Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Article

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model Masataka Kawai,1,* Jamie R. Johnston,2 Tarek Karam,1 Li Wang,1,3 Rakesh K. Singh,4 and Jose R. Pinto2 1 Departments of Anatomy and Cell Biology, and Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa; 2Department of Biomedical Sciences, College of Medicine, The Florida State University, Tallahassee, Florida; 3School of Nursing, Soochow University, Suzhou, Jiangsu, China; and 4Translational Science Laboratory, College of Medicine, The Florida State University, Tallahassee, Florida

ABSTRACT The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2þ-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25 C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3 increase in Ca2þ-sensitivity and a decrease (0.8) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5), reversal step (k4) increased (2), and the phosphate-release step (1/K5) increased (2). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in the a-myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

INTRODUCTION Hypertrophic cardiomyopathy (HCM) is the most common genetic disease involving maladaptive hypertrophy of the left ventricle (LV) and interventricular septum (1). A new study suggests that HCM affects 1 in 200 individuals (2), and the risk of sudden cardiac death in young adults annually is as high as 1% (3,4). Mutations in sarcomeric proteins are the major cause of HCM, accounting for 75% of HCM cases (5). Among several sarcomeric proteins, cardiac troponin C (cTnC) has been known to be associated with HCM in humans (6–9). The troponin complex is comprised of three subunits: TnC (encoded by gene TNNC1); an inhibitory subunit, troponin I (TnI, encoded by TNNI3); and a tropomyosin

Submitted October 17, 2016, and accepted for publication February 22, 2017. *Correspondence: [email protected] Editor: Enrique De La Cruz. http://dx.doi.org/10.1016/j.bpj.2017.02.045 Ó 2017

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binding protein, troponin T (TnT, encoded by TNNT2). TnC is a Ca2þ sensor, and its interaction with TnI is strengthened when Ca2þ is bound, and this binding weakens the inhibitory function of TnI, causing its release from actin. The troponin-tropomyosin complex then slides deeper into the groove formed by the actin helix, thereby exposing the myosin binding sites on actin and making them available for the myosin interaction (for review, see (10)). TnC is a small 180-kDa dumbbell-shaped protein, whose N- and C-terminal domains form two globular structures that are connected by a long central helix (11). Both domains contain EF-hand motifs (two for each domain), forming binding pockets for divalent cations and numbered loops I–IV starting from the N-terminus. In cardiac muscles, loop I is inactivated. Ca2þ or Mg2þ is bound to loops III and IV under the relaxing condition (~0.1 mM Ca2þ, ~1 mM Mg2þ, and ~6 mM MgATP (12)), which have a structural role. Loop II is the Ca2þ binding site that regulates the

CB Kinetics of TnC-A8V Knock-in Mouse

actomyosin interaction as the cytosolic Ca2þ concentration changes in a physiological range (13). An analysis of a cohort of 1025 patients with HCM at the Mayo Clinic (Rochester, MN) identified four cTnC mutations, and the A8V mutation was one of them (7). An analysis of eight additional genes comprising the commercially available genetic test for sarcomeric proteins revealed no other mutations in these patients. The probands displayed LV hypertrophy with significant LV outflow obstruction, dyspnea, syncope, and chest pain. Additional probands bearing the cTnC-A8V mutation have been subsequently reported (14,15). In one report, two children with restrictive cardiomyopathy (RCM) were found to be compound heterozygotes for TNNC1 HCM-associated mutations (A8V and D145E) (15). Additionally, there is an entry on ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) of a proband with RCM that is homozygote for TNNC1 A8V. It has been recognized that HCM and RCM share similarities in clinical presentations of the disease (16,17). The A8V mutation of cTnC is located in its N-helix of the N-terminal domain, a region known to affect TnC’s affinity for Ca2þ (18,19); the deletion of residues 1–14 in skeletal TnC (corresponding to 1–11 in cTnC) resulted in alterations in Ca2þ sensitivity (19,20). Skinned cardiac fibers reconstituted with the TnC A8V mutation showed an increase in Ca2þ sensitivity of force development (7). In addition, in a knock-in (KI) mouse model bearing the TnC-A8V mutation, their hearts displayed morphological and functional changes consistent to those in the proband carrying this mutation in humans, e.g., atrial enlargement, diastolic dysfunction, and LV hypercontractile phenotype (21). In this investigation, we used left-ventricular skinned cardiac muscle fibers of the TnC-A8V mouse model, and assessed the effects of the mutation on the cross-bridge (CB) kinetics and the elementary steps of the CB cycle. We applied a small length perturbation analysis method to study the molecular mechanisms underlying contraction and A8Vassociated mutation. To investigate the underlying causes of perturbed CB kinetics, we also assessed the phosphorylation profile of a-myosin heavy chain (a-MHC). Our results demonstrate how a pathogenic mutation in TnC can have sweeping effects that ripple through the myofilament. MATERIALS AND METHODS

