Nir Hus and David Dweck, JBC Manuscript G159D and L29Q CTnC Revised 3

8/3/2019 Nir Hus and David Dweck, JBC Manuscript G159D and L29Q CTnC Revised 3

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Challenging current paradigms related to cardiomyopathies: Are changes in the Ca 2+

sensitivity from myofilaments containing cardiac troponin C mutations (G159D and L29Q) goodpredictors of the phenotypic outcomes?

David Dweck, Nir Hus and James D. Potter #

Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine,Miami, Florida 33136

Running Title: Effects of L29Q and G159D-CTnC on the contractile apparatus.#corresponding author: Department of Molecular and Cellular Pharmacology, University of Miami, Miller

School of Medicine, 1600 NW 10 th Ave. (R-189), Miami, Florida, 33136, Office: 305-243-5874; Fax:305-324-6024: E-mail: [email protected]

Two novel mutations (G159D and L29Q) in cardiac troponin C (CTnC) associate theirphenotypic outcomes with dilated (DCM) and hypertrophic cardiomyopathy (HCM), respectively.Current paradigms propose that sarcomeric mutations associated with DCM decrease themyofilament Ca 2+ sensitivity while those associated with HCM increase it. Therefore, weincorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effectson the Ca 2+ sensitivities of tension and ATPase activity coincide with the current paradigms andphenotypic outcomes. The G159D-CTnC decreases the Ca 2+ sensitivity of tension and ATPaseactivation; and reduces the maximal ATPase activity when incorporated into regulated actomyosinfilaments. Under the same conditions, the L29Q-CTnC has no effect. Surprisingly, changes in theapparent G159D-CTnC Ca 2+ affinity measured by tension in fibers do not occur in the isolatedCTnC; and large changes measured in the isolated L29Q-CTnC do not manifest in the fiber. Thesecounter-intuitive findings are justified through a transition in Ca 2+ affinity occurring at the level of cardiac troponin and higher, implying that the true effects of these mutations become apparent asthe hierarchal level of the myofilament increases. Therefore, the contractile apparatus,representing a large cooperative machine, can provide the potential for dysfunction (G159D) orrepair (L29Q) in the Ca 2+-regulation of contraction. In accordance with the clinical outcomes andcurrent paradigms, the desensitization of myofilaments from G159D-CTnC is expected to weakenthe contractile force of the myocardium, while the lack of myofilament changes from L29Q-CTnCmay preserve diastolic and systolic function.

Cardiomyopathies are diseases of the myocardium that often lead to cardiac remodeling by

increasing the size and mass of the heart in order to compensate for deficiencies in cardiac output (1). Inthe case of dilated cardiomyopathy (DCM), heart failure is characterized by a systolic dysfunction (i.e.,reduced ejection fraction [EF] ); whereas, hypertrophic (HCM) and restrictive (RCM) cardiomyopathiesare characterized as having diastolic dysfunctions (i.e ., impaired relaxation) (2). In many cases, thecardiac contractile dysfunction is attributed to inherited sarcomeric gene mutations. The functional effectsof more than 40 thin filament mutations associated with cardiomyopathies assessed in vitro suggests thatthese mutations affect the Ca 2+ responsiveness of the myofilament in the absence of CTnI

phosphorylation . As the number of in vitro -characterized mutations continues to grow, a developing paradigm emerges that associates decreases in the myofilament Ca 2+ sensitivity with DCM (3-7) andincreases with HCM (4,6-12) and RCM (13-17). This suggests that distinct effects on the Ca 2+ dependent

processes of the myofilament are critical determinants of the severity and molecular pathologies of thesediseases.

Mutations in the genes encoding the cardiac troponin I (CTnI) and troponin T (CTnT) subunits of cardiac troponin (CTn) account for more than 15 % of all sarcomeric mutations associated with acardiomyopathy (4,18). In stark contrast, only three mutations in the cardiac troponin C (CTnC) subunithave been identified; suggesting for the first time that mutations in CTnC may link to cardiomyopathies.The first cardiac troponin C ( CTnC ) mutation (E59D/D75Y) was found in an explanted heart from anadult male who died from idiopathic DCM (18); the second mutation (L29Q) was found in a living 60year old male diagnosed with HCM, despite having preserved diastolic and systolic function (19); and thethird (G159D) was found by linkage analysis and cosegregation studies in more than three affectedfamilies with DCM (20,21). The lack of affected relatives and cosegregation studies associated with the


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L29Q and E59D/D75Y CTnC mutations may or may not confirm these mutations as etiological causes for the diseases; therefore, the functional and biochemical effects of these mutations must be studied in order to predict the physiological consequences of these mutations. As such, our investigations will relate thechanges in apparent CTnC Ca 2+ affinity to the myofilament response and determine if these mutationscoincide with the developing paradigm. Previously, we have reported on tT he effects of the E59D/D75Ymutation were previously reported to coincide with the aforementioned paradigms (18,22,23); therefore,this article will focus on the L29Q and G159D CTnC mutations.

Cardiac muscle contraction and relaxation is regulated by the intracellular Ca 2+ concentration viathe thin filament regulatory protein, CTnC. During systole, the rise in free calcium promotes Ca 2+ bindingto CTnC. This leads to the translocation of CTnI and tropomyosin (Tm) away from the outer domain of the actin filaments allowing for subsequent crossbridge interaction and the generation of tension. With thedecline of Ca 2+ during diastole, Ca 2+ dissociates from CTnC and the inhibitory actions of CTnI and Tmare restored [see (24,25) for review]. CTnC contains three metal ion binding sites: two C-terminal sitesthat bind Ca 2+ and Mg 2+ (Ca 2+-Mg 2+ sites) competitively ( K Ca = 1.4 X 10 7 M -1; K Mg ~ 1.0 X 10 3 M -1) andone N-terminal, Ca 2+ specific, regulatory site ( K Ca = 2.5 X 10 5 M -1) that is responsible for transmitting theCa 2+ binding signal to the rest of the thin filament and switching on contraction (26,27).

