Sarcomere Thin Filament Regulatory Isoforms

EVIDENCE OF A DOMINANT EFFECT OF SLOW SKELETAL TROPONIN I ON CARDIAC CONTRACTION*

Joseph M. MetzgerDagger §, Daniel E. MicheleDagger , Elizabeth M. RustDagger , Andrea R. BortonDagger , and Margaret V. Westfall||

From the Departments of Dagger  Physiology and  Surgery, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109-0622

Received for publication, December 10, 2002, and in revised form, January 23, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca2+ sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sarcomeric thin filament proteins form an allosteric regulatory complex that governs Ca2+-activated contraction in the myocardium (1-3). Prior to contraction, intracellular Ca2+ levels are low and the thin filament regulatory proteins block strong actin-myosin interactions to inhibit force generation. Upon release of Ca2+ from intracellular stores to initiate contraction, Ca2+ binding to troponin C (TnC) causes conformational changes within the regulatory apparatus: tropomyosin (Tm),1 troponin I (TnI), and troponin T (TnT). Consequently, strong interactions between actin and myosin are permitted and force is generated. This precise interplay between Ca2+ and the thin filament regulatory complex is essential for orchestrating normal beat-to-beat regulation of cardiac performance.

The isoform content of the thin filament regulatory complex changes during cardiac development (Fig. 1) and in numerous cardiac disease states. Specifically, alpha - and beta -Tm isoform ratios are altered during embryonic/fetal myocardial development, with the ratio of alpha -Tm gene to beta -Tm gene expression increasing from 5:1 in fetal hearts to 60:1 in adult rodent and human myocardium (4). Moreover, differential splicing of cardiac TnT gene transcripts results in expression of an embryonic TnT (eTnT/TnT2) isoform in the developing myocardium followed by a transition to a more basic, lower molecular weight adult TnT (aTnT/TnT4) in adult myocardium (5-9). In the mammalian heart two distinct TnI genes are expressed (4, 10). Early in cardiac development the slow skeletal TnI (ssTnI) isoform is expressed. In the neonatal heart, ssTnI is down-regulated and the cardiac TnI (cTnI) gene is expressed. In the adult myocardium only the cTnI isoform is detected. In concert with Tm, TnI, and TnT isoform switching during development, the cardiac contractile apparatus transitions from being highly sensitive to being less sensitive to Ca2+ (11, 12). This process effectively tunes the myofilaments to adaptive alterations in intracellular calcium handling (11). Additionally, in the diseased adult myocardium, embryonic TnT and Tm isoforms have been shown to be re-expressed. For example, in pressure overload hypertrophy beta -Tm expression is increased, and in failing human myocardium the eTnT isoform is re-expressed (5-7, 9, 13, 14). In myotonic dystrophy resulting from a CTG expansion in the TnT gene, increases in CUG-binding protein expression and binding to cardiac TnT pre-mRNA result in increased production of the longer fetal eTnT transcript (15, 16). Collectively, these studies provide correlative evidence that thin filament regulatory isoform composition influences Ca2+-activated contractile function.

The structural complexities of the multimeric thin filament assembly and the overlapping pattern of TnI, Tm and TnT isoform switching in cardiac development have made it difficult to assign specific functional outcomes to each regulatory isoform transition (4). The cellular consequences of disease-mediated disregulation in TnT and Tm isoform expression in cardiac myocytes also remain unknown. Cardiac transgenic studies have proved to be extremely valuable in understanding organ and organismal outcomes to modifications in thin filament regulatory isoforms (17, 18). One potential consideration in using this approach lies in distinguishing, at the cellular level, the primary effects of transgene expression from possible secondary/compensatory effects, including the cardiac histopathology and maladaptive growth that can arise in transgenic mice (19, 20). In addition, to date there has been no single study comparing the functional significance of a systematic manipulation of cardiac-expressed Tm, TnT, and TnI thin filament regulatory isoforms in adult cardiac myocytes.

We tested the hypothesis that systematic replacement of each embryonic isoform would uniquely modify adult myocyte contractile responses to Ca2+. To this end, recombinant viral vectors were used for acute genetic engineering of the thin filament regulatory complex. This system has been demonstrated to cause stoichiometric replacement of native thin filament regulatory proteins (TnI, TnT, or Tm) (21-29). Self-protein (e.g. expression of adult isoforms, cTnI, aTnT, alpha -Tm) or reporter viral gene transfer have further demonstrated the specificity of this approach in regard to myocyte contractile structure-function (22, 25, 26, 30). Using this system, adult cardiac myocytes were systematically transduced with eight different vectors harboring Tm, TnI, or TnT isoform expression cassettes (Fig. 1). We then directly compared Ca 2+-activated isometric force responses of single adult myocytes following specific TnT, TnI, or Tm isoform replacement. Results demonstrate that ssTnI acts in a dominant manner with respect to the functional significance of cardiac thin filament isoforms in modulating regulation: TnI > Tm and TnT.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Recombinant Adenoviral Vectors-- Eight recombinant adenoviral vectors were used in this study: cTnI; cTnIFLAG (C-terminal FLAG epitope-tagged cTnI); ssTnI, using rat cDNAs; alpha -Tm (alpha  tropomyosin); alpha -TmFLAG (C-terminal FLAG epitope-tagged alpha -Tm); beta -Tm (beta -tropomyosin), from human cDNAs; eTnT (embryonic cardiac troponin T/also termed TnT2); and aTnT (adult cardiac troponin T/also termed TnT4), from rat cDNA (Fig. 1, A and B). The general strategy and protocols for shuttle plasmid and vector construction, virus production, and purification have been described earlier (22, 25, 30, 31). In each case, transcription was driven by the CMV promoter/enhancer. The polyadenylation signal derived from SV40.


