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Ibrutinib (IB) is an oral Bruton's tyrosine kinase (BTK) inhibitor that has demonstrated benefit in B cell cancers, but is associated with a dramatic increase in atrial fibrillation (AF). We employed cell-specific differentiation protocols and optical mapping to investigate the effects of IB and other tyrosine kinase inhibitors (TKIs) on the voltage and calcium transients of atrial and ventricular human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). IB demonstrated direct cell-specific effects on atrial hPSC-CMs that would be predicted to predispose to AF. Second-generation BTK inhibitors did not have the same effect. Furthermore, IB exposure was associated with differential chamber-specific regulation of a number of regulatory pathways including the receptor tyrosine kinase pathway, which may be implicated in the pathogenesis of AF. Our study is the first to demonstrate cell-type-specific toxicity in hPSC-derived atrial and ventricular cardiomyocytes, which reliably reproduces the clinical cardiotoxicity observed., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders., (© The Author 2017. Published by Oxford University Press.)

Zebrafish, as a model for teleost fish, have two paralogous troponin C (TnC) genes that are expressed in the heart differentially in response to temperature acclimation. Upon Ca(2+) binding, TnC changes conformation and exposes a hydrophobic patch that interacts with troponin I and initiates cardiac muscle contraction. Teleost-specific TnC paralogs have not yet been functionally characterized. In this study we have modeled the structures of the paralogs using molecular dynamics simulations at 18°C and 28°C and calculated the different Ca(2+)-binding properties between the teleost cardiac (cTnC or TnC1a) and slow-skeletal (ssTnC or TnC1b) paralogs through potential-of-mean-force calculations. These values are compared with thermodynamic binding properties obtained through isothermal titration calorimetry (ITC). The modeled structures of each of the paralogs are similar at each temperature, with the exception of helix C, which flanks the Ca(2+) binding site; this region is also home to paralog-specific sequence substitutions that we predict have an influence on protein function. The short timescale of the potential-of-mean-force calculation precludes the inclusion of the conformational change on the ΔG of Ca(2+) interaction, whereas the ITC analysis includes the Ca(2+) binding and conformational change of the TnC molecule. ITC analysis has revealed that ssTnC has higher Ca(2+) affinity than cTnC for Ca(2+) overall, whereas each of the paralogs has increased affinity at 28°C compared to 18°C. Microsecond-timescale simulations have calculated that the cTnC paralog transitions from the closed to the open state more readily than the ssTnC paralog, an unfavorable transition that would decrease the ITC-derived Ca(2+) affinity while simultaneously increasing the Ca(2+) sensitivity of the myofilament. We propose that the preferential expression of cTnC at lower temperatures increases myofilament Ca(2+) sensitivity by this mechanism, despite the lower Ca(2+) affinity that we have measured by ITC., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The teleost-specific whole genome duplication created multiple copies of genes allowing for subfunctionalization of isoforms. In this study, we show that the teleost cardiac Ca2+-binding troponin C (TnC) is the product of two distinct genes: cardiac TnC (cTnC, TnnC1a) and a fish-specific slow skeletal TnC (ssTnC, TnnC1b). The ssTnC gene is novel to teleosts as mammals have a single gene commonly referred as cTnC but which is also expressed in slow skeletal muscle. In teleosts, the data strongly indicate that these are two TnC genes are different paralogs. Because we determined that ssTnC exists across many teleosts but not in basal ray-finned fish (e.g., bichir), we propose that these paralogs are the result of an ancestral tandem gene duplication persisting only in teleosts. Quantification of mRNA levels was used to demonstrate distinct expression localization patterns of the paralogs within the chambers of the heart. In the adult zebrafish acclimated at 28°C, ssTnC mRNA levels are twofold greater than cTnC mRNA levels in the atrium, whereas cTnC mRNA was almost exclusively expressed in the ventricle. Meanwhile, rainbow trout acclimated at 5°C showed cTnC mRNA levels in both chambers significantly greater than ssTnC. Distinct responses to temperature acclimation were also quantified in both adult zebrafish and rainbow trout, with mRNA in both chambers shifting to express higher levels of cTnC in 18°C acclimated zebrafish and 5°C acclimated trout. Possible subfunctionalization of TnC isoforms may provide insight into how teleosts achieve physiological versatility in chamber-specific contractile properties.

