An internal domain of β-tropomyosin increases myofilament Ca²⁺ sensitivity

Generation of chimeric α/β-TM-3 TG mice.

To generate TG mice where amino acids 175–190 of α-TM were exchanged for β-TM (α/β-TM-3 TG mice), we ligated portions of wild-type, striated muscle-specific α-TM and β-TM cDNAs (18, 19). A spliced overlap extension procedure (9) was used to generate the α/β-TM-3 cDNA construct. This strategy has been used previously to generate chimeric TM expression vectors (10). Nucleotide sequencing and comparisons with the published sequence verified the amplified sequences.

The α/β-TM-3 cDNA was ligated at the 5′-end to the 5.5-kb murine cardiac-specific α-myosin heavy chain (MHC) promoter. The resultant 6.8-kb cDNA construct was endonuclease restricted to release the transgene, purified, and microinjected into male pronuclei of fertilized ova to generate TG mice as previously described (18). TG mice were produced from the FVB/N mouse strain. PCR was used to amplify DNA isolated from ear tissue and identify mice carrying the transgene. Founder mice were identified with the use of 5′- and 3′-primers corresponding to the α-MHC 5′-untranslated region (UTR) and the α-TM coding region, respectively. Lines were established by breeding positive founder TG mice to non-TG (NTG) littermates.

Genomic Southern blot analysis.

To confirm the presence of the transgene and determine the number of copies integrated into each line, genomic DNA from tails was digested overnight with EcoRI, electrophoresis was carried out on 0.8% agarose gels, and samples were transferred to nylon membranes. Southern blots were probed with a ³²P-radiolabeled α-TM 3′-UTR cDNA fragment corresponding to the 3′-end of the transgene and the endogenous gene. Quantification of the transgene was done on an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

RNA and protein analyses.

RNA was isolated from murine hearts with TriReagent (Molecular Research Center, Cincinnati, OH). Total RNA (10 μg) from TG and NTG mouse ventricles was subject to Northern blot analyses with ³²P-radiolabeled cDNA fragments corresponding to the 5′-UTR of α-MHC exon 1 and 2, α-TM 5′-UTR, and GAPDH. TM message levels were analyzed on an ImageQuant PhosphorImager normalized to GAPDH expression levels and are presented as means ± SE.

Whole tissue and myofibrillar proteins were prepared from the ventricular myocardium as previously described (10, 18). Equal amounts of protein (25 μg) were run simultaneously on two 10% SDS-PAGE gels and subject to Coomassie blue staining or Western blot analyses. Filters were probed with a striated muscle TM-specific CH1 antibody (Sigma, St. Louis, MO) and GAPDH (MAb 6C5, Advanced Immunochemical) or sarcomeric actin (MAb 5C5, Sigma) at a 1:5,000 dilution for 2 h at room temperature. The intensity of the bands was quantified using ImageQuant 5.1 software, and results are presented as means ± SE.

Isoelectric focusing (IEF) using slab gel electrophoresis was performed on myofibrillar protein preparations as previously described (20) with some modifications. In brief, 15-cm IEF slab gels were used containing 5% Duracryl (Genomic Solutions), 9.1 mol/l urea, 2% (vol/vol) Triton X-100, and 0.05× Pharmalyte 4.2-4.9 (GE Healthcare). Total myofibrillar protein was dephosphorylated and loaded in a buffer containing 9.2 mol/l urea, 0.02× Pharmalyte 4.2-4.9, and 2% Triton X-100 with 0.025% bromophenol blue. Western blot analysis using the striated muscle TM-specific CH1 antibody was conducted using a 1:5,000 dilution (12). The intensity of the bands was quantified using ImageQuant 5.1 software, and results are presented as means ± SE.

Histological analysis.

Hearts were removed from 2- to 12-mo-old mice and immediately fixed in 10% neutral buffered formalin for 48 h. Hearts were dehydrated through a gradient of alcohols and xylene followed by embedding in paraffin. Sections were cut at 5 μm thickness and stained with either hematoxylin and eosin or with trichrome stain.

Echocardiographic analyses of TG hearts.

Left ventricular (LV) function was assessed by transthoracic echocardiography, which was performed on 5-mo-old mice (TG line 71) using an iE33 Ultrasound System (Phillips) with a 15-MHz probe. After the induction of light general anesthesia, hearts were imaged two dimensionally in a long-axis view at the level of the greatest LV diameter. This view was used to position the M-mode cursor perpendicular to the LV anterior and posterior walls. The LV internal end-diastolic dimension (LVIDd) and LV internal end-systolic dimension (LVIDs) were measured from M-mode recording according to the leading edge method. The LV ejection fraction (EF) was calculated as follows: LVEF (in %) = [(LVIDd)³ − (LVIDs)³]/(LVIDd)³ × 100. LV fractional shortening (FS) was calculated as follows: LVFS (in %) = (LVIDd − LVIDs)/LVIDd × 100. All echocardiographic measurements were averaged from at least three separate cardiac cycles. All data were analyzed using Bonferroni/Dunn statistical analyses and are presented as means ± SE.