The effects of the L29Q and G159D mutations are of particular interest because previous studiesshow that the apparent CTnC Ca 2+ affinity as measured by tension and ATPase activity are unaffectedunless CTnI (S23/S24) is phosphorylated . (28,29). TT hese studies identif yied a blunted PKA

phosphorylated myofilament response as the only determinant underlying the molecular pathologies for these mutations. However, these findings represent challenges to the developing paradigm because in the

presence of CTnI phosphorylation, the myofilament Ca 2+ sensitivities are similarly affected for bothmutations , yet lead to very different phenotypic outcomes. In additionMoreover , the clinical data from theonly L29Q proband results in a seemingly benign phenotype; yethowever , subsequent in vitro studieshave shown that in the absence of CTnI phosphorylation, the L29Q mutation can decrease (29), increase(30) or not affect (31) the myofilament Ca 2+ sensitivity. Therefore, the effects of the L29Q and G159DCTnC mutations on the various Ca 2+ dependent processes of the myofilament must be pursued further inorder to evaluate if their effects do coincide with the aforementioned paradigms.

Our results show that in the absence of CTnI phosphorylation, skinned cardiac muscle preparations reconstituted with the L29Q CTnC are all unaffectedpossibly explaining the benign

phenotypic outcome observed from this mutation. In contrast, the G159D mutation significantly decreasesthe Ca 2+ sensitivities of tension and cardiac myofibrillar ATPase activation, coinciding with the effects of other thin filament mutations associated with DCM (6,32-34). Moreover, regulated actomyosinreconstituted with G159D CTnC reduces the maximal ATPase activity despite having the inability todepress the maximal tension and cardiac myofibrillar ATPase activity. Surprisingly, the G159D CTnCstructure and apparent regulatory site Ca 2+ affinity as measured by fluorescence in the isolated state areseemingly unaffected; whereas, the structure and Ca 2+ affinity of L29Q CTnC are significantly altered.The disparity of these findings have led us to pursue how the different myofilament interactionscooperatively regulate calcium binding to the mutant CTnCs in order to reveal key interactions thatdictate the final output of tension in the muscle fiber.

Since the L29Q and G159D mutations are not located within the regulatory site, their effects onCTnC Ca 2+ occupancy are most likely communicated through CTnC inter-molecular interactions. This

rationale stems from the abilities of most myofilament protein interactions to alter the structure and Ca2+

affinity of both skeletal and cardiac isoforms of TnC (35-37). Therefore, IAANS fluorophores attached toCys 84 and/or Cys 35 of CTnC were used to measure the structural and Ca 2+ dependent changes influorescence from the mutant CTnC in the isolated, binary, ternary (35,36) and regulated thin filament(RTF) levels. Our results show that at the level of CTn, the G159D CTnC structure and Ca 2+ affinitydeviates from the WT; whereas, the L29Q CTnC structure and Ca 2+ affinity begins to converge with theWT. Therefore, the contractile apparatus, representing a large cooperative machine, can provide the

potential for dysfunction (G159D) or repair (L29Q) if any one of the proteins is mutated. Finally, our


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results show that the changes in myofilament Ca 2+ sensitivity arising from these mutations do coincidewith the developing paradigms and are indicative of the phenotypic outcomes.

EXPERIMENTAL PROCEDURERS

Mutation, Expression and Purification of CTnC The CTnC sequence was derived from a human cardiaccDNA library and subcloned into the pET3-d vector with its sequence verified. Human CTnC mutantswere generated by following Stratagenes guidelines for using the QuikChange Site-Directed Mutagenesiskit. All mutant CTnCs were sequenced to verify the correct sequences prior to expression and

purification. Standard methods previously used in this lab were utilized for expression and purification of the different CTnC mutants (37){Szczesna, 1996 #8059} .

Fluorescence labeling of CTnC Monocysteine CTnC derivatives were engineered by using cDNAs previously cloned for G159D, L29Q and WT CTnC by substituting Cys 35 for Ser in order to directspecific IAANS incorporation to C84 (denoted, CTnC(C84) IA). The CTnCs were also doubly labeledwith IAANS at Cys 35 and 84 (denoted, CTnC IA). Fluorescent incorporation and subsequent purificationof labeled CTnC followed previous methods (36){Putkey, 1997 #1392} .

Purification of tropomyosin Porcine cardiac Tm was purified from the ammonium sulfate precipitateobtained in the course of native cardiac troponin subunit preparation (38){Potter, 1982 #55} . Briefly, theTm was purified by isoelectric precipitation followed by ion exchange chromatography (Q-sepharose).Purified Tm was dialyzed against 5 mM NH 4(CO 3)2, lyophilized and stored at -20 C.

Formation of Cardiac Troponin Complex - cDNAs cloned in our laboratory from human cardiac tissuewere used for the expression and purification of CTnI (39){Zhang, 1995 #1661} and CTnT (38){Potter,1982 #55} . Formation of fluorescent binary and ternary complexes; and non-labeled ternary complexeswere carried out using recent established protocols (37,40) in the presence of 1.25 mM Mg 2+ .

Preparation of IAANS-labeled regulated thin filaments Regulatory complexes (actin-Tm-CTn) were prepared by mixing F-actin isolated from rabbit skeletal acetone powder (41){pardee, 1982 #0} and Tmin a 7:1 molar ratio in a solution containing 60 mM KCl, 0.1 mM CaCl 2, 2 mM MgCl 2, 1 mM ATP, pH7.0 on ice. The final concentration of actin was ~0.8 mg/ml. After homogenization in a glasshomogenizer, the F-actin-tropomyosin complex was combined with pre-formed IAANS-labeled CTncomplex (tropomyosin / CTn = 1 mol / mol) in a solution containing 100 mM MOPS, 75 mM KCl, 1.25

mM MgCl 2, 2 mM EGTA, 4 mM NTA (standard buffer conditions for our fluorometric titrations). After homogenization, the mixture was allowed to sit at rm. temp. for 10 min. The homogenate was centrifugedfor 1 h at 150,000 x g and the pellet resuspended in standard fluorescence buffer. The solution wascentrifuged and resuspended again in order to avoid free Tm-CTn and free CTn trapped in the pellet.Regulated thin filaments were filtered through a 0.45 m filter and stored on ice ready to use for experiments.

