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Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics

¹Cardiovascular Research Institute and Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M University System Health Science Center; and ²Department of Biophysics and Biochemistry, Texas A&M University, College Station, Texas; and ³Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital, Cincinnati, Ohio

Submitted 23 June 2005 ; accepted in final form 21 February 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the -TM carboxy terminal overlap end to the -TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of -TM in which the first 275 residues are from -TM and the last nine amino acids are from -TM (-TM9aa). Molecular analyses show that endogenous -TM mRNA and protein are nearly completely replaced with -TM9aa. Working heart preparations data show that the rates of contraction and relaxation are reduced in -TM9aa hearts. Left ventricular pressure and time to peak pressure are also reduced (–12% and –13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to –dP/dt, respectively) is increased, but is not changed significantly. Force-intracellular calcium concentration ([Ca²⁺]_i) measurements from intact papillary fibers demonstrate that -TM9aa TG fibers produce less force per given [Ca²⁺]_i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 Å, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.

force-calcium; thin filament; force-frequency; myofilament activation

Several biochemical studies have indicated that the ends of TM are crucial in determining the affinity of TM for actin (4, 6, 7, 26, 40). Removal of the first nine amino acids of TM results in the loss of actin-binding ability (6) as does removal of the last 11 amino acids (26), yet the latter is not as detrimental as the former (6). Deletion of the ends of TM in TM-troponin (Tn) complexes also lowers actin affinity; however, the cooperativity of interaction is only slightly reduced (4). These findings may indicate that the means of conferring thin filament cooperativity is due to conformational changes in actin (46) and not to the strength of end-to-end interactions in TM. Reports from Hitchcock-Degregori's group (31, 32) have also indicated that end-to-end interactions of TM are crucial for conferring the affinity of TM for actin in the "open" state of muscle contraction, and such interactions may not be necessary in the process of thin filament cooperativity (30).

To address the functional roles of TM in cardiac muscle contraction, we have used transgenesis in which one can exchange the endogenous -TM with other isoforms and mutant TM protein. In a -TM transgenic (TG) mouse model, the data show that exchanging the -TM protein for endogenous -TM protein in cardiac muscle alters sarcomere function (33, 34, 39, 51). TG myocardium expressing -TM protein exhibits a decreased maximum rate of relaxation in the isolated heart preparations (34), increased calcium sensitivity in skinned fiber preparations (39), and both decreased maximal rates of contraction and relaxation in isolated cardiomyocytes (51). We initially hypothesized that the two charge modifications in -TM, Ser229Glu and His276Asn, are responsible for these altered contractile parameters. To test this, we created a mutant form of murine-striated -TM that contains a charge change in both of these regions: -TM Ser229Glu + His276Asn or -TM DM for double mutation (8). Results showed that both maximum and minimum first derivatives of change in left ventricular systolic pressure with respect to time (+dP/dt and –dP/dt, respectively) decreased in isolated working hearts, and calcium sensitivity decreased in skinned fiber preparations (8). Furthermore, recent studies (9) with TG lines having individual mutation, i.e., TM229 (Ser229Glu) or TM276 (His276Asn), have demonstrated that a mutation at the inner-core domain of TM (TM229) primarily affects the rates of contraction, whereas a mutation at the carboxyl end of TM molecule (TM276) affects the rates of relaxation. pCa-tension relationships in skinned fiber preparations indicate decreased calcium sensitivity in -TM229 but no change in -TM276 preparations, suggesting that the function of TM is compartmentalized along its length (9).

There are three amino acid differences between the overlap ends of the last nine amino acids of the - and -TM molecules: His276Asn, Met281Ile, and Ile284Leu. Our aim in the present work was to understand the structural specificity of the end-to-end interactions of - and -TM molecules in modulating the mechanisms of cardiac muscle dynamics. We hypothesized that altering the -TM carboxy terminal overlap end to the -TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we created a mutant striated -TM in the heart in which the last nine amino acids (residues 276–284) have been altered to -TM9aa, its -TM counterpart. Results showed that the mutant TM protein -TM9aa completely replaced the endogenous TM protein in the sarcomeres and decreased the rates of contraction and relaxation in isolated working heart preparations. Force-calcium measurements from intact papillary fibers indicate that -TM9aa TG fibers produce less force per given intracellular calcium ([Ca²⁺]_i) compared with NTG fibers. Protein docking data reveal that changes in end-to-end interactions affect the overall carbon background of the molecule and the ionic and van der Waals interactions between the carboxy and amino terminal ends of TM molecules. These results clearly establish that the carboxy and amino terminal overlap regions of the TM molecule play a critical role in modulating the thin filament activation processes and thus affect cardiac muscle dynamics.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Generation of TG mice. Site-directed mutagenesis was used to generate mutations in -TM cDNA at codons 276, CAC to AAC; 281, ATG to ATC; and 284, ATT to CTT. The -TM mutant cDNAs were then ligated to clone 26, which contains the cardiac-specific -myosin heavy chain (MHC) promoter (45) and the human growth hormone (hGH) poly(A) signal sequence. Mutations were verified by sequencing.