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Fig. 1.   Vectors and thin filament isoform transitions in development. A, recombinant Tm, TnI, and TnT adenoviral vectors. Serotype 5 adenovirus linear structure is shown with the E1 region deleted and substituted with a eukaryotic expression cassette in reverse orientation. The eight cDNAs encoding the regulatory proteins are listed. B, listing of the developmentally regulated transitions in thin filament regulatory protein isoforms in the mammalian myocardium.

Adult Cardiac Myocyte Isolation, Primary Culture, and Gene Transfer-- These methods have been previously described in detail (31). Briefly, hearts were removed from adult female Sprague-Dawley rats, mounted on a modified Langendorff perfusion apparatus, perfused with Krebs-Henseleit buffer (KHB) (in mM/liter, 118 NaCl, 4.8 KCl, 25 HEPES, 1.25 K2HPO4, 1.25 MgSO4, 11 glucose) containing 1 mM Ca2+ for 5 min, and then perfused with KHB lacking added Ca2+. After 5 min, collagenase (0.5 mg/ml) and hyaluronidase (0.2 mg/ml) were added to the Ca2+-free KHB perfusate, and after 15 min of perfusion the Ca2+ concentration was gradually increased to 1 mM and perfused for an additional 15 min. The heart was removed to a sterile beaker, and the ventricles were gently minced in enzyme solution (1 mM Ca2+-KHB + 0.5 mg/ml collagenase + 0.2 mg/ml hyaluronidase). Minced tissue was gently swirled in a 37 °C water bath for 4 × 5 min. The final 2 digests were collected, centrifuged at 45 × g for 10 s, and pelleted cells were resuspended in 1 mM Ca2+-KHB + 20 mg/ml bovine serum albumin before gradually increasing the solution Ca2+ to 1.75 mM. Cells were then resuspended in Dulbecco's modified Eagle's medium + 5% fetal bovine serum + penicillin/streptomycin, counted, and plated on 18-mm2 laminin-coated glass coverslips at 2 × 104 myocytes/coverslip for 2 h. For cardiac myocyte gene transfer, medium was gently replaced with 200 µl of adenovirus diluted in Dulbecco's modified Eagle's medium + P/S to the desired titer. The multiplicity of infection ranged from 250-500 plaque forming units (pfu/rod-shaped myocyte) to optimize gene transfer efficiency for each vector tested. One hour later, 2 ml of Dulbecco's modified Eagle's medium + P/S was added to each coverslip. Medium was replaced with fresh Dulbecco's modified Eagle's medium + P/S 24 h later and every 2-3 days thereafter for up to 7 days post-gene transfer.

Immunoblotting-- Glass micropipettes were used to collect cardiac myocytes for subsequent gel analysis. The non-ionic detergent Triton X-100 (TX-100) was used to permeabilize the sarcolemma for comparison to membrane-intact myocytes prior to loading. SDS-PAGE was run at a constant current of 20 mA for 4-5 h, then transblotted onto polyvinylidene difluoride membrane (0.45 µm, Millipore) for 2000 volt-hours and fixed in PBS containing 0.25% glutaraldehyde. Western blots were carried out by initially blocking nonspecific binding sites with Tris-buffered saline containing 5% nonfat dry milk for 2 h. Blots were then incubated with either an anti-cardiac TnT monoclonal antibody (Chemicon mAb 1695, 1:500), an anti-striated muscle TnT monoclonal antibody (Sigma T-6277, clone JLT-12, 1:200), an anti-Tm monoclonal antibody (Sigma T-2780, clone TM311, 1:1 × 106), or an anti-TnI antibody (Chemicon mAb 1691, 1:500) for 2 h. To further validate isoform expression, anti-TnI antibody M32259 (Fitzgerald Industries International, Inc, Concord, MA) and anti-Tm antibodies CH-1 were used. Primary antibody was detected by peroxidase-conjugated goat anti-mouse IgG (1:1000) and an enhanced chemiluminescence detection assay (Amersham Biosciences).

