the mutation-specific HCM pathophysiology, manifesting as altered interfilament kinetics. Accordingly, the modeling efforts lend original insights into the MAVA, BLEB, and OM contributions to a new interfilament balance resulting in a cardioprotective effect.

Sarcomere dynamics revealed by a myofilament integrated FRET-based biosensor in live skeletal muscle fibers

The sarcomere is the functional unit of skeletal muscle, essential for proper contraction. Numerous acquired and inherited myopathies impact sarcomere function causing clinically significant disease. Mechanistic investigations of sarcomere activation have been challenging to undertake in the context of intact, live skeletal muscle fibers during real time physiological twitch contractions. Here, a skeletal muscle specific, intramolecular FRET-based biosensor was designed and engineered into fast skeletal muscle troponin C (TnC) to investigate the dynamics of sarcomere activation. In transgenic animals, the TnC biosensor incorporated into the skeletal muscle fiber sarcomeres by stoichiometric replacement of endogenous TnC and did not alter normal skeletal muscle contractile form or function. In intact single adult skeletal muscle fibers, real time twitch contractile data showed the TnC biosensor transient preceding the peak amplitude of contraction. Importantly, under physiological temperatures, inactivation of the TnC biosensor transient decayed significantly more slowly than the Ca2+ transient and contraction. The uncoupling of the TnC biosensor transient from the Ca2+ transient indicates the biosensor is not functioning as a Ca2+ transient reporter, but rather reports dynamic sarcomere activation/ inactivation that, in turn, is due to the ensemble effects of multiple activating ligands within the myofilaments. Together, these findings provide the foundation for implementing this new biosensor in future physiological studies investigating the mechanism of activation of the skeletal muscle sarcomere in health and disease.

In silico identification of potential calcium dynamics and sarcomere targets for recovering left ventricular function in rat heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a complex disease associated with multiple co-morbidities, where impaired cardiac mechanics are often the end effect. At the cellular level, cardiac mechanics can be pharmacologically manipulated by altering calcium signalling and the sarcomere. However, the link between cellular level modulations and whole organ pump function is incompletely understood. Our goal is to develop and use a multi-scale computational cardiac mechanics model of the obese ZSF1 HFpEF rat to identify important biomechanical mechanisms that underpin impaired cardiac function and to predict how whole-heart mechanical function can be recovered through altering cellular calcium dynamics and/or cellular contraction. The rat heart was modelled using a 3D biventricular biomechanics model. Biomechanics were described by 16 parameters, corresponding to intracellular calcium transient, sarcomere dynamics, cardiac tissue and hemodynamics properties. The model simulated left ventricular (LV) pressure-volume loops that were described by 14 scalar features. We trained a Gaussian process emulator to map the 16 input parameters to each of the 14 outputs. A global sensitivity analysis was performed, and identified calcium dynamics and thin and thick filament kinetics as key determinants of the organ scale pump function. We employed Bayesian history matching to build a model of the ZSF1 rat heart. Next, we recovered the LV function, described by ejection fraction, peak pressure, maximum rate of pressure rise and isovolumetric relaxation time constant. We found that by manipulating calcium, thin and thick filament properties we can recover 34%, 28% and 24% of the LV function in the ZSF1 rat heart, respectively, and 39% if we manipulate all of them together. We demonstrated how a combination of biophysically based models and their derived emulators can be used to identify potential pharmacological targets. We predicted that cardiac function can be best recovered in ZSF1 rats by desensitising the myofilament and reducing the affinity to intracellular calcium concentration and overall prolonging the sarcomere staying in the active force generating state.