The procedures for FVB/N TG mice generation have been described previously (8, 9). Briefly, BamH I enzyme was used to release the transgene fragment (7.2 kb) from the pBluescript II vector. The transgene was purified by using electroelution, and the purified DNA was microinjected into male pronuclei for founder mouse generation. These were identified by using PCR as described (48). PCR primers corresponding to nucleotide sequences within the second intron of the MHC promoter and the -TM cDNA were annealed to genomic DNA from ear clips producing a 234-bp fragment in TG mouse tissue. Stable lines were raised by breeding the founder TG mice with NTG cohorts. All experiments using mouse hearts were approved by the Texas A&M University Animal Care Committee.

Genomic Southern blot analysis and copy number. Genomic Southern blot analysis and copy number determination were done as previously described (34). Briefly, BamH I enzyme-digested genomic DNA from mouse tails was run onto a 0.7% agarose gel and then transferred to nitrocellulose membrane. For copy number determination, the digested DNA samples were directly blotted onto a membrane by slot-blot apparatus, and serially diluted TG construct DNA with known copy numbers was also prepared as a standard blot. A ³²P-labeled hGH DNA fragment was used as a probe for hybridization. Signal intensity was quantified by using an Imagequant PhosphorImager (Molecular Dynamics, CA).

S1 nuclease mapping. Total RNA from both NTG and TG mouse hearts was isolated with the use of RNA-Stat 60 (Tel-Test, Friendswood, TX). Quantification of endogenous versus TG transcripts was performed by using the S1 nuclease protection assay as described previously (8, 9). To distinguish -TM9aa transcripts from endogenous -TM mRNAs, a PCR probe that incorporates the second and third exon of -MHC (15 bp) and 262 nucleotides of the -TM coding region was utilized. In S1 nuclease mapping analyses, this probe protects 262 nucleotides of endogenous -TM, whereas a 277-bp fragment is protected for mutant -TM transcripts. A control GAPDH probe was also used for quantitative purposes.

DNA probes for the hybridization reaction were created using PCR. Single-stranded DNA probes used for hybridization were labeled at the 3' end with [-³²P]ATP (Amersham) via T4 kinase (Invitrogen). The sequences for the S1 probes were as follows: 5' TM primer, 5'-GCCCACACCAGAAATGACAGACAG-3'; 3' TM primer, 5'-GAGAAGCTACGTCAGCTTCAGCAT-3'; a GAPDH probe was also prepared via PCR. Hybridized RNA-DNA strands were electrophoresed on 6% polyacrylamide gels (19:1) containing 8.3 M urea. Gels were dried and autoradiographed on Kodak X-Omat AR film. Densitometry analysis of the resulting bands using Multi-Analyst software and Phosphorimaging analyses were conducted to determine RNA content. Quantification was performed by using the volume integration method with a subtraction of the tRNA control lane as a background. Three separate RNA gels were used to quantify the relative levels of transcripts.

Two-dimensional gel electrophoresis and Western blot analysis. Two-dimensional gel electrophoresis was performed according to a modification of the method of O'Farrell (36) and Jagatheesan et al. (19). Modified procedures from Pagani and Solaro (38) were used to isolate myofibrils from the heart with all solutions being supplemented with 1 µg/µl leupeptin and 1 mM phenylmethylsulfonyl fluoride. The concluding washes after Triton X-100 treatment were conducted in the following low-salt solution containing (in mM) 20 KCl, 2 KH₂PO₄, 2 EGTA, and 0.5 PMSF. Protein (25 µg) from this protein suspension was then dissolved and reduced in 300 µl of isoelectric focusing sample (rehydration) buffer containing 8 M deionized urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 2 mM tributyl phosphine, and 0.2% Bio-Lytes (4.7–5.9; Bio-Rad). Each protein sample was then used to rehydrate a 17-cm ReadyStrip (pH 4.7–5.9; Bio-Rad) for 16 h. Isoelectric focusing was then performed in three stages by utilizing the following voltage protocol: 250 V for 15 min (rapid ramp); 10,000 V for 1 h (slow ramp); and 8,000 V for 40,000 V/h (rapid ramp). Focused strips were then treated with equilibration buffer [6 M urea, 2% SDS, 0.375 M Tris·HCl (pH 8.8), and 20% glycerol] containing 2% dithiothreitol (10 min), followed by treatment with equilibration buffer containing 2.5% iodoacetamide (10-min treatment). The strips were then applied to 15% acrylamide gels for SDS-PAGE, followed by transblotting onto nitrocellulose membranes.