Indirect Immunofluorescence-- Myocytes on coverslips were fixed in 3% paraformaldehyde, washed with PBS, and treated with 50 mM ammonium chloride to remove excess aldehydes. Nonspecific binding of antibodies was minimized with blocking solution containing PBS with 20% normal goat serum (NGS) and 0.5% TX-100. Primary anti-TnI antibody (Chemicon mAb 1691) or anti-FLAG antibody were diluted in PBS with 0.5% TX-100, and 2% NGS was added to cells for 1.5 h at room temperature. Cells were then washed with PBS + 0.5% TX-100 and blocked in PBS with 20% normal goat serum in 0.5% TX-100. Secondary antibody conjugated to Texas Red, (Molecular Probes, T-862) diluted in PBS with 0.5% TX-100 and 2% NGS was added for 1 h. Coverslips were washed in PBS for 4 × 5 min, mounted, and sealed. Immunofluorescence labeling of myocytes was viewed on a Nikon Diaphot 200 microscope fitted with a Noran confocal laser imaging system and Silicon Graphics Indy work station.

Determination of Single Cardiac Myocyte Force Production-- A force transducer (Cambridge Technology, Model 403A; sensitivity, 5 µg; 1-99% response time, 1 ms) and moving coil galvanometer (Cambridge Technology 6350 optical scanner) were attached to the chamber via three-way positioners. The temperature of the experimental chamber was controlled (15 °C) using thermoelectric modules (Melcor Inc.) coupled to a recirculating water bath heat sink. The chamber was positioned on an anti-vibration table. Coverslips were washed several times in relaxing solution (see below) 2-6 days post-gene transfer, and single, rod-shaped, cardiac myocytes were then attached to the glass micropipettes (32). Sarcomere length for each cardiac myocyte was set at 2.15 µm by viewing the myocyte using light microscopy and then adjusting the overall length of the preparation by way of three-way translators. Relaxing and activating solutions contained (in mM/liter) 7 EGTA, 1 free Mg2+, 4 MgATP, 14.5 creatine phosphate, 20 imidazole, and added KCl to yield a total ionic strength of 180 mM/liter. Solution pH was adjusted to 7.00 or 6.20 with KOH/HCl. The pCa (i.e. -log Ca2+) of the relaxing solution was set at 9.0, and the pCa of the solution for maximal activation was 4.0. At each pCa, steady-state isometric tension developed, after which the fiber was rapidly (< 0.5 ms) slackened to obtain the tension baseline. The myocyte was then relaxed, and the difference between developed tension and the tension baseline following the slack step was measured as total tension. To obtain active tension, resting tension measured at pCa 9.0 was subtracted from total tension. The myocyte was transferred to relaxing solution after each activation at a given pCa. Tension-pCa relationships were constructed by expressing tensions (P) at various submaximal Ca2+ concentrations as fractions of the tension at maximal activation, Po (i.e. isometric tension at pCa 4.0), that bracketed the submaximal activations.

Statistics-- To derive values for the Hill coefficient (nH) and midpoint from the tension-pCa relationship (termed K or pCa50), data were fit using the Marquardt-Levenberg non-linear least squares fitting algorithm using the Hill equation in the form: P = (Ca2+)nH/(KnH + (Ca2+)nH), where P is tension as a fraction of maximum tension (Po), K is the Ca2+ that yields one-half maximum tension, and nH is the Hill coefficient. Analysis of variance was used to examine whether there were significant differences in pCa50 (-log Ca2+ at half maximal tension), nH, or maximum tension using Student-Neuman-Keuls multiple comparison test. A probability level of p < 0.05 indicated significance.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Troponin T Isoform Gene Transfer, Expression, and Functional Assessment-- Isolated cardiac myocytes, transduced with recombinant virus harboring adult cardiac TnT (aTnT/TnT4) (14) or embryonic cardiac TnT (eTnT, TnT2) (14), were examined for TnT expression, followed by single myocyte isometric force recordings. On average, eTnT replacement (calculated as eTnT/(eTnT+aTnT) was about 45% (Fig. 2A). Expression of eTnT was not detected in myocytes transduced with aTnT (Fig. 2A) or with reporter vectors (not shown). As demonstrated earlier (25), total TnT content was not altered in aTnT- or eTnT-transduced myocytes. In addition, in myocytes membrane-permeabilized just prior to Western analysis the detection of eTnT was unchanged compared with membrane-intact myocytes (Fig. 2, A and B). The finding of unchanged total TnT content, together with similar detection of eTnT in intact and permeabilized myocytes, provides evidence of sarcomeric replacement of TnT isoforms.


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Fig. 2.   Troponin T isoform replacement and Ca2+-activated tension assay. A, Western blot analysis of TnT isoform expression in intact and detergent-permeabilized adult cardiac myocytes. Myocytes examined at day 5-7 post-eTnT and -aTnT gene transfer. Results demonstrate eTnT expression (TnT 2) after gene transfer of eTnT but not in control myocytes or in myocytes treated with the aTnT (TnT 4) vector. B, summary of TnT replacement in membrane intact (I) and permeabilized (P) myocytes. TnT replacement calculated as eTnT/(eTnT + aTnT). Values are mean ± S.E., n = 11-17. C, tension-pCa relationships in single adult cardiac myocytes at day 5-6 post-TnT gene transfer. Values are mean ± S.E., n = 6-9. D, summary of Ca 2+ sensitivity in control, aTnT, and eTnI experimental groups. Maximum tension, kN/m2 (and Hill Coefficients, nH) for control, aTnT, and eTnT were 36.4 ± 6.3(2.6 ± 0.4), 30.7 ± 7.8(2.6 ± 0.4), and 32.1 ± 9.6(2.6 ± 0.4). Values are mean ± S.E., n = 6-9.

