INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TROPOMYOSIN (TM), an actin binding protein and a major component of the sarcomeric thin filament, forms coiled-coil dimers that assemble in a head-to-tail fashion in the major groove of actin. Numerous muscle proteins, including TM, have multiple isoforms that are expressed with developmental and tissue specificity. The TM gene family is composed of four genes [-TM, -TM, TPM 3 (-TM), and TPM 4 (-TM)], which through alternative splicing, the use of alternative promoters, and differential processing encode multiple striated muscle, smooth muscle, and nonmuscle specific transcripts (12, 17, 38). There are three primary striated muscle TM isoforms, -TM, -TM, and TPM 3, which are highly homologous and thought to exhibit unique physiological properties (28). In the mouse, -TM is expressed mainly in the heart and also in slow- and fast-twitch musculature, -TM is expressed predominantly in the developing heart and slow-twitch musculature, and TPM 3 is found only in slow-twitch musculature (27, 31).

TM, along with the troponin complex, regulates the Ca²⁺-sensitive reaction of cross bridges with actin in striated musculature. The Ca²⁺ signal is transmitted through the binding of Ca²⁺ to troponin C (TnC), one of three proteins that comprise the troponin complex. The exact mechanism by which TM and troponin work is unclear; however, it is thought to include steric, allosteric, and cooperative elements (13, 18, 36). Current models propose that there are three distinct states that are dependent on the location of TM on the thin filament (23). The "blocked state" is when the TM blocks the myosin-actin interaction in the absence of Ca²⁺. In the "closed state," cross bridges are weakly bound when the position of TM has changed due to the binding of Ca²⁺ to TnC. The "open state" is achieved following the transition of weak cross bridges into strong, force-generating cross bridges. Strong cross-bridge interactions promote additional TM movement and lead to the stability of the TM in the open position. This activates the thin filament, stimulating muscle contraction and increasing cooperativity along the length of the myofibril (reviewed in Ref. 8).

Functional differences between the -TM and -TM striated muscle TM isoforms have been demonstrated in both in vitro and in vivo (26, 35). The exchange of -TM for -TM increases the ability of strong cross-bridge binding to activate the thin filament, and the force developed by -TM myofilaments is more sensitive to calcium (29). This difference in Ca²⁺ sensitivity is more pronounced when the myofilaments are phosphorylated by cAMP-dependent protein kinase, indicating a role for TM in the modulation of myofilament activation by phosphorylation on troponin. In cardiomyocytes expressing -TM, maximum rates of contraction and relaxation are significantly reduced, and ATPase activity of myofibrils is more sensitive to Ca²⁺ (39). Furthermore, expression of -TM in transgenic (TG) mouse hearts leads to prolongation in relaxation rate and a reduction in time to half relaxation (R_1/2) (26).

Although much information has been obtained for physiological differences between the - and -TM striated muscle isoforms, less is known about the striated muscle isoform of TPM 3. Recently, Wieczorek's laboratory (31) cloned and characterized this isoform in the mouse. The TPM 3 gene, ~42 kb in length, is composed of 13 exons with its primary transcripts differentially processed to produce >11 mRNA isoforms (4, 5). Most of these isoforms are expressed in developmental and tissue-specific patterns in neurons; however, one of these isoforms is striated muscle specific with expression restricted to slow-twitch musculature (31). Unlike, -TM and -TM, the striated muscle isoform of TPM 3 is not expressed endogenously in murine cardiac tissue, but it is expressed in the adult human heart (31, 32).