Primary antibody incubations were performed with a striated muscle-specific TM antibody CH-1 (Developmental Studies Hybridoma Bank, University of Iowa) that recognizes both the endogenous and mutant forms of TM. Secondary antibody incubations were performed with goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma). Chemiluminescence (SuperSignal West Pico; Pierce) was used as the detection agent. Densitometry on the resulting bands were performed by using Multi-Analyst software. Western blot analysis of myofibrillar proteins followed by quantification was performed three times for each sample, and the resulting means ± SE were calculated.

Physiological measurements of left ventricular function. The isolated working heart preparations have been described (15). Briefly, age-matched male mice (4–6 mo) were euthanized, and the hearts immediately excised. A 20-gauge cannula was tied onto the aortic stump to allow regulation and recording of mean arterial pressure (MAP) and aortic flow (Transonic Flow Probe model T206; Transonic Systems, Ithaca, NY). A polyethylene fluid-filled catheter was inserted through the apex of the left ventricle to record intraventricular pressure. Left ventricular pressure was measured as systolic, diastolic, and end-diastolic pressure using the DigiMed Systems Analyzers BPA-2000, HPA-200, HPA-210, and LPA-200 (Micro-Med, Louisville, KY). The fluid-filled catheter system responded well within experimental requirements without distortion up to a frequency of 600 beats/min. A 20-gauge cannula was tied into the left pulmonary vein to accommodate regulation of venous return recordings (cardiac output). The catheter was completely water jacketed for improved temperature (37.4°C) regulation of the Krebs-Henseleit (KH) solution that was returned to the left side of the heart for anterograde perfusion. Custom-designed software calculated heart rate, MAP, left ventricular pressure, peak systolic pressure, time to peak pressure (TPP), half time of relaxation, ±dP/dt, left atrial pressure, and perfusate temperature. The arterial PO₂ was 650 mmHg, and the PCO₂ was 30 mmHg. The data are expressed as means ± SE. All the experiments were carried out in a blind manner without investigators knowing the genotype of the animals, and after all the experiments were done, the results were grouped according to the genotype. In addition, the heart rates were paced 400–420 beats/min; the difference (5% change) in this range of heart rates does not affect the other physiological parameters significantly.

Force frequency with calcium measurements in intact papillary muscle fibers. Force and calcium measurements were conducted as described (9, 47). Right ventricular papillary muscles were dissected from FVB/N mouse (4–5 mo) hearts in 4°C KH solution containing 20 mmol/l of 2,3-butanedione monoxime. The KH solution consisted of (in mmol/l) 119.0 NaCl, 11.0 glucose, 4.6 KCl, 25.0 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, and 1.8 CaCl_2. The KH solution was equilibrated to pH 7.4 with 95% O₂-5% CO₂. Incisions were made on the valve and a wide ventricular region around the base of the bundle so as not to damage the fiber. The muscle approximated an elliptical cone as it tapered from the right ventricular wall toward the chordae tendinae that were attached to the tricuspid valve. The width of the base was 0.45–0.6 mm, the depth at the base was 0.2–0.3 mm, and the length of the triangle was 0.6–1.2 mm long. The force was normalized per cross-sectional area at the base using approximate cross-sectional area = 0.75 x width x depth (24). All experiments were carried out at 21–22°C.

The intact bundles plus chordae tendinae were mounted by the valve and ventricular region by clips between a force transducer and a voltage-controlled motor positioner within a muscle measurement suite (Scientific Instruments). Stimulation pulse duration was 3.5 ms with an initial rate of 0.5 Hz. The papillary bundle was continuously superfused with KH maintained at room temperature. Stimulation voltage and bundle length were adjusted until maximum force was reached. The fiber bundle was then stimulated at 0.5 Hz for 45 min before executing the experimental protocol. A digital phosphor oscilloscope suite (Tektronix TDS 3014 with IEEE-488 communication module and Wavestar software) measured stimulation frequency, twitch force amplitude, averaged force amplitude within preset time windows, and continuously logged the data into the computer.

The same muscle measurement equipment suite provided all the optics and electronics needed for measuring intracellular calcium with fura-2 dye. Measurements, however, were collected through a different data acquisition suite (National Instruments A/D board and Labview software) with the digital oscilloscope suite providing continuous monitoring. A mercury lamp and filter wheel provided alternating UV pulses of 340 and 380 nm at 250 Hz with pulse duration of 1.5 ms to illuminate the bundle. A combination of microscope, dichroic mirror, filter, and photomultiplier tube collected the fura-2 fluorescence. The loading solution consisted of KH containing 10 µmol/l fura-2 AM dissolved in DMSO (KH:DMSO was 333:1) and 5.0 g/l cremophor to increase loading efficiency. A loading duration of 1.5 h gave signals of greater than threefold over background fluorescence. The ratio (R) of fluorescence (emission at 510 nm) from 340 nm excitation to fluorescence from 380 nm excitation was calculated after subtracting background fluorescence. Calcium concentration was then calculated using the following equation (14) after an in vitro calibration with 10 µM fura-2 pentapotassium salt: [Ca²⁺]_i = K_d x (R – R_min/R_max – R).

