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Am J Physiol Heart Circ Physiol 288: H1147-H1156, 2005. First published October 28, 2004; doi:10.1152/ajpheart.00140.2004
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cTnT1, a cardiac troponin T isoform, decreases myofilament tension and affects the left ventricular pressure waveform

Rashid Nassar,1 Nadia N. Malouf,3 Lan Mao,2 Howard A. Rockman,2 Annette E. Oakeley,1 James R. Frye,3 J. René Herlong,1 Stephen P. Sanders,1 and Page A. W. Anderson1

1Department of Pediatrics and 2Department of Medicine, Duke University, Durham; and 3Department of Pathology, University of North Carolina, Chapel Hill, North Carolina

Submitted 10 February 2004 ; accepted in final form 25 October 2004


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Four isoforms of cardiac troponin T (cTnT), a protein essential for calcium-dependent myocardial force development, are expressed in the human; they differ in charge and length. Their expression is regulated developmentally and is affected by disease states. Human cTnT (hcTnT) isoform effects have been examined in reconstituted myofilaments. In this study, we evaluated the modulatory effects of overexpressing one cTnT isoform on in vitro and in vivo myocardial function. A hcTnT isoform, hcTnT1, expressed during development and in heart disease but not in the normal adult heart, was expressed in transgenic (TG) mice (1–30% of total cTnT). Maximal active tension measured in skinned myocardium decreased as a function of relative hcTnT1 expression. The pCa at half-maximal force development, Hill coefficient, and rate of redevelopment of force did not change significantly with hcTnT1 expression. In vivo maximum rates of rise and fall of left ventricular pressure decreased, and the half-time of isovolumic relaxation increased, with hcTnT1 expression. Substituting total cTnT charge for hcTnT1 expression resulted in similar conclusions. Morphometric analysis and electron microscopy revealed no differences between wild-type (non-TG) and TG myocardium. No differences in isoform expression of tropomyosin, myosin heavy chain, essential and regulatory myosin light chains (MLC), TnI, or in posttranslational modifications of mouse cTnT, cTnI, or regulatory MLC were observed. These results support the hypothesis that cTnT isoform amino-terminal differences affect myofilament function and suggest that hcTnT1 expression levels present during human development and in human heart disease can affect in vivo ventricular function.

thin filaments; transgenic mice; ventricular function; myocardium

STRIATED MUSCLE CONTRACTION is regulated by the troponin (Tn) complex comprising TnI, TnC, and TnT (18, 58). Combinatorial alternative splicing of the 5' region of the cardiac (cTnT), slow skeletal (ssTnT), and fast skeletal TnT (fsTnT) primary transcripts results in multiple cTnT, ssTnT, and fsTnT isoforms that differ in amino-terminal length and charge (1, 5, 19, 23). The four cTnT isoforms (cTnT1–cTnT4), numbered according to electrophoretic mobility, are the products of the variable inclusion of two peptides, the products of exons 4 and 5. Both peptides are included in cTnT1 and are absent in cTnT4, whereas cTnT2 contains the 10-residue peptide and cTnT3 contains a 4- or 5-residue peptide (1, 19, 25). cTnT isoform expression has been shown to correlate with the calcium sensitivity of force development and ATPase activity (29, 35), suggesting that amino-terminal differences among the isoforms are functionally important (but see Ref. 46).

Previous studies of the functional effects of the cTnT isoforms have used proteins in solution, reconstituted myofilaments, and cells in culture (17, 43, 49, 52, 54). Calcium-activated ATPase activity did not differ between myofilaments reconstituted with human TnT (hcTnT) cTnT3 or hcTnT4 (52) and hcTnT1, hcTnT2, hcTnT3, or hcTnT4 (17), but calcium sensitivity of S1-ATPase activity was less in the presence of the longer bovine cTnT isoform than the shorter isoform (50). In a motility assay, velocity of myofilaments constituted with bovine cTnT3 or cTnT4 did not differ in calcium sensitivity, cooperativity, maximal velocity or in maximal force (54). In Tn extracted and reconstituted fibers, cooperativity was greater in the presence of hcTnT1-containing Tn, whereas calcium sensitivity decreased progressively from hcTnT1 to hcTnT4 (17). Expression of the embryonic rat cTnT isoform (similar to hcTnT1, see Fig. 1) in adult cardiac myocytes in culture did not affect calcium sensitivity of force development (43).



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  		Fig. 1. A comparison of the amino-terminal regions, derived by cDNA analysis, of human cardiac troponin T (cTnT1) (1), three mouse cTnT (mcTnT) isoforms (25), and a rat cTnT (24) demonstrating the similarity of the amino-terminal sequences and the similar number of negatively charged residues of the variably expressed peptides.