Determination of apparent Ca 2+ affinities Labeled proteins (isolated CTnC, binary and ternary complex)were dialyzed exhaustively in standard fluorescence buffer (see above). The isolated CTnCs weredialyzed in the absence of Mg 2+ to prevent dimerization (42){jaquet, 1987 #0} ; therefore, Mg 2+ was added

just before the titration with Ca 2+. All solutions were prepared at 21 oC. Steady state fluorescencemeasurements were made with a Jasco 6500 spectrofluorimeter. IAANS fluorescence was excited at 330nm and emission monitored at 450 nm as microliter amounts of CaCl 2 were added to a 2.0 ml mixture

containing labeled proteins. The final concentrations of isolated CTnC, binary complex, ternary complexand regulated thin filamentsRTF in each experiment were 0.25 M, 0.5 M, 0.5 M and 0.053 mg/ml,respectively. The concentration of free Ca 2+ was calculated for actual titration conditions using thecomputer program, pCa Calculator (43){Dweck, 2005 #8158} . This program corrected for dilutioneffects attributed to the incremental addition of Ca 2+. The data were fitted to the hill equation with thesoftware suite of SigmaPlot 10.0. The Ca 2+ affinities are reported as pCa 50 (-log[Ca 2+] at which 50% of maximal response is observed) values + S.D. The following modified hill equation was used to fit

biphasic binding curves:Y = {S 1 [Ca 2+]n1 / ( K d1n1 + [Ca 2+]n1)} +


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{S 2 [Ca 2+]n2 / ( K d2n2 + [Ca 2+]n2)},Where Y is the fluorescence intensity, S 1 and S 2 are the percent (%) calcium occupancies of each class of site, K d1 and K d2 are the macroscopic dissociation constants for both classes of sites and n1 and n2 are therespective hill coefficients.

Reconstituted myofilament ATPase Assays Whole porcine cardiac myosin was isolated from leftventricles (44){murakami, 1976 #0} , F-actin, tropomyosin and recombinant human CTn were prepared asdescribed above. The ATPase assays were performed in a 96 well flat bottom plate using 0.1 ml reactionvolumes. Pre-formed human cardiac Tn complexes were added (< 20 l) to the 96 wells to achieveconcentrations ranging from 0.0-2.0 M in the final reaction mixture. F-actin, myosin, and tropomyosinwere homogenized on ice in a glass tube and allowed to come to rm. temp. before addition to each well ( 9) Ca 2+ conditions. The remainder of the 0.1 ml reaction volume comprisedof: 50 l of high or low Ca 2+ ATPase buffer, x l of ddH 2O and 4 l of ATP (3.14 mM, pH 7) toinitiate the ATPase reaction. Methods for solving the free and bound metal ion equilibria were providedfor by the computer program, pCa Calculator (43){Dweck, 2005 #8158} . The final reaction conditionscontained: 50 mM KCl, 1.0 mM free Mg 2+, 2.5 mM MgATP 2-, 1.0 mM DTT, 20 mM MOPS, 1 mMEGTA (in low Ca 2+ ATPase buffer) or 0.23 mM CaCl 2 (in high Ca 2+ ATPase buffer), pH 7 at 25 oC. TheATPase reaction was initiated by the addition of ATP in a row-wise fashion and immediately mixed witha 12 channel pipettor 4-5 times and allowed to incubate at 25 oC for 20 min; thereafter, stopping thereaction by adding cold trichloroacetic acid (TCA) (4.75 % final concentration). Each row was activatedand deactivated in 30 sec intervals. After sedimenting the precipitate by centrifugation, the supernatantswere transferred to a new plate and the liberated inorganic phosphate concentration in the supernatant wasdetermined according to the method of Fiske and Subbarow (45){fiske, 1925 #0} . The ATPase rates weremeasured by single time points that were predetermined to be linear with time.

Preparation of skinned cardiac myofibrils Porcine cardiac myofibrils (CMF) were isolated form porcineleft ventricles as described by Solaro et. al. (46){solaro, 1971 #0} substituting MOPS for imidazole. AllCMF preparations were stored in 50% glycerol (v/v) at -20 oC. CDTA treatment of cardiac myofibrilswere performed by the methods of Morimoto and Ohtsuki {morimoto, 1987 #0 with the followingmodifications: CTnC extraction occurred in 5.0 mM CDTA, 5.0 mM DTT pH 8.4 adjusted with Tris-base(solid) for 15 min followed by centrifugation. This procedure was repeated 4 more times using 30 minCDTA incubations. CDTA was removed by suspending the pellet in wash buffer containing 10.0 mMMOPS, 10.0 mM KCl, 0.5% glycerol at pH 7 followed by centrifugation. These washes were repeatedtwo more times in the absence of glycerol. CTnC reconstitution was performed by mixing CDTA-treatedmyofibrils (1.5-3.0 mg/ml) in wash buffer with recombinant CTnC (~13 M final concentration) on icefor 30 min. Unbound CTnC was removed by centrifugation and the supernatant discarded. Pellets wereresuspended in wash buffer (-glycerol). This wash cycle was repeated and the final precipitateresuspended in wash buffer (-glycerol), stored on ice and used for experiments.Ca 2+ activated Myofibrillar ATPase Assays 0.1 ml reaction mixtures were carried out in a 96 well plate.First, 50 g (< 46 l volume) of myofibrils in wash buffer (-glycerol) were aliquoted per well. Theremainder of the 0.1 ml reaction volume comprised of ddH 2O, ATPase buffers (50 l) that have differentamounts of calcium added to achieve the desired free calcium concentration and 4 l of ATP (3.14 mM,

pH 7) which is added to initiate the ATPase reaction at 25 oC. The reactions were terminated 5 min later by the addition of TCA and the inorganic phosphate measured as above. Methods for solving the free and bound metal ion equilibria in our solutions were provided for by the computer program, pCa Calculator {Dweck, 2005 #8158}. The final reaction condition contained: 10 -8.0 10 -4.5 M free Ca 2+, 1 mM free Mg 2+,2.5 mM Mg-ATP 2-, 2 mM EGTA, 4 mM NTA, 20 mM MOPS, 1.0 mM DTT, 80 mM ionic strength(adjusted with KCl), pH 7 at 25 oC. The data were fitted to the hill equation using the software suite of SigmaPlot 10.0.


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Skinned Fiber preparations Skinned fibers were prepared using established protocols (37){Szczesna,1996 #8059} with the following modifications: muscle fibers were isolated from porcine left ventricular

papillary muscle; pCa solutions were generated using the pCa Calculator program (43){Dweck, 2005#8158} and endogenous CTnC was extracted with 5 mM CDTA, pH 8.4 (adjusted with solid Tris-base)until the fibers developed < 20% of the initial force (residual force) in pCa 4.0 solution. CDTA treatedfibers were reconstituted with CTnC (WT and mutant) and the Ca 2+ dependence of force generation andmaximal recovered force (in the pCa 4.0) measured. Data was analyzed using the following equation:% Change in Force =100 x [Ca 2+]n/([Ca 2+]n+[Ca 2+50]n)where "[Ca 2+50]" is the free Ca 2+ concentration which produces 50% force and "n" is the Hill coefficient(37,47){Guth, 1987 #4514; Szczesna, 1996 #8059} . WT and mutant CTnC reconstitution experimentswere repeated > 5 times and averaged together to calculate the mean + SD.Muscle fiber CTnC quantification. Five muscle fibers resulting ~20% residual force after CDTAtreatment had their proteins separated by SDS-PAGE and transferred to PDVF membranes. Anti-humancardiac troponin C, clone 7b9 (Research Diagnostics, Inc., Concord, MA), and Anti-human myosin lightchain I (ELC) (Accurate chemical, Westbury, NY) were diluted 1:500 and 1:40,000, respectively for