A microfluidic method for real-time quantitative measurement of cellular response pertaining to drug discovery is reported. This method is capable of multiple-step liquid delivery for measuring the drug response of a single cardiomyocyte, due to the improved cell retention by a newly designed chip. The chip, which consists of a cell-retention chamber with a weir structure, was fabricated just by a one-photomask microfabrication procedure followed by on-chip etching. This method differs from the conventional method, which uses two-mask photolithography to fabricate the microchannel (deep etch) and the weir structure (shallow etch). The dimensions of the weir structure have been predicted by a mathematical model, and confirmed by confocal microscopy. Using this microfluidic method, the dynamic [Ca2+]i mobilization in a single cardiomyocyte during its spontaneous contraction was quantified. Furthermore, we measured the cellular response of a cardiomyocyte on (i) a known cardiotonic agent (caffeine), (ii) a cardiotoxic chemotherapeutic drug (daunorubicin), and (iii) an herbal anticancer drug candidate - isoliquiritigenin (IQ) based on the fluorescent calcium measurement. It was found that IQ had produced a less pronounced effect on calcium mobilization( )of the cardiomyocytes whereas caffeine and daunorubicin had much stronger effects on the cells. These three experiments on cardiomyocytes pertaining to drug discovery were only possible after the improved cell retention provided by the new chip design (MV2) required for multiple-step real-time cellular analysis on a microchip, as compared with our old chip design (MV1).

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.

Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca(2+) than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca(2+) sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca(2+) affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca(2+) affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca(2+) binding to site II (F27W). The removal of the COOH terminus (amino acids 90-161) from ScTnC and McTnC maintained the difference in Ca(2+) affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln(29) and Asp(30) in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca(2+) affinity to that of McNTnC. These results demonstrate that Gln(29) and Asp(30) in ScTnC are required for the high Ca(2+) affinity of site II.

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.

The cardiac Na(+)/Ca(2+) exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30 degrees C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH(2)-terminal region of the molecule. Specifically, we found that approximately 75% of the Na(+)/Ca(2+) exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.

Knowing the mechanical properties of cardiac myofibrils isolated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can provide valuable insight into the structure and function of the heart muscle. Previous studies focused mostly on studying myofibrillar stiffness using simplified elastic models. In this study, the mechanical properties of myofibrils isolated from hiPSC-CMs were measured using atomic force microscopy (AFM). The quasi-linear viscoelastic (QLV) model was used to interpret the elastic and viscous properties of myofibrils. Since there have been no previous studies on the viscoelastic properties of myofibrils extracted from hiPSC-CMs, myofibrils extracted from porcine left-ventricular (LV) tissue were used to compare and verify experimental processes and QLV model parameters. The elastic modulus of myofibrils extracted from porcine LV tissue was determined to be 8.82 ± 6.09 kPa which is consistent with previous studies which reported that porcine LV tissue is less stiff on average than mouse and rat cardiac myofibrils. The elastic modulus of myofibrils extracted from hiPSC-CMs was found to be 9.78 ± 5.80 kPa, which is consistent with the range of 5-20 kPa reported for myofibrils extracted from the adult human heart. We found that myofibrils isolated from hiPSC-CMs relax slower than myofibrils extracted from porcine LV tissue, particularly in the first 0.25 s after the peak stress in the stress relaxation test. These findings provide important insights into the mechanical behavior of hiPSC-CMs and have implications for the development of treatments for heart diseases. [ABSTRACT FROM AUTHOR]