Development of a multiscale electromechanical computer model from cell to hemodynamics

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CVON eDETECT 2015-12 Background Patients with genetic cardiomyopathies mostly remain asymptomatic until the occurrence of life-threatening arrhythmias and sudden cardiac death. The responsible pro-arrhythmic mechanisms, as well as the indicators for pro-arrhythmia, often remain unclear. Current research approaches are commonly based on experimental animal models or clinical data obtained from mutation carriers. The translation (and bridging) of results obtained from experimental models to humans and scale integration of these findings remains challenging. As a consequence, clinical relevance is often disputed. In light of these challenges, computer modelling shows strong potential for its ability to capture the complex dynamics of the cardiovascular system across different scales. Purpose We therefore aim to develop a multiscale electromechanical computer model coupling electrophysiology, sarcomere dynamics (contractility), whole-heart function and hemodynamics to investigate mechanisms (resulting from genetic predisposition to cardiomyopathy) of arrhythmogenicity and cardiac dysfunction in these patients. Methods We coupled our previously published model of cellular electromechanics to the CircAdapt computer model of the heart and circulation. Electromechanical coupling was performed through the amount of calcium bound to troponin (as described in the cellular model). The same electrophysiological properties were assumed in all cardiac walls. Diffusion of calcium from the sarcolemmal space to the membrane was described phenomenologically through a resistance model. Results Simulated hemodynamics properties were in range with control values with LVEDV = 110.8mL, LVESV = 38.7mL, RVEDV = 99.2mL, mLAP = 10.2mmHg, mRAP = 4.0mmHg. At the cellular level, sarcolemmal calcium transients showed the effect of mechanical change as illustrated by the bump in CaT. This effect was attenuated at the membrane due to the effect of diffusion. Alterations in the functionality of molecular entities underlying calcium homeostasis recapitulated cellular experimental findings as expected illustrated by implementing changes in adrenergic signaling. Conclusions We have established a new multiscale electromechanical computer model describing electrophysiology, sarcomere mechanics and hemodynamics. The model simulates normal values in the control situation. Current work focusses on parameter sensitivity analysis and validation. Feeding this new model with experimentally obtained data from patient-specific models (ranging from individual cells to e.g. transgenic mouse models based on relevant mutations) therefore has potential to bridge the gap between pathogenic experimental models and patient data. This will add to the understanding of underlying mechanisms responsible for disease onset and progression, and furthermore could develop into a tool to identify patients/mutation carriers at risk for severe disease outcome. Abstract Figure.

Exercise-induced alterations of myocardial sarcomere dynamics are associated with hypophosphorylation of cardiac troponin I.

Although the knowledge of sports cardiology advanced significantly in the recent years, the molecular mechanisms by which exercise training augments cardiac performance is poorly understood. Here we aimed at determining left ventricular (LV) myocardial sarcomeric protein modifications in a rat model of exercise training and detraining. Young male Wistar rats were divided into exercised (Ex) and control (Co) groups. Trained rats swam 200 min/day for 12 weeks. Detrained (DEx) and control (DCo) rats remained sedentary for 8 weeks after completion of the 12-week-long protocol. Ca2+-regulated active force production (Fa⁢c⁢t⁢i⁢v⁢e), its Ca2+-sensitivity (pCa50) and Ca2+-independent passive tension (Fp⁢a⁢s⁢s⁢i⁢v⁢e) were determined in isolated permeabilized cardiomyocytes and phosphorylation levels of sarcomeric proteins were assayed by biochemical methods. Means of maximal Ca2+-activated isometric force (Fm⁢a⁢x) and pCa50 values were higher (p < 0.05) in the Ex group (28.0 ± 1.4 kN/m2 and 5.91 ± 0.03, respectively, mean ± SEM) than those in the Co group (15.8 ± 0.8 kN/m2 and 5.81 ± 0.03, respectively). Fp⁢a⁢s⁢s⁢i⁢v⁢e did not differ between these two groups. The level of cardiac troponin I (cTnI) phosphorylation decreased upon exercise (from 1.00 ± 0.02 to 0.66 ± 0.06, p < 0.05; in relative units). Site specific phosphorylation assays revealed cTnI hypophosphorylations at the protein kinase A (PKA)-specific Ser-22/23 sites and at the protein kinase C (PKC)-specific Thr-143 site. Mechanical and biochemical parameters of the DEx and DCo groups did not differ from each other following the detraining period. Exercise-induced hypertrophy is associated with reversible increases in Ca2+-dependent force production and its Ca2+-sensitivity in LV cardiomyocytes, which can be associated with changes in cTnI phosphorylation.