In this study, we sought to determine the functional significance of the striated muscle isoform of TPM 3 by its overexpression in murine hearts. Murine hearts possess a uniform myofiber composition with a well-defined complement of sarcomeric proteins; thus the effects of ectopic expression of TPM 3 in the absence of other myofibrillar protein changes are readily ascertained. Six TG mouse lines were generated, which express varying levels of the TPM 3 RNA. Results show that the TPM 3 transgene is expressed at high levels, with the TPM 3 protein accounting for 40-60% of the cardiac muscle TM. Morphological analyses employing both light and electron microscopy demonstrate there are no structural changes in the heart or in the sarcomeres that are associated with the increased TPM 3 expression. However, extensive physiological analyses of these TG hearts using in situ and in vitro measurements reveal myocardial contraction and relaxation parameters are altered; there are increased rates of relaxation (dP/dt) and contraction (+dP/dt). Also, measurement of force generated by skinned fiber bundles demonstrates a decreased sensitivity to Ca²⁺ and a decrease in the length dependence of myofilament Ca²⁺ activation. This functional phenotype is unique to the TPM 3 mice and differs from wild-type mice and -TM TG mice. This is the first report demonstrating that a novel functional property is associated with TPM 3 expression, namely increased cardiac performance associated with perturbations in Ca²⁺ sensitivity and activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of the -myosin heavy chain/TPM 3 TG construct and production of TG mice. The TPM 3 cDNA was generated from murine skeletal muscle by using PCR primers corresponding to the human TPM 3 sequence (4, 31). The forward primer begins at the translational start position and maintains the highly conserved length of the striated muscle TMs. Its nucleotide sequence is 5'-ATGGAGGCCATCAAGAAA-3'. The reverse primer includes the 3'-untranslated region (3'-UTR) of the cDNA and is 5'-TTTCCAGCAGCTTAACAT-3'. The 1.1-kb TPM 3 cDNA fragment was ligated into the HindIII/SalI sites of a vector containing the -myosin heavy chain (-MyHC) promoter and its 5'-UTR. The 600-bp human growth hormone (hGH) polyadenylation signal was added to ensure correct transcript processing of the transgene (gift from Dr. Jeff Robbins). The 7.2-kb fragment containing the -MyHC promoter, the TPM 3 cDNA, and the hGH polyadenylation signal was released from the vector by NotIdigestion (Fig. 1). The resulting linear DNA fragment was isolated on a low-melting point agarose gel, followed by purification in a cesium chloride gradient. The resulting DNA was suspended in 5 mM Tris · HCl, pH 7.4, 0.1 mM EDTA at a final concentration of 2 µg/ml. Single-cell embryos derived from superovulated FVB/N mouse strain females were used in the microinjection. Purified DNA was microinjected into the pronuclei, and the surviving embryos were implanted into pseudopregnant foster mothers.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Cardiac-specific tropomysin (TM) isoform TPM 3 construct. TPM 3 cDNA [including the coding region and the 3'-untranslated region (3'-UTR)] was ligated to the -myosin heavy chain (-MyHC) promoter and a human growth hormone (hGH) poly A signal. Construct was released with Not I digestion and injected into male pronuclei to generate the transgenic (TG) mice.

Genomic Southern blot analysis. To confirm the presence of the transgene and to determine the number of copies integrated into each line, genomic DNA from tails was digested overnight with EcoRI, and electrophoresis was carried out on 0.8% agarose gels. The Southern blots were probed with a ³²P-radiolabeled hGH fragment specific to the 3' end of the transgene. Quantification of the transgene was done on Imagequant phosphorimager version 5.1 (Molecular Dynamics; Sunnyvale, CA).

Northern blot analysis. RNA was isolated from murine hearts using TriReagent (Molecular Research Center; Cincinnati, OH). Total RNA (15 µg) from each line was electrophoresed on 1% formaldehyde gels and transferred to nylon membranes. Hybridization was performed with ³²P-radiolabeled -TM 3'-UTR, TPM 3 3'-UTR, and GAPDH probes. Blots were washed in 2× SSC and 0.05% SDS for 20 min at 50°C, followed by 0.1× SSC and 0.1% SDS for 30 min at 60°C. TM message levels were analyzed on Imagequant phosphorimager version 5.1 and normalized to GAPDH expression levels. Results are presented as a percentage of the highest expressing line.

Western blot analysis. Whole tissue and myofibrillar homogenates were prepared as previously described (24). Protein samples were run simultaneously on two 3.4 M urea, 8% SDS-PAGE gels. One gel was stained with Coomassie blue to ensure equal loading, and the other was transferred to nitrocellulose for Western blot analyses. Filters were probed with a striated muscle, TM-specific CH1 antibody, and cardiac-specific troponin T antibody (Sigma; St. Louis, MO) at a 1:1,000 dilution for 2 h at room temperature (19). After washing was completed, an anti-mouse IgG antibody conjugated with peroxidase (Roche; Indianapolis, IN) was incubated with the blot for 1 h at a dilution of 1:10,000. Detection was carried out with the Super Signal West Pico Chemiluminescent Substrate Detection System (Pierce; Rockford, IL). Band intensity was analyzed on Imagequant phosphorimager Version 5.1. Results are presented as means ± SE.

