Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 µm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 µM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

A novel kinetic model to demonstrate the independent effects of ATP and ADP/Pi concentrations on sarcomere function.

Understanding muscle contraction mechanisms is a standing challenge, and one of the approaches has been to create models of the sarcomere-the basic contractile unit of striated muscle. While these models have been successful in elucidating many aspects of muscle contraction, they fall short in explaining the energetics of functional phenomena, such as rigor, and in particular, their dependence on the concentrations of the biomolecules involved in the cross-bridge cycle. Our hypothesis posits that the stochastic time delay between ATP adsorption and ADP/Pi release in the cross-bridge cycle necessitates a modeling approach where the rates of these two reaction steps are controlled by two independent parts of the total free energy change of the hydrolysis reaction. To test this hypothesis, we built a two-filament, stochastic-mechanical half-sarcomere model that separates the energetic roles of ATP and ADP/Pi in the cross-bridge cycle's free energy landscape. Our results clearly demonstrate that there is a nontrivial dependence of the cross-bridge cycle's kinetics on the independent concentrations of ATP, ADP, and Pi. The simplicity of the proposed model allows for analytical solutions of the more basic systems, which provide novel insight into the dominant mechanisms driving some of the experimentally observed contractile phenomena.

A two-segment model for thin filament architecture in skeletal muscle.

Correct specification of myofilament length is essential for efficient skeletal muscle contraction. The length of thin actin filaments can be explained by a novel 'two-segment' model, wherein the thin filaments consist of two concatenated segments, which are of either constant or variable length. This is in contrast to the classic 'nebulin ruler' model, which postulates that thin filaments are uniform structures, the lengths of which are dictated by nebulin. The two-segment model implicates position-specific microregulation of actin dynamics as a general principle underlying actin filament length and stability.

Modeling thick filament activation suggests a molecular basis for force depression.

Multiscale models aiming to connect muscle's molecular and cellular function have been difficult to develop, in part due to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single, skinned rabbit psoas muscle fibers to ramp shortenings and step stretches performed on the plateau region of the force-length relationship. We isolated myosin from the same muscles and, under similar conditions, performed single-molecule and ensemble measurements of myosin's ATP-dependent interaction with actin using laser trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of an inhibited state. The model also includes a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force after stretch (residual force enhancement). By optimizing the model fit to a subset of our fiber measurements, we specified seven unknown parameters. The model then successfully predicted the remainder of our fiber measurements and also our molecular measurements from the laser trap and in vitro motility. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale models. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combines with thick filament activation to decrease the number of cycling cross-bridges.

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.

Differences in thick filament activation in fast rodent skeletal muscle and slow porcine cardiac muscle.

There is a growing appreciation that regulation of muscle contraction requires both thin filament and thick filament activation in order to fully activate the sarcomere. The prevailing mechano-sensing model for thick filament activation was derived from experiments on fast-twitch muscle. We address the question whether, or to what extent, this mechanism can be extrapolated to the slow muscle in the hearts of large mammals, including humans. We investigated the similarities and differences in structural signatures of thick filament activation in porcine myocardium as compared to fast rat extensor digitorum longus (EDL) skeletal muscle under relaxed conditions and sub-maximal contraction using small angle X-ray diffraction. Thick and thin filaments were found to adopt different structural configurations under relaxing conditions, and myosin heads showed different changes in configuration upon sub-maximal activation, when comparing the two muscle types. Titin was found to have an X-ray diffraction signature distinct from those of the overall thick filament backbone, and its spacing change appeared to be positively correlated to the force exerted on the thick filament. Structural changes in fast EDL muscle were found to be consistent with the mechano-sensing model. In porcine myocardium, however, the structural basis of mechano-sensing is blunted suggesting the need for additional activation mechanism(s) in slow cardiac muscle. These differences in thick filament regulation can be related to their different physiological roles where fast muscle is optimized for rapid, burst-like, contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned, graded response to allow for their substantial functional reserve. KEY POINTS: Both thin filament and thick filament activation are required to fully activate the sarcomere. Thick and thin filaments adopt different structural configurations under relaxing conditions, and myosin heads show different changes in configuration upon sub-maximal activation in fast extensor digitorum longus muscle and slow porcine cardiac muscle. Titin has an X-ray diffraction signature distinct from those of the overall thick filament backbone and this titin reflection spacing change appeared to be directly proportional to the force exerted on the thick filament. Mechano-sensing is blunted in porcine myocardium suggesting the need for additional activation mechanism(s) in slow cardiac muscle. Fast skeletal muscle is optimized for rapid, burst-like contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned graded response to allow for their substantial functional reserve.

Live imaging of adult zebrafish cardiomyocyte proliferation ex vivo.

Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.

A low-cost 2-D sarcomere model to demonstrate titin-related mechanisms for force production.

