Electronic Supplementary Material
Journal of Muscle Research and Cell Motility
L71F mutation in rat cardiac Troponin T augments crossbridge recruitment and detachment dynamics against α-myosin heavy chain, but not against β-myosin heavy chain
Sherif M. Reda, Sampath K. Gollapudi, and Murali Chandra*
Department of Integrative Physiology and Neuroscience (IPN), Washington State University, Pullman, WA, USA
*Address correspondence to: Murali Chandra, Ph.D, PO Box 647620, 251 Veterinary and Biomedical Research Building, Department of IPN, Washington State University, Pullman, WA-99164. Tel: +1 (509) 335-7561; Fax: +1 (509) 335-4650; Email:
SUPPORTING MATERIALS & METHODS
Propylthiouracil(PTU) treatment: PTU was administered in both drinking water (0.2 g L-1) and solid feed (0.15% PTU, Harlan Laboratories, Madison, WI) for approximately 5 weeks. PTU silences the α-myosin heavy chain (MHC) promoter, leading to activation of the β-MHC promoter in the cardiac ventricle (Chizzonite and Zak 1984). After 5 weeks of treatment with PTU, there was a complete switch of MHC isoform from α- to β-MHC in the ventricles (Michael and Chandra 2016; Michael et al. 2014). Previous studies have shown that this switch to β-MHC occurs without any alterations in thick or thin filament proteins, their stoichiometry, or phosphorylation status(Fitzsimons et al. 1998; Herron et al. 2001; Krenz et al. 2007; Locher et al. 2009; Metzger et al. 1999).
Detergent-skinned cardiac muscle fibers: Detergent-skinned cardiac muscle fibers were prepared as described previously (Chandra et al. 2006; Tschirgi et al. 2006). Hearts were quickly removed from deeply anesthetized (isofluorane) rats and immersed in an ice-cold high relaxing (HR) solution containing (in mM): 20 2,3-butanedione monoxime (BDM), 50 N,N-bis (2-hydroxyethyl)-2-amino-ethane-sulfonic acid (BES), 20 EGTA, 6.29 MgCl2, 6.09 Na2ATP, 30.83 potassium propionate, 10 sodium azide, 1.0 DTT, and 4 benzamidine-HCl. The pH of the HR solution was adjusted to 7.0 with KOH. HR also included protease inhibitors (in μM: 5 bestatin, 2 E-64, 10 leupeptin, 1 pepstatin, and 200 PMSF). Left ventricular papillary muscle bundles were dissected into smaller muscle fibers of ~0.15 mm in width and 2-2.5 mm in length in ice-cold HR solution. Muscle fibers were detergent-skinned overnight at 4°C in HR solution containing 1% Triton X-100.
Purification of recombinant rat cardiac Tn subunits: Previously described methods for purifying recombinant rat cardiac TnT (TnTWT and TnTL71F), rat cardiac TnI, and rat cardiac TnC were used(Chandra et al. 1999; Gollapudi and Chandra 2012; Mamidi et al. 2013a). Both TnTWT and TnTL71Fweretagged with the c-myc epitope at the N-terminus. The expression vector was pSBETa and the expression cell was BL21 DE (STAR). In brief, TnTWT and TnTL71F were purified by ion-exchange chromatography on a DEAE-fast sepharose column (GE Healthcare Biosciences, Pittsburgh, PA) (Chandra et al. 1999; Chandra et al. 2006). TnIwas purified by ion exchange chromatography on a CM sepharose column (Gollapudi and Chandra 2012; Guo et al. 1994; Mamidi et al. 2013a). TnCwas purified by ion exchange chromatography on a DEAE-fast sepharose column (Gollapudi and Chandra 2012; Mamidi et al. 2013a; Pan and Johnson 1996), followed by phenyl sepharose column. Samples from eluted fractions were run on a 12.5% SDS gel to determine their purity. Pure protein fractions were pooled and dialyzed extensively against deionized water containing 15 mM β-mercaptoethanol, lyophilized, and stored at -80°C.
