Component Of Filaments Research Articles

Mutual dependence between tropomodulin and tropomyosin in the regulation of sarcomeric actin assembly in Caenorhabditis elegans striated muscle

Tropomodulin and tropomyosin are important components of sarcomeric thin filaments in striated muscles. Tropomyosin decorates the side of actin filaments and enhances tropomodulin capping at the pointed ends of the filaments. Their functional relationship has been extensively characterized in vitro, but in vivo and cellular studies in mammals are often complicated by the presence of functionally redundant isoforms. Here, we used the nematode Caenorhabditis elegans, which has a relatively simple composition of tropomodulin and tropomyosin genes, and demonstrated that tropomodulin (unc-94) and tropomyosin (lev-11) are mutually dependent on each other in their sarcomere localization and regulation of sarcomeric actin assembly. Mutation of tropomodulin caused sarcomere disorganization with formation of actin aggregates. However, the actin aggregation was suppressed when tropomyosin was depleted in the tropomodulin mutant. Tropomyosin was mislocalized to the actin aggregates in the tropomodulin mutants, while sarcomere localization of tropomodulin was lost when tropomyosin was depleted. These results indicate that tropomodulin and tropomyosin are interdependent in the regulation of organized sarcomeric assembly of actin filaments in vivo.

Alpha B-Crystallin in Muscle Disease Prevention: The Role of Physical Activity.

HSPB5 or alpha B-crystallin (CRYAB), originally identified as lens protein, is one of the most widespread and represented of the human small heat shock proteins (sHSPs). It is greatly expressed in tissue with high rates of oxidative metabolism, such as skeletal and cardiac muscles, where HSPB5 dysfunction is associated with a plethora of human diseases. Since HSPB5 has a major role in protecting muscle tissues from the alterations of protein stability (i.e., microfilaments, microtubules, and intermediate filament components), it is not surprising that this sHSP is specifically modulated by exercise. Considering the robust content and the protective function of HSPB5 in striated muscle tissues, as well as its specific response to muscle contraction, it is then realistic to predict a specific role for exercise-induced modulation of HSPB5 in the prevention of muscle diseases caused by protein misfolding. After offering an overview of the current knowledge on HSPB5 structure and function in muscle, this review aims to introduce the reader to the capacity that different exercise modalities have to induce and/or activate HSPB5 to levels sufficient to confer protection, with the potential to prevent or delay skeletal and cardiac muscle disorders.

Filament coalescence and hub structure in Mon R2

There is growing evidence of the role of hub-filament systems (HFS) in the formation of stars from low to high masses. As of today, however, the detailed structures of these systems are still not well described. Here we study the Mon R2 star-forming region, which has a rich network of filaments joining in a star cluster forming hub, and aim to understand the hub structure and to examine the mass fraction residing in the hub and in the filaments, which is a key factor that influences massive star formation. We conducted a multi-scale, multi-component analysis of the Herschel column density maps (resolution of 18.2′′ or ~0.07 pc at 830 pc) of the region using a newly developed algorithm getsf to identify the structural components, namely, extended cloud, filaments, and sources. We find that cascades of lower column density filaments coalesce to form higher-density filaments eventually merging inside the hub (0.8 pc radius). As opposed to the previous view of the hub as a massive clump with ~1 pc radius, we find it to be a network of short high-density filaments. We analyse the orientations and mass per unit length (M∕L) of the filaments as a function of distance from the hub centre. The filaments are radially aligned towards the centre of the hub. The total mass reservoir in the Mon R2 HFS (5 pc × 5 pc) is split between filaments (54%), an extended cloud (37%), and sources (9%). The M∕L of filaments increases from ~10 M⊙ pc−1 at 1.5 pc from the hub to ~100 M⊙ pc−1 at its centre, while the number of filaments per annulus of 0.2 pc width decreases from 20 to two in the same range. The observed radial column density structure of the HFS (filament component only) displays a power-law dependence of NH2 ∝ r−2.17 up to a radius of ~2.5 pc from the central hub, resembling a global collapse of the HFS. We present a scenario where the HFS can be supported by magnetic fields which interact, merge, and reorganise themselves as the filaments coalesce. We plotted the plane-of-the-sky magnetic field line geometry using archival Planck data to support our scenario. In the new view of the hub as a network of high-density filaments, we suggest that only the stars located in the network can benefit from the longitudinal flows of gas to become massive, which may explain the reason for the formation of many low-mass stars in cluster centres. We show the correlation of massive stars in the region to the intertwined network-like hub, based on which we updated the implications of the filaments to clusters (F2C) model for massive star formation.