stained first with Pro-Q Diamond phosphoprotein stain (Cat. No. P33301; Molecular Probes, Eugene, OR) according to the manufacturer’s protocol. Subsequently, the gels were restained by SYPRO-Ruby (Cat. No. S12001) to detect the total protein content. Both stains were visualized using a UV trans-illuminator (Bio-Rad, Hercules, CA), their digital images were obtained, and band density of each protein was determined with the software ImageJ (National Institutes of Health, Bethesda, MD). Relative phosphorylation of each protein was calculated as the ratio (phosphorylated protein)/(total protein). For mechanical analyses, strips of muscles, 0.1–0.5 mm in diameter and 1.0–1.5 mm in length, were similarly dissected from the left papillary muscle and skinned overnight in the relaxation solution. The preparations were then transferred to the glycerol relaxing solution (45% relaxing, 55% glycerol by volume), shipped to the University of Iowa, and kept at 20 C. The strips were further dissected to small fibers of ~100 mm in diameter and 1.0–1.5 mm in length. The fibers were mounted to the experimental apparatus with each end fixed with a tiny amount of nail polish to two hooks. One was connected to a length driver, and the other to a tension transducer. Relaxing solution (0 C) was applied immediately and fibers were equilibrated for ~10 min until the nail polish dried and the connections were stable. Fibers were skinned further with 1% Triton X-100 in the relaxing solution for 20 min at 25 C.

Solution compositions The experiments included the standard activation study, the rigor study, the ATP study, the phosphate (Pi) study, and the Ca study. All experiments were performed at 25 C. The solution compositions used for these studies were published previously in Table 1 of Wang et al. (22). Acetate (Ac) was the major anion, because it preserves muscle fibers better than most other anions (23). The fibers were first tested with the standard activating solution that contained 6 mM K2CaEGTA, 6.06 mM Na2H2ATP, 6.62 mM MgAc2, 4 mM KH2PO4, 4 mM K2HPO4, 15 mM Na2CP (phosphocreatine), 13 mM NaAc, 54 mM KAc, 12 mM KCl, 10 mM MOPS, 80 unit/mL creatine kinase, and pH was adjusted to 7.00. Our equilibrium analysis showed that Mg2þ was 1 mM, MgATP2 5 mM, Pi 8 mM, ionic strength 200 mM, and pCa 4.55. The Naþ concentration was minimized at 55 mM, for it comes in as Na2H2ATP and Na2CP, and intracellular Naþ was reported to be in the low micromolar range (12). After the first standard activation, solutions containing varied [MgATP], [Pi], or [Ca2þ] were applied. For the ATP study, [MgATP] tested was 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM, in which 8 mM Pi was present. For the Pi study, its concentration was 0, 2, 4, 8, 16, and 30 mM, in which 5 mM MgATP was present. For the Ca2þ study, its pCa (¼ log10[Ca2þ]) was 8.0, 7.0, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, and 4.55, in which 8 mM Pi and 5 mM MgATP were present. The standard activating solution has the same composition as the 5 mM MgATP solution in the ATP study, the 8 mM Pi solution in the Pi study, and the pCa 4.55 solution in the Ca study. The reproducibility of the tension at the end of the experiment was tested with the standard activating solution, and only the data that reproduced R90% of the first tension were used. In the rigor study, the standard activating solution was first applied, followed by induction of the rigor state with two solution changes.

Fiber preparations Adult homozygous KI TnC A8V and wild-type (WT) mice (7–8 months old) were euthanized and sacrificed by cervical dislocation in accordance with the animal protocol approved by the Florida State University Animal Care and Use Committee. As we have reported, diastolic dysfunction and HCM develops fully by 3 months of age in KI TnC A8V homozygous mice (21). Papillary muscles bundles were isolated from the LV and skinned overnight in the relaxing solution (20 mM MOPS, 2.5 mM MgATP, 7 mM EGTA, 150 mM ionic strength, 1% Triton X-100, pH 7.0) at 4 C. For biochemical analysis, fiber bundles with 0.2–0.3 mm diameter, 1 mm length were loaded onto 10% SDS-PAGE gels, and electrophoresed. Gels were

Sinusoidal analysis The sinusoidal analysis method was previously described in detail in the literature (22,24–26). In brief, during a steady tension plateau, the length of the fibers was oscillated in a sinusoidal waveform with 17 discrete frequencies (f): 0.13, 0.25, 0.35, 0.5, 0.7, 1, 1.4, 2, 3.1, 5, 7, 11, 17, 25, 35, 50, 70, and 100 Hz. This corresponds to 1.6 ms to 1.2 s (¼ 1/(2pf)) in the time domain analysis; actual measurements were carried out from high to low frequencies, because the high-frequency range has more interesting information than the low-frequency range. Also, it takes longer to