 
We approached the question by using mouse transgenesis to determine in vivo and in vitro the effects of hcTnT1, which is expressed in the human fetus and in the failing adult human but not in the normal adult human heart (2, 53). The mouse is an ideal model to test the effects of hcTnT1 on heart function because hcTnT1 is longer and more negatively charged than any of the endogenous cTnT isoforms expressed in the adult mouse heart.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animals used in this study were handled according to the animal welfare regulations of Duke University (Durham, NC). The protocol was approved by the Institutional Animal Care and Use Committee.

Transgene Construct

hcTnT1 cDNA, containing the complete coding region, was excised from a Bluescript vector (1) by digestion with XbaI and KpnI and cloned at these sites in pGEM-3Zf(–), allowing SalI restriction sites proximal and distal to the cTnT1 sequence. The hcTnT1 transcript was then cloned into the single SalI site in pGEM-5Z(–) containing an {alpha}-myosin heavy chain (MHC) promoter-simian virus 40 (SV40) poly(A) signal [kindly provided by Dr. J. Robbins (47) and modified by Dr. W. Koch (41)]. The cDNA and its flanking sequences were sequenced to ensure proper orientation and fidelity of the sequence (1). The DNA transcript ({alpha}MHC-hTnT1-SV40) was linearized with SfiI and NotI, purified, and microinjected into the pronucleus of single egg cells from Bl6SJLF1/J mice (Jackson Laboratories) and implanted into pseudopregnant foster mothers (Duke Comprehensive Cancer Center and Transgenic Facility).

Screening for Transgenic Mice

Screening mice by PCR was done using the primers 5'-TCCACATTCTTCAGGATTCTCT-3' and 5'-ATCGGGGATCTTGGGAGGC-3', which amplify the 3' portion of the {alpha}-MHC promoter and the proximal portion of the hcTnT1 coding region, yielding a 560-bp amplicon. Genomic DNA was prepared from tail DNA. Southern blots were prepared by electrophoresing the DNA in 0.7% agarose, transferring it to a nitrocellulose filter, probing the filter with a 32P-labeled SV40 fragment, and exposing X-ray film to the filter.

Generation of Transgenic Mice

The founder mice were bred with C57BL/J6 mice. Three founder lines, F19, F27, and F35, were generated. F1 transgenic (TG) mice from a given founder line were crossbred. The resulting F2s were used in this study. Wild-type [non-TG (NTG)] mice were mice from the three founder lines that did not express hcTnT1.

In Vitro Myocardial Properties

Ventricular bundles [diameter 70–400 (median 180) µm, length 0.5–2.1 (median 0.93) mm] were obtained and detergent skinned as described previously (35). Briefly, the mice were anesthetized (100 mg/kg ketamine-10 mg/kg xylazine ip), and the heart was washed in physiological solution (in some preparations, the initial washing solution contained 25 mmol/l 2,3-butanedione monoxime) (13, 22, 34). The aorta was cannulated, and the heart was perfused for 3 min with physiological solution containing 2 mmol/l EGTA, followed by 3 min of skinning solution (relaxing solution containing 0.5% Triton X-100, see below). The heart was kept on ice, in skinning solution for 30 min, and then in relaxing solution, and a preparation was obtained for study.

Relaxing (pCa 9) and activating (pCa 4.5) solutions contained 10 mmol/l EGTA, 30 mmol/l BES, 30 mmol/l Na+, 12 mmol/l phosphocreatine, 15 U/ml creatine phosphokinase, 2.5 mmol/l pMgATP (3.0 pMg), 2% polyvinylpyrrolidone, and 100 µg/l pepstatin A, with pH 7.10 adjusted with KOH and an ionic strength of 190 mmol/l (adjusted with KCl). The compositions of these solutions were calculated using a computer program based on the study of Fabiato and Fabiato (15). The apparent Ca-EGTA stability constant (log10K = 6.568) included corrections for temperature and ionic strength (21). Solutions of various pCa were prepared by mixing appropriate volumes of pCa 9 and pCa 4.5 solutions (15).

The preparations were placed in the 30-µl cuvette (temperature controlled at 22°C) of a Güth OPT1M system (20). The force transducer had a resonance frequency of 715 Hz. Data were recorded using Digidata 1200 analog-to-digital board and pCLAMP 6 software (Axon Instruments) using a sampling frequency of 100 Hz for the force versus pCa curve and 2 kHz for the rate constant of tension redevelopment (ktr) (see below). One preparation was studied from each mouse. The preparation was stretched to a sarcomere length of 2.2–2.3 µm measured using light diffraction (preparations that did not produce a diffraction pattern were excluded from the analysis). The dimensions of the muscle (free length and diameter) were recorded.