Values for the equations were as follows: K_d, 283 nM, as taken from Backx and Ter Keurs (2); , 12.29, calculated as the ratio of fluorescence from 380 nm excitation at zero calcium to the fluorescence of 380 nm excitation at saturating calcium; R_min, 0.11; and R_max, 2.91. R_max is defined as the 340-to-380 ratio at saturating calcium (10 mmol/l Ca²⁺), whereas R_min is the 340-to-380 ratio in the absence of calcium (0 mM Ca²⁺ + 10 mmol/l EGTA).

Data analyses of force-calcium loop. From the calcium and force values, time to peak Ca²⁺ amplitude and time to peak tension (TPT), decay time for Ca²⁺ amplitude, and relaxation time for force were first calculated. The data were fitted to a sigmoidal-dose response equation with a nonlinear curve fit by using Prism 3.0 version to derive the EC₅₀ value. Second, force-calcium loop data were analyzed at specific points during a twitch cycle, as we have recently described (9, 47). In addition, the maximum rate of isometric tension development (+dF/dt) and rate of relaxation (–dF/dt) were calculated as described previously (47).

Protein docking. The COOH- and NH₂-terminal ends of TM molecule were docked by using the Global Range Molecular Matching (GRAMM) program (20, 21). This geometric-based program performs an exhaustive six-dimensional grid-based search through the relative translation and rotations of the molecules. It identifies the area of the global minimum of intermolecular energy for structures of different accuracy. At a given angular orientation of the molecular structures, all the relative translations of the structures are tested by a correlation procedure using fast Fourier transformation. The search is repeated in all angular orientations. This geometric-based algorithm predicts the structure of complexes formed between the two interacting molecules by using their atomic coordinates, without any prior information as to their binding sites.

The coordinates for both the NH₂-terminal and COOH-terminal TM were retrieved from the Protein Data Bank (PDB) (accession codes 1IC2 and 1KQL, respectively). 1KQL PDB contains residues 253–283 in the A chain and 253–282 in the B chain of the carboxy terminal ends of TM. We built one residue (Ile) on the A chain and two residues (Ser and Ile) on the B chains based on the 7-Å TM structure. 1IC2 PDB contains residues 1–81 in both the A and B chains of the amino terminal end of TM. We performed a low-resolution helix docking procedure using the GRAMM program. In the input parameter file, matching mode, grid step, potential range, angel rotation, and other relevant parameters were modified according to the helix-helix docking protocol (20, 21). In the molecular description input file, we specified the interacting region [NH₂ terminal (1–14) and COOH terminal (271–284)] for both fragments and used the parallel mode of interaction. In the docking procedure, the COOH-terminal fragment was considered as the fixed molecule, and no constraints were placed on the positioning of the NH₂-terminal fragment. GRAMM performed an exhaustive grid-based search for surface complementary molecular fragments. The search is performed at all angular orientations. For each pair of docked molecular structures, GRAMM outputs a list of low-energy matches sorted according to the energy. Lower energy values correspond to a better geometric fit.

The energy minimization was performed for the best-matched coordinates using Discovery-3 program in Insight-II suite (Accelrys). Before minimization hydrogen atoms were added in the coordinates (low-energy conformers), the pH was set to 7.0. In addition, partial charge and potential energy were assigned according to the cff91 force field. After the force field and potential energy were fixed, the model underwent minimization for 2,000 iterative cycles.

To generate the mutant TM, first the IKQL coordinates were read in Xtalview (28). The mutations were introduced in the following residues: His at 276 was mutated to Asn (His276Asn), Met281Ile, and Ile284Leu. The mutated COOH-terminal PDB and normal NH₂-terminal PDB were then used for the docking analyzes as described above.

