Single Sarcomere Research Articles

Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

Association between Hypertrophic Cardiomyopathy and Variations in Sarcomere Gene and Calcium Channel Gene in Adults

Introduction: Calcium channel gene variations have been reported to be associated with hypertrophic cardiomyopathy (HCM) in family, but the relationship between calcium channel gene variations and HCM remains undefined in the population. Methods: A total of 719 HCM unrelated patients were initially enrolled. Finally, 371 patients were identified based on inclusion and exclusion criteria, including 145 patients with gene negative, 28 patients with a single rare calcium channel gene variation (calcium gene variation), 162 patients with a single pathogenic/likely pathogenic sarcomere gene variation (sarcomere gene variation) and 36 patients with a single pathogenic/likely pathogenic sarcomere gene variation and a single rare calcium channel gene variation (double gene variations). Then the demographic, electrocardiographic, echocardiographic, and follow-up data were collected. Results: Patients with double gene variations were at an earlier age and had more percent of family history of HCM, and had thicker walls, higher left ventricular outflow tract pressure gradient, more pathological Q waves, and more bundle branch blocks as compared with those with single sarcomere gene variation. During the follow-up period, patients with double gene variations had more primary endpoints than the other three groups (p = 0.0013). Multivariate analysis showed that double gene variations were the independent predictor of primary endpoint events in patients (HR: 4.82, 95% CI: 1.77–13.2; p = 0.002). Conclusion: We found that patients with double gene variations had more severe HCM phenotype and prognosis. The pathogenesis effects of sarcomere gene variation and calcium channel gene variation may be cumulative in HCM populations.

Visualization of cardiac thick filament dynamics in ex vivo heart preparations

RationaleCardiac muscle cells are terminally differentiated after birth and must beat continually throughout one's lifetime. This mechanical process is driven by the sliding of actin-based thin filaments along myosin-based thick filaments, organized within sarcomeres. Despite costly energetic demand, the half-life of the proteins that comprise the cardiac thick filaments is ∼10 days, with individual molecules being replaced stochastically, by unknown mechanisms. ObjectivesTo allow for the stochastic replacement of molecules, we hypothesized that the structure of thick filaments must be highly dynamic in vivo. Methods and resultsTo test this hypothesis in adult mouse hearts, we replaced a fraction of the endogenous myosin regulatory light chain (RLC), a component of thick filaments, with GFP-labeled RLC by adeno-associated viral (AAV) transduction. The RLC-GFP was properly localized to the heads of the myosin molecules within thick filaments in ex vivo heart preparations and had no effect on heart size or actin filament siding in vitro. However, the localization of the RLC-GFP molecules was highly mobile, changing its position within the sarcomere on the minute timescale, when quantified by fluorescence recovery after photobleaching (FRAP) using multiphoton microscopy. Interestingly, RLC-GFP mobility was restricted to within the boundaries of single sarcomeres. When cardiomyocytes were lysed, the RLC-GFP remained strongly bound to myosin heavy chain, and the intact myosin molecules adopted a folded, compact configuration, when disassociated from the filaments at physiological ionic conditions. ConclusionsThese data demonstrate that the structure of the thick filament is highly dynamic in the intact heart, with a rate of molecular exchange into and out of thick filaments that is ∼1500 times faster than that required for the replacement of molecules through protein synthesis or degradation.

Association between clinical phenotypes of hypertrophic cardiomyopathy and Ca2+ gene variation gene variation