Calcium sensitivity of tension. Test solutions (see above) were applied in steps of increasing [Ca2+], and the developed force was recorded. pCa 9 was reapplied, usually at every third step, to assess resting tension and allow later adjustment for any baseline drift. We used a release-restretch protocol (7) during the acquisition of the force versus pCa curve. Nonlinear regression was used to fit the force (F)-pCa data with the Hill equation, , where nH is the Hill coefficient, Fmax is the maximum force obtained at saturating calcium, and Ca50 is the [Ca2+] required for half Fmax (Fig. 2).



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  		Fig. 2. Force (filled symbols and solid curve) and rate constant of tension redevelopment (ktr; open symbols and dashed curve) normalized to values at saturating [Ca2+] vs. pCa from one transgenic (TG) preparation (F/Fmax and ktr/ktr,max). Curves are Hill equation fits to the data. Force: pCa50 5.78, Hill coefficient (nH) 4.6; ktr: pCa50 5.58, nH 4.7. In both non-TG (NTG) and TG preparations, the calcium sensitivity for force was higher than that for ktr (16) (NTG by 0.095 ± 0.021 pCa units, n = 6; TG by 0.098 ± 0.034 pCa units, n = 9).

 
Rate constant of tension redevelopment. ktr, a measure of fapp+ gapp where fapp and gapp are the apparent rates of cross-bridge attachment and detachment (6, 37), was determined as a function of pCa. The muscle was released by 20% of its free length at a rate of 4 muscle lengths/s, followed, after 25 ms, by a quick restretch to original length (see Fig. 3) (8, 39, 57). ktr was obtained by fitting a single exponential to the postrestretch force vs. time record. ktr,max was ktr at saturating [Ca2+]. We analyzed the effect of hcTnT1 on the ktr versus pCa relation by fitting the Hill equation to the data (Fig. 2). Because sarcomere length was not held constant by feedback control, the values obtained for ktr are likely to underestimate ktr (11, 57).



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  		Fig. 3. Force records as a function of time at different [Ca2+] obtained from the quick release-restretch protocol used for estimating ktr from a skinned TG preparation. Curves (bottom to top) are pCa 5.6: ktr = 6.6 s–1, pCa 5.4: ktr = 10.3 s–1, pCa 5.2: ktr = 11.5 s–1, and pCa 4.75: ktr = 11.9 s–1.

 
In Vivo Ventricular Function

Cardiac catheterization. Cardiac catheterization was performed as reported previously (32, 41, 42). Briefly, mice were anesthetized (100 mg/kg ketamine-2.5 mg/kg xylazine ip) and ventilated (room air, 0.3–0.5 ml; rate 104 breaths/min), and their body temperature maintained at ~37°C. After a bilateral vagotomy, a 1.4-Fr micromanometer catheter (Millar Instruments; Houston, TX) was inserted into the aorta and left ventricle (LV) via the right carotid artery, and isoproterenol was infused through a polyethylene-50 catheter into the jugular vein. LV pressure (LVP) was recorded at baseline and after isoproterenol infusions of 50, 500, and 1,000 pg (41). The data were recorded using data-acquisition software (Sonometrics) and analyzed using pCLAMP software (Axon Instruments). The maximum rates of the rise and fall of LVP (dP/dtmax and dP/dtmin; mean of ~10 consecutive beats) and the half-time of isovolumic relaxation [T1/2; the time required for the pressure at dP/dtmin to drop to half its value (33), mean of 4 consecutive beats] were determined. The heart was excised and weighed. Part of the LV was fixed in 2% glutaraldehyde or 4% paraformaldehyde, and the remainder was frozen in liquid nitrogen or placed in sample buffer.

Echocardiography. Mouse hearts were imaged using an Acuson Sequoia C256 Imaging System (Mountainview, CA) and a 15L8Tx linear transducer at 13 MHz. The mice were anesthetized (45 mg/kg etomadate-50 µg/kg fentanyl ip), and body temperature was maintained at ~37°C. The LV was imaged from the parasternal short-axis view at the level of the papillary muscles (sweep speed 100–200 mm/s). M mode was used to measure LV end-systolic and end-diastolic dimensions (EDD and ESD) and LV posterior wall and ventricular septal thickness (45). Pulsed-wave Doppler spectral tracings of the mitral inflow and LV outflow were obtained from an apical four-chamber view with the sample volume placed at the papillary muscle tips.

Myocardial Structure

LV longitudinal and cross sections were stained with hematoxylin-eosin and Masson’s trichrome. Morphometric data were captured using a Nikon Microphot FXA microscope/Optronics DEI-750 charge-coupled device video camera system and Apple Power Macintosh G3 computer using a Scion CG-7 capture card and analyzed using NIH Image. LV myocardium was processed for electron microscopy (31), and myofibril organization and sarcomere structure were examined.

