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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Interleukin-27 signalling induces stem cell antigen-1 expression in T lymphocytes in vivo.

Stem cell antigen-1 (Sca-1/Ly6A/E) is a cell surface glycoprotein that is often used as a biomarker for stem cells and cell stemness. However, it is not clear what factors can directly induce the expression of Sca-1/Ly6A/E in T lymphocytes in vivo, and if induction of Sca-1 is associated with T cell stemness. In this study, we show that interleukin-27 (IL-27), a member of the IL-12 family of cytokines, directly induces Sca-1 expression in T cells in vivo. We found that mice-deficient for IL-27 (either P28 or EBI3) or its signalling (IL-27Rα) had profound reduction of Sca-1 expression in naive (CD62L+  CD44- ), memory (CD62L+  CD44+ ) and effector (CD62L-  CD44+ ) T cells. In contrast, in vivo delivery of IL-27 using adeno-associated viral vectors strongly induced the expression of Sca-1 in naive and memory/effector T-cell populations in an IL-27 receptor- or signal transducer and activator of transcription 1-dependent manner. Interestingly, IL-27-induced Sca-1+ T cells do not express or up-regulate classic stem cell-associated genes such as Nanog, Oct4, Sox2 and Ctnnb1. However, IL-27-induced Sca-1+ T cells had increased expression of effector/memory-associated transcription factor T-bet, Eomes and Blimp1. Hence, IL-27 signalling directly induces the expression of Sca-1/Ly6A/E expression in T cells. Direct expansion of Sca-1+  CD62L+  CD44- T memory stem cells may explain why IL-27 enhances T-cell memory.

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a relaxed complex scheme (RCS). In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute's Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds, 3-(4-methoxyphenyl)-6,7-chromanediol (NSC600285) and 3-(4-methylphenyl)-7,8-chromanediol (NSC611817), that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of NSC600285 and NSC611817 on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Thus, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Building on this finding is of interest to researchers working on development of drugs for calcium sensitization.

Effects of Modified Parvalbumin EF-Hand Motifs on Cardiac Myocyte Contractile Function.

Cardiac gene delivery of parvalbumin (Parv), an EF-hand Ca(2+) buffer, has been studied as a therapeutic strategy for diastolic heart failure, in which slow Ca(2+) reuptake is an important contributor. A limitation of wild-type (WT) Parv is the significant trade-off between faster relaxation and blunted contraction amplitude, occurring because WT-Parv sequesters Ca(2+) too early in the cardiac cycle and prematurely truncates sarcomere shortening in the facilitation of rapid relaxation. We recently demonstrated that an E → Q substitution (ParvE101Q) at amino acid 12 of the EF-hand Ca(2+)/Mg(2+) binding loop disrupts bidentate Ca(2+) binding, reducing Ca(2+) affinity by 99-fold and increasing Mg(2+) affinity twofold. ParvE101Q caused faster relaxation and not only preserved contractility, but unexpectedly increased it above untreated myocytes. To gain mechanistic insight into the increased contractility, we focused here on amino acid 12 of the EF-hand motif. We introduced an E → D substitution (ParvE101D) at this site, which converts bidentate Ca(2+) coordination to monodentate coordination. ParvE101D decreased Ca(2+) affinity by 114-fold and increased Mg(2+) affinity 28-fold compared to WT-Parv. ParvE101D increased contraction amplitude compared to both untreated myocytes and myocytes with ParvE101Q, with limited improvement in relaxation. Additionally, ParvE101D increased spontaneous contractions after pacing stress. ParvE101D also increased Ca(2+) transient peak height and was diffusely localized around the Z-line of the sarcomere, suggesting a Ca(2+)-dependent mechanism of enhanced contractility. Sarcoplasmic reticulum Ca(2+) load was not changed with ParvE101D, but postpacing Ca(2+) waves were increased. Together, these data show that inverted Ca(2+)/Mg(2+) binding affinities of ParvE101D increase myocyte contractility through a Ca(2+)-dependent mechanism without altering sarcoplasmic reticulum Ca(2+) load and by increasing unstimulated contractions and Ca(2+) waves. ParvE101D provides mechanistic insight into how changes in the Ca(2+)/Mg(2+) binding affinities of parvalbumin's EF-hand motif alter function of cardiac myocytes. These data are informative in developing new Ca(2+) buffering strategies for the failing heart.