primary detection. Secondary detection for quantitative western blots was performed with goat anti-mouse Alexa-680 (1:4000) from Rockland Immunologicals (Gilbertsville, PA). Visualization andquantification of gel bands was performed with the Odyssey Infrared Imaging System (LI-Cor Biosystems, Homburg, Germany). Normalization of gel bands was accomplished by ratiometric analysisusing ELC as a loading control. The changes in CTnC content from the CDTA-treated fiber wereestimated by comparing the ratios of CTnC/ELC between the experimental and control fibers

RESULTS

Effect of CTnC mutations on the Ca 2+ sensitivity of tension. Skinned papillary muscle fibers weretreated with CDTA to remove the endogenous porcine CTnC followed by reconstituting them back withthe recombinant CTnCs (WT, G159D and L29Q). Figure 1A shows that reconstituting fibers with G159DCTnC significantly decreases the Ca 2+ sensitivity of tension by 0.08 pCa units. This is made evident as arightward shift in the curve with respect to the WT. Moreover, this mutant can generate less tension in the

pCa 6.0-5.6 range (Fig. 1A and 1C ), but does not affect the ability to restore the maximal activatedtension in pCa 4.0 when compared to the WT1 control (Fig. 1C). Alternatively, the L29Q CTnC has noeffect on the Ca 2+ sensitivity (Fig. 1B) or maximal tension (Fig. 1C) when reconstituted into skinnedfibers. Figure 1C shows that theCTnC reconstituted fibers are able to restore ~70% of the maximaltension in accordance with similarly published values (37,48). After CDTA-treatment, the residual force(in pCa 4) maximal tensions of muscle fib ersers after CDTA treatment (residual force) from twodifferent heart s preparations are 19.4 % (WT1 and L29Q) and 12.3 % (WT2 and G159D), respectively.This implies that the residual force is attributed to nearly the same number of remaining CTnC regulatorysites. Western blot analysis showed that 14.0 % + 5.3% of the endogenous CTnC remains after CDTAtreatment and that incubation of fibers with WT or mutant CTnC is able to restore the full complement of CTnC back into the CDTA treated fibers (see supplemental Fig. 1). Moreover, the addition of excessCTnC would compete for CTnC sites on the thin filament and further reduce the amount of endogenous

CTnC during the reconstitution phase.Treatment of porcine fibers with CDTA that yielded ~20 % of theinitial maximal tension (residual force) was attributed to the removal of more than 86 % of the totalendogenous CTnC (data not shown) as reported in our earlier work (23). After CTnC reconstitution, all of the CTnC mutants are able to restore the full complement of CTnC back into the CDTA treated fibers andrestore ~70% of the maximal tension in accordance with similarly published values (23,38,49). Therefore,the changes in Ca 2+ sensitivity or lack ofare due to the specific CTnC mutation, rather than the inabilityto incorporate the CTnC into the muscle fibers. The force and pCa relationships are summarized in Table1.


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Effect of CTnC mutations on cardiac myofibrillar and regulated actomyosin ATPase activities.Cardiac myofibrils (CMF) isolated from porcine left ventricles were treated with CDTA followed byreconstitution with recombinant CTnCs. Figure 2A shows the Ca 2+ dependence of ATPase activity fromthe WT, G159D and L29Q CTnC reconstituted CMF. The maximal output for each mutant and WTCTnC were normalized to 100% in Fig. 2A to better illustrate the ability of the G159D reconstituted CMFto reduce the Ca 2+ sensitivity of ATPase activity by 0.08 pCa units, and the inability of the L29Q mutantto generate statistical differences from the WT controls ( pCa = +0.02, p>0.05). Figure 2B shows thatafter CDTA treatment, the myofibrillar ATPase activity in pCa 4.5 is nearly inactivated (1.59 foldactivation); whereas, after the mutant CTnCs are reconstituted, the maximal myofibrillar ATPase isactivated ~ 4 fold and statistically insignificant in comparison to the WT (Fig. 2B, Table 2).

Preformed cardiac troponin complexes reconstituted with WT, G159D or L29Q CTnC weremixed with F-actin, tropomyosin and myosin in order to determine if the different CTnC mutations canaffect the ability to inhibit or activate the regulated actomyosin ATPase activity. The ATPase activity of the unregulated filaments (i.e., in the absence of CTn) is defined as 100%. Figure 2C shows that in theabsence of Ca 2+ (Ca 2+), the ATPase activities of regulated actomyosin filaments containing the G159Dand L29Q mutants are statistically insignificant in comparison to the WT (~27%) at all CTnconcentrations. However, in pCa 4.0 solution (+Ca 2+), the incorporation of G159D CTnC into regulatedactomyosin filaments significantly lowers the ATPase activation (144%) in comparison to the WTcounterpart (163%) when CTn is sufficiently present to saturate the thin filament. Table 2 summarizes thevalues obtained in Fig. 2.

Effect of metal ion binding on fluorescence from IAANS-labeled CTnC. All cloned CTnCs ( WT,G159D and L29Q CTnC clones) were used to generate monocysteine mutants to direct specificincorporation of the IAANS fluorophore onto residue Cys-84 [CTnC(C84) IA] which. Previousinvestigations show that this configuration solely reports Ca 2+ dependent changes in fluorescence thatreflect metal ion binding to the regulatory site of CTnC(C84) IA in isolation and in binary complex (36)(37) . In addition , to labeling the monocysteine derivatives of CTnC, we conjugated the IAANSfluorophore to both cysteine s at residues (35 and 84 ) were labeled with IAANS [( CTnC IA]) which. Thelatter configuration reports metal ion binding to both classes of binding sites the Ca 2+-specific and ( Ca 2+-Mg 2+ and Ca 2+-specificsites ) in the isolated CTnC IA CTnC IA, and to the Ca 2+-specific site of CTnC IA withinCTn (CTn IA) complex (35,36). (35,37).