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Kawai et al. collect low-frequency data. The amplitude of the oscillations was kept at 0.125%, which corresponds to 51.5 nm per half sarcomere (50.8 nm/ CB with 50% series compliance) and smaller than the CB step size. If this is larger than the step size, the elementary steps of the CB cycle could not be resolved. The tension and length time course data were digitized (16 bits), and the complex modulus data Y(f) were calculated as the ratio of tension change to length change at each frequency. Y(f) is a frequency response function relating the length change (strain) to the tension change (stress) expressed in the frequency domain, and consists of two components: the viscous modulus (imaginary part of Y(f)) and the elastic modulus (real part of Y(f)). The complex modulus data were fitted to Eq. 1, which consists of two exponential processes B and C, that are involved in active CB cycling (24,26,27).

2pb ¼ sk4 þ

K5 P k4 ; 1 þ K5 P

(3)

where

s ¼

K2 K1 S 1 þ ð1 þ K2 ÞK1 S

(4)

and P ¼ [Pi], k4 and k4 are the forward and reversal rate constants of the force-generation step 4, K4 ¼ k4/k4 is its equilibrium constant, and K5 is the Pi association constant.

Ca study To determine the Ca2þ sensitivity and cooperativity, pCa was changed from 8 to 4.55 in the presence of 5 mM MgATP and 8 mM Pi, and isometric tension was measured. The tension results were fitted to: pffiffiffiffiffiffiffi where i ¼ 1; b and c (b < c) are the characteristic frequencies of processes B and C, respectively; and 2pb and 2pc are their respective apparent rate constants. Process B is a medium frequency-exponential delay (delayed tension), where the muscle generates oscillatory work on the length driver; process C is a high frequency-exponential advance (fast tension recovery), where the muscle absorbs work. B and C are their respective magnitudes (amplitudes), and H is a constant that represents the elastic modulus extrapolated to zero frequency (f / 0). The two exponential processes are absent in relaxed fibers, or in fibers in which rigor is induced (24,26). Hence the exponential processes are manifestations of actively cycling CBs. From Eq. 1, the elastic modulus extrapolated to the infinite frequency (f / N) is: YN ¼ H  B þ C, which is generally called ‘‘stiffness’’ in muscle mechanics literature. In step analysis, YN corresponds to phase 1, process C corresponds to phase 2, and process B corresponds to phase 3 (24,28,29). A slow process A (phase 4), which is prominent in skeletal muscle fibers (24), has not been observed in cardiac fibers at temperatures %25 C, but it is evident at higher temperatures (30).

ATP study The result of the ATP study was fitted to Eq. 2, which was derived from CB scheme in Fig. 1, steps 1 and 2 (31):

2pc ¼

K1 S k2 þ k2 ; 1 þ K1 S

(2)

where K1 is the ATP association constant, S (substrate) ¼ [MgATP]; k2 is the rate constant of CB detachment step 2; k2 is its reversal rate constant; and K2 ¼ k2/k2 is its equilibrium constant.

Pi study The result of the Pi study was fitted to Eq. 3, which was derived from CB scheme in Fig. 1, steps 4 and 5 (31):

Tension ¼

Tact n ; Ca50 H 1þ ½Ca2þ  

(5)

where Tact is the Ca2þ activated tension measured at saturating Ca2þ (pCa 4.55), Ca50 is the apparent Ca2þ dissociation constant, and pCa50 ¼ log10(Ca50) is the Ca2þ sensitivity. Each experiment was fitted to Eq. 5, tension was normalized by Tact, and the data were averaged and plotted. For summary plots, individual pCa50 and nH were averaged.

Mass-spectrometry analysis In-gel digest was performed using ProteoExtract All-in-One Trypsin Digestion Kit (Cat. No. 650212; Calbiochem, EMD Millipore, Billerica, MA) according to manufacturer’s specifications. Briefly, excised gel pieces were destained with wash buffer, and dried at 90 C for 15 min. Gel pieces were rehydrated with digest buffer, and treated with reducing agent for 10 min at 37 C. Samples were cooled to room temperature and then blocked using blocking reagent for 10 min at room temperature. Trypsin at the final concentration of 8 ng/mL was added and incubated for 2 h at 37 C with shaking. Peptides were eluted in 50 mL 0.1% formic acid and run on liquid chromatography-mass spectrometry (LC-MS) as described below: An externally calibrated high-resolution electrospray tandem mass spectrometer (Thermo Q Exactive HF; Thermo Fisher Scientific, Waltham, MA) was used in conjunction with a Dionex UltiMate 3000 RSLCnano System (Thermo Fisher Scientific). A 5 mL sample was aspirated into a 50 mL loop and loaded onto the trap column (Thermo m-Precolumn 5 mm, with nanoViper tubing of 30-mm inner diameter, 10 cm length; Thermo Fisher Scientific). The flow rate was set to 300 nL/min for separation on the analytical column (Acclaim pepmap RSLC 75 mm, and 15 cm nanoViper; Thermo Fisher Scientific). Mobile phase A solution was composed of 0.1% formic acid in H2O (EMD Omni Solvent; EMD Millipore), and mobile phase B solution was composed of 0.1% formic

FIGURE 1 The six-state CB scheme, where A is actin, M is myosin, S is MgATP, D is MgADP, and P is Pi, which is phosphate. K indicates the equilibrium constants, and k indicates the rate constants of the elementary step. These are called ‘‘kinetic constants’’ as a whole.

