Molecular Insights into the MLCK Activation by CaM.

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca2+) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally, we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

Mouse Models of Cardiomyopathies Caused by Mutations in Troponin C.

Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms.

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscle and Structure Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering systolic calcium concentration, thereby strengthening cardiac function. Here, we examined the effect of our previously identified calcium sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This led to the identification of three novel low affinity binders, which had similar binding affinities to known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM .

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling.

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

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

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

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity.

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-ß4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6-24 µM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 µM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 µM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia.

BACKGROUND: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+ homeostasis, impairing luminal Ca2+ regulation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process. METHODS: A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca2+ and reactive oxygen species imaging in isolated ventricular myocytes (VMs). RESULTS: The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca2+ transient amplitude and SR Ca2+ content, and reduced proarrhythmic spontaneous Ca2+ waves in TAB VMs under ß-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca2+-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. CONCLUSIONS: A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca2+ release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

Non-Canonical Interaction between Calmodulin and Calcineurin Contributes to the Differential Regulation of Plant-Derived Calmodulins on Calcineurin.

Calmodulin (CaM) serves as an important Ca2+ signaling hub that regulates many protein signaling pathways. Recently, it was demonstrated that plant CaM homologues can regulate mammalian targets, often in a manner that opposes the impact of the mammalian CaM (mCaM). However, the molecular basis of how CaM homologue mutations differentially impact target activation is unclear. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isoforms, sCaM-1 and sCaM-4, share >90 and ∼78% identity with the mCaM, respectively, and activate CaN with comparable or reduced activity relative to mCaM. We used molecular dynamics (MD) simulations and fluorometric assays of CaN-dependent dephosphorylation of MUF-P to probe whether calcium and protein-protein binding interactions are altered by plant CaMs relative to mCaM as a basis for differential CaN regulation. In the presence of CaN, we found that the two sCaMs' Ca2+ binding properties, such as their predicted coordination of Ca2+ and experimentally measured EC50 [Ca2+] values are comparable to mCaM. Furthermore, the binding of CaM to the CaM binding region (CaMBR) in CaN is comparable among the three CaMs, as evidenced by MD-predicted binding energies and experimentally measured EC50 [CaM] values. However, mCaM and sCaM-1 exhibited binding with a secondary region of CaN's regulatory domain that is weakened for sCaM-4. We speculate that this secondary interaction affects the turnover rate (kcat) of CaN based on our modeling of enzyme activity, which is consistent with our experimental data. Together, our data describe how plant-derived CaM variants alter CaN activity through enlisting interactions other than those directly influencing Ca2+ binding and canonical CaMBR binding, which may additionally play a role in the differential regulation of other mammalian targets.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes.

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart's biology in health and disease.

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.

Corrigendum: Muscle Twitch Kinetics Are Dependent on Muscle Group, Disease State, and Age in Duchenne Muscular Dystrophy Mouse Models.

[This corrects the article DOI: 10.3389/fphys.2020.568909.].

Muscle Twitch Kinetics Are Dependent on Muscle Group, Disease State, and Age in Duchenne Muscular Dystrophy Mouse Models.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by the lack of functional dystrophin protein. In muscular dystrophy preclinical research, it is pertinent to analyze the force of the muscles affected by the disease to assess pathology and potential effectiveness of therapeutic interventions. Although muscles function at sub-maximal levels in vivo, maximal tetanic contractions are most commonly used to assess and report muscle function in muscular dystrophy studies. At submaximal activation, the kinetics of contraction and relaxation are heavily impacted by the kinetics of the single twitch. However, maximal tetanic force is often the main, if not sole, outcome measured in most studies, while contractile kinetics are rarely reported. To investigate the effect of muscle disease on twitch contraction kinetics, isolated diaphragm and extensor digitorum longus (EDL) muscles of 10-, 20-week, "het" (dystrophin deficient and utrophin haplo-insufficient), and 52-week mdx (dystrophin deficient) mice were analyzed and compared to wild-type controls. We observed that twitch contractile kinetics are dependent on muscle type, age, and disease state. Specific findings include that diaphragm from wildtype mice has a greater time to 50% relaxation (RT50) than time to peak tension (TTP) compared to the het and mdx dystrophic models, where there is a similar TTP compared to RT50. Diaphragm twitch kinetics remain virtually unchanged with age, while the EDL from het and mdx mice initially has a greater RT50 than TTP, but the TTP increases with age. The difference between EDL contractile kinetics of dystrophic and wildtype mice is more prominent at young age. Differences in kinetics yielded greater statistical significance compared to previously published force measurements, thus, using kinetics as an outcome parameter could potentially allow for use of smaller experimental groups in future study designs. Although this study focused on DMD models, our findings may be applicable to other skeletal muscle conditions and diseases.