Statistical analyses. All data are expressed as means ± SE. Statistical analyses were done using a Student's t-test or a two-way ANOVA, where appropriate. P 0.05 was regarded as statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Generation of mutant -TM9aa mice. The TG -TM9aa DNA construct was generated as described in MATERIALS AND METHODS, and the schematic DNA construct is shown in Fig. 1A. Germ-line transmission of the transgene was confirmed by PCR analysis of DNA from mice ear clips. Southern blot analysis was conducted to verify the presence of the 7.2-kb transgene within the genomic DNA of TG mice (data not shown), and the copy number determined in the three established TG mouse lines reveals that 28, 21, and 30 copies of the transgene are present in TM TG mouse lines 9aa-9, 9aa-12, and 9aa-A, respectively. None of the founder mice or their progeny demonstrated any gross phenotypic alterations or reduced viability, and heart weight-to-body weight ratios were not significantly different from those of NTG mice.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Mutant form of -tropomyosin (TM), in which the first 275 residues are from -TM and the last nine amino acids are from -TM (-TM9aa), construct, and mRNA expression. A: schematic representation of transgenic (TG) construct. Mutant TM (His276Asn + Met281Ile + Ile284Leu) was linked to the -myosin heavy chain (MHC) promoter (45) and used to generate multiple lines of TG mice. Human growth hormone polyadenylation site (hGH poly A) was placed downstream of TM cDNA. Black boxes denote the exons derived from -MHC that encode the 5'-untranslated region. Construct was released using BamH I enzyme. B: RNA expression in TG mice. Total cardiac RNA (20 µg) was hybridized to 3'-radioactively labeled DNA probes specific for either TM (see small diagram below RNA gel) or GAPDH. TM probe is 320 bp in length and yields fragments of 277 bp for -TM TG and 262 bp for endogenous TM RNA after S1 nuclease treatment. Electrophoresed samples shown here contained both mutant and endogenous bands for -TM 9aa samples and an endogenous band only for nontransgenic (NTG) hearts. tRNA was used as a negative control. WT, wild-type; MWM, molecular weight marker. C: quantification of RNA. NTG and -TM 9aa RNA levels were quantified on a PhosphorImager and via densitometry using Bio-Rad Multi-Analyst software. Percentages of mutant transcripts in TG lines 9aa-9 and 9aa-A are significantly different from line 9aa-12 (P < 0.05; n = 3 experiments); however, the relative levels of total TM transcript in all TG lines are not significantly different from NTG group (**P > 0.05; n = 3 experiments).

Replacement of -TM with -TM9aa protein in hearts of TG mice.

Protein analyses of myofibrillar protein fractions from both -TM9aa TG and NTG hearts were accomplished by using two-dimensional gel electrophoresis. Initial experiments using SDS-PAGE either with (3.4 M or 8 M) or without urea (44) could not resolve endogenous -TM from -TM9aa protein (data not shown); thus two-dimensional gels were utilized. Results show that the -TM protein spot from NTG heart samples (Fig. 2A) falls near its isoelectric point (pI) value of 5.1 and that the mutant -TM9aa protein spot (Fig. 2, B–D) shifts toward a more acidic pI (i.e., closer to 4.7) due to the loss of a basic residue at amino acid 276 as we have shown in our previous studies (9). The spot corresponding to endogenous protein was seen in the TG line 9aa-12 sample (Fig. 2D) but not in the TG lines 9aa-A and 9aa-9 samples, even after prolonged film exposure (data not shown). We surmise that replacement of endogenous -TM with -TM 9aa-A and 9aa-9 is nearly complete in the -TM9aa mouse hearts, although we cannot rule out that a very small percentage of endogenous -TM is still present. Quantitative determination showed that 100% mutant TM protein is present in TG -TM 9aa-A and -TM 9aa-9 samples, whereas 80% mutant TM protein is present in the -TM 9aa-12 TG line, which is statistically significant from the TG lines 9aa-A and 9aa-9 (P < 0.001; n = 3) that corroborates well with the mRNA data. Although the differences in the transcript levels between these TG lines are smaller (but significant), the protein levels differ by 20% in the 9aa-12 TG line compared with the other two TG lines. These data indicate that the translation is not exactly proportional to the amount of available transcripts; this might be due to a posttranscriptional and/or translational control mechanism that exists between the transgenic and endogenous TM transcripts to maintain the total TM protein in the sarcomere, which might be dependent on the relative levels of mutant and endogenous TM message levels.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Two-dimensional gel analyses of NTG and TG -TM 9aa myofibrillar proteins. Total myofibrillar proteins from both NTG (A) and TG -TM 9aa (B, C, and D, representing TG lines 9aaA, -9, and -12, respectively) hearts were subjected to reduced-reduced two-dimensional electrophoresis and transferred to nitrocellulose membranes, which were then probed with CH-1, a striated muscle-specific TM antibody. Isoelectric point (pI) values (4.7–5.9) are indicated (A–D, top). Notice in the TG samples the shift toward a more acidic pI (4.7) and the lack of endogenous -TM spot (B and C), whereas TG sample (D) has two spots. Quantification analyses show that relative levels of mutant protein present in TG line 9aa-12 (80 ± 1.8%) are significantly different from TG lines 9aa-9 and -A (100%).

-TM9aa hearts are hypodynamic. To determine the function of -TM9aa TG hearts apart from neurohumoral input, the isolated working heart technique was utilized in age-matched, male NTG and -TM9aa TG hearts. Table 1 shows the results of the contractile parameters of NTG and TG lines 9aa-9 and 9aa-12. Results indicate that -TM 9aa-9 hearts exhibit a significantly lower (–29%) rate of ventricular pressure development (+dP/dt) and (–32%) pressure relaxation (–dP/dt) compared with NTG hearts (Table 1). In addition, other cardiac function parameters, left ventricular pressure, and TPP are also significantly reduced (–12% and –13%, respectively) (Table 1). Furthermore, the ratio of +dP/dt to –dP/dt is significantly increased in -TM9aa hearts. To further verify these alterations in the TG mouse heart, we analyzed the TG line 9aa-12 that expresses 80% of the mutant TM protein. Results show that TG 9aa-12 hearts also exhibit significantly slower +dP/dt and –dP/dt compared with NTG hearts (Table 1); however, these effects are smaller compared with TG 9aa-9 hearts that express 100% of the mutant TM protein. These data indicate that the resultant phenotype due to the effects of mutant TM protein depends on the amount present in the cardiac muscle, which correlates well with our previous studies with -TM mouse hearts (33, 34).