Identification and Quantitation of Sarcomere Proteins

Myocardial proteins were resolved with SDS-PAGE using a modified Laemmli protocol (3). hcTnT1 expression was examined by loading two to five lanes with different amounts of myocardium. Staining of the gels and Western blots were performed as described previously (2, 26). Two-dimensional (2-D) gels were run using Invitrogen Zoom Strips (pH 4–7) for the first dimension. The cTnT isoforms were identified on Western blots using monoclonal antibody (MAb) 13-11, which recognizes a cTnT-specific epitope conserved across species (26). The relative amounts of the cTnT isoforms were determined, using densitometric analysis, for each mouse as a fraction of total cTnT. The expression of total TnT was compared with the expression of TnI and tropomyosin in TG and NTG mice using Western blots stained with MAb 3350 and Sigma T9283 for TnI and tropomyosin, respectively.

The isoforms of TnI, tropomyosin, myosin light chain (MLC)1, and MLC2 were resolved in 8.5%, 9.5%, and 12.5% acrylamide gels using SDS-PAGE as described previously (3). For cTnI and MLC2, 2-D gels were run using Invitrogen Zoom Strips (pH 3–10 and pH 4–7, respectively) for the first dimension. The myosin heavy chain (MHC) isoforms were resolved after the method of Warren and Greaser (55) and stained with Invitrogen "SimplyBlue Safestain." The Western blots were probed with cTnI-specific MAb 3350 (4); MAb 3309, which recognizes cTnI and ssTnI (4); MAbs 3A8 and 10C6, which are specific for MLC1 and MLC2 isoforms (12); and a MAb (Sigma CH1, T9283) specific for striated muscle tropomyosin.

Statistical Analysis

Except where indicated, values are given as means ± SE. The measures of LV and myofilament function were evaluated as a function of the percent hcTnT1 expression using linear regression, and the results are reported as the change in the variable (slopes ± SE) for an increase in hcTnT1 expression of 10%. Force and stiffness were corrected for cross-sectional area of the preparations by regression. Except as noted, we found no significant effects of age or sex. Statistical analysis was carried out using R software (38) and package NLME (36) for repeated measurements.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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cTnT Isoform Expression in Wild-Type Mouse Myocardium

We examined cTnT isoform expression in C57BL/6 mouse hearts at 15 and 17 days of gestation and in neonatal and adult mice. Western blots probed with MAb 13-11 demonstrated four bands (Fig. 4, A and B), numbered mouse cTnT1–4 (mcTnT1–4) to correspond to the four hcTnT isoforms (2). In the fetal mouse heart, mcTnT1 and mcTnT2 were the most highly expressed isoforms. In the neonate, mcTnT1 and mcTnT2 expression decreased and mcTnT3 and mcTnT4 increased. In the adult mouse heart, only mcTnT3 and mcTnT4 were expressed (Fig. 4A). This pattern of cTnT isoform expression in the mouse heart is consistent with combinatorial alternative splicing of two exons of cTnT cDNA (25) yielding four isoforms, cTnT1, cTnT2, cTnT3, and cTnT4 (Fig. 1); the charges of the peptides encoded by these two exons are –2 and –8 in the mouse and –2 and –7 in the human.



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  		Fig. 4. A: developmental changes in cTnT expression. Four cTnT isoforms were identified in Western blots of mouse myocardium probed with monoclonal antibody (MAb) 13-11, a cTnT-specific MAb (26). The expression of the two largest cTnT isoforms decreased with development, whereas that of the two smallest increased. Lane 1, 15 days of gestation; lane 2, newborn; lane 3, adult. The numbering convention follows that used to identify human cTnT isoforms (2). B: expression of human cTnT (hcTnT1) in TG myocardium. The electrophoretic mobility of hcTnT1 allowed its easy identification in adult mouse TG myocardium. Lane 1, fetal human myocardium (2); lane 2, adult mouse NTG myocardium; lane 3, adult mouse TG myocardium.

 
hcTnT1 Expression in TG Mice

The ranges of hcTnT1 expression in the three founder lines, F19, F35, and F27, were 1–7%, 4–16%, and 14–34% of total cTnT expression (see also Table 1 and Fig. 5). There were no significant difference between NTG and TG mice in total cTnT-to-TnI (P = 0.7) and cTnT-to-tropomyosin (P = 0.4) ratios. In the TG mice, the expression of hcTnT1 correlated negatively with the expression of mcTnT4 but appeared to be independent of the expression of mcTnT3 (Fig. 5, A and B). hcTnT1 was more negatively charged than mcTnT3 or mcTnT4 (Fig. 6). The 2-D electrophoretic profiles were similar, suggesting that there were no differences in posttranslational modifications of mcTnT3 and mcTnT4 (Fig. 7).


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  		Table 1. TG and NTG mouse characteristics

 


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  		Fig. 5. The wide range of hcTnT1 expression in F2 TG mice. The founder lines are identified by symbols: F19, {square}; F35, {triangleup}; and F27, {triangledown}. The relative expression of hcTnT1 in TG mice appeared to have been at the expense of the dominant isoform, mcTnT4 (A), with no effect on the relative expression of mcTnT3 (B).