Discovery of Novel Small-Molecule Calcium Sensitizers for Cardiac Troponin C: A Combined Virtual and Experimental Screening Approach.

Heart failure is a leading cause of death throughout the world and is triggered by a disruption of the cardiac contractile machinery. This machinery is regulated in a calcium-dependent manner by the protein complex troponin. Calcium binds to the N-terminal domain of cardiac troponin C (cNTnC) setting into motion the cascade of events leading to muscle contraction. Because of the severity and prevalence of heart failure, there is a strong need to develop small-molecule therapeutics designed to increase the calcium sensitivity of cardiac troponin in order to treat this devastating condition. Molecules that are able to stabilize an open configuration of cNTnC and additionally facilitate the binding of the cardiac troponin I (cTnI) switch peptide have the potential to enable increased calcium sensitization and strengthened cardiac function. Here, we employed a high throughput virtual screening methodology built upon the ability of computational docking to reproduce known experimental results and to accurately recognize cNTnC conformations conducive to small molecule binding using a receiver operator characteristic curve analysis. This approach combined with concurrent stopped-flow kinetic experimental verification led to the identification of a number of sensitizers, which slowed the calcium off-rate. An initial hit, compound 4, was identified with medium affinity (84 ± 30 µM). Through refinement, a calcium sensitizing agent, compound 5, with an apparent affinity of 1.45 ± 0.09 µM was discovered. This molecule is one of the highest affinity calcium sensitizers known to date.

Calmodulin-Calcineurin Interaction beyond the Calmodulin-Binding Region Contributes to Calcineurin Activation.

Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation is in part driven by the binding of calmodulin (CaM) to a CaM recognition region (CaMBR) within CaN's regulatory domain (RD). However, secondary interactions between CaM and the CaN RD may be necessary to fully activate CaN. Specifically, it is established that the CaN RD folds upon CaM binding and a region C-terminal to CaMBR, the "distal helix", assumes an α-helix fold and contributes to activation [Dunlap, T. B., et al. (2013) Biochemistry 52, 8643-8651]. We hypothesized in that previous study that this distal helix can bind CaM in a region distinct from the canonical CaMBR. To test this hypothesis, we utilized molecular simulations, including replica-exchange molecular dynamics, protein-protein docking, and computational mutagenesis, to determine potential distal helix-binding sites on CaM's surface. We isolated a potential binding site on CaM (site D) that facilitates moderate-affinity interprotein interactions and predicted that mutation of site D residues K30 and G40 on CaM would weaken CaN distal helix binding. We experimentally confirmed that two variants (K30E and G40D) indicate weaker binding of a phosphate substrate p-nitrophenyl phosphate to the CaN catalytic site by a phosphatase assay. This weakened substrate affinity is consistent with competitive binding of the CaN autoinhibition domain to the catalytic site, which we suggest is due to the weakened distal helix-CaM interactions. This study therefore suggests a novel mechanism for CaM regulation of CaN that may extend to other CaM targets.

The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of NaV1.5 and RyR2 function.

Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM-protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.

Impact of heart rate on cross-bridge cycling kinetics in failing and nonfailing human myocardium.

The force-frequency relationship (FFR) is an important regulatory mechanism that increases the force-generating capacity as well as the contraction and relaxation kinetics in human cardiac muscle as the heart rate increases. In human heart failure, the normally positive FFR often becomes flat, or even negative. The rate of cross-bridge cycling, which has been reported to affect cardiac output, could be potentially dysregulated and contribute to blunted or negative FFR in heart failure. We recently developed and herein use a novel method for measuring the rate of tension redevelopment. This method allows us to obtain an index of the rate of cross-bridge cycling in intact contracting cardiac trabeculae at physiological temperature and assess physiological properties of cardiac muscles while preserving posttranslational modifications representative of those that occur in vivo. We observed that trabeculae from failing human hearts indeed exhibit an impaired FFR and a reduced speed of relaxation kinetics. However, stimulation frequencies in the lower spectrum did not majorly affect cross-bridge cycling kinetics in nonfailing and failing trabeculae when assessed at maximal activation. Trabeculae from failing human hearts had slightly slower cross-bridge kinetics at 3 Hz as well as reduced capacity to generate force upon K+ contracture at this frequency. We conclude that cross-bridge kinetics at maximal activation in the prevailing in vivo heart rates are not majorly impacted by frequency and are not majorly impacted by disease.NEW & NOTEWORTHY In this study, we confirm that cardiac relaxation kinetics are impaired in filing human myocardium and that cross-bridge cycling rate at resting heart rates does not contribute to this impaired relaxation. At high heart rates, failing myocardium cross-bridge rates are slower than in nonfailing myocardium.

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.