Figure 3A represents the Ca 2+ dependent changes in fluorescence arising from the WT, G159Dand L29Q CTnC IA labeled proteins in the presence ( inset ) and absence of Mg 2+. As the free Ca 2+ increasesfrom pCa 8.0 to 3.6, the graphs are biphasic, representing metal ion binding to two classes of bindingsites. Therefore, the pCa 50 and Hill values obtained with the CTnC IA configuration in Fig. 3A arewerefitted to a two- site Hill equation and are summarized in Table 3. As the free Ca 2+ increases from pCa 8.0to pCa 6.6 in the absence of Mg 2+, the isolated WT CTnC IA fluorescence decreases 11.3 % with a pCa 50of 7.04, indicating Ca 2+ binding to the Ca 2+-Mg 2+ sites. In the presence of Mg 2+ (inset ), the WT CTnC IACa 2+-Mg 2+ sites decrease their fluorescence intensity ( 4.8 %) with a concomitant increase in affinity for Ca 2+ by +0.08 pCa units (pCa 50 = 7.12). Increasing the concentration of free Ca 2+ from pCa 6.6 to 3.8leads to an increase in the fluorescence intensity for the WT CTnC IA protein, indicating Ca 2+ binding tothe Ca 2+-specific (regulatory) site with a pCa 50 of 5.23 (Mg 2+) and 4.94 (+Mg 2+).

On Ca 2+ binding to the Ca 2+-Mg 2+ sites of isolated L29Q CTnC IA (Fig. 3A), there are larger

decreases in fluorescence in the absence (72.5 %) and presence (28.1 %) of Mg 2+; however, the fitted pCa 50 values for these sites are unaffected in comparison to the WT counterpart. Moreover, Increasing thefree Ca 2+ from pCa 6.6 to 3.6 increases the L29Q CTnC IA fluorescence, indicating Ca 2+ binding to theregulatory site. However, tt he pCa 50 values for this the regulatory site is markedly reduced in the absenceof Mg 2+ ( pCa 50 = 0.15), and to a lesser extent, in the presence of Mg 2+ ( pCa 50 = 0.06) whencompared to the WT counterpart. On Ca 2+ binding to the Ca 2+-Mg 2+ sites of G159D CTnC IA, in the pCarange of 8.0 to 6.6, significant reductions in the pCa 50 values of the Ca 2+-Mg 2+ sites occur are reduced inthe presence ( pCa 50 = 0.19) and absence ( pCa 50 = 0.17) of Mg 2+. However, increasing the


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concentration of free Ca 2+ from from pCa 6.6 to 3.8, results in the G159D CTnC IA regulatory site affinityCTnC IA fluorescence intensity to increase without affecting the pCa 50 values for the is unaffected Ca 2+-specific site ( pCa 50 < 0.03 in + or presence and absence of Mg 2+). Table 3 summarizes the fittedvalues obtained in Fig. 3A.

Figure 3B represents the calcium dependent changes in fluorescence from labeled mono-cysteine derivatives of CTnC ( WT, L29Q and G159D CTnC(C84) IA) in the absence and presence ( inset )of Mg 2+. As the free Ca 2+ increases from pCa 6.8 to 3.6, the isolated WT CTnC(C84) IA fluorescenceintensity increases monophasically, indicating Ca 2+ binding to one class of sites, namely the regulatorysite. The pCa 50 of these Ca 2+ dependent changes in fluorescencevalues are 5.18 (Mg 2+) and 4.84 (+Mg 2+).In this same pCa range, the G159D CTnC(C84) IA regulatory site affinity protein monophasically increaseits fluorescence intensity without affecting the pCa 50 is unaffecte d. This is made evident by the WT andG159D CTnC(C84) IA curves overlapping each other in the presence ( inset ) and absence of Mg 2+. On theother hand, when on Ca 2+ bind ings to the isolated L29Q CTnC(C84) IA protein (+/ Mg 2+),, thefluorescence intensity unexpectedly increases biphasically, revealing that this labeled protein is sensitiveto Ca 2+ binding to the two classes of sites in the presence and absence of Mg 2+ . Therefore, the L29QCTnC(C84) IA proteins structural effects greatly differ from the previous CTnCs tested herein , and may

preclude its comparison to the WT control. Despite the inconsistencies between the isolated mono-labeledL29Q and WT CTnC(C84) IA CTnC proteins, these data were fitted with a two-site hill equation in order to calculate the pCa 50 values of both classes of sites (summarized in Table 3). These results show thatwhen fitted, the pCa 50 for the L29Q CTnC(C84) IA Ca 2+ specificregulatory site increas ese +0.12 pCa units(Mg 2+) when compared to the WT CTnC(C84) IA protein.

Effect of Ca 2+ binding on fluorescence from IAANS-labeled CTnC in complex. WT, L29Q andG159D Monocysteine derivatives of CTnC ( CTnC(C84) IA) were mixed with recombinant CTnI to formfluorescent binary complexes. Figure 4A shows the Ca 2+ dependent changes in fluorescence arising fromthe WT and mutant binary CTnC(C84) IA complexes. As the free Ca 2+ rises from pCa 7.6 to 4.8, the WT

binary complex increases its fluorescence intensity 1.24 fold with a pCa 50 of 6.24. The results from theG159D binary complex are statistically insignificant in comparison to the WT; whereas, the fluorescenceintensity of the L29Q binary complex increases 1.33 fold with a significant reduction in Ca 2+ affinity( pCa 50 = 0.19). Table 4 summarizes the pCa 50 and Hill values calculated from the data obtained in Fig.4A.

WT, L29Q and G159D CTnC IA were mixed together with recombinant CTnI and CTnT togenerate IAANS-labeledfluorescent cardiac troponin complexes (CTn IA). Figure 4B illustrates the Ca 2+

dependent changes in fluorescence arising from the IAANS-labeled CTn IA complexes. In the pCa range of 8.8 to 5.2, Ca 2+ binding to the WT and mutant and WT CTn IA complexes are accompanied by a 1.4 folddecrease in fluorescence intensity. The G159D CTn IA Ca 2+ affinity is significantly reduced ( pCa 50 = 0.15 p


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DISCUSSION

Since the heart is a dynamic pump, thoracic imaging techniques are essential in identifying thespecific systolic and diastolic dysfunctions underlying the pathologies of familial cardiomyopathies.However, different individuals affected by the same mutation can show variable penetrance (e.g., gradedwall thickening or dilation) suggesting that, cardiomyopathies are complex multi-factorial diseases.Therefore, steady state measurements have the benefit of observing the influence of these mutations oncontractility in the absence of long-term neurohumoral and autonomic stimulation. This has led to the invitro investigation of numerous thin filament mutations linked to cardiomyopathies and the discovery of their characteristic effects on the Ca 2+ dependent processes of contraction in the absence of CTnI

phosphorylation. Within the context of thin filament mutations, a unifying paradigm emerges thatassociates decreases in the myofilament Ca 2+ sensitivity with DCM-linked mutations and increases withHCM- and RCM-linked mutations (4,7,14)(4,7,14) . However, does this imply that small insignificantchanges in the myofilament Ca 2+ sensitivity are going to be associated with a benign phenotype? This

poses a difficult challenge to the developing paradigm because these mutations are rare polymorphisms making it difficult to determine if the clinical outcome is primarily due to a disease causing mutation inthe myocardium, or physiological adaptations to conditions such as, hypertension, diabetes, obesity andalcoholism to name a few (2)(2).