Western blot: Cardiac muscle fibers were solubilized in 2.5% SDS solution (10 µl/fiber) and an equal volume of protein gel loading dye (125 mM Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 0.01% bromophenol blue, and 50 mM β-mercaptoethanol) was added. Proteins were separated on an 8% SDS gel and transferred onto a polyvinylidenedifluoride membrane for Western blot analysis using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories Inc., Hercules, CA). The incorporation of TnTWT or TnTL71F was assessed using a monoclonal anti-TnT primary antibody (M401134, Fitzgerald Industries Int, Concord, MA), followed by a HRP-labeled anti-mouse secondary antibody (RPN 2132, Amersham Biosciences, Piscataway, NJ). Densitometric analysis was performed using the Image J software (acquired from NIH at:
Steady-state isometric force and ATPase activity: We measured steady-state isometric force and ATPase activity, as described previously (Chandra et al. 2007; de Tombe and Stienen 1995; Stienen et al. 1995). T-shaped aluminum clips were used to attach the muscle fiber between a motor arm (322C, Aurora Scientific Inc., Ontario, Canada) and a force transducer (AE 801, Sensor One Technologies Corp., Sausalito, CA). The sarcomere length (SL) of the muscle fibers was set to 2.3 µm under relaxing conditions (Chandra et al. 2007; de Tombe and Stienen 1995; Stienen et al. 1995). The SL was readjusted to 2.3 µm after two cycles of maximal activation and relaxation, if necessary. Detergent-skinned muscle fibers were exposed to various Ca2+ solutions in a constantly-stirred chamber. The concentration of Ca2+ in the test solutions ranged from pCa 4.3 to 9.0 (pCa = -log of [Ca2+]free). The composition of pCa solutions was calculated using methods described previously (Fabiato and Fabiato 1979). The composition of the maximal Ca2+ activating solution (pCa 4.3) was: (in mM) 31 potassium propionate, 5.95 Na2ATP, 6.61 MgCl2, 10 EGTA, 10.11 CaCl2, 50 BES, 5 sodium azide, and 10 phosphoenol pyruvate (PEP). The relaxing solution (pCa 9.0) contained: (in mM) 51.14 potassium propionate, 5.83 Na2ATP, 6.87 MgCl2, 10 EGTA, 0.024 CaCl2, 50 BES, 5 NaN3, and 10 PEP. The activating and relaxing solutions also contained 0.5 mg/ml pyruvate kinase (500 U/mg), 0.05 mg/ml lactate dehydrogenase (870 U/mg), 20 µM diadenosine pentaphosphate, 10 µM oligomycin, and a cocktail of protease inhibitors. The pH and ionic strength of maximal activating and relaxing solutions were adjusted to 7.0 and 180 mM, respectively. All measurements were made at 20°C.
We measured steady-state isometric ATPase activity using a protocol described previously (de Tombe and Stienen 1995; Gollapudi et al. 2012; Kirk et al. 2009; Rodgers et al. 2009; Stienen et al. 1995). Briefly, near-UV light (340 nm) was projected through the muscle chamber, then split (50:50) via a beam splitter and detected at 340 nm (sensitive to changes in NADH) and at 400 nm (insensitive to changes in NADH). ATPase activity was measured as follows: ATP regeneration from ADP was coupled to the breakdown of PEP to pyruvate and ATP catalyzed by pyruvate kinase, which was linked to the synthesis of lactate catalyzed by lactate dehydrogenase. The breakdown of NADH was proportional to the ATP consumption and was measured by changes in UV absorbance at 340 nm. The signal for NADH was calibrated by multiple injections of 250 pmol of ADP.
Measurement of rate of tension redevelopment, ktr: ktr was estimated at pCa 4.3 using a modification to the large slack/restretch maneuver originally described by Brenner and Eisenberg (Brenner and Eisenberg 1986). The modification was described in our earlier works (Ford and Chandra 2013; Gollapudi et al. 2012; Michael et al. 2014). Soon after reaching the steady-state isometric force, the motor arm was commanded to rapidly slacken the muscle fiber by 10% of the ML using a high-speed length-control device (322C, Aurora Scientific Inc., Ontario, Canada). After a brief shortening period of 25 ms at the decreased ML, the motor arm was commanded to rapidly (within 0.5 ms) swing past the original set point by a 10% stretch. The 10% stretch was applied to disengage any remaining bound XBs. A large release-restretch length transientimposed on a muscle fiber and the corresponding force response are shown in Fig. S1a and S1b, respectively. ktr was determined by fitting the following mono-exponential equation to the rising phase of the force response.
Where F is the force at time t, Fss is the steady-state isometric force, and Fres is the residual force from which force starts to redevelop.
Muscle fiber mechano-dynamics: Upon attainment of steady-state force at pCa 4.3, the muscle fiber was subjected various amplitude stretche/release perturbations (in the order of ± 0.5%, ± 1.0%, ± 1.5%, and ± 2.0% of the initial muscle length (ML)) and the resulting force responses were recorded, as described previously (Ford et al. 2010). A nonlinear recruitment distortion (NLRD) model was fit to the elicited force responses to estimate the following model parameters (Ford et al. 2010): the magnitude of the instantaneous muscle fiber stiffness brought about by a sudden change in ML (ED); the rate of dissipation of strain within bound XBs(c); the parameter describing the XB strain-mediated negative impact on other force-bearing XBs (γ); the rate by which new XBs are recruited into the force-bearing state (b). Below, we describe the relationship between model parameters and physiological phenomena.