Objective: To observe the association between clinical phenotypes of hypertrophic cardiomyopathy (HCM) patients and a rare calcium channel and regulatory gene variation (Ca2+ gene variation) and to compare clinical phenotypes of HCM patients with Ca2+ gene variation, a single sarcomere gene variation and without gene variation and to explore the influence of rare Ca2+ gene variation on the clinical phenotypes of HCM. Methods: Eight hundred forty-two non-related adult HCM patients diagnosed for the first time in Xijing Hospital from 2013 to 2019 were enrolled in this study. All patients underwent exon analyses of 96 hereditary cardiac disease-related genes. Patients with diabetes mellitus, coronary artery disease, post alcohol septal ablation or septal myectomy, and patients who carried sarcomere gene variation of uncertain significance or carried>1 sarcomere gene variation or carried>1 Ca2+ gene variation, with HCM pseudophenotype or carrier of ion channel gene variations other than Ca2+ based on the genetic test results were excluded. Patients were divided into gene negative group (no sarcomere or Ca2+ gene variants), sarcomere gene variation group (only 1 sarcomere gene variant) and Ca2+ gene variant group (only 1 Ca2+ gene variant). Baseline data, echocardiography and electrocardiogram data were collected for analysis. Results: A total of 346 patients were enrolled, including 170 patients without gene variation (gene negative group), 154 patients with a single sarcomere gene variation (sarcomere gene variation group) and 22 patients with a single rare Ca2+ gene variation (Ca2+ gene variation group). Compared with gene negative group, patients in Ca2+ gene variation group had higher blood pressure and higher percentage of family history of HCM and sudden cardiac death (P<0.05); echocardiographic results showed that patients in Ca2+ gene variation group had thicker ventricular septum ((23.5±5.8) mm vs. (22.3±5.7) mm, P<0.05); electrocardiographic results showed that patients in Ca2+ gene variation group had prolonged QT interval ((416.6±23.1) ms vs. (400.6±47.2) ms, P<0.05) and higher RV5+SV1 ((4.51±2.26) mv vs. (3.50±1.65) mv, P<0.05). Compared with sarcomere gene variation group, patients in Ca2+ gene variation group had later onset age and higher blood pressure (P<0.05); echocardiographic results showed that there was no significant difference in ventricular septal thickness between two groups; patients in Ca2+ gene variation group had lower percentage of left ventricular outflow tract pressure gradient>30 mmHg (1 mmHg=0.133 kPa, 22.8% vs. 48.1%, P<0.05) and the lower early diastolic peak velocity of the mitral valve inflow/early diastolic peak velocity of the mitral valve annulus (E/e') ratio ((13.0±2.5) vs. (15.9±4.2), P<0.05); patients in Ca2+ gene variation group had prolonged QT interval ((416.6±23.1) ms vs. (399.0±43.0) ms, P<0.05) and lower percentage of ST segment depression (9.1% vs. 40.3%, P<0.05). Conclusion: Compared with gene negative group, the clinical phenotype of HCM is more severe in patients with rare Ca2+ gene variation; compared with patients with sarcomere gene variation, the clinical phenotype of HCM is milder in patients with rare Ca2+ gene variation.

Thick filament dynamics: A mechanism for protein replacement within the sarcomere.

The heart contracts continually throughout one's lifetime through antiparallel sliding of actin-based thin filaments along myosin-based thick filaments, organized within muscle sarcomeres. A recent biochemical study from our lab demonstrated that the half-life of cardiac thick filament proteins is ∼10 days in adult mice, suggesting that continual protein replacement supports structural and functional fidelity. These data further suggest that newly synthesized molecules are randomly incorporated into preexisting thick filaments by a stochastic mechanism. Therefore, we hypothesize that myosin replacement involves the dynamic exchange of single molecules into thick filaments in vivo. To test this hypothesis, we generated an adeno-associated virus that replaced 27±7% of the endogenous regulatory light chain (RLC) on myosin molecules with a fluorescent GFP-RLC. We then visualized myosin within intact hearts using two-photon microscopy. At high magnification, individual sarcomeres were observed with GFP-RLC incorporated into thick filaments. Next, we measured the movement of fluorescently labeled myosin within the heart by irreversibly photobleaching limited areas of the cells and quantifying fluorescence recovery after photobleaching (FRAP). No FRAP occurred within 30 minutes, when photobleaching regions encompassed entire sarcomeres. However, rapid FRAP occurred after photobleaching small portions of single sarcomeres at rates orders of magnitude faster than protein synthesis. Recovery appeared to come from the exchange of molecules in the unbleached portion of the sarcomere and was fastest near the tips of the thick filaments, suggesting enhanced exchange where myosin is less densely packed in the filament. Collectively, these data demonstrate dynamic thick filament structure in vivo with myosin molecules freely exchanging between thick filaments within single sarcomeres. Therefore, these large macromolecular complexes appear to be designed to allow for the rapid incorporation and release of myosin molecules while maintaining the structural integrity that is essential to support contractility.