Following TnT gene transfer, steady-state isometric force production was determined in single cardiac myocytes. Maximum isometric tension and the slope (Hill coefficient) and position (pCa50) of the tension-pCa relationship were not significantly different among the control, aTnT, or eTnT groups (Fig. 2, C and D).

Troponin I Isoform Gene Transfer, Expression, and Functional Assessment-- Adult cardiac myocytes were transduced with vectors harboring rat ssTnI or cTnI expression cassettes. In agreement with previous work (27-29), ssTnI replacement of endogenous cTnI was greater than 90% at day six post-gene transfer (Fig. 3, A and B) with no detected alterations in the expression pattern or stoichiometry of other sarcomeric proteins (data not shown). In addition, ssTnI or cTnI expression did not alter total TnI content and is evidence of stoichiometric replacement of TnI in the sarcomere. To further establish the efficiency and localization of newly expressed TnIs in this setting, we expressed an epitope-tagged TnI in the myocytes (cTnIFLAG). Results showed cTnIFLAG was detected in greater than 95% of the cardiac myocytes (Fig. 3C) with expression discretely localized to the cardiac thin filament as shown previously (22).


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Fig. 3.   TnI replacement dose-response relationship in adult cardiac myocytes. A, Western blot analysis of TnI isoform expression in adult cardiac myocytes at days 2, 3, and 6 post-ssTnI gene transfer. The soleus muscle sample provides a positive control for ssTnI expression. B, replacement of cTnI by ssTnI as a function of time post-gene transfer (ssTnI replacement = ssTnI/(ssTnI + cTnI)). Values are mean ± S.E., n = 6. Asterisks indicate control and cTnI-transduced myocytes at day 6. C, immunofluorescence detection of the replacement of native cTnI by epitope-tagged cTnI. Panels a and c are control, and panels b and d are following cTnIFLAG gene transfer. Antibodies are anti-troponin I (panels a and b) and anti-FLAG (panels c and d). Greater than 95% of cTnIFLAG-transduced myocytes were positive for cTnIFLAG expression and localization in the thin filament. D, summary of pCa50 values at days 2 and 3 post-ssTnI gene transfer. Maximum tension, kN/m2 (and Hill coefficients, nH) for control, ssTnI(d2) and ssTnI(d3) were 14.6 ± 6.2(2.2 ± 0.2), 27.4 ± 5.4(3.0 ± 0.4), 15.5 ± 2.8(1.5 ± 0.3). Values are mean ± S.E., n = 4-5. Asterisks indicate significantly different from control, p < 0.01. Gene transfer of cTnI, with data collected at days 2 and 3 (not different), served as an additional control. Maximum tension (kN/m2) and Hill coefficients were 19.6 ± 3.0 and 3.0 ± 0.3. E, silver-stained SDS-PAGE gel of myocytes post-ssTnI gene transfer. Lanes 1 and 2, control, day 2; lanes 3 and 4, ssTnI, day 2; lanes 5 and 6, control, day 4; lanes 7 and 8, ssTnI, day 4. Odd lanes are membrane-intact samples; even lanes are membrane-permeabilized samples (gave comparable results). F, TnI antibody comparison. Upper blot is probed with Chemicon anti-TnI antibody 1691 (same as in panel A); the same blot was then stripped and reprobed with the Fitzgerald anti-TnI antibody (lower blot). In both blots, lanes 1-6 are ssTnI, day 2; lanes 7 and 8 are control, day 2; lanes 9-14 are ssTnI day, 3; lanes 15 and 16 are control, day 3.