Sarcomere length non-uniformities dictate force production along the descending limb of the force-length relation.

The force-length relation is one of the most defining features of muscle contraction, and yet a topic of debate in the literature. The sliding filament theory predicts that the force produced by muscle fibres is proportional to the degree of overlap between myosin and actin filaments, producing a linear descending limb of the active force-length relation. However, several studies have shown forces that are larger than predicted, especially at long sarcomere lengths (SLs). Studies have been conducted with muscle fibres, preparations containing thousands of sarcomeres that make measurements of individual SL challenging. The aim of this study was to evaluate force production and sarcomere dynamics in isolated myofibrils and single sarcomeres from the rabbit psoas muscle to enhance our understanding of the theoretically predicted force-length relation. Contractions at varying SLs along the plateau (SL = 2.25-2.39 µm) and the descending limb (SL > 2.39 µm) of the force-length relation were induced in sarcomeres and myofibrils, and different modes of force measurements were used. Our results show that when forces are measured in single sarcomeres, the experimental force-length relation follows theoretical predictions. When forces are measured in myofibrils with large SL dispersions, there is an extension of the plateau and forces elevated above the predicted levels along the descending limb. We also found an increase in SL non-uniformity and slowed rates of force production at long lengths in myofibrils but not in single sarcomere preparations. We conclude that the deviation of the descending limb of the force-length relation is correlated with the degree of SL non-uniformity and slowed force development.

Biophysically detailed mathematical models of multiscale cardiac active mechanics.

We propose four novel mathematical models, describing the microscopic mechanisms of force generation in the cardiac muscle tissue, which are suitable for multiscale numerical simulations of cardiac electromechanics. Such models are based on a biophysically accurate representation of the regulatory and contractile proteins in the sarcomeres. Our models, unlike most of the sarcomere dynamics models that are available in the literature and that feature a comparable richness of detail, do not require the time-consuming Monte Carlo method for their numerical approximation. Conversely, the models that we propose only require the solution of a system of PDEs and/or ODEs (the most reduced of the four only involving 20 ODEs), thus entailing a significant computational efficiency. By focusing on the two models that feature the best trade-off between detail of description and identifiability of parameters, we propose a pipeline to calibrate such parameters starting from experimental measurements available in literature. Thanks to this pipeline, we calibrate these models for room-temperature rat and for body-temperature human cells. We show, by means of numerical simulations, that the proposed models correctly predict the main features of force generation, including the steady-state force-calcium and force-length relationships, the length-dependent prolongation of twitches and increase of peak force, the force-velocity relationship. Moreover, they correctly reproduce the Frank-Starling effect, when employed in multiscale 3D numerical simulation of cardiac electromechanics.

Abstract 522: Impaired Inside-out Force Transmission in Hipsc-cardiomyocyte Model of Duchenne Muscular Dystrophy Cardiomyopathy

Duchenne Muscular Dystrophy (DMD) is a lethal X-chromosome linked disease that affects ~1:3500 boys and culminates in heart failure in early adulthood. DMD results from &gt;200 possible dystrophin mutations. The lack of dystrophin disrupts the anchoring of the sarcomere to the extracellular matrix (ECM), impairing cardiomyocyte function and cardiac contraction. With disease progression, the heart tissue stiffness increases as fibrosis progresses and a dilated cardiomyopathy phenotype develops. We hypothesize that disease progression accelerates because of a positive feedback loop involving fibrotic stiffening and mechanosensing. Our preliminary results show that single human-derived pluripotent stem cell (hiPSC) cardiomyocytes (CMs) with DMD mutations have a dramatically reduced ability to produce force compared to isogenic controls on stiffer substrates mimicking a fibrotic state, but not on soft healthy substrates. This stiffness-dependent contractile deficiency correlates with an increase in reactive oxygen species (ROS) and mitochondrial dysfunction, as well as other markers of cellular stress response and senescence. Here, we ask how stiffness affects the progression of DMD cardiomyopathy and aim to draw a link between the lack of dystrophin and force production deficiency in hiPSC-CMs with or without DMD mutations in an isogenic background. Using a novel assay and algorithm, we simultaneously measure extracellular force production by traction force microscopy (TFM) and single sarcomere dynamics (SSD) in single hiPSC-CMs adhered onto ECM micropatterns on top of a hydrogel of stiffness matching that of healthy (~10kPa) or fibrotic (~35kPa) tissue. We induce the single hiPSC-CMs to adopt a physiologic elongated 1:7 aspect ratio using microcontact printing of ECM protein. Using this platform, we determine how the lack of dystrophin impacts sarcomere architecture, sarcomere contractility, and force generation in the etiology of DMD cardiac disease.