Isolated anterograde perfused heart preparation.

Control and TG age-matched mice (TG lines 1 and 71) were anesthetized with 100 mg/kg Nembutal (pentobarbital sodium) and injected with 1.5 units of heparin to prevent intracardiac blood coagulation. Hearts were rapidly excised, and the aorta was cannulated with a 20-gauge needle and connected to a perfusion apparatus (10, 12). Retrograde perfusion with modified Krebs-Henseleit buffer [containing (in mM) 118 NaCl, 2.5 CaCl₂, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.5 Na₂EDTA, 25 NaHCO₃, and 11 glucose] was started to clear the coronary vasculature, and a polyethylene-50 catheter was inserted intraventricularly for pressure measurements. The perfusion fluid, oxygenation, and temperature were constant throughout the experiment.

After a short period of stabilization on retrograde perfusion, the pulmonary vein was cannulated, and perfusion of the heart was switched from retrograde to anterograde as previously described (10, 12). Signals were digitized and analyzed using Biobench software. Heart rate, LV pressure (LVP), and mean coronary perfusion pressure were continuously monitored. The pressure curve was used to calculate the rate of pressure development (+dP/dt) and decline (−dP/dt). Parameters of cardiac performance were compared between the NTG and TG groups with a two-tailed t-test. Experimental values are expressed as means ± SE.

The response of isolated work-performing hearts to changes in afterload and β-adrenergic stimulation were studied using the same hearts. Pressure loading is a short-term functional test without long-term effects on cardiac functional performance and does not affect β-adrenoreceptor-dependent transduction and subsequently functional responsiveness. As such, pressure loading and adrenergic responsiveness are independent tests that are carried out to evaluate possible alterations in cardiac intrinsic regulatory mechanisms (pressure loading) and functional responsiveness to a sympathetic agonist. In our experimental setting, β-adrenergic functional responses were studied after pressure loading measurements followed by a 20-min stabilization period at basal loading conditions. The β-adrenoagonist isoproterenol (Sigma) was added to the Krebs-Henseleit solution in varying concentrations (10⁻¹¹–10⁻⁷ mol/l) and entered the heart using a microperfusion pump (MasterFlex L/S). Functional analyses of cardiac performance were performed as described above.

Curves reflecting afterload dependence were generated by linear regression with Origin software (version 4.0, Microcal Software). For the regression lines, the average y-intercepts and slopes were calculated using only the initial part of the LVP loading curve over a range of cardiac work that could be generated by both experimental groups. Experimental values are expressed as means ± SE.

Fiber bundle preparation and force measurements.

We measured force developed by bundles of detergent-extracted fibers obtained from papillary muscle as previously described (19, 33). Isometric tension was plotted as a function of pCa (−log[Ca²⁺]) and fitted to the Hill equation as previously described (33) by applying nonlinear least-squares regression analysis using Prism software (version 2.0, GraphPad). Isometric tensions measured at submaximally activating Ca²⁺ concentrations were expressed as a percentage of the maximum tension. Half-maximally activating concentrations of Ca²⁺ (pCa₅₀) were determined from individual Hill fits of each pCa-tension relation and then averaged. Data in which measurements were made in the same fiber bundle at different sarcomere lengths were analyzed individually using Student's paired t-test. When three or more groups were analyzed, one-way repeated-measures ANOVA was used with a Newman-Keuls multiple-comparison test. The criterion for statistical significance was set at P < 0.05. Experimental values are expressed as means ± SE.

Bacterial recombinant protein expression.

Recombinant TM was expressed and purified using the Champion pET SUMO Expression System (Invitrogen). Both wild-type α-TM and chimeric α/β-TM-3 cDNA constructs were designed to include a NH₂-terminal Ala-Ser dipeptide to functionally compensate for the lack of acetylation of bacterially expressed TM (15). The coding sequences of the expression plasmids were confirmed by automated DNA sequencing.

Circular dichroism measurements.