.Our results indicate that the G159D mutation reduces the Ca 2+ sensitivity of tension and cardiac

myofibrillar ATPase activation without affecting the capability to generate maximal output (< pCa 4.5).Despite this mutants inability to reduce the maximal output, the myofilament Ca 2+ desensitization isexpected to recruit fewer strongly attached crossbridges at submaximal Ca 2+ concentrations, leading to thereduction of both ATPase activation and the subsequent generation of tension (1,3,49) (1,3,50) . In supportof this argument, independent studies have shown that the reconstitution of the K210 CTnT mutation(associated with DCM) into skinned fibers also reduces the myofilament Ca 2+ sensitivity without affectingthe maximal tension (3,34).(3,34). Moreover, the K210 CTnT mutation is still able to recapitulate theDCM phenotype (e.g, reduced EF and dilation) in a knock-in mouse model (3).

(3).Previous investigations have reported that the G159D mutation does not alter the Ca 2+ sensitivity

of tension and ATPase activation in the absence of CTnI phosphorylation (51, 28). These reports showthat ~50% (51) (50) to ~68% (28) (28) of the endogenous CTnC is replaced by the mutant in comparisonto the >86% that is exchanged in our fibers . (23) . Therefore, an increase in the ratio of incorporatedmutant to endogenous CTnC in along the thin filament may revealexplain why some of the changes inthe Ca 2+ sensitivity arise in our system . In addition, the pCa 50 values for ATPase activity reported byBiesiadecki et al. (28)(28) are presented with large standard error values (as much as + 0.2 pCa units),suggesting that small significant changes in the myofilament Ca 2+ sensitivity cannot be uncovered by theCa 2+ buffering capabilities of their solutions. Therefore, we employed the combinatorial use of NTA andEGTA to improve the Ca 2+ buffering capabilities of our solutions which has previously been shown toincrease the sensitivity and reproducibility of the experiment (43).

(44).Considering the indirect two-way communication between crossbridge attachment and Ca 2+

binding to CTnC, it is expected that a perturbation due to a CTnC mutation can affect the Ca 2+ sensitivityand dynamics of contraction through the following mechanisms: 1. directly modifying the Ca 2+ binding

properties of the regulatory site, 2. changing CTnC intra-molecular interactions and/or 3. changing CTnCinter-molecular interactions. Measuring the shifts in the Ca 2+ sensitivity and maximum tension (or ATPase) is not sufficient to suggest the mechanism(s). Therefore, covalently attached fluorophores onCTnC can help to identify key intra- and inter-molecular interactions that dictate the final output of tension in the muscle fiber (35,36).

(35,37).


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The isolated G159D (-CTnC IA and -CTnC(C84) IA) regulatory site apparent affinities arestatistically insignificant in comparison to their respective WT counterparts (Table 3). Therefore, theexistence of mechanism 1 may be excluded. Nevertheless, a reduction in the affinity for the Ca 2+-Mg 2+

sites (+/ Mg 2+) indicates that intra-molecular interactions within the C-domain are altered (mechanism2). Measuring the Ca 2+ dependent changes in G159D binary complexes did not uncover the changes seenin the Ca 2+ sensitivity of tension (or ATPase). Rather, within the ternary complex, significant reductionsin the regulatory site Ca 2+ affinity begin to emerge ( pCa = 0.15) which persist up to the level of theRTF and to a lesser extent, in the skinned fiber. These results show that a C-terminal mutation in CTnCcan indirectly affect the N-terminus through altered CTnT inter-molecular interactions (mechanism 3).Based on all of the above, we expect that the ability of the G159D mutant to decrease the myofilamentCa 2+ sensitivity would preserve or facilitate muscle relaxation in the intact heart; yet, lead to systolicdysfunction by weakening the contractile force (i.e., reduced EF).

Echocardiographic examination of the only L29Q CTnC proband shows that the septal and freeventricular walls are concentrically hypertrophied ( 15 mm; cutoff = 15 13 mm) (2,19)(20) and close to theupper limits of normal wall thickness (2). . Notably, in the absence of any cardiomyopathy, many manyclinical studies strongly correlate concentric hypertrophy with hypertension (51,52)(52,53) .MoreoverNevertheless , the amount of left ventricle (LV) hypertrophy may be overestimated in the L29Q

proband because the authors did not adjust the y do not adjust LV mass to body surface area (i.e. height,surface area or skin fold) or mass indices in order to assess the possible impact of obesity andhypertension (53,54)(54,55) . Despite the mild hypertrophy, cardiac catheterization showed a normalejection fractionEF with preserved diastolic heart function (20)(19).. Based upon the available clinicaldata, we hypothesized that if the L29Q mutation is benign, that then significant changes in themyofilament Ca 2+ sensitivity should not arise in the muscle fiber , thereby, excluding a paradigmcontradiction . When we assessed the effects of the L29Q CTnC mutation on tension and myofibrillar myofibrillar ATPase activation (in the absence of CTnI phosphorylation), neither the Ca 2+ sensitivities nor the maximal generating capabilities were significantly different from the WT controls (Tables 1-2) .Moreover, regulated actomyosin reconstituted with preformed CTn containing the recombinant L29Qmutant did not affect the ability to inhibit or maximally activate the ATP hydrolysis at all levels of CTn(Fig. 2C, Table 2) . By taking into account the clinical pathology of the L29Q proband, the authorsconcluded that the L29Q mutation may simply be a rare polymorphism without any phenotypicalrelevance ((19).20) , in which case, our in vitro results would coincide with their findings. Therefore, thecombined clinical and in vitro data should be weighed equally in order to determine more accurately the

potential severity of specific mutations. Under this premise, the E244D CTnT mutation associated withHCM would be considered a benign polymorphism because it does not affect the Ca 2+ regulation in vitro(i.e., pCa 50) (56,57) (55,56) or present with any cardiac dysfunction in the only proband (58).