Simultaneous single-molecule imaging of RNA polymerase II dynamics and chromatin nanostructures in living cells

Transcription in eukaryotic cells is precisely regulated by the interaction between the transcription machinery and highly organized chromatin structures in the nucleus. Recent superresolution imaging techniques, such as single-molecule localization microscopy, have helped in visualization of the organization of nanometer-sized chromatin, including nanodomains, clutches, and filamentous components. However, how transcriptional molecules interact dynamically with these nanoscale chromatin structures in living cells is still unclear.

3D Printing Technology in Aerospace Industry – A Review

In the modern era, several new technologies are being developed for the most convenient and easy usage of materials. Due to its effortless and quick usage properties, 3D printing technology has a vast application range nowadays in different fields. The application of 3D printing technology in the aerospace industry has been developed in the past few decades, but now it has been enhanced to a new level. 3D printing technology now has several applications in the aeronautical and space sectors. Soon, 3D printing technology will be used for building habitations on other planets and for the printing of spacecraft and satellite parts directly in space. Currently, several components of aircraft and spacecraft are manufactured using 3D printers. 3D printing technology can be used to print components using all the filament materials. The main advantage of 3D printing technology is the low-cost creation of components ranging from simple to complex designs. Because of its ability to print any small-to-large complex designs, this technology is currently primarily used in the research sector. The review article aims to give an overview of 3D printing technologies and their current and futuristic uses in the aerospace industry. The types of 3D printers and their extruder nozzles are discussed in detail. The advantages and limitations of 3D printing technology as well as their application are presented. Filament materials and components along with their properties are discussed, especially those developed for aerospace industry applications. This review article mainly shows 3D printing technology as one of the core technologies for the aerospace industry.

Possible Equivalent Circuit Model and Physical Structures of Sputter-Deposited Silicon Oxide Film Showing Resistive Switching

Based on the results of experiments on the resistive switching behaviors of sputter-deposited silicon oxide films, this paper proposes a possible equivalent circuit model to characterize the switching behavior at room temperature. It is revealed that frequency dispersion of the conductance component and capacitance component in the equivalent circuit model dominate the physical interpretation of the frequency-dependence of the components. The validity of the model and its physical interpretation are examined based on a theoretical model of the dielectric function of the conductive filament region. The polarizability of the conductive filament region suggests that the capacitance component of the conductive filament is insensitive to frequency in the low frequency range, whereas the conductance component of the conductive filament is proportional to frequency in the low frequency range. These theoretical results match experimental findings, and it is revealed that the equivalent circuit models and the frequency dispersion models for the capacitance and conductance component of the silicon oxide film are acceptable. In addition, this paper reveals the importance of the volume of the sub-oxide region and the Si precipitate region in determining the resistive switching behaviors of sputter-deposited silicon oxide film.

Evolution of Archaellum Rotation Involved Invention of a Stator Complex by Duplicating and Modifying a Core Component.

Novelty in biology can arise from opportunistic repurposing of nascent characteristics of existing features. Understanding how this process happens at the molecular scale, however, suffers from a lack of case studies. The evolutionary emergence of rotary motors is a particularly clear example of evolution of a new function. The simplest of rotary motors is the archaellum, a molecular motor that spins a helical propeller for archaeal motility analogous to the bacterial flagellum. Curiously, emergence of archaellar rotation may have pivoted on the simple duplication and repurposing of a pre-existing component to produce a stator complex that anchors to the cell superstructure to enable productive rotation of the rotor component. This putative stator complex is composed of ArlF and ArlG, gene duplications of the filament component ArlB, providing an opportunity to study how gene duplication and neofunctionalization contributed to the radical innovation of rotary function. Toward understanding how this happened, we used electron cryomicroscopy to determine the structure of isolated ArlG filaments, the major component of the stator complex. Using a hybrid modeling approach incorporating structure prediction and validation, we show that ArlG filaments are open helices distinct to the closed helical filaments of ArlB. Curiously, further analysis reveals that ArlG retains a subset of the inter-protomer interactions of homologous ArlB, resulting in a superficially different assembly that nevertheless reflects the common ancestry of the two structures. This relatively simple mechanism to change quaternary structure was likely associated with the evolutionary neofunctionalization of the archaellar stator complex, and we speculate that the relative deformable elasticity of an open helix may facilitate elastic energy storage during the transmission of the discrete bursts of energy released by ATP hydrolysis to continuous archaellar rotation, allowing the inherent properties of a duplicated ArlB to be co-opted to fulfill a new role. Furthermore, agreement of diverse experimental evidence in our work supports recent claims to the power of new structure prediction techniques.