Thermal stability measurements were made by following the ellipticity of TM at 222 nm as a function of temperature, beginning at 5°C in 0.5 mol/l NaCl, 10 mmol/l sodium phosphate (pH 7.5), 1 mmol/l EDTA, and 0.5 mmol/l DTT using an Aviv model 215 spectropolarimeter. Data were obtained at 2°C intervals with a protein concentration of 3 μmol/l. The apparent melting temperature and thermodynamic parameters for TM unfolding were calculated based on the assumption that the unfolding could be fit by up to three independent helix-coil transitions with dissociation accompanying the helix-coil transition at the highest temperature, as previously described (3).

Generation of chimeric α/β-TM-3 TG mice.

To elucidate the relation between the putative TM isoform-specific internal TnT-binding region and contractile behavior in striated muscle, we developed a TG mouse model to overexpress a chimeric α/β-TM protein. The chimeric cDNA (α/β-TM-3) encoded the following amino acids: α-TM 1–174, β-TM 175–190, and α-TM 191–284 + 3′-UTR. This chimeric TM sequence was ligated at the 5′-end to the murine α-MHC promoter and the 5′-UTR, which confers cardiac-specific expression (Fig. 1A). A simian virus (SV)40 polyadenylation and termination cassette is located 3′ of the TM cDNA to ensure correct transcript processing. Founder mice were identified by PCR and confirmed by Southern blot analysis (Fig. 1B). Three lines of transgenic mice were produced with one or five copies: line 1, one copy; line 71, five copies, and line 90, one copy.

α/β-TM-3 TM transcript and protein expression in TG mice.

We measured TM expression using Northern blot analysis after RNA isolation from hearts of TG and NTG littermates. RNA from each TG line was isolated and probed with ³²P-labeled sequences from α-MHC 5′-UTR, α-TM 5′-UTR, or GAPDH to assess expression from the transgene, endogenous α-TM, or control RNA, respectively. We used the α-MHC 5′-UTR as a probe of transgene RNA expression because this UTR is present in the construct mRNA but not found in endogenous TM. [Although the α-MHC 5′-UTR is present in α-MHC mRNA, the message size of α-MHC (∼6.5 kb) is readily distinguishable from transgene mRNA (∼1.3 kb).] As shown in Fig. 1, C and D, RNAs from the transgene are expressed in TG lines 1 and 71, and endogenous α-TM is expressed in all hearts. For quantification, TM levels were normalized to GAPDH expression to account for loading differences. PhosphorImager quantitative analysis showed that endogenous α-TM mRNA was expressed in equivalent levels in NTG and TG hearts. In TG hearts, the percentage of endogenous α-TM RNA to α−/β-TM-3 RNA was as follows: 93.5:6.5% for line 1, 67:33% for line 71, and 100:0% for line 90. The varied expression of α/β-TM-3 mRNA correlated with the copy number of the transgene in the different lines except for line 90, where no transgenic RNA was detected despite the presence of one copy of the transgene. We hypothesize that this lack of expression in line 90 is due to the effect of positional integration of the transgene.

To examine the production of exogenous chimeric α/β-TM-3 protein in the hearts of TG mice, we isolated myofibrillar proteins from the hearts of TG and NTG mice. Western blot analyses showed that the amount of TM in total cardiac lysates and myofilament preparations was constant (Fig. 2, A–D). Coomassie blue staining of myofibrillar proteins showed that there were no gross differences in the expression of other cardiac contractile proteins (including actin, TnI, and myosin light chains 1 and 2) between TG and NTG mice (data not shown). Under these conditions, endogenous and chimeric TM proteins cannot be separated electrophoretically and their quantity cannot be determined. To enhance the sensitivity of the assay, we ran an IEF gel followed by Western blot analysis with the striated muscle TM-specific CH1 antibody (Fig. 2E). Quantification of myofilament TM content showed that lines 1 and 71 expressed 16% and 57% chimeric α/β-TM-3 transgenic protein, respectively (Fig. 2F). We speculate that the increased levels of chimeric TM protein may be due to the differences in the 5′- and 3′-UTR; the transgene incorporates an α-MHC 5′-UTR and a SV40 poly A 3′-UTR, which may account for the increases in transcription, RNA stability, and/or translation of the transgene transcripts or proteins. Also, this phenomenon has been previously observed with other TM constructs (11).

To negate the possibility that an unexpected mutation or deletion of α/β-TM-3 DNA sequences occurred during transgenesis, we conducted PCR analysis of TG mouse tail DNA. With oligonucleotide primers corresponding to the α-MHC and α-TM 3′-UTR sequences, a PCR fragment was generated, amplified, cloned, and sequenced. The results showed there were no mutations or deletions in the α/β-TM-3 nucleotide sequence (or the corresponding amino acid sequence) in the integrated transgene construct.