 


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  		Fig. 6. Western blots of two-dimensional gels probed with MAb 13-11. Left: adult TG mouse myocardium expressing hcTnT1. Middle: newborn NTG myocardium. Right: adult NTG myocardium. hcTnT1 is more negatively charged than the two mouse cTnT isoforms expressed in the adult.

 


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  		Fig. 7. Western blots of one- and two-dimensional gels probed with MAb 13-11. A: adult TG mouse myocardium expressing hcTnT1. B: adult mouse NTG myocardium. mcTnT3 and mcTnT4 resolved into the same patterns in NG and NTG myocardium.

 
Myofilament Proteins

{alpha}-Tropomyosin was the only tropomyosin isoform recognized in NTG and TG hearts (n = 12; Fig. 8). {alpha}-MHC was the only MHC isoform expressed in NTG and TG ventricular myocardium (Fig. 8). cTnI, but not ssTnI (n = 45), and only the ventricular isoforms of MLC1 and MLC2 (n = 4) were expressed in NTG and TG ventricular myocardium (Fig. 8). mcTnT, cTnI, and MLC2 were resolved into similar patterns for NTG and TG myocardium using 2-D gel electrophoresis (Figs. 7 and 8).



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  		Fig. 8. Contractile protein isoform expression in NTG and TG myocardium. A: {alpha}-tropomyosin (Tm), cTnI, and ventricular myosin light chain 1 (MLC1) were expressed in NTG and TG myocardium. There was no evidence of {beta}-tropomyosin, slow skeletal TnI (ssTnI), or atrial MLC1 expression. Western blots were probed with Sigma T-9283, which recognizes striated muscle tropomyosin, MAb 3309, which recognizes cTnI and ssTnI (4), and MAb 3A8, which recognizes atrial and ventricular MLC1 (12). B: myosin heavy chain (MHC) isoforms in the pig atrium ({alpha}-MHC) and ventricle ({beta}-MHC) and mouse NTG and TG ventricular myocardium were resolved and stained with Invitrogen SimplyBlue Safestain. {alpha}-MHC, but not {beta}-MHC, expression was recognized in NTG and TG mouse ventricular myocardium. C and D: Western blots of two-dimensional gels probed with MAb 3350 (4), which recognizes cTnI, and MAb 10C6 (12), which recognizes MLC2, respectively.

 
TG and NTG Mouse Characteristics

Table 1 gives a summary of the characteristics of mice from the three founder lines. TG mice appeared normal and healthy. LV myocardium of NTG and TG mice had normal morphology (Fig. 9), and examination under the electron microscope demonstrated normal sarcomere and myofibril structure. Morphometric examination of LV myocardium (3 sections/mouse) from NTG and TG mice showed no significant difference in the percentage of myocardium made up of myocytes: 98.3 ± 1.3% for NTG (mean ± SD, n = 5 mice) and 96 ± 5.4% for TG (n = 7). Myocyte cross-sectional area (~100 cells/mouse) was also similar: 363 ± 30 µm2 for NTG (mean ± SD, n = 5) and 374 ± 73 µm2 for TG (n = 6).



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  		Fig. 9. Light micrographs of ventricular sections from NTG (A) and TG (B) myocardium stained with Masson’s trichrome demonstrating normal structure. Calibration bar = 100 µm.

 
In Vitro Myofilament Properties

Force. At 0% hcTnT1 expression (NTG), Fmax was 25.9 ± 2.4 (n = 10) vs. 16.5 ± 1.2 mN/mm2 (n = 15) for mice expressing >20% hcTnT1 (P = 0.011, Welch test). With the use of all the data, regression analysis gave a similar result: Fmax decreased significantly with increasing hcTnT1 expression (R2 = 0.65, n = 30, P = 0.019; see Fig. 10A).



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  		Fig. 10. Results from skinned preparations plotted as a function of hcTnT1 expression. A: Fmax was lower in TG than NTG mice (P = 0.01). The solid line is a regression line of Fmax versus %hcTnT1. B: pCa50 did not change significantly with hcTnT1 expression (P = 0.76). C: nH appeared to increase slightly with hcTnT1 expression, but the increase was not statistically significant (P = 0.12). D: ktr at saturating [Ca2+] did not change significantly with hcTnT1 expression (P = 0.15). {circ}, NTG; {square}, F19; {triangleup}, F35; {triangledown}, F27.