Given the recently proposed three-filament theory of muscle contraction, we present a low-cost physical sarcomere model aimed at illustrating the role of titin in the production of active force in skeletal muscle. With inexpensive materials, it is possible to illustrate actin-myosin cross-bridge interactions between the thick and thin filaments and demonstrate the two different mechanisms by which titin is thought to contribute to active and passive muscle force. Specifically, the model illustrates how titin, a molecule with springlike properties, may increase its stiffness by binding free calcium upon muscle activation and reducing its extensible length by attaching itself to actin, resulting in the greater force-generating capacity after an active than a passive elongation that has been observed experimentally. The model is simple to build and manipulate, and demonstration to high school students was shown to result in positive perception and improved understanding of the otherwise complex titin-related mechanisms of force production in skeletal and cardiac muscles.NEW & NOTEWORTHY Our physical sarcomere model illustrates not only the classic view of muscle contraction, the sliding filament and cross-bridge theories, but also the newly discovered role of titin in force regulation, called the three-filament theory. The model allows for easy visualization of the role of titin in muscle contraction and aids in explaining complex muscle properties that are not captured by the traditional cross-bridge theory.

Characterizing residual and passive force enhancements in cardiac myofibrils.

Residual force enhancement (RFE), an increase in isometric force after active stretching of a muscle compared with the purely isometric force at the corresponding length, has been consistently observed throughout the structural hierarchy of skeletal muscle. Similar to RFE, passive force enhancement (PFE) is also observable in skeletal muscle and is defined as an increase in passive force when a muscle is deactivated after it has been actively stretched compared with the passive force following deactivation of a purely isometric contraction. These history-dependent properties have been investigated abundantly in skeletal muscle, but their presence in cardiac muscle remains unresolved and controversial. The purpose of this study was to investigate whether RFE and PFE exist in cardiac myofibrils and whether the magnitudes of RFE and PFE increase with increasing stretch magnitudes. Cardiac myofibrils were prepared from the left ventricles of New Zealand White rabbits, and the history-dependent properties were tested at three different final average sarcomere lengths (n = 8 for each), 1.8, 2, and 2.2 µm, while the stretch magnitude was kept at 0.2 µm/sarcomere. The same experiment was repeated with a final average sarcomere length of 2.2 µm and a stretching magnitude of 0.4 µm/sarcomere (n = 8). All 32 cardiac myofibrils exhibited increased forces after active stretching compared with the corresponding purely isometric reference conditions (p < 0.05). Furthermore, the magnitude of RFE was greater when myofibrils were stretched by 0.4 compared with 0.2 µm/sarcomere (p < 0.05). We conclude that, like in skeletal muscle, RFE and PFE are properties of cardiac myofibrils and are dependent on stretch magnitude.

Small molecule drugs to improve sarcomere function in those with acquired and inherited myopathies.

Muscle weakness is a hallmark of inherited or acquired myopathies. It is a major cause of functional impairment and can advance to life-threatening respiratory insufficiency. During the past decade, several small-molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small-molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin. We also discuss their use in the treatment of skeletal myopathies. The first of three classes of drugs discussed here increase contractility by decreasing the dissociation rate of calcium from troponin and thereby sensitizing the muscle to calcium. The second two classes of drugs directly act on myosin and stimulate or inhibit the kinetics of myosin-actin interactions, which may be useful in patients with muscle weakness or stiffness.NEW & NOTEWORTHY During the past decade, several small molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin.

How to estimate the sarcomere size based on oblique sections of skeletal muscle.

Ultrastructural analysis of muscular biopsy is based on images of longitudinal sections of the fibers. Sometimes, due to experimental limitations, the resulting sections are instead oblique, and no accurate morphological information can be extracted with standard analysis methods. Thus, the biopsy is performed again, but this is too invasive and time-consuming. In this study, we focused our attention on the sarcomere's shape and we investigated which is the structural information that can be obtained from oblique sections. A routine was written in MATLAB to allow the visualization of how a sarcomere's section appears in ultrastructural images obtained by Transmission Electron Microscopy (TEM) at different secant angles. The routine was used also to analyze the intersection between a cylinder and a plane to show how the Z-bands and M-line lengths vary at different secant angles. Moreover, we explored how to calculate sarcomere's radius and length as well as the secant angle from ultrastructural images, based only on geometrical considerations (Pythagorean theorem and trigonometric functions). The equations to calculate these parameters starting from ultrastructural image measurements were found. Noteworthy, to obtain the real sarcomere length in quasi-longitudinal sections, a small correction in the standard procedure is needed and highlighted in the text. In conclusion, even non-longitudinal sections of skeletal muscles can be used to extrapolate morphological information of sarcomeres, which are important parameters for diagnostic purposes.

The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium.

[Figure: see text].

High hydrostatic pressure induces slow contraction in mouse cardiomyocytes.

Cardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.

Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range.

Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1-2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1-2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1-2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.

An increase in force after stretch of diaphragm fibers and myofibrils is accompanied by an increase in sarcomere length nonuniformities and Ca2+ sensitivity.

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca2+ sensitivity as shown by a change in the force-pCa2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.

Quantitative mapping of force-pCa curves to whole-heart contraction and relaxation.