Sarcomere Lengths Become More Uniform Over Time in Intact Muscle-Tendon Unit During Isometric Contractions

The seemingly uniform striation pattern of skeletal muscles, quantified in terms of sarcomere lengths (SLs), is inherently non-uniform across all hierarchical levels. The SL non-uniformity theory has been used to explain the force creep in isometric contractions, force depression following shortening of activated muscle, and residual force enhancement following lengthening of activated muscle. Our understanding of sarcomere contraction dynamics has been derived primarily from in vitro experiments using regular bright-field light microscopy or laser diffraction techniques to measure striation/diffraction patterns in isolated muscle fibers or myofibrils. However, the collagenous extracellular matrices present around the muscle fibers, as well as the complex architecture in the whole muscles may lead to different contraction dynamics of sarcomeres than seen in the in vitro studies. Here, we used multi-photon excitation microscopy to visualize in situ individual sarcomeres in intact muscle tendon units (MTUs) of mouse tibialis anterior (TA), and quantified the temporal changes of SL distribution as a function of SLs in relaxed and maximally activated muscles for quasi-steady state, fixed-end isometric conditions. The corresponding muscle forces were simultaneously measured using a force transducer. We found that SL non-uniformity, quantified by the coefficient of variation (CV) of SLs, decreased at a rate of 1.9–3.1%/s in the activated muscles, but remained constant in the relaxed muscles. The force loss during the quasi-steady state likely did not play a role in the decrease of SL non-uniformity, as similar force losses were found in the activated and relaxed muscles, but the CV of SLs in the relaxed muscles underwent negligible change over time. We conclude that sarcomeres in the mid-belly of maximally contracting whole muscles constantly re-organize their lengths into a more uniform pattern over time. The molecular mechanisms accounting for SL non-uniformity appear to differ in active and passive muscles, and need further elucidation, as do the functional implications of the SL non-uniformity.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

Personalized Perioperative Multi-scale, Multi-physics Heart Simulation of Double Outlet Right Ventricle.

For treatment of complex congenital heart disease, computer simulation using a three-dimensional heart model may help to improve outcomes by enabling detailed preoperative evaluations. However, no highly integrated model that accurately reproduces a patient's pathophysiology, which is required for this simulation has been reported. We modelled a case of complex congenital heart disease, double outlet right ventricle with ventricular septal defect and atrial septal defect. From preoperative computed tomography images, finite element meshes of the heart and torso were created, and cell model of cardiac electrophysiology and sarcomere dynamics was implemented. The parameter values of the heart model were adjusted to reproduce the patient's electrocardiogram and haemodynamics recorded preoperatively. Two options of in silico surgery were performed using this heart model, and the resulting changes in performance were examined. Preoperative and postoperative simulations showed good agreement with clinical records including haemodynamics and measured oxyhaemoglobin saturations. The use of a detailed sarcomere model also enabled comparison of energetic efficiency between the two surgical options. A novel in silico model of congenital heart disease that integrates molecular models of cardiac function successfully reproduces the observed pathophysiology. The simulation of postoperative state by in silico surgeries can help guide clinical decision-making.

Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live Drosophila melanogaster larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres. Therefore, wide-field SHG microscopy has applications in basic contractility research and studying arrhythmias, muscular dystrophies and pharmaceutical effects on the muscle contraction dynamics of sarcomeres.