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 (&gt;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 (&gt;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 (&gt;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.

Developmental Regulation of Sarcomere Branching in Cardiomyocytes

Cardiac muscles undergo constant contractions to continuously supply oxygenated blood to both central and peripheral organs. To sustain such large demand, cardiac muscles must convert biological fuel to functional force via the contraction of their sarcomeres. Using nanoscale 3D imaging analysis, our group demonstrated that sarcomeres within the cardiac muscle of mature mice (≥ 2 months) are frequently branched to form a unified myofibrillar matrix across the entire cell, thereby providing the architecture needed for both longitudinal and lateral transmission of active force. How these contractile networks are formed during postnatal development remains unclear. Here we used 3D focused‐ion beam scanning electron microscopy (FIB‐SEM) to evaluate myofibrillar ultrastructure within cardiac muscle cells in mice at postnatal (P) days 1, 7, 14, and 42, respectively. 3D reconstruction of myofibrillar structures showed highly connected networks at all time points. Quantitative assessment of sarcomere branch‐points revealed that branching frequency is varied by developmental process. The percentages of sarcomeres with at least one branch‐point are as follows: 57 ± 2.4% (mean ± SE, 14 cells, 129 myofibrils, 802 sarcomeres) in P1, 64 ± 2.1% (17 cells, 161 myofibrils, 795 sarcomeres) in P7, 49 ± 3.9% (4 cells, 50 myofibrils, 250 sarcomeres) in P14, and 27 ± 1.1 % (7 cells, 236 myofibrils, 2320 sarcomeres) in P42. Data was further separated based on whether a single sarcomere is branched into two (single‐branch, Fig 1E, F) or more (multi‐branch, Fig 1G, H) structures to delineate the nature and overall magnitude of branching within cardiomyocytes (Fig 2B‐ D). Across the postnatal development (P1 to P42), there was a 0.4‐fold change in the magnitude of branching within ten sarcomeres (Fig 2D; 7.9±0.41, 11±0.45, 6.5±0.58, and 3.3±0.16 for P1, P7, P14, and P42, respectively). All changes were statistically significant at P&lt;0.05. In conclusion, the frequency of sarcomere branching was highest at P7 and progressively declined until maturation. Findings indicate that while the architecture needed for active force transmission across the length and width of the cell is already formed at birth, the connectivity of cardiac contractile networks is dynamic throughout postnatal development. These results suggest that sarcomere branching dynamics may provide a target for modulating the contractility of cardiac muscle cells.

What Can We Learn from Single Sarcomere and Myofibril Preparations?

Sarcomeres are the smallest functional contractile unit of muscle, and myofibrils are striated muscle organelles that are comprised of sarcomeres that are strictly aligned in series. Furthermore, passive forces in sarcomeres and myofibrils are almost exclusively produced by the structural protein titin, and all contractile, regulatory, and structural proteins are in their natural configuration. For these mechanical and structural reasons single sarcomere and myofibril preparations are arguably the most powerful to answer questions on the mechanisms of striated muscle contraction. We developed and optimized single myofibril research over the past 20 years and were the first to mechanically isolate and test single sarcomeres. The results from this research led to the uncovering of the crucial role of titin in muscle contraction, first molecular explanations for the origins of the passive and the residual force enhancement properties of skeletal and cardiac muscles, the discovery of sarcomere length stability on the descending limb of the force-length relationship, and culminating in the formulation of the three-filament theory of muscle contraction that, aside from actin and myosin, proposes a crucial role of titin in active force production. Aside from all the advantages and possibilities that single sarcomere and myofibril preparations offer, there are also disadvantages. These include the fragility of the preparation, the time-consuming training to master these preparations, the limited spatial resolution for length and force measurements, and the unavailability of commercial systems for single sarcomere/myofibril research. Ignoring the mechanics that govern serially linked systems, not considering the spatial resolution and associated accuracies of myofibril systems, and neglecting the fragility of myofibril preparations, has led to erroneous interpretations of results and misleading conclusions. Here, we will attempt to describe the methods and possible applications of single sarcomere/myofibril research and discuss the advantages and disadvantages by focusing on specific applications. It is hoped that this discussion may contribute to identifying the enormous potential of single sarcomere/myofibril research in discovering the secrets of muscle contraction.