Characterization of paramyosin protein structure and gene expression during myogenesis in Pacific oyster (Crassostrea gigas).

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas), and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas, which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks.

Abstract 649: Classics to diagnostics: Assessing EMT biomarkers in colorectal cancer prognosis

Abstract Colorectal cancer (CRC) is a second most common cause of cancer death in the United States and exhibits disparities among racial and ethnic minorities. Colon polyps are abnormal growth in the epithelium that can rapidly invade surrounding mesenchyme and metastasize to form multiple secondary tumor sites. Such Epithelial-Mesenchymal Transition (EMT) is the fundamental event that results in lower survival rate for CRC patients. Altered EMT processes are also indicative of health disparity as observed for aggressive nature of prostate cancer in African Americans. In search of potential diagnostic markers that enable us to capture this transition in situ in CRC, we focused on two major biomarkers, E-Cadherin, an epithelial specific surface protein, and Vimentin, a mesenchymal specific cellular filament component. We obtained de-identified biopsy tissues samples of different stages in CRC, classified as precancerous and cancerous, along with the normal tissues, that were collected during colonoscopies for routine histopathological evaluation. In parallel, these formalin fixed paraffin embedded tissues were immunostained for the expression of E-Cadherin and Vimentin. As expected, normal tissue specimens expressed E-Cadherin exclusively in the mucosal epithelial cells while mesenchymal cells in the stroma specifically stained for vimentin expression. Interestingly, in some precancerous and cancerous specimens, we observed vimentin-positive foci within epithelial cells that lack E-Cadherin expression. This suggests a proof-of principal that an initiation of epithelial to mesenchymal transition can be detected at the clinics by performing selective immunofluorescence staining of these markers. More importantly, such detection can be correlated with the stage of the CRC that can be very insightful for the progression of the disease. Early detection in CRC through these biomarkers to supplement histopathological evaluation is promising and would provide more predictive and detailed analysis of cancer progression in tissue samples to aid in the choice of appropriate treatment plan. The accurate pre-treatment patient evaluation is pivotal to the modern push for personalized therapy. The array of early detection biomarkers will also enhance our understanding in health disparity in CRC when a large set of samples are analyzed for these markers. Citation Format: Rohin Patel, Angelique Robertson, Anjana Saxena, Mintoo Patel. Classics to diagnostics: Assessing EMT biomarkers in colorectal cancer prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 649.

Multiscale mechanics and temporal evolution of vimentin intermediate filament networks

The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics-single-filament mechanics, filament length, and interactions between filaments-including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament-filament interactions.

Vimentin intermediate filaments stabilize dynamic microtubules by direct interactions

The cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components—actin filaments, intermediate filaments, and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a reconstituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism by which cells establish the fundamental cross talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

Molecular dynamics provides new insights into the mechanism of calcium signal transduction and interdomain interactions in cardiac troponin.

Understanding the regulation of cardiac muscle contraction at a molecular level is crucial for the development of therapeutics for heart conditions. Despite the availability of atomic structures of the protein components of cardiac muscle thin filaments, detailed insights into their dynamics and response to calcium are yet to be fully depicted. In this study, we used molecular dynamics simulations of the core domains of the cardiac muscle protein troponin to characterize the equilibrium dynamics of its calcium-bound and calcium-free forms, with a focus on elements of cardiac muscle contraction activation and deactivation, that is, calcium binding to the cardiac troponin Ca2+ -binding subunit (TnC) and the release of the switch region of the troponin inhibitory subunit (TnI) from TnC. The process of calcium binding to the TnC binding site is described as a three-step process commencing with calcium capture by the binding site residues, followed by cooperative residue interplay bringing the calcium ion to the binding site, and finally, calcium-water exchange. Furthermore, we uncovered a set of TnC-TnI interdomain interactions that are critical for TnC N-lobe hydrophobic pocket dynamics. Absence of these interactions allows the closure of the TnC N-lobe hydrophobic pocket while the TnI switch region remains expelled, whereas if the interactions are maintained, the hydrophobic pocket remains open. Modification of these interactions may fine-tune the ability of the TnC N-lobe hydrophobic pocket to close or remain open, modulate cardiac contractility and present potential therapy-relevant targets.