Abstract 860: Functional Characterization of a Novel Human Heart-specific Microprotein With a Potential Mitochondrial Localization and Role in Sarcomere Dynamics

Cardiac microproteins are an emerging class of important regulators of cellular homeostasis, often encoded by presumed long noncoding RNAs. With only few described so far, their prevalence and biological significance largely remains unclear. To create an atlas of cardiac-expressed microproteins, we performed ribosome profiling on 80 human heart samples. This revealed the translation of hundreds of previously undetected microproteins, which we validated in vivo by targeted mass spectrometry. A single heart-specific microprotein caught our attention as it showed strong expression coregulation with components of mitochondrial OXPHOS pathway. We next deleted this microprotein's lncRNA host gene in hiPSCs using CRISPR/Cas9, and, upon differentiation into 30-d old cardiomyocytes, find a strong expression upregulation of genes involved in oxidative phosphorylation and calcium handling. To investigate the impact of the microprotein KO on cardiomyocyte mechanical function, we assessed sarcomere dynamics using an endogenous TTN-GFP tag, enabling live cell imaging analysis with the SarcTrack algorithm. Preliminary analysis sowes a putative increase in sarcomere contraction and relaxation times, suggesting a repressive regulatory function of the eliminated microprotein encoding lncRNA.

Abstract 807: Rad Ablation as a Treatment to Target Cardiac Inotropy via L-type Calcium Channel Function

The L-type Ca 2+ channel current (I Ca,L ) provides trigger Ca 2+ to contribute to cardiac contraction. Rad GTPase associates with the L-type Ca 2+ channel (LTCC) and serves as an endogenous inhibitor of LTCC activity. Overexpression of Rad blocks I Ca,L ; absence of Rad increases I Ca,L . Rad attenuates β-adrenergic receptor (β-AR) signaling. Chronic β-AR stimulation associates with Ca 2+ mishandling and can promote signaling that progresses towards heart failure. Early studies of global, constitutive Rad-knockout mice (gRadKO) suggested that elevated Ca 2+ dynamics leads to pathological cardiac hypertrophy; however, Rad is also expressed in non-cardiac tissues. Our objective is to test the hypothesis that increased myocardial I Ca,L via Rad deletion safely enhances cardiac function without driving pathological remodeling. We created a cardiac-restricted inducible Rad knockout mouse (Rad Δ/Δ ). In vivo function was measured with echocardiography. We examined I Ca,L through whole cell configuration of the patch clamp technique, assessed Ca 2+ handling, and sarcomere dynamics. Unlike gRadKO, Rad Δ/Δ showed no elevation of fetal gene program, nor fibrosis, and no change to aortic pressure. Rad Δ/Δ had a sustained increase of inotropy without structural or functional remodeling (EF: Rad Δ/Δ =76 ± 2%, n=16; Rad fl/fl =59 ± 4%, n=7; p=0.001.) I Ca,L was significantly increased, with Rad loss mirroring a β-AR modulated phenotype on basal I Ca,L (max. conductance: Rad Δ/Δ =254 ± 19 pS/pF, n=15; Rad fl/fl =144 ± 12 pS/pF, n=18; p&lt;10 -4 ). Contrary to models of chronic β-AR stimulation, Rad Δ/Δ retained β-AR signaling shown in vivo using isoproterenol, and by preserved phosphorylation of protein regulators of Ca 2+ reuptake and contractility. Rad Δ/Δ cardiomyocytes show enhanced cytosolic Ca 2+ handling (Decay of Ca 2+ transient: Rad Δ/Δ = 0.07 ± 0.003 (F 340 /F 380 )/s, n=67, Rad fl/fl = 0.10 ± 0.005, n=69; p&lt;10 -4 ), increased contractile function, and elevated SERCA2a expression. These new findings challenge the canonical assumption that increased myocardial Ca 2+ necessarily promotes pathology. We conclude that cardiac hypertrophy in gRadKO was caused by non-cardiac tissue effects, and myocardial Rad deletion is a promising cardiac inotropic therapeutic direction.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.