View this table: [in this window] [in a new window]   Table 1. Contractile parameters of NTG and TG -TM 9aa mouse hearts

Myofilament activation mechanisms are altered in -TM9aa fibers.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Ca2+ transients and tension. Traces of the Ca2+ amplitude (A) and tension (B) for NTG and TG fibers are shown. Time to peak Ca2+ (C) amplitude and Ca2+ decay time (E), and time to peak tension (TPT; D) and relaxation (F) time are the times representing EC50 values. Data are presented as means ± SE. *P < 0.05, paired t-test.

Figure 4A shows typical force-calcium loops for NTG and -TM9aa TG papillary fibers. Note the positive force-frequency relationship in both groups. Figure 4B shows the loop with three distinct points labeled as points A, B, or C. Point A represents the point of minimal force/calcium, point B represents maximal [Ca²⁺]_i, and point C represents maximal force. Figure 4, C and D, shows the maximum [Ca²⁺]_i, maximum active force, and maximum active force divided by the change in [Ca²⁺]_i (force/[Ca²⁺]_i) for each of the four stimulation frequencies: 0.5, 1, 2, and 3 Hz. Force/[Ca²⁺]_i is defined as the active force divided by the difference in [Ca²⁺]_i between points A and C. Because force/[Ca²⁺]_i quantifies changes in force per unit calcium, the alteration in this parameter could represent changes in the myofilament activation processes. Results demonstrate that maximum [Ca²⁺]_i and active force decrease in the -TM 9aa-9 TG fibers over the range of frequencies studied (P = 0.0255 and P < 0.0001, respectively), although they both decrease more dramatically at the higher frequencies (Fig. 4C). Furthermore, a significant decrease (P = 0.0002) in force/[Ca²⁺]_i in the -TM 9aa-9 TG fibers indicates, the myofilament activation mechanisms are altered. Figure 4D shows that -TM 9aa-12 TG fibers also exhibit a significant decrease in the maximum [Ca²⁺]_i, active force, and force/[Ca²⁺]_i compared with NTG fibers. However, these values are not significantly different among the two TG lines (maximum [Ca²⁺]_i at 3.0 Hz for 9aa-9 = 410 ± 15 nM and for 9aa-12 = 430 ± 12 nM; and maximum active force at 3.0 Hz is 13.9 ± 1.1 and 14.0 ± 1.1 mN/mm² for 9aa-9 and 9aa-12, respectively).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Analyses of force-intracellular calcium concentration ([Ca2+]i) loops from both NTG and 9aa TG papillary fibers. A: force-calcium traces of NTG and TG papillary fibers. Both force and [Ca2+]i increase with increasing stimulation frequency (0.5–3 Hz). B: example of force-to-[Ca2+]i ratio (force/[Ca2+]i) loop used in data analysis. Point A, B, and C are described in RESULTS. C and D: maximum [Ca2+]i, maximum active force, and force/[Ca2+]i from both NTG and -TM 9aa-9 (C) and NTG and 9aa-12 (D) fibers. All data were analyzed with two-way ANOVA with Bonferroni-Dunn's post hoc test across the range of frequencies (Freq) shown (0.5–3 Hz).

-TM9aa hearts are not morphologically different from NTG hearts.