 
Sensitivity of the myofilaments to calcium. Mean pCa50 at 0% hcTnT1 was 5.70 ± 0.02; the change in pCa50 with hcTnT1 expression was not significantly different from 0 (–0.004 ± 0.012 pCa units/10% increase in expression, R2 = 0.003, n = 31, P = 0.76; Fig. 10B). Mean nH for NTG mice was 4.02 ± 0.51. The increase in nH with hcTnT1 expression was not significantly different from 0 (0.40 ± 0.25 for a 10% increase in expression, R2 = 0.08, n = 31, P = 0.12; Fig. 10C).

Maximum rate of redevelopment of tension. ktr was sensitive to calcium (57) (Fig. 2), but the ktr versus pCa relation was not significantly affected by hcTnT1 expression: mean pCa for one-half ktr,max for NTG mice was 5.63 ± 0.03 and did not change significantly with hcTnT1 expression (slope –0.01 ± 0.02/10% increase in hcTnT1, R2 = 0.014, n = 15, P = 0.7). At saturating calcium, mean ktr,max was 10.0 ± 0.8 s–1 at 0% hcTnT1 expression. Its increase, by 0.57 ± 0.38 s–1/10% increase in hcTnT1, was not significantly different from 0 (R2 = 0.086, n = 26, P = 0.15; Fig. 10D).

In Vivo Myocardial Function

Echocardiography. Fractional shortening (FS) was calculated from the measured dimensions as FS = (EDD – ESD)/EDD. Mean FS at 0% hcTnT1 expression (NTG) was 27.6 ± 2.2%. FS increased by 2.0 ± 1.5%/10% increase in hcTnT1 expression (Fig. 11); the increase was not significantly different from 0 (R2 = 0.10, n = 19, P = 0.19). The E wave-to-A wave ratio (E/A; Fig. 12) was also not significantly affected by hcTnT1 expression: at 0% hcTnT1, E/A was 1.09 ± 0.12, and it increased by 0.028 ± 0.076 for a 10% increase in hcTnT1 expression (R2 = 0.014, n = 12, P = 0.72). Heart rate (mean ± SD) under etomadate-fentanyl anesthesia was 470 ± 67 beats/min.



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  		Fig. 11. Fractional shortening as a function of hcTnT1 expression obtained from echo data. The slope of the regression line was not significantly different from 0 (R2 = 0.1, P = 0.2, n = 19). {circ}, NTG; {square}, F19; {triangleup}, F35; {triangledown}, F27.

 


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  		Fig. 12. E wave-to-A wave ratio obtained from Doppler measurements did not change significantly as a function of hcTnT1 expression (R2 = 0.01, P = 0.7, n = 12). {circ}, NTG; {square}, F19; {triangleup}, F35; {triangledown}, F27.

 
Physiological measurements. Thirty-six randomly selected F2 mice (248–450 days old) underwent cardiac catheterization; one mouse died during catheter insertion. The remaining mice were distributed as follows: 11 NTG, 5 TG-F19, 8 TG-F35, and 11 TG-F27. Summary statistics for LV and aortic pressures and heart rate are shown in Table 1. Both dP/dtmax and –dP/dtmin decreased and T1/2 increased as a function of hcTnT1. Mean dP/dtmax at 0% hcTnT1 expression (NTG mice) was 9,660 ± 400 mmHg/s; dP/dtmax decreased by 740 ± 300 mmHg/s per 10% increase in hcTnT1 expression (n = 35 mice, P = 0.019; Fig. 13). –dP/dtmin at 0% hcTnT1 was 8,790 ± 300 mmHg/s; it decreased by 670 ± 240 mmHg/s per 10% increase in hcTnT1 expression (P = 0.009). The mean –dP/dtmin response in male mice was 856 mmHg/s higher than in females (P = 0.043). Mean T1/2 at 0% expression was 5.91 ± 0.20 ms; it increased by 0.39 ± 0.14 ms (P = 0.009) per 10% increase in hcTnT1 expression. Infusion of 1,000 pg isoproterenol increased dP/dtmax by 2,260 mmHg/s (23%, P < 0.001) and –dP/dtmin by 1,680 mmHg/s (19%, P < 0.001) and decreased T1/2 by 0.55 ms (9%, P = 0.001). The effects of isoproterenol did not depend on hcTnT1 expression level (P values for %cTnT1x isoproterenol interaction were 0.22 for dP/dtmax, 0.85 for –dP/dtmin, and 0.14 for T1/2).