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CB Kinetics of TnC-A8V Knock-in Mouse acid in acetonitrile. A 60-min linear gradient from 3 to 45% phase B solution (in phase A solution) was performed. The LC eluent was directly nanosprayed into a Q Exactive HF Mass Spectrometer (Thermo Fisher Scientific). During the chromatographic separation, the Q Exactive HF was operated in a data-dependent mode and under direct control of the Thermo Excalibur 3.1.66 (Thermo Fisher Scientific). The MS data were acquired using the following parameters: 20 data-dependent collisionalinduced-dissociation MS/MS scans per full scan (350–1700 m/z) at the 1:60,000 resolution. MS2 were acquired in centroid mode at the 1:15,000 resolution. Ions with single charge or those with more than seven charges, as well as unassigned charges, were excluded to only select for peptides. A 15 s dynamic exclusion window was used. All measurements were performed at room temperature. Resultant raw data files were searched with Proteome Discoverer 1.4 using Sequest HT (Thermo Fisher Scientific) as the search engine, with the modified mouse FASTA database and percolator as peptide validator. Phosphorylation was verified by including the PhosphoRS 3.1 (http://ms.imp.ac.at/? goto¼phosphors) node in the Proteome Discoverer work flow. PhosphoRS is a software that enables automated and precise localization of phosphorylation sites within a peptide sequence (32).

Immunoblotting Muscle fibers were prepared following the same method used for the Pro-Q analysis. For detection of MHC phosphorylation, the fiber preparations were resolved on a 6% Acrylamide-20 mM Phos-tag gel at 15 mA for 2 h at room temperature (33). The gel was transferred to PVDF membrane overnight at 30 V, 4 C. The membrane was probed with rabbit polyclonal anti-phosphothreonine (Fitzgerald Industries International, Acton, MA) at 1:1250 and then mouse monoclonal MF-20 (Developmental Studies Hybridoma Bank, University of Iowa) at 1:800 to detect total MHC. The blot was probed with anti-rabbit (IRDye 800 RD; LI-COR Biosciences, Lincoln, NE) and anti-mouse secondary (IRDye 680 RD; LI-COR Biosciences) antibodies at a 1:10,000 dilution and imaged using the Odyssey infrared imaging system (LI-COR Biosciences). Image Studio Lite (LICOR Biosciences) was used to quantify the signals obtained specifically from MHC-phospho-threonine and we divided this by the total MHC to obtain the ratio to quantitate the amount of phosphorylated threonine residues.

Statistical analysis All values are presented as mean 5 SE. Significance was determined by one-way ANOVA using Student-Newman-Keuls post hoc analysis. A comparison was made between mutant and WT, and an asterisk (*) is placed if significantly different (0.01 < p % 0.05), or a double asterisk (**) if highly significantly different (p % 0.01).

RESULTS Active tension and rigor stiffness To determine how Ca2þ-activated tension differs between WT mice and TnC KI A8V mice, the fibers were maximally activated with the standard activating solution (pCa 4.55) and the results are plotted in Fig. 2 A. This figure shows that the active tension was slightly less in the A8V than in the WT, but their difference was not significant. To determine whether the in-series compliance is affected by the mutation, rigor stiffness was compared among two groups of muscle preparations. First, the fibers

FIGURE 2 (A) Active tension in WT and A8V papillary muscle fibers, as measured in standard activating solution (5 mM MgATP, 8 mM Pi, pCa 4.55). Mean 5 SE are shown. N ¼ 13–20. (B) Elastic modulus of the rigor state in the WT and A8V papillary muscle fibers, measured at 100 Hz, is given. N ¼ 11–13.