Carboxy and amino terminal interactions of TM molecules are altered in -TM9aa protein. To further investigate the effects of alterations in the carboxy terminal end of -TM in -TM9aa, interactions between the COOH-terminal and NH₂-terminal TM molecules were computationally investigated by using the GRAMM program. GRAMM performs an exhaustive six-dimensional search through relative translations and rotations of the molecules. This technique locates the area of the global minimum of intermolecular energy for structures of different accuracy. Figure 5, A,I and A,II, shows an overview of the normal carboxy and amino terminal molecules of TM interactions. The possible interacting residues of the two molecules in this complex that are detected by GRAMM are highlighted (Fig. 5B). The two chains of the carboxy terminal are marked as chains A and B, whereas chains C and D represent the two chains of the amino terminus of TM. Table 2 lists the favorable amino acid contacts at the COOH-terminal and NH₂-terminal TM interface. There are several attractive features of this model. First, the nitrogen atom (N₂) in the imidazole ring of His276B of COOH-terminal residue is positioned to favorably interact with the side chain of Gln9D of the NH₂-terminal fragment. Second, the N- group of Lys7C (NH₂-terminal fragment) is within ion-pairing distance of the carboxylate group of Asp280B (COOH-terminal). In addition, the side chain of Ser283B and one of the carboxyl oxygens of Ile284B are close enough to form an ionic interaction with the N- of Lys12D (NH₂ terminal). Third, the side chain of Glu273B (COOH-terminal) and the N- of Lys5D (NH₂ terminal) permits close approach of the two alpha helixes. Fourth, the side chain of Asp275A (COOH terminal) is positioned in close vicinity to interact with the side chain of Lys6C (NH₂ terminal). In general, basic residues (Lys5, -6, -7, and -12; and Asp2 and -9) in the NH₂ terminal of TM are positioned favorably to form ionic interactions with the charged residues of the COOH terminal of TM.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Docking of the carboxy and amino terminal ends of TMs. All figures were made and rendered using ICM-Browser (see Ref. 1). A,I: ribbon diagram showing structure of both carboxy (red) and amino (violet) terminal ends of TM used for docking studies. The A and B represent the two chains of carboxy terminal end of TM, whereas the C and D represent the two chains of amino terminal end of TM. The numbers of amino acid (aa) residues used are marked. A,II: docking structure of normal carboxy and amino terminal ends of TM. A,III: docking structure of the mutant carboxy end (yellow) with normal amino terminal ends (pink) of TM. A,IV: superimposition of both normal and mutant docking structures of TM molecules. B: interactions between residues that are predicted by Global Range Molecular Modeling (GRAMM) program are highlighted in normal TM molecule structure. C: interactions between residues that are predicted by GRAMM program are highlighted in mutant TM molecule structure.

	DISCUSSION

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The data presented here demonstrate that specific changes in the end-to-end interactions between contiguous TMs, i.e., three amino acid changes at the overlap regions of TMs, alter the structure and function of the thin filament. The amino terminus overlap regions of TM isoforms are highly conserved among vertebrates and thus remain the same for both - and -TM. However, the last coding exon and noncoding untranslated sequences of these isoforms are different. In this study we have created a TG mouse model that overexpresses -TM9aa mutant protein, a mutation that mimics the overlap region of -TM homodimers. In the TG mouse hearts, there is nearly a complete replacement of the endogenous -TM with -TM9aa protein, and the data show that changes in the end-to-end interactions of -TM affect the activation mechanisms of thin filament regulatory strand and modulate the cardiac muscle dynamics.

The structure of the TM overlap region in -TM9aa TG protein is altered (Fig. 5). Normally, the amino terminus of -TM is helical, as seen at the atomic level in NMR and circular dichroism studies (12) and in the crystal structure (3). The structure of the COOH terminus, on the other hand, is unclear. In solution, NMR analysis revealed the following: a coiled-coil helix through residues 269; parallel, linear helixes from residues 270–279; and flexible, nonhelical strands from residues 280–284 (13). The crystal structure, at a resolution of 2.7 Å, indicates that the two -helixes of TM splay apart at residue 263 (25) in TMs that interact in a tail-to-tail manner. We have used the latter structure to predict the effects of altering the carboxy terminus of -TM with its -TM counterpart. Our results show that, in TM, His276 may provide a charge neutralization role or act as a proton donor because its N₂ interacts with the carboxylate of Asp280B and the distance between N₁-nitrogen and Gln9D is 3.63 Å. In addition, N₁ also interacts with the side chain of Asn279B. These interactions tend to favor a constrained coil-coil structure. In the mutated TM structure, Asn276B is not favorably positioned to interact with either with Gln9D or Asn279B. In contrast, it is in close vicinity to Lys12D and may form an ionic interaction with this residue. This difference in interaction is due to the shift in the C- helix of the mutant COOH-terminal fragment. However, if we used TM protein with only the His276Asn mutation in the COOH terminal, the shift in the C- helix is not very significant (data not shown).

The data from the isolated heart preparations and the fiber studies demonstrate that changes in the end-to-end interactions of TM molecule affect the rates of contraction and relaxation that are supported by the presented data. The decreased tension, developed from the -TM9aa TG myofibers (Figs. 3 and 4), is well correlated with the reduced left ventricular (systolic) pressure in these TG hearts (Table 1). Although one would expect that an increased TPP in the working-heart preparations and an increased TPT in the myofilament studies would be associated with the observed decrease in +dP/dt and +dF/dt, respectively, these TG hearts (line 9aa-9) exhibit a decreased TPP and TPT (Table 1 and Fig. 3); because in the TG hearts the pressure and force development are reduced by 8–12% and 20–25%, respectively, it is possible to obtain a decreased TPP or TPT with the slower contractile dynamics. In addition, the ratio of maximum-to-minimum dP/dt is significantly increased in -TM 9aa-9 TG hearts, indicating impaired relaxation. A tendency of increase in the parameter is correlated with this TG line. However, in TG line 9aa-12, the ratio of maximum-to-minimum dP/dt and are not changed significantly. These data indicate that the rate of relaxation is not primarily affected in these TG hearts. Furthermore, the data suggest that the physiological responses of these 9-aa TG hearts are fine-tuned with the concentration of mutant TM protein, and further in vitro biochemical studies with different concentrations of mutant TM proteins should be warranted to address the mechanisms.