In our previous work (27, 28, 30, 33), we showed that replacement of >90% of cTnI by ssTnI causes marked increases in the Ca2+ sensitivity of tension development, with an average increase in Ca2+ sensitivity of 0.26 pCa units. Left unknown is whether ssTnI replacement can be titrated to <50%, which is similar to maximum replacement obtained for eTnT (Fig. 2, A and B), and still cause alterations in Ca2+ -activated tension. Based on the efficiency and synchronization of myofilament gene transfer (Fig. 3C) and the normal turnover rate of sarcomeric proteins (34), it is possible to construct a functional dose-response relationship for ssTnI by examining single myocytes at specific time points after gene transfer. Accordingly, in myocytes at days 2 and 3 post-gene transfer, ssTnI replacement averaged at the single myocyte level 14.4 and 20.4%, respectively (Fig. 3, A and B). Even at these relatively low levels of replacement there was a significant increase (p < 0.01) in the Ca2+ sensitivity of tension, indicating a dominant effect of ssTnI to modulate myofilament regulation of contraction (Fig. 3D). The magnitude increase in Ca2+ sensitivity of tension was 0.12 and 0.20 pCa units at days 2 and 3, respectively (p < 0.01). For controls, we examined calcium-activated tension in untreated myocytes and myocytes after cTnI gene transfer at days 2 and 3 in primary culture. Results showed that control and cTnI myocytes were unchanged and not different from each other (Fig. 3D). This is in agreement with our earlier finding of stable contractile function in adult myocytes in short-term primary culture (26). In separate experiments, we evaluated the extent of ssTnI replacement by SDS-PAGE/silver staining and through use of a second anti-TnI antibody. In broad agreement with our main findings (Fig. 3, A and B), SDS-PAGE showed 27.5% replacement of cTnI by ssTnI at day 2 and 67% replacement at day 4. Similarly, blots probed with a second TnI antibody (Fitzgerald) showed 29% replacement of cTnI by ssTnI at day 2 after gene transfer (Fig. 3, E and F). Collectively, our findings, using two different TnI antibodies (Chemicon and Fitzgerald) and SDS-PAGE, demonstrate that the extent of TnI replacement is between 14-29% at day 2 and that this extent of TnI replacement achieves nearly the same magnitude shift in Ca2+ sensitivity of tension as we have reported in several previous studies with > 90% replacement (0.26 pCa units, Refs. 27, 28, 30, 33).

Tropomyosin Isoform Gene Transfer, Expression, and Functional Assessment-- Tropomyosin isoforms alone or together with different TnT isoforms have been postulated to play a role in determining Ca2+ sensitivity of tension (35-38). In general agreement with the 5.5-day half-life for Tm (34), gene transfer of beta -Tm to adult cardiac myocytes resulted in 30-40% replacement of beta -Tm at day six post-gene transfer (Fig. 4, A and B). These results were obtained by using two different anti-Tm antibodies (Fig. 4). In earlier work, total Tm protein content and the stoichiometry of Tm within the sarcomere were shown to be unchanged after Tm gene transfer (21, 22). Expression of beta -Tm also was not different in intact and permeabilized myocytes (Fig. 4, A and B), an indication that beta -Tm incorporated into the myofilaments. In functional studies on permeabilized single cardiac myocytes, neither maximum isometric tension nor the Ca2+ sensitivity of tension (Hill coefficient/pCa50) were affected by alpha -Tm, alpha -TmFLAG, or by beta -Tm gene transfer (Fig. 4C legend). Thus, at this level of alpha -Tm replacement by beta -Tm, which is similar to the Tm isoform switching observed during normal myocardial development, myofilament Ca2+ sensitivity is not significantly altered.


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Fig. 4.   Tropomyosin isoform replacement and cardiac myocyte function. A, Western blot of Tm isoform expression in intact and detergent-permeabilized adult cardiac myocytes after beta -Tm gene transfer. Myocytes collected at days 5-7 post-gene transfer. B, summary of beta -Tm replacement in control and alpha - and beta -Tm-transduced myocytes. beta -Tm replacement calculated as beta -Tm/(beta -Tm + alpha -Tm). Values are mean ± S.E., n = 7-9. Blots probed with TM311 antibody. CH-1 anti-Tm antibody gave similar results (data not shown) C, summary of pCa50 values in control and alpha -Tm-, beta -Tm-, and alpha -TmFLAG-transduced myocytes. The control pCa50 is different from other control pCa50s (e.g. Fig. 3) because these experiments were conducted separately and, over time, the absolute pCa50s can vary from study to study. Maximum tension, kN/m2 (and Hill Coefficients, nH) for control, alpha -Tm, and beta -Tm were 19.8 ± 7.1(1.5 ± 0.1), 15.2 ± 5.8 (1.6 ± 0.1), 10.9 ± 1.6(1.9 ± 0.3). Values are mean ± S.E., n = 7-9. No significant differences between groups by analysis of variance.

Influence of Thin Filament Isoform Expression on pH Sensitivity of Tension Development-- Acidosis is an important contributor to cardiac dysfunction during myocardial ischemia (39). Differential thin filament isoform expression in cardiac development, and among slow and fast skeletal fibers, is thought to play an important role in defining the magnitude in acidic pH-mediated contractile dysfunction (30, 40). We therefore compared the relative effects of Tm, TnI, and TnT isoforms on contractile function at pH 6.20, a level of acidification that accrues during acute myocardial ischemia (39). In concert with results obtained under physiological conditions, ssTnI replacement, but not Tm or TnT isoform replacement, had a significant effect on the Ca2+ sensitivity of tension under acidic pH conditions (Fig. 5).