Designing proteins to combat disease: Cardiac troponin C as an example.

Throughout history, muscle research has led to numerous scientific breakthroughs that have brought insight to a more general understanding of all biological processes. Potentially one of the most influential discoveries was the role of the second messenger calcium and its myriad of handling and sensing systems that mechanistically control muscle contraction. In this review we will briefly discuss the significance of calcium as a universal second messenger along with some of the most common calcium binding motifs in proteins, focusing on the EF-hand. We will also describe some of our approaches to rationally design calcium binding proteins to palliate, or potentially even cure cardiovascular disease. Considering not all failing hearts have the same etiology, genetic background and co-morbidities, personalized therapies will need to be developed. We predict designer proteins will open doors for unprecedented personalized, and potentially, even generalized medicines as gene therapy or protein delivery techniques come to fruition.

Insights into length-dependent regulation of cardiac cross-bridge cycling kinetics in human myocardium.

Cross-bridge cycling kinetics play an essential role in the heart's ability to contract and relax. The rate of tension redevelopment (ktr) slows down as a muscle length is increased in intact human myocardium. We set out to determine the effect of rapid length step changes and protein kinase A (PKA) and protein kinase C-ßII (PKC-ßII) inhibitors on the ktr in ultra-thin non-failing and failing human right ventricular trabeculae. After stabilizing the muscle either at L90 (90% of optimal length) or at Lopt (optimal length), we rapidly changed the length to either Lopt or L90 and measured ktr. We report that length-dependent changes in ktr occur very rapidly (in the order of seconds or faster) in both non-failing and failing muscles and that the length at which a muscle had been stabilized prior to the length change does not significantly affect ktr. In addition, at L90 and at Lopt, PKA and PKC-ßII inhibitors did not significantly change ktr. Our results reveal that length-dependent regulation of cross-bridge cycling kinetics predominantly occurs rapidly and involves the intrinsic properties of the myofilament rather than post-translational modifications that are known to occur in the cardiac muscle as a result of a change in muscle/sarcomere length.

Obligatory role of neuronal nitric oxide synthase in the heart's antioxidant adaptation with exercise.

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.

The Frank-Starling mechanism involves deceleration of cross-bridge kinetics and is preserved in failing human right ventricular myocardium.

Cross-bridge cycling rate is an important determinant of cardiac output, and its alteration can potentially contribute to reduced output in heart failure patients. Additionally, animal studies suggest that this rate can be regulated by muscle length. The purpose of this study was to investigate cross-bridge cycling rate and its regulation by muscle length under near-physiological conditions in intact right ventricular muscles of nonfailing and failing human hearts. We acquired freshly explanted nonfailing (n = 9) and failing (n = 10) human hearts. All experiments were performed on intact right ventricular cardiac trabeculae (n = 40) at physiological temperature and near the normal heart rate range. The failing myocardium showed the typical heart failure phenotype: a negative force-frequency relationship and ß-adrenergic desensitization (P < 0.05), indicating the expected pathological myocardium in the right ventricles. We found that there exists a length-dependent regulation of cross-bridge cycling kinetics in human myocardium. Decreasing muscle length accelerated the rate of cross-bridge reattachment (ktr) in both nonfailing and failing myocardium (P < 0.05) equally; there were no major differences between nonfailing and failing myocardium at each respective length (P > 0.05), indicating that this regulatory mechanism is preserved in heart failure. Length-dependent assessment of twitch kinetics mirrored these findings; normalized dF/dt slowed down with increasing length of the muscle and was virtually identical in diseased tissue. This study shows for the first time that muscle length regulates cross-bridge kinetics in human myocardium under near-physiological conditions and that those kinetics are preserved in the right ventricular tissues of heart failure patients.

Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca(2+) sensitivity and accelerates deactivation in an acidic environment.

The binding of Ca(2+) to troponin C (TnC) in the troponin complex is a critical step regulating the thin filament, the actin-myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate that phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca(2+) to TnC and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca(2+) binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca(2+) from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylations to maintain Ca(2+) binding while accelerating Ca(2+) dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca(2+) sensitive activation. These data suggest that TnI Ser-150 phosphorylation induced attenuation of the pH-dependent decrease in Ca(2+) sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated thin filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.