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  		Fig. 13. Maximum rates of the rise and fall of LVP (dP/dtmax and dP/dtmin) and the half-time of isovolumic relaxation (T1/2) obtained from in vivo measurements plotted as a function of hcTnT1 expression at baseline and after an infusion of 1,000 pg isoproterenol. {circ}, NTG; {square}, F19; {triangleup}, F35; {triangledown}, F27. For clarity, all postisoproterenol data are plotted as +. Solid and dashed lines are the fitted regression lines for baseline and isoproterenol data, respectively. A: fitted values for dP/dtmax were 9,660 (baseline) and 11,920 mmHg/s (isoproterenol) at 0% hcTnT1 and 7,430 (baseline) and 9,690 mmHg (isoproterenol) at 30% hcTnT1. B: fitted values for dP/dtmin were 8,790 (baseline) and 10,470 mmHg/s (isoproterenol) at 0% hcTnT1 and 6,780 (baseline) and 8,460 mmHg/s (isoproterenol) at 30% hcTnT1. C: fitted values for T1/2 were 5.9 (baseline) and 5.4 ms (isoproterenol) at 0% hcTnT1 and 7.1 (baseline) and 6.5 ms (isoproterenol) at 30% hcTnT1.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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We examined the effects of hcTnT1 expression on the function of the heart in vivo in TG mice that overexpress hcTnT1 and on the myocardium isolated from these hearts in vitro. Myocardial functional properties were altered as the level of hcTnT1 expression increased.

cTnT Isoforms

A developmental shift in expression of TnT isoforms to those with shorter and less negatively charged amino-terminal regions occurs in cTnT, fsTnT, and ssTnT and in indirect flight muscle TnT (1, 5, 19, 23, 27). The similarity in this shift across muscle types strongly suggests that alterations in TnT isoform expression contribute to the plasticity of muscle function.

Of the four developmentally regulated mcTnT isoforms, the larger, more negatively charged isoforms are expressed early in development, whereas the smaller, less negatively charged isoforms are expressed in the adult. Combinatorial alternative splicing of two 5' cTnT exons provides the basis for four cTnT cDNAs in the human and other mammals (1, 19). Three previously cloned mouse cDNAs variably contain those two 5' exons (25). As in hcTnT1, they encode two negatively charged peptides with charges of –2 and –8. The successively less negative isoelectric points of mcTnT1, mcTnT2, mcTnT3, and mcTnT4 (Fig. 6) suggest that these isoforms are products of combinatorial alternative splicing of these two exons.

hcTnT1 is expressed in the fetal heart and in infants with congenital heart defects (2, 44). It is absent from the normal adult human heart and is reexpressed in the failing adult heart (2, 53). The proximity of the cTnT amino-terminal region to the overlap of adjoining tropomyosin molecules may enable cTnT isoform amino-terminal differences to modulate the Tn-tropomyosin calcium-regulated switch where activation with calcium results in movement of tropomyosin and exposure of myosin binding sites on actin leading to strong cross-bridge formation (18, 56, 58).

Functional Consequences of Transgenic Expression of hcTnT1

Correlations between the force-pCa and myofibrillar ATPase-pCa relations and cTnT isoform expression during development have been studied (29, 35, 46). For example, in vitro studies of rabbit neonatal myocardium demonstrated a positive correlation between cTnT2 expression and the sensitivity of the myofilaments to calcium (35). A review of the effects of development on cTnT isoform expression, which involves a change in expression from longer, more acidic cTnT isoforms (cTnT1 and cTnT2) to shorter, less acidic ones (cTnT3 and cTnT4), indicates that the calcium sensitivity of both the myofibrillar ATPase activity (29) and force (14, 40) decreased with development. Because the adult mouse expresses only cTnT isoforms that are smaller and less negatively charged than hcTnT1, we expected that hcTnT1 expression in the adult mouse would result in an increase in the sensitivity of the myofilaments in vitro.

In a recent study, Gomes et al. (17) described the results of in vitro experiments in which they replaced endogenous cTnT in pig myocardial fibers with each of the four recombinant human isoforms and show a nearly linear relationship between pCa50 and charge (hcTnT1 being the most negatively charged isoform). Using their values and assuming complete cTnT replacement in their preparations, we can calculate, based on charge, the expected difference in pCa50, pCa50(NTG – TG), between NTG and TG mice expressing 24.3% hcTnT1 (the mean expression level of founder line F27) as –0.042. This expected difference is well within the 95% confidence interval (–0.084 to +0.117) for the difference in pCa50 obtained from our mice.

Our in vitro measurement of force in the skinned preparation showed a decrease of Fmax with increased hcTnT1 expression. The three-state model of thin filament activation of McKillop and Geeves (30) provides a possible explanation for the apparent effect of hcTnT1 expression on Fmax. According to the model, the thin filaments are in a blocked, closed, or open activation state. In the blocked position, cross-bridge formation is prevented; in the closed state, myosin can weakly bind actin; and in the open state, myosin can strongly bind actin. In the presence of saturating calcium, ~20% of the actin sites are in the open state and 80% in the closed state (30). Different cardiac and skeletal muscle TnT fragments have different effects on thin filament activation (28, 51). The sequence differences between these fragments, which are in the TnT amino-terminal region, and their relation to the overlap of adjoining tropomyosin molecules may modulate thin filament activation. If increased hcTnT1 expression causes a shift in the average position of tropomyosin towards the closed state, a decrease in Fmax could be expected.