Molecular Genetic Basis of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the myocardium characterized by a hypertrophic left ventricle with a preserved or increased ejection fraction. Cardiac hypertrophy is often asymmetrical, which is associated with left ventricular outflow tract obstruction. Myocyte hypertrophy, disarray, and myocardial fibrosis constitute the histological features of HCM. HCM is a relatively benign disease but an important cause of sudden cardiac death in the young and heart failure in the elderly. Pathogenic variants (PVs) in genes encoding protein constituents of the sarcomeres are the main causes of HCM. PVs exhibit a gradient of effect sizes, as reflected in their penetrance and variable phenotypic expression of HCM. MYH7 and MYBPC3, encoding β-myosin heavy chain and myosin binding protein C, respectively, are the two most common causal genes and responsible for ≈40% of all HCM cases but a higher percentage of HCM in large families. PVs in genes encoding protein components of the thin filaments are responsible for ≈5% of the HCM cases. Whereas pathogenicity of the genetic variants in large families has been firmly established, ascertainment causality of the PVs in small families and sporadic cases is challenging. In the latter category, PVs are best considered as probabilistic determinants of HCM. Deciphering the genetic basis of HCM has enabled routine genetic testing and has partially elucidated the underpinning mechanism of HCM as increased number of the myosin molecules that are strongly bound to actin. The discoveries have led to the development of mavacamten that targets binding of the myosin molecule to actin filaments and imparts beneficial clinical effects. In the coming years, the yield of the genetic testing is expected to be improved and the so-called missing causal gene be identified. The advances are also expected to enable development of additional specific therapies and editing of the mutations in HCM.

Multiscale, multiwavelength extraction of sources and filaments using separation of the structural components:getsf

High-quality astronomical images delivered by modern ground-based and space observatories demand adequate, reliable software for their analysis and accurate extraction of sources, filaments, and other structures, containing massive amounts of detailed information about the complex physical processes in space. The multiwavelength observations with highly variable angular resolutions across wavebands require extraction tools that preserve and use the invaluable high-resolution information. Complex fluctuating backgrounds and filamentary structures appear differently on various scales, calling for multiscale approaches for complete and reliable extraction of sources and filaments. The availability of many extraction tools with varying qualities highlights the need to use standard model benchmarks for choosing the most reliable and accurate method for astrophysical research. This paper presentsgetsf, a new method for extracting sources and filaments in astronomical images using separation of their structural components, designed to handle multiwavelength sets of images and very complex filamentary backgrounds. The method spatially decomposes the original images and separates the structural components of sources and filaments from each other and from their backgrounds, flattening their resulting images. It spatially decomposes the flattened components, combines them over wavelengths, detects the positions of sources and skeletons of filaments, and measures the detected sources and filaments, creating the output catalogs and images. The fully automated method has a single user-defined parameter (per image), the maximum size of the structures of interest to be extracted, that must be specified by users. This paper presents a realistic multiwavelength set of simulated benchmark images that can serve as the standard benchmark problem to evaluate qualities of source- and filament-extraction methods. This paper describeshires, an improved algorithm for the derivation of high-resolution surface densities from multiwavelength far-infraredHerschelimages. The algorithm allows creating the surface densities with angular resolutions that reach 5.6″ when the 70 μm image is used. If the shortest-wavelength image is too noisy or cannot be used for other reasons, slightly lower resolutions of 6.8−11.3″ are available from the 100 or 160 μm images. These high resolutions are useful for detailed studies of the structural diversity in molecular clouds. The codesgetsfandhiresare illustrated by their applications to a variety of images obtained with ground-based and space telescopes from the X-ray domain to the millimeter wavelengths.

CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

The precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2’s function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2’s multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.

RanBP2/Nup358 enhances miRNA activity by sumoylating Argonautes.

Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.

Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis.

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

Nucleoskeleton proteins for nuclear dynamics.

The eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins. It has been believed that such a dense nuclear environment prevents the formation of a cytoskeleton-like network of protein filaments. However, accumulating evidence suggests that the cell nucleus also possesses structural filamentous components to support nuclear organization and compartments, which are referred to as nucleoskeleton proteins. Nucleoskeleton proteins including lamins and actin influence nuclear dynamics including transcriptional regulation, chromatin organization and DNA damage responses. Furthermore, these nucleoskeleton proteins play a pivotal role in cellular differentiation and animal development. In this commentary, we discuss how nucleoskeleton-based regulatory mechanisms orchestrate nuclear dynamics.

The nuclear lamina is a hub for the nuclear function of Jacob

Jacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy and proximity ligation assay combined with STED imaging revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with the INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export.

Binding of calcium and magnesium to human cardiac troponin C

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.