(57).Two independent investigations havereports have suggested shown that the L29Q mutation may

not lead to a benign outcome because their results indicate thatcan alter the myofilament Ca 2+ sensitivityis significantly altered in a reconstituted S1-ATPase system (29) and in the skinned rat cardiomyocyte(30)(29) and in the skinned rat cardiomyocyte (30) . Unexpectedly, the first investigation reports a

paradigm contradiction because the HCM associated L29Q mutation decreases the myofilament Ca 2+

sensitivity (29)} . However, caution must be given when interpreting these data because their statisticalanalysis for the control and mutant mean pCa 50 values are presented with SD error values greater thantheir respective mean values. Moreover, their use of skeletal actin, Tm and myosin may disrupt somecardiac specific myofilament interactions that govern the Ca 2+ regulation of contraction (or ATPaseactivity). Therefore, to deter the possible contribution of errors from above, we used EGTA incombination with NTA in our solutions (43)(44) to accurately assess the effects of CTnC mutations onthe Ca 2+ sensitivity activated profiles of cardiac myofibrillar ATPase activ ityation .

The second study shows that L29Q CTnC is able to increase the apparent CTnC Ca 2+ affinity inthe isolated state and in the skinned rat cardiomyocyte (30)(30) . Although in our hands, the changes in theCa 2+ sensitivities measured in fibers and cardiac myofibrils reconstituted with L29Q CTnC are


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statistically insignificant with respect to the WT, we do observe tendencies to increase the Ca 2+ sensitivityof tension ( pCa 50 = +0.01 , Table 1 ), myofibrillar ATPase activity ( pCa 50 = +0.02 , Table 2 ) andlabeled regulated thin filaments RTF ( pCa 50 = +0.05 , Table 3 ). It is expected that the different proteinexpression profiles among higher mammals and lower rodents will affect the biophysical properties thatgovern CTnC. Therefore, it is possible that similar force values can not determine if the ratios of endogenous to mutant CTnC are equivalent in both systems after EDTA/CDTA treatment (residual) or after the ratio of mutant to endogenous CTnC after the CTnC reconstitution phase is higher in the ratcardiomyocyte.even though both systems are treated with EDTA/CDTA to achieve similar residualforce values. Moreover, a single cardiomyocyte would require more sensitive transducer(s) to measurechanges in the Ca 2+ sensitivity of tension that w ould go undetected on the scale of the skinned fiber.

Based on the above, our results may not show significant increases in the myofilament Ca 2+

sensitivity because the skinned porcine fiber is less sensitive to changes in tension and may limit theremoval of the endogenous CTnC from the fiber while not affecting the ability to restore the fullcomplement of CTnC back into the myofilament.

Since the L29Q mutation does not alter the Ca 2+ sensitivity of tension in porcine fibers and othershave reported an increased Ca 2+ affinity in the isolated CTnC level (30), we rationalized that the regulatedthin filament can adjust to those changes in the isolated L29Q CTnC and dissipate its effects through thedifferent myofilament interactions. Measuring the structural and Ca 2+ dependent changes from the isolatedL29Q (-CTnC IA and -L29Q CTnC(C84) IA)A proteins confirms that this mutation has the ability to alter theregulatory site binding properties and N- and C - domain intra-molecular interactions . and affect theregulatory site binding properties. Interestingly, the L29Q CTnC IA reports a significant decrease in affinityat the regulatory site; whereas, the L29Q CTnC(C84) IA protein unexpectedly reports Ca 2+ binding to twoclasses of sites with an increased Ca 2+ affinity at the regulatory site (Fig. 3). Although these resultssuggest that the combined effects of the L29Q mutation and IAANS fluorophore on Cys-84 maydramatically perturb the structure of isolated L29Q CTnC(C84) IA, it also provides the opportunity todetermine if the addition of different myofilament proteins and their interactions have the ability to repair these processes. When we assess the effects of L29Q CTnC in binary complex, the structural changes arestill significant and the apparent Ca 2+ affinity is reduced by 0.19 pCa units in comparison to the WT.Further addition Formation of CTnT to generate ternary complexes shows that the changes in Ca 2+

affinity are diminishinggetting smaller ( pCa = 0.07, Table 4). Finally, and when regulated thinfilaments are reconstituted with L29Q CTnC, the apparent changes in Ca 2+ affinity are almostnearlystatistically insignificant in the RTF when compar edison to the WT counterpart ( pCa = +0.05, 0.04< p< 0.05). The above results show that the regulated thin filamentRTF , representing a highly cooperativesystem can restore the Ca 2+ affinity of L29Q CTnC to that of the WT via intra- and inter-molecular myofilament CTnC interactions and can may explain why significant changes in the Ca 2+ sensitivity of tension in the porcine fiber may not occur.

By virtue of having numerous flexible linkers and motile helices, the molecular architecture of CTn is well equipped to adapt to the demands of the working heart (58).(59) . This plasticity assures thatthe structural and inter-molecular changes that occur during contraction (e.g.,after CTnI phosphorylationand Ca 2+ binding ) are restored while the muscle is relaxing . Therefore, the cardiac contractile apparatus,representing a large, elastic, and cooperative machine can provide the potential for dysfunction (G159D)or repair (L29Q) if any one of the proteins is mutated. Myofilament dysfunction can be illustrated throughthe manifestation of alterations in the Ca 2+ sensitivity of tension, despite the apparent G159D CTnC Ca 2+affinity (site II) measured in the isolated state is unaffected. In contrast, myofilament repair can beillustrated through the lack of changes in the Ca 2+ sensitivity of tension measured in the fiber even thoughthe isolated apparent L29Q CTnC Ca 2+ affinity is severely affected. The dysfunction or repair seemingly

predominates at the level of CTn or higher, implying that the true effects of the mutations becomeapparent as the hierarchal level of the myofilament increases. This is illustrated by the tendency of regulated thin filamentsRTFs to follow those changes (+ or ) in the apparent CTnC Ca 2+ affinity that aremeasured by tension and ATPase. Considering that the in vitro -characterizations of over 40 RTF


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mutations assessed (in absence of CTnI phosphorylation) have led to the emergence of current paradigmsrelated to familial cardiomyopathies, we measured the various Ca 2+ dependent processes of contraction todetermine if these new mutations challenge or support these paradigms. Our results indicate that themyofilament Ca 2+ desensitization arising from G159D CTnC and the lack of effects arising from L29QCTnC do coincide with the current paradigms and are indicative of their respective clinical outcomes ..