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.

Nanoscopic changes in the lattice structure of striated muscle sarcomeres involved in the mechanism of spontaneous oscillatory contraction (SPOC)

Muscles perform a wide range of motile functions in animals. Among various types are skeletal and cardiac muscles, which exhibit a steady auto-oscillation of force and length when they are activated at an intermediate level of contraction. This phenomenon, termed spontaneous oscillatory contraction or SPOC, occurs devoid of cell membranes and at fixed concentrations of chemical substances, and is thus the property of the contractile system per se. We have previously developed a theoretical model of SPOC and proposed that the oscillation emerges from a dynamic force balance along both the longitudinal and lateral axes of sarcomeres, the contractile units of the striated muscle. Here, we experimentally tested this hypothesis by developing an imaging-based analysis that facilitates detection of the structural changes of single sarcomeres at unprecedented spatial resolution. We found that the sarcomere width oscillates anti-phase with the sarcomere length in SPOC. We also found that the oscillatory dynamics can be altered by osmotic compression of the myofilament lattice structure of sarcomeres, but they are unchanged by a proteolytic digestion of titin/connectin—the spring-like protein that provides passive elasticity to sarcomeres. Our data thus reveal the three-dimensional mechanical dynamics of oscillating sarcomeres and suggest a structural requirement of steady auto-oscillation.

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.

Simulating electromechanical delay across the scales – relating the behavior of single sarcomers on a sub‐cellular scale and the muscle‐tendon system on the organ scale

AbstractThe time lag between the activation of a muscle and the development of force is commonly denoted as electromechanical delay (EMD). Within this work, a multi‐scale model of an idealized muscle‐tendon system is used to analyze EMD on different characteristic anatomical length scales. Thereby, the simluated EMD‐stretch curves for a complete muscle‐tendon system and a single sarcomere are significantly different. While for an isolated muscle EMD is shown to be strongly determined by excitation‐contraction coupling (ECC), the EMD‐stretch curve for a complete muscle‐tendon system is influenced by additional factors, such as, e.g., the tendon. Further, it is shown that the action potential conduction velocity (APCV) has minor influence on EMD.

Single sarcomere contraction dynamics in a whole muscle

The instantaneous sarcomere length (SL) is regarded as an important indicator of the functional properties of striated muscle. Previously, we found greater sarcomere elongations at the distal end compared to the mid-portion in the mouse tibialis anterior (TA) when the muscle was stretched passively. Here, we wanted to see if SL dispersions increase with activation, as has been observed in single myofibrils, and if SL dispersions differ for different locations in a muscle. Sarcomere lengths were measured at a mid- and a distal location of the TA in live mice using second harmonic generation imaging. Muscle force was measured using a tendon force transducer. We found that SL dispersions increased substantially from the passive to the active state, and were the same for the mid- and distal portions of TA. Sarcomere length non-uniformities within a segment of ~30 serial sarcomeres were up to 1.0 µm. We conclude from these findings that passive, mean SLs obtained from a single location are not necessarily representative of the distribution of SL in active muscle, and thus may be misinterpreted when deriving muscle mechanical properties, such as the force-length relationship. In view of these findings, it seems crucial to determine how SL distributions within a muscle relate to the most fundamental properties of muscle, such as the maximal isometric force.