were activated using the standard activating solution, and then ATP was removed from the solution. The rigor state developed in ~3 min, and the stiffness was determined at 100 Hz. The rigor stiffness was almost constant in frequency (24). The results are plotted in Fig. 2 B; the rigor stiffness did not differ significantly among the two groups of preparations. We infer from this result that there are no differences in the series compliance, including the actomyosin interface among the two groups of muscle fibers. Kinetic studies with sinusoidal analysis To determine how CB kinetics are affected by the mutation, a small-amplitude sinusoidal analysis was performed during the standard activation. The complex modulus data Y(f) were collected and plotted in Fig. 3 with fibers from LV of WT and A8V mutants. Fig. 3 A is a plot of elastic modulus [¼ Real Y(f)] versus frequency (f), and Fig. 3 B is a plot of viscous modulus [¼ Imag Y(f)] versus frequency. The data were fitted to Eq. 1, and the smooth curves represent values calculated based on best fit of the parameters to the data. As seen in Fig. 3, A and B, curves are shifted to the right for the A8V mutant compared to WT, indicating that the mutation results in an acceleration of CB kinetics. The right shift can be observed as the shift of the frequency that generates minimum elastic modulus (Fig. 3 A), and a shift of the frequency that generates the maximum viscous modulus (Fig. 3 B). These observations indicate that the CB kinetics become faster for the mutant. The effect of ATP To characterize elementary steps involved in binding of MgATP to the myosin head and the subsequent actomyosin dissociation, we changed [MgATP] and studied the apparent rate constants. Fig. 4 A plots the results of 2pc versus [MgATP] in the log scale. For both preparations, 2pc

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rated for R16 mM. Notably, A8V had a higher level of saturation than WT by 1.7 (Fig. 4 B). The data were fitted to Eq. 3 to characterize the kinetic constants associated with steps 4 and 5. The results are plotted in Fig. 5, C and D. The rate constant of the force-generation step 4 (k4) is significantly less in A8V than WT, and its reversal step (k4) is significantly larger than WT (Fig. 5 D), resulting in a large and significant decrease in its equilibrium constant (K4) compared to WT (Fig. 5 C). Fig. 5 C shows that the Pi binding (K5) is somewhat less for A8V than for WT, but this effect is not significant. Ca2D sensitivity and cooperativity To determine the Ca2þ sensitivity and cooperativity, the [Ca2þ] concentration was changed across the pCa range of 8 through 4.55. The results of tension are plotted in Fig. 6 A for the pCa range 5–7; there was no tension at pCa 8, and it was saturated for pCa < 5. These plots show that Ca2þ sensitivity increased by 0.5 pCa unit (3 increase in the apparent Ca2þ sensitivity) for A8V compared to WT. pCa50 and nH for each pCa study were averaged and plotted in Fig. 6, B and C. Fig. 6 B shows that pCa50 increased by 0.5 pCa units. Fig. 6 C shows that cooperativity (nH) was significantly decreased in A8V compared to WT. FIGURE 3 Plots of complex modulus Y(f) as measured in the standard activating solution. (A) Elastic modulus [¼ Real Y(f)] versus frequency (f), and (B) viscous modulus [¼ Imag Y(f)] versus frequency are shown. (A and B) Here, solid symbols represent WT; open symbols represent the TnC-A8V mutant; and smooth curves represent best fit of the data to Eq. 1. Average of 13 (WT) and 21 (A8V) experiments. Baseline is not subtracted.

Phosphorylation status of sarcomeric proteins by Pro-Q Diamond stain of skinned fibers

increased from 0.05 to 2 mM [MgATP], but then saturated at R2 mM. Among the two groups, A8V showed a higher level of saturation by 1.5 (Fig. 4 A). The data were fitted to Eq. 2 to characterize the kinetic constants for ATP binding (step 1) and subsequent CB detachment (step 2). As shown in Fig. 5 A, the ATP binding (K1) was somewhat less in WT than in A8V, but the difference was not significant. The rate constant of the CB detachment step 2 (k2) was significantly larger for A8V, and its reversal step (k2) was smaller (not significant) (Fig. 5 B), resulting in a significantly large increase in its equilibrium constant (K2) (Fig. 5 A).

CB kinetics and Ca2þ sensitivity may be affected by phosphorylation of sarcomeric proteins. For this reason, proteins from skinned fibers were separated by SDS-PAGE, and the degree of phosphorylation was assessed by Pro-Q Diamond and SYPRO-Ruby staining (Fig. 7). Fig. 8 is quantification of the phosphorylation levels of MyBP-C, TnTþactin, TnI, and regulatory light chain (RLC). Because the separation between TnT and actin was poor, they were combined and analyzed; and because muscle actin is poorly phosphorylated (Fig. 7 B), hence the actin band of the SYPRO staining is used for normalization of the loading difference of each lane. As seen in Fig. 8, no significant differences were observed in the relative abundance of phosphorylation of these proteins (MyBP-C, TnTþactin, TnI, and RLC) between A8V versus WT.