Gene duplication results in extra copies of genes that must coevolve with their interacting partners in multimeric protein complexes. The cardiac troponin (Tn) complex, containing TnC, TnI, and TnT, forms a distinct functional unit critical for the regulation of cardiac muscle contraction. In teleost fish, the function of the Tn complex is modified by the consequences of differential expression of paralogs in response to environmental thermal challenges. In this article, we focus on the interaction between TnI and TnC, coded for by genesthat have independent evolutionary origins, but the co-operation of their protein products has necessitated coevolution. In this study, we characterize functional divergence of TnC and TnI paralogs, specifically the interrelated roles of regulatory subfunctionalization and structural subfunctionalization. We determined that differential paralog transcript expression in response to temperature acclimation results in three combinations of TnC and TnI in the zebrafish heart: TnC1a/TnI1.1, TnC 1 b/TnI1.1, and TnC 1a/TnI1.5. Phylogenetic analysis of these highly conserved proteins identified functionally divergent residues in TnI and TnC. The structural and functional effect of these Tn combinations was modeled with molecular dynamics simulation to link divergent sites to changes in interaction strength. Functional divergence in TnI and TnC were not limited to the residues involved with TnC/TnI switch interaction, which emphasizes the complex nature of Tn function. Patterns in domain-specific divergent selection and interaction energies suggest that substitutions in the TnI switch region are crucial to modifying TnI/TnC function to maintain cardiac contraction with temperature changes. This integrative approach introduces Tn as a model of functional divergence that guides the coevolution of interacting proteins. [ABSTRACT FROM AUTHOR]

The amino-terminal domain of cardiac troponin C (cNTnC) is an essential Ca2+ sensor found in cardiomyocytes. It undergoes a conformational change upon Ca2+ binding and transduces the signal to the rest of the troponin complex to initiate cardiac muscle contraction. Two classical EF-hand motifs (EF1 and EF2) are present in cNTnC. Under physiological conditions, only EF2 binds Ca2+; EF1 is a vestigial site that has lost its function in binding Ca2+ owing to amino-acid sequence changes during evolution. Proteins with EF-hand motifs are capable of binding divalent cations other than calcium. Here, the crystal structure of wild-type (WT) human cNTnC in complex with Cd2+ is presented. The structure of Cd2+-bound cNTnC with the disease-related mutation L29Q, as well as a structure with the residue differences D2N, V28I, L29Q and G30D (NIQD), which have been shown to have functional importance in Ca2+ sensing at lower temperatures in ectothermic species, have also been determined. The structures resemble the overall conformation of NMR structures of Ca2+-bound cNTnC, but differ significantly from a previous crystal structure of Cd2+-bound cNTnC in complex with deoxycholic acid. The subtle structural changes observed in the region near the mutations may play a role in the increased Ca2+ affinity. The 1.4 Å resolution WT cNTnC structure, which is the highest resolution structure yet obtained for cardiac troponin C, reveals a Cd2+ ion coordinated in the canonical pentagonal bipyramidal geometry in EF2 despite three residues in the loop being disordered. A Cd2+ ion found in the vestigial ion-binding site of EF1 is coordinated in a noncanonical `distorted' octahedral geometry. A comparison of the ion coordination observed within EF-hand-containing proteins for which structures have been solved in the presence of Cd2+ is presented. A refolded WT cNTnC structure is also presented. [ABSTRACT FROM AUTHOR]