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Fig. 5.   Summary of altered Ca2+ sensitivity (pCa50) under acidic pH conditions. Values are mean ± S.E., average of 5 myocytes per group. Because these data were collected separately over time (denoted by vertical dashed line) two control groups are shown, one for the ssTnI and one for the TnT/Tm groups. These controls values were not different. Measurements obtained at days 2 and 3 for ssTnI and days 5-7 for TnT/Tm. beta -Tm, n = 2. Asterisk indicates significantly different from control, p < 0.01.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Function of Thin Filament Regulatory Isoforms-- Our results demonstrate a functional difference among thin filament regulatory protein isoforms for modulation of Ca2+ sensitivity of tension. Stoichiometric replacement of < 30% of native cTnI with ssTnI caused significant alterations in myofilament Ca2+ sensitivity of tension development. By contrast, 30-50% isoform replacement with either eTnT or beta -Tm had no significant influence on the Ca2+ sensitivity of tension. This comparative analysis provides direct evidence that ssTnI has a dominant modulatory effect on the regulation of contraction in cardiac muscle.

In its simplest form, the cardiac thin filament can be considered a linear array of 24 regulatory units. A regulatory unit is defined as seven actin monomers, one Tm dimer, and one heterotrimeric troponin complex consisting of troponin C, troponin T, and TnI (41). There is considerable evidence that these discrete regulatory units are coupled functionally. Nearest neighbor and long-range interactions have been demonstrated among thin filament regulatory units (41-43). Our finding that 14-29% TnI isoform replacement can cause significant alterations in function supports the concept of long-range regulatory unit interactions. We previously showed that TnI replacement is a stochastic process along the thin filaments, even at early time points after gene transfer (22). As an example, 14% TnI isoform replacement would represent an average replacement of 3-4 regulatory units with ssTnI of a total of 24 in each thin filament. Stated differently, 1 in 7 regulatory units, on average, is switched from cTnI to ssTnI. With stochastic TnI replacement, a dominant effect of ssTnI is proposed to extend across 3-4 native cTnI regulatory units from both sides of each new ssTnI regulated unit. Similar types of long-range interaction have been inferred by the partial removal of troponin C subunits, where as little as 5% manipulation in troponin C content has been hypothesized to exert an effect on calcium-activated tension (41, 42). Mechanistically, the near neighbor effects of TnI could be mediated via tropomyosin. Recent studies on thin filament regulation reveal multiple transition states/binding interactions of tropomyosin on actin under the influence of calcium and myosin binding (44). In the present study, there were further increases in Ca2+ sensitivity as TnI isoform replacement rose from day 2 to day 3 after ssTnI gene transfer (Fig. 3). The calcium sensitivity data at physiological and acidic pH at < 30% TnI replacement are in general agreement with previous gene transfer studies in which nearly all cTnI had been replaced by ssTnI (27-29). This suggests apparent saturation of TnI isoform-dependent functional effects even though 70-80% regulatory units remained coupled with native cTnI. Collectively, these results establish a dominant gain-in-function by ssTnI. Because a change in only about 1 in 7 regulatory units is required to bring about functional effects, this may impact on strategies to influence contractile performance in diseased myocardium.

Comparisons to Earlier Work-- Our finding that up to 45% replacement of alpha -Tm with the beta -Tm isoform has no significant effect on Ca2+-activated tension is in apparent contrast to earlier studies on transgenic mice (45). Cardiac bundles isolated from beta -Tm transgenic mice show increased Ca2+ sensitivity when activated under physiological conditions; however, in agreement with our findings, beta -Tm had no significant effect on myofilament pH sensitivity (46). One factor to consider in comparing these results is the extent of Tm replacement. In transgenic studies, about 55% or greater Tm replacement was achieved in vivo (17, 45), a value greater than we have obtained by Tm gene transfer. Based on biochemical and transgenic studies (45), it appears that significant alpha beta heterodimer formation is necessary for Tm isoform-dependent effects on myofilament Ca2+ sensitivity. Another issue to consider is that transgenic manipulation of Tm can and does result in organ-level maladaptive growth (19, 20), and this could impact subsequent assays of myocyte function. Similarly, eTnT replacement of 40-50% did not alter Ca2+-activated tension. Previously, correlations have been reported between developmental/disease-mediated transitions in cardiac TnT isoform expression and myocardial function (35, 37). As with Tm isoforms, higher levels of replacement of TnT isoforms may be necessary to detect changes in myofilament function. In this light, it is interesting to note that single missense mutations associated with hypertrophic cardiomyopathy (HCM) and placed in eTnT can significantly affect calcium sensitivity, even at very low levels (about 8%) of TnT replacement (25). Similarly, HCM mutant Tms produce alterations in function at replacement levels comparable with the beta -Tm replacement achieved in the present study (21). There are 39 amino acid differences between alpha - and beta -Tm, including many in the C terminus thought to be critical for Tm and TnT interactions (47). In our functional assay, these 39 amino acid differences appear less significant than HCM-associated single missense mutations in Tm. However, regarding the developmental regulation of thin filament isoform expression, it is apparent that TnI has a much more dominant effect than TnT or Tm isoforms on developmental tuning/modulation of myofilament Ca2+ sensitivity. We cannot exclude the possibility that if higher levels of TnT or Tm replacement could be achieved in this system this would then cause alterations in the Ca2+ sensitivity of tension. This result, if obtained, would suggest that the threshold for thin filament isoforms to have a modulatory effect on regulation is markedly higher for Tm and TnT than for TnI isoforms.