Sarcomeric Auto-Oscillations in Single Myofibrils From the Heart of Patients With Dilated Cardiomyopathy.

Left ventricular wall motion is depressed in patients with dilated cardiomyopathy (DCM). However, whether or not the depressed left ventricular wall motion is caused by impairment of sarcomere dynamics remains to be fully clarified. We analyzed the mechanical properties of single sarcomere dynamics during sarcomeric auto-oscillations (calcium spontaneous oscillatory contractions [Ca-SPOC]) that occurred at partial activation under the isometric condition in myofibrils from donor hearts and from patients with severe DCM (New York Heart Association classification III-IV). Ca-SPOC reproducibly occurred in the presence of 1 μmol/L free Ca2+ in both nonfailing and DCM myofibrils, and sarcomeres exhibited a saw-tooth waveform along single myofibrils composed of quick lengthening and slow shortening. The period of Ca-SPOC was longer in DCM myofibrils than in nonfailing myofibrils, in association with prolonged shortening time. Lengthening time was similar in both groups. Then, we performed Tn (troponin) exchange in myofibrils with a DCM-causing homozygous mutation (K36Q) in cTnI (cardiac TnI). On exchange with the Tn complex from healthy porcine ventricles, period, shortening time, and shortening velocity in cTnI-K36Q myofibrils became similar to those in Tn-reconstituted nonfailing myofibrils. Protein kinase A abbreviated period in both Tn-reconstituted nonfailing and cTnI-K36Q myofibrils, demonstrating acceleration of cross-bridge kinetics. Sarcomere dynamics was found to be depressed under loaded conditions in DCM myofibrils because of impairment of thick-thin filament sliding. Thus, microscopic analysis of Ca-SPOC in human cardiac myofibrils is beneficial to systematically unveil the kinetic properties of single sarcomeres in various types of heart disease.

Coupling Langevin Dynamics With Continuum Mechanics: Exposing the Role of Sarcomere Stretch Activation Mechanisms to Cardiac Function.

High-performance computing approaches that combine molecular-scale and macroscale continuum mechanics have long been anticipated in various fields. Such approaches may enrich our understanding of the links between microscale molecular mechanisms and macroscopic properties in the continuum. However, there have been few successful examples to date owing to various difficulties associated with overcoming the large spatial (from 1 nm to 10 cm) and temporal (from 1 ns to 1 ms) gaps between the two scales. In this paper, we propose an efficient parallel scheme to couple a microscopic model using Langevin dynamics for a protein motor with a finite element continuum model of a beating heart. The proposed scheme allows us to use a macroscale time step that is an order of magnitude longer than the microscale time step of the Langevin model, without loss of stability or accuracy. This reduces the overhead required by the imbalanced loads of the microscale computations and the communication required when switching between scales. An example of the Langevin dynamics model that demonstrates the usefulness of the coupling approach is the molecular mechanism of the actomyosin system, in which the stretch-activation phenomenon can be successfully reproduced. This microscopic Langevin model is coupled with a macroscopic finite element ventricle model. In the numerical simulations, the Langevin dynamics model reveals that a single sarcomere can undergo spontaneous oscillation (15 Hz) accompanied by quick lengthening due to cooperative movements of the myosin molecules pulling on the common Z-line. Also, the coupled simulations using the ventricle model show that the stretch-activation mechanism contributes to the synchronization of the quick lengthening of the sarcomeres at the end of the systolic phase. By comparing the simulation results given by the molecular model with and without the stretch-activation mechanism, we see that this synchronization contributes to maintaining the systolic blood pressure by providing sufficient blood volume without slowing the diastolic process.