L-type Ca2+ channel-mediated, Ca2+-induced Ca2+ release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the mature mammalian myocardium but is thought to be absent in the fetal and newborn mammalian myocardium. Furthermore, the characteristics and contributors of E-C coupling at the earliest developmental stages are poorly understood. In this study, we measured [³H](+)PN200-110 dihydropyridine binding capacity, functionality and expression of the L-type Ca2+ channel, and cytosolic [Ca2+1 ([Ca2+]i) at various developmental stages (3, 6, 10, 20, and 56 days old) to characterize ontogenetic changes in E-C coupling. We found that 1) the whole cell L-type Ca2+ channel peak current (ICa) density increased slightly in parallel with cell growth, but the current-voltage relationship, the steady-state activation, and the maximum DHP binding and binding affinity did not exhibit significant developmental changes; 2) sarcoplasmic reticulum Ca2+ dependence of inactivation rates of L-type Ca2+ channel and peak of ICa density were only observed after 10 days of age, which temporally coincides with transverse (T)-tubule formation; 3) the relationship between [Ca2+]i and voltage changed from a linear relationship at the earliest developmental stages to a "bell-shaped" relationship at the later developmental stages, presumably corresponding to a switch from reverse-mode Na/Ca exchange-dependent to IQ-dependent E-C coupling; and 4) the expression of two different splice variants of Cay 1.2, IVS3A and IVS3B, switched from predominantly IVS3A at the earliest stages to IVS3B at the later developmental stages. Our data suggest that whereas the density of functional dihydropyridine receptors (DHPRs) increases only slightly during ontogeny, the enhancement of functional coupling between DHPR and ryanodine receptor is dramatic between the second and third weeks after birth. Furthermore, we found that the differential expression of splice variants during development temporally correlated with the appearance of ICa-dependent E-C coupling and T-tubule formation. [ABSTRACT FROM AUTHOR]

The sensitivity of the cardiac myocyte contractile element for Ca2+ decreases with temperature. As myocyte contractility is regulated by changes in cytosolic [Ca2+], this desensitizing effect represents a challenge for temperate fish such as the rainbow trout, Oncorhynchus mykiss, living in environments where temperatures are low and variable. To allow cardiac function in a temperate environment it is thought that the comparatively high Ca2+ sensitivity of trout cardiac myocytes compensates for the effects of low temperature on myocyte contractility. The high Ca2+ sensitivity of the trout myocyte is due, at least in part, to changes in the amino acid sequence of the thin filament protein, cardiac troponin C (cTnC). cTnC is the Ca2+-activated switch that triggers myocyte contraction. The isoform of cTnC cloned from trout ventricle (ScTnC) is 92% identical to mammalian cTnC (McTnC) and is significantly more sensitive to Ca2+. This result suggests that ScTnC has evolved in trout to allow cardiac function at low temperatures. cTnC also appears to play a role in maintaining cardiac function when temperatures change. Increasing myofibrillar pH according to α-stat regulation, as would occur when temperature decreases, increases Ca2+ sensitivity. A similar increase in pH also sensitizes cTnC to Ca2+. ScTnC therefore appears critical in maintaining cardiac function in trout at low temperatures as well as during changes in temperature. [Copyright &y& Elsevier]

• hiPSC-CM model can be used to recapitulate phenotypes of various cardiac arrhythmias. • hiPSC-CM model can enhibit drug-induced changes in functional behavior of cardiomyocytes. • Microelectrode arrays assay cardiac electrophysiology at the level of functional syncytium. • Microelectrode arrays allow to study cardiac field potentials, propagation and contractility. • Optical mapping of population or individual clusters of hiPSC-CMs underlies macro- and micro-OM systems. • Optical mapping of Ca2+ and V m transients allows to assess excitation-contraction coupling. • Patch clamping provides a single-cell approach to study cardiac electrophysiology. • Patch clamping allows to investigate individual currents involved in generation of action potential. Fundamental to the functional behavior of cardiac muscle is that the cardiomyocytes are integrated as a functional syncytium. Disrupted electrical activity in the cardiac tissue can lead to serious complications including cardiac arrhythmias. Therefore, it is important to study electrophysiological properties of the cardiac tissue. With advancements in stem cell research, protocols for the production of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been established, providing great potential in modelling cardiac arrhythmias and drug testing. The hiPSC-CM model can be used in conjunction with electrophysiology-based platforms to examine the electrical activity of the cardiac tissue. Techniques for determining the myocardial electrical activity include multielectrode arrays (MEAs), optical mapping (OM), and patch clamping. These techniques provide critical approaches to investigate cardiac electrical abnormalities that underlie arrhythmias. [ABSTRACT FROM AUTHOR]

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 Nterminal 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. [ABSTRACT FROM AUTHOR]