Histological examination. For light microscopy, hearts were removed from 2- to 12-mo-old mice and immediately fixed in 10% neutral buffered formalin. Sections were cut at 5-µm thickness and stained with hematoxylin-eosin. For electron microscopy, hearts were fixed in 2% glutaraldehyde and then transferred to cacodylate buffer. Heart tissue was postfixed in osmium tetroxide. Sections (70-80 Å) were stained with lead acetate and examined on a Hitashi H600 microscope.

Working heart preparations. Perfusion and analysis of mouse hearts were done as previously described (10, 11). Briefly, mice were anesthetized with pentobarbital sodium intraperitoneally, and the hearts were removed and cannulated. Five TG mice and four NTG, age- and sex-matched littermates were examined. Cardiac performance was monitored by a six-channel P7 Grass polygraph. Intraventricular pressure, aortic pressure, and heart rate recordings were digitized via a TL-1 DMA interface board (Axon Instruments; Foster City, CA). Rates of +dP/dt, dP/dt, time to peak pressure (TPP), and RT_1/2 were derived. Isoproterenol was added to the perfusion fluid close to the heart at increasing concentrations (8 × 10¹¹ to 8 × 10⁸ M) with a multispeed, microperfusion pump (model 600, Harvard Apparatus). Individual points were recorded and summarized as means ± SD.

Closed-chest preparations. Closed-chest analysis was carried out as previously described (20). Six TG and six NTG littermates were examined. After anesthesia (Inactin, 100 µg/g body wt and ketamine, 50 µg/g body wt intraperitoneally), a 1.4-Fr Millar MIKRO-TIP transducer (SPR-671, Millar Instruments; Houston, TX) was placed in the right carotid artery and threaded down into the ascending aorta and into the left ventricle to record the heart's performance. A cannula was placed into the right femoral vein to deliver dobutamine using a syringe pump, and a catheter that was connected to a pressure transducer was placed in the right femoral artery to monitor blood pressure. Dobutamine was delivered to challenge the heart. Measurements are recorded and analyzed on a MacLab 4/s data acquisition system (ADInstruments; Mountain View, CA).

Force and X-ray diffraction measurements of skinned fiber bundle preparations. Mechanical experiments in which we determined relations between free Ca²⁺ and tension and interfilament spacing were done as previously described (1, 14, 29). The mice were anesthetized with ethyl ether, and their hearts were removed and incubated in a cold high-relaxing solution (53 mM KCl, 10 mM EGTA, 20 mM MOPS, 1 mM free Mg²⁺, 5 mM MgATP², 12 mM creatine phosphatase, and 10 IU/ml creatine phophokinase and protease inhibitors), adjusted to pH 7.0. Left ventricular papillary muscles were removed, and bundles of fibers were prepared (~4-5 mm in length and 150-200 µm in width). Fiber bundles were attached to a micromanipulator and a force transducer with cellulose-acetate glue and extracted in high-relaxing solution containing 1% Triton X-100. Sarcomere lengths (SLs) were set at 1.9 and 2.3 µm as determined by laser diffraction patterns. The fiber bundles were first bathed in a low-relaxing solution (high-relaxing solution with 0.1 mM instead of 10 mM EGTA). Force was then measured in high-relaxing solution containing varying levels of free Ca²⁺.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of TPM 3 TG mice. To elucidate relations between sarcomeric TM isoforms and contractile behavior in striated muscle, we developed a TG mouse model to overexpress the striated muscle-specific TPM 3 isoform in the heart. The transgene construct was generated by ligating the murine TPM 3 cDNA (containing the entire amino acid coding region and the 3'-UTR) into an expression vector regulated by the murine -MyHC promoter, which directs cardiac-specific expression (Fig. 1). A hGH polyadenylation sequence is located 3' of the TPM 3 cDNA to ensure correct transcript processing. Founder mice were identified by PCR and confirmed by Southern blot analysis. Six lines of TG mice were produced with copy numbers ranging from 2 to 60.