ED: Phase 1: a sudden increase in ML (Fig. S2a) leads to an instantaneous increase in force, from Fss to F1(Fig. S2b). This rise in force results from the distortion of elastic elements in the strongly-bound XBs. Therefore, an increase in F1suggests an increase in the number of strongly-bound XBs. Conversely, a decrease in F1suggests a decrease in the number of strongly-bound XBs. Because ED is estimated as the slope of the relationship (F1-Fss) and change in muscle length (L), it is an approximate measure of the number of strongly-bound XBs (Ford et al. 2010).
c: Phase 2: force decays rapidly to a minimum (nadir; Fig. S2b), as the muscle fiber is held at the increased ML (Fig. S2a). Force decays rapidly because strained XBs detach and equilibrate into a non-force bearing state. The dynamic rate at which the force decays (c) is an index of XB detachment rate, g (Campbell et al. 2004; Ford et al. 2010).
γ:When the negative effect of strained XBs on the state of other force-bearing XBs is more pronounced, the magnitude of force decline is greater; that is, nadir is more pronounced and γ is augmented. Negative effect of strained XBs on other force-bearing XBs is transmitted along the thin filament via nonlinear (cooperative) effects because XBs do not interact directly with other XBs. Therefore, γ represents the allosteric/cooperative mechanisms by which strained XBs negatively impact other force-bearing XBs.
b: Phase 3: immediately after reaching a minimum (nadir), force rises gradually with a characteristic dynamic rate, b (Fig. S2b). This rise in force is due torecruitment of additional XBs into force-bearing state in response to an increase in ML.
Data Analysis: Steady-state and dynamic contractile parameters were analyzed using a two-way analysis of variance (ANOVA); one factor in this analysis was TnT (TnTWT and TnTL71F) and the other factor was MHC (α-MHC and β-MHC). When the interaction effect was significant, it suggested that the effect of TnT on a given contractile parameter was dissimilar in fibers containing α- versus β-MHC. When the interaction effect was not significant, we interpreted the main effect of TnT. To probe the cause for a significant interaction or a main effect, post-hoc multiple comparisons were made using Fisher’s uncorrected Least Significant Differences (LSD) method. Before analyzing the data, we verified that all NLRD model-derived parameters followed a normal distribution pattern within each group and that the variance in the parameter was uniform among groups. Measurements made from several muscle fibers in each group were averaged and presented as mean ± SEM. The criterion for statistical significance was set at P<0.05.
SUPPORTING RESULTS
TnTL71F-mediated impact on ED in α- and β-MHC fibers: Muscle length-perturbation experiments were performed to determine if EDcorroborated maximal tension (Fig. 1) in α- and β-MHC fibers. We have previously shown that EDis an approximation of the number of strongly-bound XBs (Ford et al. 2010), and that ED is positively correlated to maximal tension (Chandra et al. 2015; Gollapudi et al. 2015; Michael et al. 2014). ED was estimated as the slope of the linear relationship between changes in the instantaneous forces, ∆F, and the imposed ML changes, ∆L (Fig. S4a). Two-way ANOVA did not reveal a significant interaction effect (P=0.23) on ED, although, the main effect of TnT was significant (P<0.001). Post-hoc analysis revealed that TnTL71F significantly decreased ED by 27% in α-MHC fibers (P<0.001; Fig. S4b) and by 13% in β-MHC fibers (P<0.05; Fig S4b). Thus, EDcorroborated our observations on maximal tension; this substantiated that greater attenuation of maximal tension in α-MHC+TnTL71F fibers was due to greater decrease in the number of force-bearing XBs.
TnTL71F -mediated impact on ktr in α- and β-MHC fibers: To support our observations on the NLRD model parameter b (Fig. 3a), we also analyzed ktr, which is another index of XB turnover rate. Previous studies have shown correlation between b and ktr(Campbell et al. 2004). Two-way ANOVA revealed a significant interaction effect on ktr (P<0.05), suggesting that the TnTL71F-mediated effect was differently modulated in α- and β-MHC fibers. Post-hoc tests revealed that TnTL71F increased ktrby 15% in α-MHC fibers (P<0.001; Fig. S5a) but showed no effect on ktrin β-MHC fibers (P=0.71; Fig. S5a). Thus,ktrcorroborated our findings in b.
TnTL71F-mediated impact on tension cost in α- and β-MHC fibers: To support our observations on the NLRD model parameter c (Fig. 3b),we also assessed tension cost. Tension cost is strongly correlated to c and both are valuable indices of XB detachment rate, g(Campbell et al. 2004). Tension cost was estimated as the slope of the linear relationship between tension and ATPase activity at various pCa (Fig. S6a). Two-way ANOVA revealed a significant interaction effect on tension cost (P<0.05), suggesting that TnTL71F-mediated effect on tension cost was differently modulated by α- and β-MHC. Post-hoc analysis showed that TnTL71F increased tension cost by 17% in α-MHC fibers (P<0.01; Fig. S6b), but revealed no effect in β-MHC fibers (P=0.81; Fig. S6b). Our observations on tension cost corroboratec, suggesting that TnTL71F augments g in α-MHC fibers but not in β-MHC fibers.
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