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi

The second phase of the biphasic force decay upon release of phosphate from caged phosphate was previously interpreted as a signature of kinetics of the force-generating step in the cross-bridge cycle. To test this hypothesis without using caged compounds, force responses and individual sarcomere dynamics upon rapid increases or decreases in concentration of inorganic phosphate [Pi] were investigated in calcium-activated cardiac myofibrils. Rapid increases in [Pi] induced a biphasic force decay with an initial slow decline (phase 1) and a subsequent 3–5-fold faster major decay (phase 2). Phase 2 started with the distinct elongation of a single sarcomere, the so-called sarcomere “give”. “Give” then propagated from sarcomere to sarcomere along the myofibril. Propagation speed and rate constant of phase 2 (k+Pi(2)) had a similar [Pi]-dependence, indicating that the kinetics of the major force decay (phase 2) upon rapid increase in [Pi] is determined by sarcomere dynamics. In contrast, no “give” was observed during phase 1 after rapid [Pi]-increase (rate constant k+Pi(1)) and during the single-exponential force rise (rate constant k−Pi) after rapid [Pi]-decrease. The values of k+Pi(1) and k−Pi were similar to the rate constant of mechanically induced force redevelopment (kTR) and Ca2+-induced force development (kACT) measured at same [Pi]. These results indicate that the major phase 2 of force decay upon a Pi-jump does not reflect kinetics of the force-generating step but results from sarcomere “give”. The other phases of Pi-induced force kinetics that occur in the absence of “give” yield the same information as mechanically and Ca2+-induced force kinetics (k+Pi(1) ∼ k-Pi ∼ kTR ∼ kACT). Model simulations indicate that Pi-induced force kinetics neither enable the separation of Pi-release from the rate-limiting transition f into force states nor differentiate whether the “force-generating step” occurs before, along, or after the Pi-release.

Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140.

In cardiac muscle, contraction is triggered by sarcolemmal depolarization, resulting in an intracellular Ca(2+) transient, binding of Ca(2+) to troponin, and subsequent cross-bridge formation (excitation-contraction [EC] coupling). Here, we develop a novel experimental system for simultaneous nano-imaging of intracellular Ca(2+) dynamics and single sarcomere length (SL) in rat neonatal cardiomyocytes. We achieve this by expressing a fluorescence resonance energy transfer (FRET)-based Ca(2+) sensor yellow Cameleon-Nano (YC-Nano) fused to α-actinin in order to localize to the Z disks. We find that, among four different YC-Nanos, α-actinin-YC-Nano140 is best suited for high-precision analysis of EC coupling and α-actinin-YC-Nano140 enables quantitative analyses of intracellular calcium transients and sarcomere dynamics at low and high temperatures, during spontaneous beating and with electrical stimulation. We use this tool to show that calcium transients are synchronized along the length of a myofibril. However, the averaging of SL along myofibrils causes a marked underestimate (∼50%) of the magnitude of displacement because of the different timing of individual SL changes, regardless of the absence or presence of positive inotropy (via β-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that β-adrenergic stimulation with 50 nM isoproterenol accelerated Ca(2+) dynamics, in association with an approximately twofold increase in sarcomere lengthening velocity. We conclude that our experimental system has a broad range of potential applications for the unveiling molecular mechanisms of EC coupling in cardiomyocytes at the single sarcomere level.

In vivo Sarcomere Lengths and Sarcomere Elongations Are Not Uniform across an Intact Muscle.

Sarcomere lengths have been a crucial outcome measure for understanding and explaining basic muscle properties and muscle function. Sarcomere lengths for a given muscle are typically measured at a single spot, often in the mid-belly of the muscle, and at a given muscle length. It is then assumed implicitly that the sarcomere length measured at this single spot represents the sarcomere lengths at other locations within the muscle, and force-length, force-velocity, and power-velocity properties of muscles are often implied based on these single sarcomere length measurements. Although, intuitively appealing, this assumption is yet to be supported by systematic evidence. The objective of this study was to measure sarcomere lengths at defined locations along and across an intact muscle, at different muscle lengths. Using second harmonic generation (SHG) imaging technique, sarcomere patterns in passive mouse tibialis anterior (TA) were imaged in a non-contact manner at five selected locations (“proximal,” “distal,” “middle,” “medial,” and “lateral” TA sites) and at three different lengths encompassing the anatomical range of motion of the TA. We showed that sarcomere lengths varied substantially within small regions of the muscle and also for different sites across the entire TA. Also, sarcomere elongations with muscle lengthening were non-uniform across the muscle, with the highest sarcomere stretches occurring near the myotendinous junction. We conclude that muscle mechanics derived from sarcomere length measured from a small region of a muscle may not well-represent the sarcomere length and associated functional properties of the entire muscle.