TPM3 transcript and protein expression in the TG mice. We measured TM expression by using Northern blot analysis following RNA isolation from hearts of TG and NTG littermates. RNA from each TG line was isolated and probed with ³²P-radiolabeled 3'-UTR sequences from endogenous -TM and the TPM 3 transgene construct; these nucleotide regions are specific to the particular striated muscle isoform and do not cross react with other striated muscle TM isoforms (31). As seen in Fig. 2A, endogenous -TM is expressed in all hearts, whereas TPM3 expression is restricted to TG hearts. For quantification, TPM 3 transcript levels are normalized against GAPDH expression in the hearts to account for any loading differences. Maximum expression of the transgene occurs in line 89; this quantified value was set at 100%. Expression of the transgene varies from ~40% in line 71 to 100% in line 89 (Fig. 2B).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   A: RNA expression in TPM 3 TG mice. Northern blot analyses in hearts from TG and nontransgenic (NTG) mice. Total RNA (15 µg; pooled from 5 mice from each line) was electrophoresed and probed with 32P-radiolabeled TPM 3 3'-UTR, -TM 3'-UTR, or GAPDH. B: graphic representation of the levels of TPM 3 message in TG mice. TPM 3 is not endogenously expressed in the murine heart. Levels from each TG line were compared with the highest expressing line (line 89), which was set at 100%. Three sets of experiments were averaged and normalized against GAPDH expression.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Contractile protein expression in TPM 3 TG mice. A: total myofibrillar proteins in TG and NTG hearts. Total cardiac myofibrillar protein (20 µg) from NTG or TPM 3 TG mice was electrophoresed on SDS-PAGE gels and stained with Coomassie blue dye. TnI, troponin I; MLC-2, myosin light chain-2; TnT, troponin T. B: Western blot analysis of TM protein expression in TG and NTG hearts. Whole cell homogenate (20 µg) was run on 3.4 M urea and 8% SDS-PAGE gels. Blots were probed with the striated TM-specific antibody CH1. Top band is -TM and the lower band is TPM 3 as determined by a bacterially expressed protein (29). C: quantified TM protein levels in TG and NTG hearts. -TM and TPM 3 are shown as the percentage of total striated TM. Results are quantified from 3 different blots. D: Western blot analysis of cardiac TnT from myofibrillar preparations of TPM 3 TG and control mice. SKM, skeletal muscle preparation.

Histological and physiological studies. None of the founder mice or their progeny demonstrates any gross phenotypic alterations or reduced viability. Over 30 TG mice ranging in age from 2 mo to 1 yr were examined for morphological changes. Hematoxylin and eosin-stained heart sections show no signs of hypertrophy, fibrosis, thrombi, necrosis, or any other pathological condition in any of the six lines examined (data not shown). Electronmicrographs also show no disruption of normal sarcomeric structure. Furthermore, no differences in percent heart-to-body weight ratios from the TG to the NTG mice are observed. Thus incorporation of TPM3 into the cardiac myofibers does not lead to morphological or pathological alterations in the sarcomere.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A: Ca2+-force relations of skinned fiber bundle preparations from NTG and TPM 3 TG mouse hearts at sarcomere lengths (SL) 1.9 and 2.3 µm. The log of free [Ca2+] required for half-maximum activation (pCa50) in the NTG fibers (solid lines) at SL 1.9 µm (n = 7, ) was 5.75 ± 0.01 and at SL 2.3 µm (n = 7, ) was 5.98 ± 0.03. In the TPM 3 TG fiber bundle preparations (dashed lines), the pCa50 at SL 1.9 µm was 5.68 ± 0.01 (n = 10, ) and at SL 2.3 µm 5.80 ± 0.02 (n = 10, ). P < 0.05 between SL 1.9 and 2.3 µm in NTG group. Maximum steady-state tension development in skinned fiber bundle preparations from NTG and TPM 3 TG mouse hearts at two different sarcomere lengths. B: maximum steady-state tension development at pCa 4.5 (highest log of free [Ca2+] concentration) in the NTG fibers (open bars, n = 5) at SL 1.9 µm was 59.79 ± 11.92 mN/mm2, and at SL 2.3 µm was tension 80.60 ± 11.85 mN/mm2. In the TPM 3 TG groups (solid bars, n = 7) at SL 1.9 µm tension was 61.50 ± 10.93 mN/mm2, and at SL 2.3 µm was tension 72.01 ± 13.96 mN/mm2. All data are presented as means ± SE; *P value <0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Average myofilament lattice spacing as a function of sarcomere length in skinned cardiac fiber bundles from NTG () and TPM 3 TG () mouse hearts (n = 8) for each group. Relationships were obtained by averaging the data in 0.05-µm wide sarcomere length bins. There was no significant difference in slope between the two groups. HR, heart rate; MAP, mean arterial pressure; LVP, left ventricular pressure. Values are means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Results of experiments reported here provide new and important insights into structure-function relations of TM and their influence on cardiac function. TM has three main striated muscle isoforms: -TM, -TM, and TPM 3. We have employed a transgenic approach to determine the functional significance of these three highly homologous isoforms. Our data are the first to demonstrate that TM isoform switching from -TM to TPM 3 is associated with a decrease in myofilament Ca²⁺ sensitivity and an attenuation of length-dependent activation. Moreover, the change in myofilament activation correlates with hyperdynamic ventricular function. Previous studies indicated that specific thin filament alterations are able to modify length-dependent activation but not in the case of TM isoform switching. We reported that specific and complete switching of slow skeletal troponin I (ssTnI) for cardiac troponin I (cTnI) attenuated length-dependent activation (1). In this case, cardiac myofilaments with ssTnI were significantly more sensitive to Ca²⁺ than controls. Switching of -TM for -TM in TG mice also increased myofilament Ca²⁺ sensitivity but had no effect on length-dependent activation (39).