The in vivo study demonstrated the effects of increased hcTnT1 expression on cardiac function: dP/dtmax and –dP/dtmin fell and T1/2 increased. When we omitted the NTG mice and analyzed only the data from the TG mice (n = 24), we reached the same conclusions: dP/dtmax decreased at a rate of 1,120 ± 490 mmHg/s per 10% increase in %hcTnT1 (P = 0.030), –dP/dtmin decreased by 980 ± 360 mmHg/s per 10% hcTnT1 (P = 0.013), and T1/2 increased by 0.54 ± 0.24 ms for a 10% increase in hcTnT1 (P = 0.034). These data are consistent with the effect of hcTnT1 expression on Fmax and the force-pCa relation. We found a decrease in Fmax with no change in pCa50; such a decrease would be expected to scale the force-pCa relation downward and result in a decrease in dP/dtmax and –dP/dtmin, assuming other properties, e.g., cytosolic calcium concentration as a function of time during the contraction-relaxation cycle, remains constant.

The in vivo variables could have been affected by preload or afterload and subject to other potential compensatory changes, e.g., in cytosolic intracellular [Ca2+]. When we controlled statistically for end-diastolic pressure (as a surrogate for preload), the changes in dP/dtmax as a function of hcTnT1 expression remained the same. Although the Western blots of the 2-D gels showed similar patterns of resolution of mcTnT, mcTnI, and MLC2, differences in phosphorylation of contractile and calcium-regulating proteins could have contributed to the differences observed between NTG and TG hearts (9, 48). The effect of isoproterenol on the physiological variables, however, did not depend on the hcTnT1 expression level, leading us to speculate that the in vivo baseline level of cAMP-dependent phosphorylation is similar in NTG and TG myocardium. In our in vitro study, both sarcomere length and [Ca2+] were under our control, and therefore the differences in Fmax are unlikely to be due to differences in fiber length or [Ca2+]. The effects of hcTnT1 expression observed are unlikely to be due to abnormal structure because we saw no evidence of morphometric or structural differences between NTG and TG mice.

cTnT Isoform Charge and Function

To explore the potential effects of differences in charge resulting from the expression of hcTnT1, we refitted the regression models using charge in place of hcTnT1 expression and obtained similar results. The effect of charge brought about by hcTnT1 expression on Fmax was statistically significant, as were the slopes of dP/dtmax, –dP/dtmin, and T1/2 as a function of charge. The expression of hcTnT1 in TG mice appeared to be at the expense of mcTnT4 and not mcTnT3 (Fig. 5), leading to the potential ambiguity that the functional effects can be ascribed to either an increase in hcTnT1 or a decrease in mcTnT4 expression. However, ascribing these functional effects to differences in charge eliminates this ambiguity and at the same time invokes a more general concept.

In summary, our in vivo and in vitro results suggest that a change in cTnT isoform expression has a modulatory role in cardiac function and are consistent with the interpretation that cTnT amino-terminal charge and length may be the basis of this effect. Although human and mouse cTnT are highly homologous, they are not identical. Therefore, it is possible that some of the functional changes may have resulted from sequence differences other than the 15-residue peptide in the amino-terminal region of hcTnT1. We were interested in hcTnT1 because levels of hcTnT1 expression of 26–30% of total cTnT are present in the human fetal heart [scans of previously published Western blots (2)], and levels up to 15% are present in infants with severe congenital cardiac defects that require surgery (44). Our findings suggest that hcTnT1 expression at these levels affects myofilament function. This effect of cTnT1 could be potentially protective of the heart by decreasing its steady-state level of contractility whereby increasing its functional reserve. Future studies will assess whether hcTnT1 expression protects against or exacerbates the effects of pathological hemodynamic loading on survival and ventricular function.

Because the expression of hcTnT1 was almost exclusively at the expense of expression of mcTnT4 (Fig. 5), it is not possible to distinguish whether the effects were due to an increase in hcTnT1 or a decrease in mcTnT4. However, inasmuch as hcTnT1 and mcTnT4 differ in charge, the effect of the cTnT isoforms on ventricular and myofilament function can be stated in terms of a net increase in cTnT negative charge.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-67385 and HL-42250.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Jeffrey Robbins for providing the plasmid containing the murine {alpha}-myosin heavy chain promoter. We thank the Duke Comprehensive Cancer Center Transgenic Facility for the technical assistance in generating the transgenic founder lines. We are grateful to Dr. Scott Buck for many valuable suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. W. Anderson, Division of Cardiology, Dept. of Pediatrics, Duke Univ. Medical Center, Research Park Bldg. II, Rm. 113, PO Box 3218, Durham, NC 27710 (E-mail: ander005{at}mc.duke.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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