Oxidative stress decreases microtubule growth and stability in ventricular myocytes

Microtubules (MTs) have many roles in ventricular myocytes, including structural stability, morphological integrity, and protein trafficking. However, despite their functional importance, dynamic MTs had never been visualized in living adult myocytes. Using adeno-associated viral vectors expressing the MT-associated protein plus end binding protein 3 (EB3) tagged with EGFP, we were able to perform live imaging and thus capture and quantify MT dynamics in ventricular myocytes in real time under physiological conditions. Super-resolution nanoscopy revealed that EB1 associated in puncta along the length of MTs in ventricular myocytes. The vast (~80%) majority of MTs grew perpendicular to T-tubules at a rate of 0.06μm∗s−1 and growth was preferentially (82%) confined to a single sarcomere. Microtubule catastrophe rate was lower near the Z-line than M-line. Hydrogen peroxide increased the rate of catastrophe of MTs ~7-fold, suggesting that oxidative stress destabilizes these structures in ventricular myocytes. We also quantified MT dynamics after myocardial infarction (MI), a pathological condition associated with increased production of reactive oxygen species (ROS). Our data indicate that the catastrophe rate of MTs increases following MI. This contributed to decreased transient outward K+ currents by decreasing the surface expression of Kv4.2 and Kv4.3 channels after MI. On the basis of these data, we conclude that, under physiological conditions, MT growth is directionally biased and that increased ROS production during MI disrupts MT dynamics, decreasing K+ channel trafficking.

Simultaneous High-Precision Imaging of Local Calcium and Single Sarcomere Length in Rat Neonatal Cardiomyocytes via Expression of Yellow Cameleon-Nano140 in Z-Discs

In the present study, we developed a novel experimental system for simultaneous measurement of changes in the intracellular Ca2+ concentration ([Ca2+]i) at local areas of the T-tubules / Z-discs and single sarcomere length (SL) via expression of FRET-based Ca2+ indicator Yellow Cameleon (YC)-Nano140 in Z-discs (α-actinin) in rat neonatal cardiomyocytes. Under dual-view fluorescence microscopy, local [Ca2+]i was determined by the YFP/CFP fluorescence ratio and SL by the peak-to-peak distance of the YFP fluorescence profile (see Shintani et al., J Gen Physiol 2014 for SL nanometry: S.D., 12 nm in the present study). During spontaneous beating, although [Ca2+]i changed uniformly in a localized area (5 consecutive sarcomeres) of a myocyte, sarcomere shortening / lengthening was not synchronized, but rather occurred in an asynchronous fashion, suggesting that the length of each sarcomere is determined via tug-of-war forces of sarcomeres along the myofibril. Likewise, we successfully quantified amplifications of systolic [Ca2+]i and single sarcomere contraction upon β-adrenergic stimulation. Furthermore, α-actinin-YC-Nano140 enabled high-precision simultaneous measurements of local [Ca2+]i and single sarcomere contraction under electric field stimulation at 5 Hz. The present experimental system has a broad range of application possibilities for analyzing cardiac excitation-contraction (E-C) coupling in detail at the single sarcomere level. Here, we discuss 1) the technical aspects of the application of YC-Nano140 for the analysis of cardiac E-C coupling, and 2) mechanistic implications of single sarcomere dynamics in relation to local [Ca2+]i changes.