JP, Tibbits GF. Familial hypertrophic cardiomyopathy-related cardiac troponin C mutation L29Q affects Ca 2ϩ binding and myofilament contractility. Physiol Genomics 33: 257-266, 2008. First published February 19, 2008 doi:10.1152/physiolgenomics.00154.2007.-The cardiac troponin C (cTnC) mutation, L29Q, has been found in a patient with familial hypertrophic cardiomyopathy. We previously showed that L29, together with neighboring residues, Asp2, Val28, and Gly30, plays an important role in determining the Ca 2ϩ affinity of site II, the regulatory site of mammalian cardiac troponin C (McTnC Interestingly, the change in Ca 2ϩ sensitivity of force generation in response to an SL change (1.9, 2.1, and 2.3 m) was significantly reduced in myocytes containing L29Q McTnC or NIQD McTnC. These results demonstrate that the L29Q mutation enhances the Ca 2ϩ -binding characteristics of cTnC and that when incorporated into cardiac myocytes, this mutant alters myocyte contractility.

Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca TnC binding Ca initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnI). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca and TnI Surprisingly the Ca affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca-binding site of cTnC.

Since current experimental models of Atrial Fibrillation (AF) have significant limitations, we used human embryonic stem cells (hESCs) to generate an atrial-specific tissue model of AF for pharmacologic testing. We generated atrial-like cardiomyocytes (CMs) from hESCs which preferentially expressed atrial-specific genes, and had shorter action potential (AP) durations compared to ventricular-like CMs. We then generated confluent atrial-like CM sheets and interrogated them using optical mapping techniques. Atrial-like CM sheets (~1 cm in diameter) showed uniform AP propagation, and rapid re-entrant rotor patterns, as seen in AF could be induced. Anti-arrhythmic drugs were tested on single atrial-like CMs and cell sheets. Flecainide profoundly slowed upstroke velocity without affecting AP duration, leading to reduced conduction velocities (CVs), curvatures and cycle lengths of rotors, consistent with increased rotor organization and expansion. By contrast, consistent with block of rapid delayed rectifier K+ currents (Ikr) and AP prolongation in isolated atrial-like CMs, dofetilide prolonged APs and reduced cycle lengths of rotors in cell sheets without affecting CV. In conclusion, using our hESC-derived atrial CM preparations, we demonstrate that flecainide and dofetilide modulate reentrant arrhythmogenic rotor activation patterns in a manner that helps explain their efficacy in treating and preventing AF.

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations and biological reagents for functional interrogation. The similarity of the zebrafish (Danio rerio) cardiac action potential with that of humans further enhances the relevance of this model. In spite of this, little is known about excitation-contraction coupling in the zebrafish heart. To address this issue, adult zebrafish cardiomyocytes were isolated by enzymatic perfusion of the cannulated ventricle and were subjected to amphotericin-perforated patch-clamp technique, confocal calcium imaging, and/or measurements of cell shortening. Simultaneous recordings of the voltage dependence of the L-type calcium current (I(Ca,L)) amplitude and cell shortening showed a typical bell-shaped current-voltage (I-V) relationship for I(Ca,L) with a maximum at +10 mV, whereas calcium transients and cell shortening showed a monophasic increase with membrane depolarization that reached a plateau at membrane potentials above +20 mV. Values of I(Ca,L) were 53, 100, and 17% of maximum at -20, +10, and +40 mV, while the corresponding calcium transient amplitudes were 64, 92, and 98% and cell shortening values were 62, 95, and 96% of maximum, respectively, suggesting that I(Ca,L) is the major contributor to the activation of contraction at voltages below +10 mV, whereas the contribution of reverse-mode Na/Ca exchange becomes increasingly more important at membrane potentials above +10 mV. Comparison of the recovery of I(Ca,L) from acute and steady-state inactivation showed that reduction of I(Ca,L) upon elevation of the stimulation frequency is primarily due to calcium-dependent I(Ca,L) inactivation. In conclusion, we demonstrate that a large yield of healthy atrial and ventricular myocytes can be obtained by enzymatic perfusion of the cannulated zebrafish heart. Moreover, zebrafish ventricular myocytes differed from that of large mammals by having larger I(Ca,L) density and a monophasically increasing contraction-voltage relationship, suggesting that caution should be taken upon extrapolation of the functional impact of mutations on calcium handling and contraction in zebrafish cardiomyocytes.

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Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (load(SR)) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 microM) inhibited load(SR) at rest. Interestingly, inhibition of forward mode NCX with 30 microM KB-R7943 stimulated SOCE significantly and resulted in enhanced load(SR). In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis.

Much less is known about the contributions of the Na(+)/Ca(2+) exchanger (NCX) and sarcoplasmic reticulum (SR) Ca(2+) pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca(2+) decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I(NCX)) and the Ca(2+) transient. We found that the integral of I(NCX), an indicator of SR Ca(2+) content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca(2+) extrusion by NCX (V(NCX)) was linear with [Ca(2+)] and did not indicate saturation kinetics until [Ca(2+)] reached 1-3 microM for each age group. There was a significantly greater time delay between the peaks of I(NCX) and the Ca(2+) transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age.

ated muscle contraction is initiated when, following membrane depolarization, Ca 2ϩ binds to the low-affinity Ca 2ϩ binding sites of troponin C (TnC). The Ca 2ϩ activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle. phylogenetic analysis; protein evolution; temperature; muscle TROPONIN C (TnC), present in all striated muscle, is the Ca 2ϩ -activated trigger that initiates myocyte contraction. The binding of Ca 2ϩ to TnC initiates a cascade of conformational changes through the component proteins of the thin filament, leading to the formation of cross bridges (CBs) between actin and myosin and the generation of force by the myocyte. Therefore, the functional properties of TnC, including its ability to be activated by Ca 2ϩ , have significant regulatory influence on the contractile reaction of the myocyte. Myocyte contractility is also influenced by the strength of interaction between actin and myosin, the rate of CB cycling, and the rate of ATP hydrolysis by myosin ATPase (24). There are two muscle-specific TnC proteins found in vertebrate striated muscle. The first, skeletal TnC (sTnC), is expressed in fast skeletal muscle and the second, cardiac TnC (cTnC), is expressed in cardiac and slow skeletal muscle. A critical difference between these two paralogs is that sTnC is activated by Ca 2ϩ binding to two low-affinity sites on the NH 2 terminus of the protein (sites I and II), while cTnC is activated by Ca 2ϩ binding to a single low-affinity site (site II). Site I is nonfunctional in cTnC due to sequence manipulations that have disrupted its ability to coordinate Ca 2ϩ ion binding. cTnC and sTnC have been cloned from a variety of vertebrate species across a range of phylogenic groups. The most ancient of these is the arctic lamprey, Lampetra japonica, a member of the earliest diverged vertebrate taxon. L. japonica sTnC and cTnC are 70 and 83% identical to human sTnC and cTnC, respectively. The presence of distinct cTnC and sTnC paralogs in L. japonica suggests that these ...