However, the decreased TPP and the rates of pressure development and relaxation from the isolated heart preparations data strongly agree with the reduced TPT and the rates of force development and relaxation in the papillary fibers of these TG mice. It has been suggested that the alterations in the end-to-end interactions of TM might alter the affinity of TM for actin in the "open" state of muscle contraction (31, 32). This idea is supported by our data that the maximum tension developed by -TM9aa TG fibers is reduced, suggesting that the number of force-generating cross bridges is decreased. In addition, the force/[Ca²⁺]_i is reduced in the -TM9aa TG fibers (Fig. 4, C and D), indicating that the process of strong cross bridges formation between thick and thin filaments is altered to reduce the force development. These data indicate that fibers containing -TM9aa may not be able to stay locked in the "open" state because of structural perturbations that lead to depression of thin filament activation. This would lead to decreases in the rates of contraction and left ventricular pressure, which is consistent with the isolated heart data. This is also consistent with Campbell's (5) steady-state model that has explained that changes in strong cross-bridge activation modulate the rates of contraction. Taken together, our data demonstrate that the alterations in the end-to-end interactions of TM molecules primarily affect the rate of contraction, and the decrease in rate of relaxation observed in the isolated heart preparations could be the consequence of the decrease in the rate of contraction. Thus the alterations in the end-to-end interactions of TM molecules affect the thin filament activation mechanisms and modulate the cardiac muscle dynamics both at the whole heart and myofilament levels.

It is interesting to see the different functional phenotype between the -TM9aa and -TM276 TG mice (9). In a recent study from Muthuchamy's laboratory (9), it has been shown that charge residue alteration at residue 276 in the TM molecule mainly affects the diastolic function along with increased calcium sensitivity and strong cross-bridge activation. Because -TM9aa and -TM276 TG proteins are similar, except for the additional Met281Ile and Ile284Leu conversions in the -TM9aa protein, the functional differences seen in these two TG groups can be attributed to these amino acid differences in the carboxy terminal end overlap region. Met281 is known to play a role in head-to-tail polymerization of TM molecule due to its polarity and its space-filling methionyl side chain, which occupies the center of the overlap region. Johnson and Smillie (22) have shown that the removal of amino acids 281–284 abolishes the ability of TM to polymerize. Because the Ile284Leu change is a conservative one, alteration of Met281Ile might comparatively play a larger role in modulating the thin filament activation.

Furthermore, the -TM9aa TG mouse data correlate well with that of Jagatheesan et al. (19), who have shown that replacement of the entire ninth carboxy terminal exon of -TM (residues 258–284) with the ninth exon of -TM also severely impairs contractile function in mouse hearts. In their work, the rightward shift in the pCa-force relationship was quite substantial (nearly 0.3 pCa units), and the diminished rates of contraction and relaxation were comparable to our -TM9aa data. The lack of correlation in the calcium sensitivity index may be due to the inclusion of the other two amino acid changes at positions 265 (Leu265Met) and 260 (Leu260Val) in their model. These changes may affect the binding of TnT to the carboxy terminus of TM.

Taken together, the results presented in this study demonstrate that perturbing the end-to-end interactions of TM reduces both +dP/dt and –dP/dt in the -TM9aa TG hearts. In addition, the three amino acid changes present in the carboxy terminus of - and -TM play an essential role in the structure of its end-to-end interactions. The data indicate that the mechanisms of myofilament activation in the TG mouse fibers are altered, leading to depressed contractile and relaxation dynamics of -TM9aa TG mouse hearts. This, along with other recent findings (29, 37) that point mutations in TM cause familial hypertrophic cardiomyopathy or dilated cardiomyopathy, suggests that changes in key residues affect interactions of TM with neighboring molecules, such as actin, troponin, and intramolecularly via head-to-tail interactions. Thus our studies support the idea that the localized flexibility present in the coiled-coil structure of various TM isoforms plays an important role in interacting with neighboring thin filament regulatory proteins and differentially modulating the myofilament activation processes.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grant HL-60758 (to M. Muthuchamy).

	ACKNOWLEDGMENTS

 
We thank Dr. Michael Davis for help in developing data analysis software for intact fiber studies and Drs. Katalin Kiss and James Samuels for technical assistance and use of equipment concerning the two-dimensional gel electrophoresis. We also acknowledge the National Institute of Child Health and Human Development transgenic mouse development facility (contract number: NO1-HD-5-3229) and the University of Alabama at Birmingham for generating the transgenic mice.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Muthuchamy, Cardiovascular Research Institute, 336 Reynolds Medical Bldg., College Station, TX 77843-1114 (e-mail: marim{at}tamu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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