Histological analysis.

To determine whether histological or pathological alterations occurred after the expression of the TM transgene, we examined 20 TG mice ranging in age from 2 to 12 mo. At 4.5 mo, nine TG hearts (from lines 1 and 71) and 5 NTG littermate control hearts were isolated, weighed, sectioned, and stained with hematoxylin and eosin or with trichrome. The results showed there were no morphological changes associated with the TG hearts at this age. Additional results demonstrated that at 3, 7, 10, and 12 mo of age, there were no gross cardiac phenotypic alterations in any of the mice, nor any indication of fibrosis, necrosis, inflammation, or cardiomyocyte hypertrophy. For each of these ages, we examined three hearts each from lines 1, 71, and 90 along with littermate NTG controls. Also, there were no significant differences in the percentage of heart weight-to-body weight ratios (in mg·g⁻¹·100) in TG mice at 3 mo or 1 yr of age. At 3 mo of age, the heart weight-to-body weight ratios were 5.6 ± 0.9 versus 4.9 ± 0.8 mg·g⁻¹·100 for TG line 1 versus NTG mice, respectively, and 5.8 ± 1.0 versus 4.9 ± 0.9 mg·g⁻¹·100 for TG line 71 versus NTG mice, respectively. At 1 yr, the heart weight-to-body weight ratios were 6.3 ± 1.4 versus 6.4 ± 0.4 mg·g⁻¹·100 for TG line 1 versus NTG mice, respectively, and 6.3 ± 0.5 versus 6.4 ± 0.4 mg·g⁻¹·100 for TG line 71 versus NTG mice, respectively. Thus, expression of the chimeric α/β-TM-3 protein in the hearts of these TG mice does not lead to morphological or pathological changes.

Physiological analyses of chimeric α/β-TM-3 TG mice.

To obtain a better functional understanding of the putative internal TnT-binding domain of TM, we conducted analyses of cardiac performance using echocardiography, isolated working-heart preparations, and skinned fiber bundles. An in vivo physiological assessment of cardiac function was conducted on 5-mo-old TG mice from line 71 by M-mode echocardiographic analyses. The results determined there were no morphological changes in the LV dimensions during systolic or diastolic phases (Fig. 3 and Table 1); these results confirm the findings from the morphological analysis. Additional results showed there were no differences in EF or FS in TG mouse hearts (Table 1). In total, the echocardiographic results confirmed the morphological analyses showing that no pathological alterations occurred with the substitution of the internal region of α-TM amino acids by β-TM sequences.

The work-performing heart model was used to obtain an ex vivo assessment of cardiac performance. These measurements were conducted on TG animals from line 71 with confirmation of the results in mice from line 1. At 4 mo of age, TG hearts were subjected to an anterograde-perfused heart preparation and showed significant changes in the rates of contraction and relaxation. There was a decrease in +dP/dt for contraction from 3,931 ± 123 mmHg/s in wild-type hearts to 3,307 ± 147mmHg/s in TG hearts (P < 0.05; Table 2). Coupled with the decreased contractility, −dP/dt was also decreased from 3,855 ± 275 mmHg/s in wild-type hearts to 3,055 ± 187 mmHg/s in TG hearts (P < 0.05).

To evaluate the ability of α/β-TM-3 and NTG mice to tolerate increased work, LVP loading functional curves were generated in the hearts. Hearts were loaded with increasing afterload (mean aortic pressure), resulting in varied cardiac minute work. The plots of cardiac work versus the rates of pressure development in TG and NTG hearts are shown in Fig. 4. The slopes of the regression curves were not significantly different, indicating that the maximal +dP/dt and −dP/dt were sensitive to afterload-dependent regulation to an extent similar to those of NTG hearts. Although TG hearts displayed a positive response to length-dependent regulation, they did not attain the levels of cardiac performance seen in NTG hearts.

Response to β-adrenergic stimulation.

Because there was a significant decrease in the rates of both contraction and relaxation, we checked the isoproterenol response of these TG hearts to see whether this may be due to blunting of the β-adrenergic response. The reduced contractile and lusitropic performance by α/β-TM-3 TG hearts was assessed during stimulation with isoproterenol, a β-adrenergic agonist that augments muscle contraction and relaxation by cAMP/PKA-dependent phosphorylation by increasing the rate of Ca²⁺ cycling (Fig. 5). TG hearts responded to increased concentrations of isoproterenol by attaining control rates of contraction and relaxation at intermediate concentrations of isoproterenol.