Implications-- The finding of a dominant effect of ssTnI isoform replacement even at low levels on Ca2+ sensitivity may be useful in designing TnI proteins for addressing cardiac contractile dysfunction in disease. Based on our results, modified TnI appears to be the best candidate for improving myofilament Ca2+ sensitivity of tension under acidotic conditions during acute myocardial ischemia. In failing myocardium, or in cardiomyopathies associated with altered diastolic Ca2+ levels, TnI chimeras with specified regulatory functions may be advantageous (27-29). Modified TnIs may also serve as a potential strategy for correcting inherited diseases associated with mutated sarcomeric proteins because these mutations have been associated with changes in myofilament Ca2+ sensitivity (17, 21). It may be possible to affect disease progression if these Ca2+ sensitivity shifts are prevented by targeted TnI replacement. Alternatively, pharmaceutical-based strategies to manipulate TnI function may be another approach for future investigation. Finally, although re-expression of embryonic isoforms of TnT and Tm may provide useful bio-markers of failing adult myocardium, our findings indicate that these alterations, at least up to 30-50% replacement, do not appear to contribute to altered calcium-activated tension of the diseased heart.

    FOOTNOTES

* This work is supported by grants from the National Institutes of Health and the American Heart Association (to J. M. M.).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.

§ To whom correspondence should be addressed: Dept. of Physiology, 7730 Medical Science Bldg. II, University of Michigan, Ann Arbor, MI 48109-0622. Tel.: 734-763-0560; Fax: 734-647-6461; E-mail: metzgerj@umich.edu.

|| Recipient of a Scientist Development grant from the American Heart Association.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212601200