The effect of Pi

Phosphorylation profile of MHC by MS

To characterize elementary steps associated with the force generation (step 4 in Fig. 1) and Pi release (step 5), [Pi] was changed from 0 to 30 mM, and the apparent rate constants were studied. Fig. 4 B plots the results of 2pb against [Pi]. In all preparations, the apparent rate constant 2pb increased over the range 0–16 mM Pi, and then was satu-

Because the resolution of MHC bands in SYPRO-Ruby and Pro-Q Diamond (Fig. 7) was unsatisfactory, we examined the phosphorylation profile of MHC by MS. The gel in Fig. 7 was Coomassie-stained, MHC bands were excised, trypsin digested, and the peptides were detected by liquidchromatography tandem-MS (LC-MS/MS). Table 1 lists

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FIGURE 4 (A) Effects of [MgATP] on the apparent rate constant 2pc in A8V and WT papillary muscle fibers. Smooth curves represent best fit of the data to Eq. 2. (B) Effects of [Pi] on the apparent rate constant 2pb. Smooth curves represent best fit of the data to Eq. 3.

the peptide sequences with their respective phosphorylation sites that were identified with R75% highest PTM score. Fig. 9 shows phosphorylated sites of a-MHC from WT (in A) and A8V mutant (in B). As indicated in the figure and table, we were able to identify 31 phosphorylation sites in a-MHC, and many sites (S393, T666, Y717, S881, T1021/ T1023/S1025, T1129, S1201, S1301/T1304, S1337/ S1338, T1655, T1791, and T1853/Y1854/T1856) are not

phosphorylated in TnC-A8V mutant, particularly in the rod domain. Reduced threonine phosphorylation in MHC Out of the 11 total phospho-Thr residues identified in the MHC rod by LC-MS/MS, eight of them were found unphosphorylated in A8V fibers compared to WT fibers (Table 1).

FIGURE 5 The kinetic constants of the CB cycle (Fig. 1) as deduced from ATP and Pi studies (Fig. 4). (A and C) Equilibrium constants and (B and D) rate constants are shown. *, Significantly different (p < 0.05) from WT.

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FIGURE 6 (A) Effects of Ca2þ on tension. Data were collected for pCa 8.0–4.55 to confirm saturation, but point in either end are not shown; all the points were used to fit the data to Eq. 5 (smooth curves). The data were also fitted individually and pCa50 and nH were averaged for 13–15 preparations, and plotted in (B) and (C), respectively. *, Significantly different (0.01 % p < 0.05) and **, highly significantly different (p < 0.01) from WT.

To further validate the MS results, we carried out immunoblotting (Fig. 10 A). In agreement with the results in Table 1, we observed a significant reduction in the relative abundance of phosphorylated MHC-Thr residues in A8V muscle fibers compared to WT (Fig. 10 B).

DISCUSSION The purpose of this study was to investigate the alterations of CB kinetics in heart muscle of a TnC-A8V KI mouse model that exhibits hypercontractility and diastolic dysfunction (21). Our results confirm that the A8V mutation in TnC results in an increase in Ca2þ sensitivity (pCa50; Fig. 6) as reported previously (7,21). Moreover, our examination of changes in the kinetics of the CB cycle caused by this mutation revealed specific changes for the mutant: significant changes were observed for many of the kinetic constants (Fig. 5). This effect can be seen in the frequency plots (Fig. 3) and in the plots of the apparent

rate constants (Fig. 4), where significant speeding of the elementary steps of the CB cycle is evident in the mutant. Phosphorylation is one of the most common post translational modifications known to regulate the activity of proteins. Therefore, we investigated the relative abundance of phosphorylated sarcomeric proteins. To accomplish this, we examined the phosphorylation status of MyBP-C, TnT, TnI, and RLC by SDS-PAGE using the Pro-Q staining method (Fig. 7). Our results show that the total phosphorylation of each protein is not significantly affected by the mutation (Figs. 7 and 8). For the determination of TnT phosphorylation, we used TnTþActin (due to poor separation), which is justified because actin phosphorylation level (at Y53) is small and limited to <10% (Fig. 7 B shows almost no phosphorylation), and actin phosphorylation is known not to alter muscle function (34–36). Consequently, this method can be used for a quantification of the TnT phosphorylation when A8V is compared to WT. Since we observed no significant differences in the total phosphorylation status of sarcomeric proteins using the

FIGURE 7 10% SDS polyacrylamide gel demonstrating phosphorylation status of sarcomeric proteins in WT versus TnC-A8V of skinned cardiac muscle fiber bundles. (A) SYPRO-Ruby stain for total proteins and (B) Pro-Q Diamond stain for total phosphorylated proteins are shown.

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FIGURE 8 Quantification of gels in Fig. 7. The density of bands were measured by the software ImageJ (National Institutes of Health) with background subtraction. Then the density value of Pro-Q Diamond stain was divided by the density value of SYPRO-Ruby stain and plotted. (A) Myosin binding protein-C (MyBP-C), (B) summation of TnT and actin, (C) TnI, and (D) myosin RLC are given as an average of four measurements.