These results will aid us and other laboratories in the development of therapeutic strategiestailored to modulate the myofilament sensitivity in the treatment of specific cardiomyopathies.


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FOOTNOTES

This work was supported by NIH HL67415 and HL42325 (JDP) and by NIH T32-HL07188 and an awardfrom the American Heart Association, AHA 0315164B (DD).

The abbreviations used are: CDTA, trans -1,2-cyclohexane- N,N,N',N'- tetraacetic acid; CMF,cardiac myofibrils; CTn, cardiac troponin; CTn IA, cardiac troponin reconstituted fromCTnC IA, CTnI and CTnT; CTnC, cardiac troponin C; CTnC(C84) IA, CTnC with one IAANSmolecule covalently attached to Cys 84; CTnC IA, CTnC with two IAANS moleculescovalently attached to Cys 35 and 84; CTnI, cardiac troponin I; CTnT, cardiactroponin T; DTT, dithiothreitol; DCM, Dilated cardiomyopathy; EDTA, ethylene glycol

bis( -aminoethyl ether)-N,N,N',N'-tetraacetic acid; EGTA , ethylene glycol bis( -aminoethylether)- N,N,N',N'-tetraacetic acid; HCM, hypertrophic cardiomyopathy; IAANS, 2-(4'-(iodoacetamido)anilino)-naphthalene-6-sulfonic acid; LV, left ventricle; MOPS, 4-morpholinepropanesulfonic acid; NTA, Nitrilotriacetic acid; pCa, -log[Ca 2+ ]; RCM,restrictive cardiomyopathy; RTF, regulated thin filament; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; Tm, tropomyosin; TRIS-HCl, tris(hydroxyl-methyl) aminomethane hydrochloride.

Keywords: Muscle Contraction, Troponin C, Cardiac Troponin, DCM, HCM,Cardiomyopathy, Skinned Muscle Fiber, Actomyosin, ATPase Activity, CardiacMyofibril, Fluorescence, IAANS-labeled CTnC, Troponin Mutation, Ca 2+ Binding, Ca 2+

Affinity, Ca 2+ Sensitivity, Myofilament, CDTA, EDTA, L29Q CTnC, G159D CTnC,Regulated Thin Filament.

FIGURE LEGENDS

Figure 1: The effects of CTnC mutations associated with cardiomyopathies on the Ca 2+ sensitivityand maximum tension in skinned muscle fibers. The Ca 2+ dependencies of tension from porcine musclefibers reconstituted with (a) G159D CTnC and (b) L29Q CTnC. (c) A comparison of the relative tensionsfrom muscle fibers after CDTA treatment in pCa 4.0 (residual tension, white), and after CTnCreconstitution in pCa 4.0 (black) and 5.8 (grey). 100% relative tension indicates the maximal tension (in

pCa 4.0) of fibers before CDTA treatment. All CMF concentrations were 0.5 mg/ml. Data summarized inTable 1.

Figure 2: The effects of skinned cardiac myofibrils and regulated actomyosin filamentsreconstituted with CTnC mutants. (a) The Ca 2+ sensitivity of ATPase activity from skinned cardiacmyofibrils reconstituted with the G159D and L29Q CTnC. (b) A comparison of the CDTA treated andCTnC reconstituted myofibrillar ATPase activities in pCa 8.0 (black) and pCa 4.5 (grey) solutions. (c)Effect of CTnC mutations on the activation (+Ca 2+) and inhibition (Ca 2+) of regulated actin-Tm activatedmyosin ATPase activity. CTn containing G159D, L29Q or WT CTnC was mixed with actin, Tm andmyosin to reconstitute the regulated actomyosin filaments. The protein concentrations used are as followsin M: 3.5 F-actin, 1.0 Tm, 0.6 myosin and 0-2.0 CTn. At all concentrations of CTn (02.0 M), thespecific ATPase activities are measured in the presence of 0.1 mM free Ca 2+ (+Ca 2+) or 1.0 mM EGTA ( Ca 2+). All experiment were done in the presence 1.0 mM free Mg 2+. Data summarized in table 2.

Figure 3: The Ca 2+ dependent changes in fluorescence arising from IAANS labeled CTnC mutantsassociated with cardiomyopathy. (a) A comparison of the Ca 2+ dependent changes in fluorescence fromthe double labeled L29Q and G159D CTnC IA. (b) The Ca 2+ dependent changes in fluorescence from the


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mono-labeled L29Q and G159D CTnC(C84) IA. Microliter amounts of Ca 2+ were added to 2.0 ml of labeled CTnCs (0.25 M) in the presence and absence of 1.25 mM Mg 2+ (insets ). Fluorescence changesare expressed as a percent of the total change. Data summarized in Table 3.

Figure 4: The Ca 2+ dependent changes in fluorescence arising from IAANS labeled CTnC mutantsin complex. Effect of thin filament proteins on the Ca 2+ dependence of fluorescence from L29Q andG159D mutations in CTnC(C84) IA binary complexes (a), CTnC IA ternary complexes (b) and CTnC(C84) IAregulated thin filaments (c). Microliter amounts of Ca 2+ were added to 2.0 ml of labeled regulatorycomplexes in the presence of 1.25 mM Mg 2+. The protein concentrations used were as follows: 0.5 M

binary complex, 0.5 M ternary complex and 0.053 mg/ml regulated thin filaments. Fluorescencechanges are expressed as a percent of the total change. Data summarized in Table 4.


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Table 1: Summary of pCa-tension relationships of skinned cardiacfibers reconstituted with mutant CTnCs

CTnC reconstituted

fiber pCa 50 a

pCa 50b n H

% Residual Tension

% Restored Tension

WT1 5.69 + 0.05 4.07 + 0.68 21.4 + 3.5 74.9 + 8.2

L29Q 5.70 + 0.01 + 0.01 3.89 + 0.18 19.4 + 6.3 66.0 + 5.6

WT2 5.70 + 0.04 2.26 + 0.29 12.3 + 2.5 66.1 + 4.6

G159D *5.62 + 0.02 * 0.08 2.23 + 0.10 12.3 + 1.4 73.4 + 7.7a The pCa 50 values were obtained by fitting the data in the range from pCa 7 to 4.5.b pCa 50 = pCa 50 of CTnC mutant fiber pCa 50 of WT fiber. *p value

Nir Hus and David Dweck, JBC Manuscript G159D and L29Q CTnC Revised 3

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