The force-pCa (F-pCa) curve is used to characterize steady-state contractile properties of cardiac muscle cells in different physiological, pathological and pharmacological conditions. This provides a reduced preparation in which to isolate sarcomere mechanisms. However, it is unclear how changes in the F-pCa curve impact emergent whole-heart mechanics quantitatively. We study the link between sarcomere and whole-heart function using a multiscale mathematical model of rat biventricular mechanics that describes sarcomere, tissue, anatomy, preload and afterload properties quantitatively. We first map individual cell-level changes in sarcomere-regulating parameters to organ-level changes in the left ventricular function described by pressure-volume loop characteristics (e.g. end-diastolic and end-systolic volumes, ejection fraction and isovolumetric relaxation time). We next map changes in the sarcomere-regulating parameters to changes in the F-pCa curve. We demonstrate that a change in the F-pCa curve can be caused by multiple different changes in sarcomere properties. We demonstrate that changes in sarcomere properties cause non-linear and, importantly, non-monotonic changes in left ventricular function. As a result, a change in sarcomere properties yielding changes in the F-pCa curve that improve contractility does not guarantee an improvement in whole-heart function. Likewise, a desired change in whole-heart function (i.e. ejection fraction or relaxation time) is not caused by a unique shift in the F-pCa curve. Changes in the F-pCa curve alone cannot be used to predict the impact of a compound on whole-heart function. KEY POINTS: The force-pCa (F-pCa) curve is used to assess myofilament calcium sensitivity after pharmacological modulation and to infer pharmacological effects on whole-heart function. We demonstrate that there is a non-unique mapping from changes in F-pCa curves to changes in left ventricular (LV) function. The effect of changes in F-pCa on LV function depend on the state of the heart and could be different for different pathological conditions. Screening of compounds to impact whole-heart function by F-pCa should be combined with active tension and calcium transient measurements to predict better how changes in muscle function will impact whole-heart physiology.

Cisd2 is essential to delaying cardiac aging and to maintaining heart functions.

CDGSH iron-sulfur domain-containing protein 2 (Cisd2) is pivotal to mitochondrial integrity and intracellular Ca2+ homeostasis. In the heart of Cisd2 knockout mice, Cisd2 deficiency causes intercalated disc defects and leads to degeneration of the mitochondria and sarcomeres, thereby impairing its electromechanical functioning. Furthermore, Cisd2 deficiency disrupts Ca2+ homeostasis via dysregulation of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) activity, resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay cardiac aging and attenuate age-related structural defects and functional decline. In addition, it results in a younger cardiac transcriptome pattern during old age. Our findings indicate that Cisd2 plays an essential role in cardiac aging and in the heart's electromechanical functioning. They highlight Cisd2 as a novel drug target when developing therapies to delay cardiac aging and ameliorate age-related cardiac dysfunction.

Force loss induced by inhibiting cross-bridge cycling is mitigated in eccentric contraction.

We examined whether the force loss induced by 2,3-butanedione monoxime affects isometric and eccentric forces differently. Single skinned muscle fibers were activated at an average sarcomere length of 2.4 µm and then stretched to 3.0 µm. This trial was performed with and without 2,3-butanedione monoxime to calculate the magnitude of force loss attained at several time points: pre-stretch phase at 2.4 µm, eccentric phase, end of eccentric contraction, and post-stretch phase at 3.0 µm. The magnitude of force loss was significantly larger in the pre-stretch phase than at the other time points. Further, the mitigated force loss in the eccentric contraction was more prominent in the long condition than in the short condition. We suggest that the eccentric force is relatively preserved compared with the reference isometric force (pre-stretch) when cross-bridge cycling is inhibited, possibly because of the contribution of the elastic force produced by titin.

Fast stretching of skeletal muscle fibres abolishes residual force enhancement.

The steady-state isometric force of a muscle after active stretching is greater than the steady-state force for a purely isometric contraction at the same length and activation level. The mechanisms underlying this property, termed residual force enhancement (rFE), remain unknown. When myofibrils are actively stretched while cross-bridge cycling is inhibited, rFE is substantially reduced, suggesting that cross-bridge cycling is essential to produce rFE. Our purpose was to further investigate the role of cross-bridge cycling in rFE by investigating whether fast stretching that causes cross-bridge slipping is associated with a loss of rFE. Skinned fibre bundles from rabbit psoas muscles were stretched slowly (0.08 µm s-1) or rapidly (800 µm s-1) while activated, from an average sarcomere length of 2.4 to 3.2 µm. Force was enhanced by 38±4% (mean±s.e.m) after the slow stretches but was not enhanced after the fast stretches, suggesting that proper cross-bridge cycling is required to produce rFE.

AP-1 Contributes to Chromatin Accessibility to Promote Sarcomere Disassembly and Cardiomyocyte Protrusion During Zebrafish Heart Regeneration.

RATIONALE: The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur. OBJECTIVE: To identify transcription factor regulators of the chromatin landscape, which promote cardiomyocyte regeneration in zebrafish, and investigate their function. METHODS AND RESULTS: Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that activator protein-1 (AP-1) binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the fbxl22 and ilk loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the AP-1 family members Junb and Fosl1 can promote changes in mammalian cardiomyocyte behavior in vitro. CONCLUSIONS: AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.