Pro-Q staining method, yet there are functional differences at pCa 4.55 as described above, we hypothesized that there could be a shift in the phosphorylation status of a-MHC. This is the protein chosen for the investigation of the phosphorylation status for two reasons: 1) modifications of MHC may have the potential to affect the kinetics; and 2) at maximal activation (pCa 4.55), there should not be a direct link between TnC and altered CB kinetics. To explore this possibility, we performed LC-MS/MS to analyze the phosphorylation profile of a-MHC in WT versus A8V muscle fibers. Quite interestingly, we observed a remarkable difference in the phosphorylation patterns between the two groups. Specifically, the rod domain of a-MHC in TnC-A8V fibers appears to be hypophosphorylated compared to WT fibers, predominantly at Thr residues (Table 1). To corroborate the MS results, we carried out immunoblotting studies, which demonstrated a significant reduction in the relative abundance of phospho-Thr residues on MHC in A8V muscle fibers compared to WT (Fig. 10). The rod forms the backbone of the thick filament structure, hence it is possible that the hypophosphorylation causes a rearrangement of the backbone structure. However, such rearrangement seems not to affect series compliance of the thick filament, because the stiffness measured during rigor induction did not change with the mutation (Fig. 2 B).

Although there is a paucity of research that has examined the effects of myosin rod phosphorylation on cardiac contractile function, it has been reported that changes in phosphorylation of the myosin tail domain in smooth muscles affect its intermolecular interactions within the thick filament (e.g., coiled-coil assembly) and elicit conformational rearrangements that modulate contraction (37,38). It has also been reported that the myosin rod mediates specific interactions with the head region, which are important for maintaining the inactive state of vertebrate smooth muscle myosin (39). Therefore, there is a possibility that the signal of hypophosphorylation of the rod domain may be transmitted to the myosin head domain and contribute to the acceleration of CB kinetics as seen in mutant fibers. Because adult murine ventricles predominantly express the fast aMHC isoform rather than slow b-MHC isoform (40–43), it is not likely that myosin isoform switching takes place. We infer from these observations that a Ca2þ-sensitizing mutation in TnC causes a shift in myosin rod phosphorylation profile. Identifying the constitutively phosphorylated residues of sarcomeric proteins in the heart is essential for understanding the regulation of contraction. In our studies, residues Y724, S740, and T925 of a-MHC were found phosphorylated (in five to six out of six samples, Table 1) with the MS analysis, which may underscore their importance for the basic function of myosin during contraction. It is important to note that these three residues are clustered in the neck region of myosin, which is a region known to interact with the regulatory light chain. Furthermore, to the best of our knowledge, Y724, S740, and T925 have not been identified as phosphorylation sites in a-MHC, suggesting novel phosphorylated residue identifications that could potentially provide a platform for innovative research questions with regards to myosin’s function. We recognize, however, that not all of these phosphorylation sites may impact the function of cardiomyocytes or the CB kinetics. Since the TnC A8V mutation is located in a region known to affect Ca2þ binding affinity (18,19), it is reasonable to assume that the increase in Ca2þ sensitivity (Fig. 6, A and B, pCa50) is the primary effect of the mutation (21). This effect is large, corresponding to 3 in Ca2þ sensitivity and DpCa50 ~ 0.5. If we assume that pCa is ~6.0 during diastole, the muscle in WT mice is only 12% active, whereas the muscle in mutant A8V mice is 84% active, based on active tension (Fig. 6 A), i.e., incomplete relaxation for the mutant. The high level of activity during the diastole results in an inefficient usage of energy that may cause a problem. Another problem is a decreased pumping ability. If we assume pCa is ~5.5 during systole, WT muscle is 88% active, whereas A8V muscle is 99% active (Fig. 6 A). Thus, their difference is 8812% ¼ 76% for WT, and 9984% ¼ 15% for A8V. This differential force is responsible for pumping the blood from LV to systemic circulation. This situation becomes worse when the

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Kawai et al. TABLE 1

Phosphorylation Profile of a-MHC

Phospho-Peptides VIQYFASIAAIGDRSK FGKFIRIHFGATGK IHFGATGKLASADIETYLLEKSR NYHIFYQILSNK SAYLMGLNSADLLK GQSVQQVYYSIGALAKSVYEK TTHPHFVR KGFPNRILYGDFR YRILNPAAIPEGQFIDSRK Subtotal for Subfragment 1 (S1) IKPLLKSAETEKEMANMK DALEKSEAR KELEEKMVSLLQEK IQLEAKVKEMTER LTKEKK VNTLTKSKVK TARAKVEK KHADSVAELGEQIDNLQR QLEEKEALISQLTR NALAHALQSSR DTQLQLDDAVHANDDLK VQLLHSQNTSLINQKKK KNMEQTIKDLQHR ELTYQTEEDKKNLMR Subtotal for Rod Total

Phosphorylated Residue(s)