    ABBREVIATIONS

The abbreviations used are: TM, tropomyosin; TnT, troponin T; TnI, troponin I; ssTnI, slow skeletal TnI; cTnI, cardiac TnI; cTnIFLAG, C-terminal, FLAG epitope-tagged cTnI; eTnT, embryonic eTnT; aTnT, adult TnT; NGS, normal goat serum; TX-100, Triton X-100.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Tobacman, L. S. (1996) Annu. Rev. Physiol 58, 447-481[CrossRef][Medline] [Order article via Infotrieve]
2. Gordon, A. M., Homsher, E., and Regnier, M. (2000) Physiol. Rev. 80, 853-924[Abstract/Free Full Text]
3. Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755-767[Abstract/Free Full Text]
4. Schiaffino, S., and Reggiani, C. (1996) Physiol. Rev. 76, 371-423[Abstract/Free Full Text]
5. Anderson, P. A., Moore, G. E., and Nassar, R. N. (1988) Circ. Res. 63, 742-747[Abstract]
6. Anderson, P. A., Malouf, N. N., Oakeley, A. E., Pagani, E. D., and Allen, P. D. (1991) Circ. Res. 69, 1226-1233[Abstract]
7. Anderson, P. A., Malouf, N. N., Oakeley, A. E., Pagani, E. D., and Allen, P. D. (1992) Basic Res. Cardiol. 87 Suppl 1, 117-127[Medline] [Order article via Infotrieve]
8. McAuliffe, J. J., and Robbins, J. (1991) Pediatr. Res. 29, 580-585[Abstract]
9. Townsend, P. J., Barton, P. J., Yacoub, M. H., and Farza, H. (1995) J. Mol. Cell. Cardiol. 27, 2223-2236[Medline] [Order article via Infotrieve]
10. Murphy, A. M., Jones, L., Sims, H. F., and Strauss, A. W. (1991) Biochemistry 30, 707-712[Medline] [Order article via Infotrieve]
11. Fabiato, A., and Fabiato, F. (1978) Ann. N. Y. Acad. Sci. 307, 491-522[Medline] [Order article via Infotrieve]
12. Metzger, J. M., Lin, W. I., and Samuelson, L. C. (1994) J. Cell Biol. 126, 701-711[Abstract]
13. Mesnard-Rouiller, L., Mercadier, J. J., Butler-Browne, G., Heimburger, M., Logeart, D., Allen, P. D., and Samson, F. (1997) J. Mol. Cell Cardiol. 29, 3043-3055[CrossRef][Medline] [Order article via Infotrieve]
14. Greig, A., Hirschberg, Y., Anderson, P. A., Hainsworth, C., Malouf, N. N., Oakeley, A. E., and Kay, B. K. (1994) Circ. Res. 74, 41-47[Abstract]
15. Ladd, A. N., Charlet, N., and Cooper, T. A. (2001) Mol. Cell. Biol. 21, 1285-1296[Abstract/Free Full Text]
16. Philips, A. V., Timchenko, L. T., and Cooper, T. A. (1998) Science 280, 737-741[Abstract/Free Full Text]
17. Muthuchamy, M., Pieples, K., Rethinasamy, P., Hoit, B., Grupp, I. L., Boivin, G. P., Wolska, B., Evans, C., Solaro, R. J., and Wieczorek, D. F. (1999) Circ. Res. 85, 47-56[Abstract/Free Full Text]
18. James, J., and Robbins, J. (1997) Am. J. Physiol. 273, H2105-H2118[Abstract/Free Full Text]
19. MacGowan, G. A., Du, C., Wieczorek, D. F., and Koretsky, A. P. (2001) Am. J. Physiol. Heart Circ. Physiol. 281, H2539-H2548[Abstract/Free Full Text]
20. Muthuchamy, M., Boivin, G. P., Grupp, I. L., and Wieczorek, D. F. (1998) J. Mol. Cell Cardiol. 30, 1545-1557[CrossRef][Medline] [Order article via Infotrieve]
21. Michele, D. E., Albayya, F. P., and Metzger, J. M. (1999) Nat. Med. 5, 1413-1417[CrossRef][Medline] [Order article via Infotrieve]
22. Michele, D. E., Albayya, F. P., and Metzger, J. M. (1999) J. Cell Biol. 145, 1483-1495[Abstract/Free Full Text]
23. Michele, D. E., and Metzger, J. M. (2000) Trends Cardiovasc. Med. 10, 177-182[CrossRef][Medline] [Order article via Infotrieve]
24. Michele, D. E., and Metzger, J. M. (2000) J. Mol. Med. 78, 543-553[CrossRef][Medline] [Order article via Infotrieve]
25. Rust, E. M., Albayya, F. P., and Metzger, J. M. (1999) J. Clin. Invest. 103, 1459-1467[Abstract/Free Full Text]
26. Rust, E. M., Westfall, M. V., and Metzger, J. M. (1998) Mol. Cell. Biochem. 181, 143-155[CrossRef][Medline] [Order article via Infotrieve]
27. Westfall, M. V., Albayya, F. P., and Metzger, J. M. (1999) J. Biol. Chem. 274, 22508-22516[Abstract/Free Full Text]
28. Westfall, M. V., Albayya, F. P., Turner, I. I., and Metzger, J. M. (2000) Circ. Res. 86, 470-477[Abstract/Free Full Text]
29. Westfall, M. V., and Metzger, J. M. (2001) News Physiol. Sci. 16, 278-281[Abstract/Free Full Text]
30. Westfall, M. V., Rust, E. M., and Metzger, J. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5444-5449[Abstract/Free Full Text]
31. Westfall, M. V., Rust, E. M., Albayya, F., and Metzger, J. M. (1997) Methods Cell Biol. 52, 307-322[Medline] [Order article via Infotrieve]
32. Metzger, J. M. (1995) Biophys. J. 68, 1430-1442[Abstract]
33. Westfall, M. V., Turner, I., Albayya, F. P., and Metzger, J. M. (2001) Am. J. Physiol. Cell Physiol. 280, C324-C332[Abstract/Free Full Text]
34. Martin, A. F. (1981) J. Biol. Chem. 256, 964-968[Abstract/Free Full Text]
35. Greaser, M. L., Moss, R. L., and Reiser, P. J. (1988) J. Physiol. 406, 85-98[Abstract]
36. Reiser, P. J., Greaser, M. L., and Moss, R. L. (1992) J. Physiol. 449, 573-588[Abstract]
37. Schachat, F. H., Diamond, M. S., and Brandt, P. W. (1987) J. Mol. Biol. 198, 551-554[Medline] [Order article via Infotrieve]
38. McAuliffe, J. J., Gao, L. Z., and Solaro, R. J. (1990) Circ. Res. 66, 1204-1216[Abstract]
39. Lee, J. A., and Allen, D. G. (1991) J. Clin. Invest. 88, 361-367[Medline] [Order article via Infotrieve]
40. Solaro, R. J., Lee, J. A., Kentish, J. C., and Allen, D. G. (1988) Circ. Res. 63, 779-787[Abstract]
41. Moss, R. L. (1992) Circ. Res. 70, 865-884[Abstract]
42. Brandt, P. W., Roemer, D., and Schachat, F. H. (1990) J. Mol. Biol. 212, 473-480[Medline] [Order article via Infotrieve]
43. Brandt, P. W., Diamond, M. S., Rutchik, J. S., and Schachat, F. H. (1987) J. Mol. Biol. 195, 885-896[Medline] [Order article via Infotrieve]
44. Lehman, W., Hatch, V., Korman, V., Rosol, M., Thomas, L., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606[CrossRef][Medline] [Order article via Infotrieve]
45. Muthuchamy, M., Grupp, I. L., Grupp, G., O'Toole, B. A., Kier, A. B., Boivin, G. P., Neumann, J., and Wieczorek, D. F. (1995) J. Biol. Chem. 270, 30593-30603[Abstract/Free Full Text]
46. Palmiter, K. A., Kitada, Y., Muthuchamy, M., Wieczorek, D. F., and Solaro, R. J. (1996) J. Biol. Chem. 271, 11611-11614[Abstract/Free Full Text]
47. Smillie, L. (1996) Biochemistry of Smooth Muscle Contraction , Academic Press, San Diego


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