Cardiac diastolic function can also be assessed by measuring the time constant for isovolumic relaxation (τ). Measurements of diastolic function during the isovolumic stages of relaxation have been used to reflect active or intrinsic relaxation properties of the heart independent of changes in volume (4). τ is independent of peak aortic pressure and is a reliable measure of early diastolic function. Values for τ were derived from the work-performing heart preparations when the hearts were subjected to isoproterenol (10⁻⁷–10⁻¹⁰ M). The results showed there were no significant differences between NTG and TG values in the range of 10⁻¹⁰–10⁻⁸ M (10⁻¹⁰ M: 35.6 ± 0.8 vs. 40.2 ± 1.7 for NTG vs. TG hearts; 10⁻⁹ M: 39.0 ± 7.5 vs. 31.5 ± 1.4 for NTG vs. TG hearts; and 10⁻⁸ M: 30.8 ± 3.7 vs. 37.3 ± 3.5 for NTG vs. TG hearts). The value for τ was significantly greater with 10⁻⁷ M isoproterenol (24.2 ± 2.4 vs. 39.9 ± 6.8 for NTG vs. TG, P < 0.01). Although there were significant differences between NTG and chimeric α/β-TM-3 mice in +dP/dt and −dP/dt, the τ measurements indicated there may not be differences in isovolumic relaxation except under conditions with high cardiac performance.

Ca²⁺-force measurements in skinned fiber bundles.

To examine the correlation between physiological results from the whole heart and chimeric α/β-TM-3 TG expression at the sarcomere level, a series of experiments was conducted using detergent-extracted (skinned) fiber bundles. These experiments compared the relation between Ca²⁺ and tension developed by myofilaments obtained from control α-TM versus α/β-TM-3 LV fiber bundles of 4-mo-old mice from TG line 71; confirmatory results were also obtained with TG line 1 mice. Measurements were made at both short and long sarcomeric lengths. As shown in Fig. 6A, pCa₅₀ values were significantly different (P < 0.001) at a sarcomere length of 1.9 μm between NTG (pCa₅₀: 5.65 ± 0.02) and TG (pCa₅₀: 5.82 ± 0.02) fiber bundles (n = 12). With a sarcomere length of 2.3 μm, there was also a significant difference (P < 0.001) between pCa₅₀ values from NTG (5.84 ± 0.03) and TG (6.11 ± 0.01) fiber bundles (n = 12). There were no significant differences in the maximum tension that was developed between NTG and TG fibers at sarcomere lengths of either 1.9 or 2.3 μm (data not shown).

To examine whether the β-TM 175–190 amino acid sequences would affect cooperative activation of the thin filament, we controlled the population of strongly bound cross-bridges by varying the MgATP concentration at pCa 9.0 in detergent-treated fiber bundles (Fig. 6B). At relatively high concentrations of MgATP, tension was low, reflecting relaxing conditions and a high population of cross-bridges with bound nucleotide; there were no differences in the percent normalized tension between chimeric α/β-TM-3 and control myofilaments. As the MgATP concentration was lowered, tension increased, even at pCa 9.0, as strongly bound rigor cross-bridges (nucleotide free) activated the thick filament. Maximum tension was a function of the number of cycling and rigor cross-bridges as well as the relative activation of the thin filament. As shown in Fig. 6B, at pMgATP concentrations of >5.0, chimeric α/β-TM-3 myofilaments developed significantly more tension than myofilaments containing only α-TM. Assuming that the number of rigor cross-bridges is the same in both preparations, our results indicate that switching the putative internal TnT-binding region from α- to β-TM increased the ability of strong cross-bridge binding to activate the thin filament.

Analysis of the thermal stability of TM isoforms.

To investigate whether the increased Ca²⁺ sensitivity and associated physiological alterations observed in α/β-TM-3 TG mice is due to the effect of altered protein structure, we used circular dichroism to study the thermal stability of this chimeric TM compared with α-TM and β-TM proteins. These bacterially expressed TMs have a NH₂-terminal Ala-Ser tag to simulate acetylation and promote polymerization (14, 15). Thermal stability measurements were made following the ellipticity of TMs at 222 nm as a function of temperature. The results showed that homodimers of α-TM and α/β-TM-3 have similar thermal denaturation curves, which indicates that the substituted β-TM amino acid 175–190 region does not induce destabilization (Fig. 7). Also, the results demonstrated that β-TM exhibits a downward shift at intermediate temperatures compared with either α-TM or α/β-TM-3 proteins (Fig. 7). This result implies that as a TM homodimer, the exchanged β-TM domain may not dramatically influence the flexibility of the α/β-TM-3 protein from its α-TM backbone (31).