Number of Sites

WT Counts

A8V Counts

Differential Counts

S205 T256 S261 Y284 S393 Y433 T666 Y717 Y724, S740

1 1 1 1 1 1 1 1 2 10 2 1 1 1 1 3 1 1 2 2 1 1 1 3 21 31

1/3 2/3 — — 1/3 — 1/3 1/3 2/3 8/3 2/3 1/3 1/3 3/3 1/3 1/3 1/3 1/3 1/3 1/3 1/3 — 1/3 1/3 16/3 24/3

1/3 2/3 1/3 1/3 — 1/3 — — 3/3 9/3 1/3 1/3 — 3/3 1/3 — — — — — — 1/3 — — 7/3 16/3

— — 1/3 1/3 1/3 1/3 1/3 1/3 1/3 1/3 1/3 — 1/3 — — 1/3 1/3 1/3 1/3 1/3 1/3 1/3 1/3 1/3 9/3 8/3

S844, T847 S868 S881 T925 T995 T1021, T1023, S1025 T1129 S1201 S1301, T1304 S1337, S1338 T1655 S1720 T1791 T1853, Y1854, T1856

MYH6 is cardiac MHC-a gene, UniProt accession number Q02566 (Mus musculus). Column 1 lists the phospho-peptides detected in a run of 3 replicates. In Column 1, bold and underlined letters denote the phosphorylated residues, which are tabulated in column 2. Counts in columns 3 and 4 represent the number of times the phospho-peptide was detected out of 3 biological replicates. Only sites with R75% site probabilities (PTM score) for phosphorylation as determined by proteome discoverer 1.4 (PhosphoRS node) are reported. See the Supporting Material for a comprehensive list of all phosphorylation sites detected with a PTM score of <75%.

cooperativity (nH) is decreased as observed (Fig. 6 C). The system of the body tries to compensate for the impaired function by enhanced adrenergic and other cellular signaling mechanisms. This apparently causes an overworking of the heart, leading to hypophosphorylation in the rod domain of MHC, and eventually results in HCM/RCM. The changes we have observed in CB kinetics at maximal Ca2þ activation (Fig. 5) may be the consequences of the change in the phosphorylation status of myosin. We have observed changes in the following kinetic parameters: k2 (rate constant of the CB detachment step 2, Fig. 1) increased; k2 (the reversal step of k2) decreased (Fig. 5 B); k4 (rate constant of the force-generation step 4) decreased; k4 (reversal step of k4) increased (Fig. 5 D);

and the Pi release step 5 (1/K5) increased (Fig. 5 C) when A8V is compared to WT. Despite the fact that the CB kinetics are affected by A8V mutation, neither the actomyosin interface nor series compliance, including that of CBs, is affected by the mutation as judged by rigor stiffness (elastic modulus), which does not differ between A8V and WT (Fig. 2 B). The fact that Ca2þ-activated tension is not affected by the mutation (Fig. 2 A) also implies that the actomyosin interface is unlikely to be affected by this TnC mutation. These insights are reasonable, because the mutation is located at a site distant from the actomyosin interface, and once the system is fully activated by Ca2þ, there appears to be no difference between the mutant and WT. Similarly, the ATP association

FIGURE 9 Schematic representation of compiled phosphorylated residues on a-MHC obtained by LC-MS/MS analysis. Phosphorylated residues on alpha-MHC in WT fibers (A) and TnC-A8V fibers (B). S1 is subfragment 1 of MHC and Rod is subfragment 2þlight meromyosin.

1734 Biophysical Journal 112, 1726–1736, April 25, 2017

CB Kinetics of TnC-A8V Knock-in Mouse

ACKNOWLEDGMENTS This work was supported by the National Heart, Lung and Blood Institute (grants No. HL103840 and No. HL128683 to J.R.P.) and the American Heart Association (grants No. 13GRNT16810043 to M.K. and No. 15GRNT25280004 to J.R.P.). Additional support was from the Florida Heart Research Institute (to J.R.P.).

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constant (K1) is minimally affected by the mutation and not significantly different from WT (Fig. 5 A). This finding suggests that the contour of the ATP binding pocket is not affected by the TnC mutation. In conclusion, we found that the TnC-A8V mutation increases Ca2þ sensitivity of force development and decreases the cooperativity of thin filament activation, which are the primary functional consequences of the mutation in the regulation of the actomyosin interaction. These changes result in less differential force development with Ca2þ activation. However, the overall pumping of the blood by LV is not decreased (21), because the adrenergic and other cellular signaling mechanisms compensate for the lowered force. Consequently, the heart overworks to result in decreased phosphorylation in the myosin rod domain as a secondary effect, resulting in pathogenesis of HCM/RCM. At the same time, some of the elementary steps of the CB cycle are accelerated, presumably due to the decreased phosphorylation level of MHC. SUPPORTING MATERIAL Six tables are available at http://www.biophysj.org/biophysj/supplemental/ S0006-3495(17)30295-3.

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