Mechanisms for length-dependent activation that may be modified by switching of TPM 3 for -TM include alterations in interfilament spacing as well as modulation of the cooperative feedback of strong cross bridges on myofilament activation. As reported in Fig. 5, results from our experiments using X-ray diffraction show that skinned fiber bundles from TPM 3 and control hearts both demonstrate the same change in interfilament spacing as SL is changed. This result indicates that the response of myofilament activation to the change in SL and interfilament spacing must be different in TPM 3 containing myofilaments versus controls.

What are the differences between TPM 3 and -TM that could account for an alteration in cooperative feedback of strong cross bridges on thin filament activation? The feedback effects of strong cross bridges on myofilament activation appear certain to involve the flexibility of TM as well as its interactions with TnT, actin, and with contiguous TMs along the thin filament. Differences in hydrophobic amino acids between -TM and TPM 3 may account for the functional effects of isoform switching. There are 26 amino acid differences between TPM 3 and -TM, of which 15 are highly conservative. Of the remaining 11 amino acid differences, 10 are associated with the gain or loss of a hydrophobic amino acid (Ala³¹Gln, Ser⁴⁵Ala, Gln¹³⁵Leu, Ser¹⁷⁴Gly, Gly¹⁸⁸, Ser, Ser¹⁸⁶Ala, Ala¹⁹¹Ser, Thr¹⁹⁹Asn, Ser²²⁹Thr, and Ser²⁵²Thr), whereas just one polar change is found (Lys²⁹Gln). Seven of the nonconservative amino acid differences between -TM and TPM 3 are present after codon 185 in the TPM 3 molecule, a region that influences thin filament cooperativity and TnT binding. Our hypothesis is that these differences in hydrophobic amino acids alter the structure of TM, thereby altering the transition from myofilament "on" to "off" states as well as "off" to "on" states. Interestingly, functional differences between muscle and nonmuscle TM isoforms include modulation of myosin binding to actin. The difference in myosin binding is apparently due to both steric effects as well as induction of changes in actin structure by both TM and myosin binding (37). The importance of hydrophobic residues in this differential functional effect was pointed out by the work of Brown et al. (2), who suggested that the hydrophobic core consisting of seven alanine clusters is important in the flexibility of the TM that is key in its role in regulation. A mutation (V95A) involving this hydrophobic core has been linked to a penetrant form of hypertrophic cardiomyopathy associated with alterations in thin filament cTnC Ca²⁺ binding and thin filament activation.

Amino acid differences between -TM and TPM3 may also change the stoichiometry of dimer formation and thereby alter myofilament activation by Ca²⁺. For example, TPM 3 may preferentially form dimers with -TM in slow-twitch muscle to modulate its effects, but when expressed in the heart, TPM 3 forms dimers with -TM leading to a unique "hypercontractile" phenotype. Amino acid analysis of the coiled-coil TM structure shows repetitive segments of seven amino acids of which the "first" and "fourth" (a and d positions) are hydrophobic and the "fifth" and "seventh" (e and g positions) are polar (24). Analysis of the -helical repeat of the coiled-coil structure of TM shows two TPM 3 hydrophobic residues are in the "fifth" and "seventh" polar position. These properties may change the stoichiometry of dimer formation. The one polar change observed in TPM 3 (Lys²⁹Gln) located in a "first" hydrophobic position might destabilize the coiled-coil structure and alter TM flexibility in this portion of the NH₂-terminus of TM because evidence shows that a determining factor affecting the stability of TM dimers is the hydrophobicity of the residues at the "first"/"fourth" coiled-coil interface (9). Furthermore, it is known that the NH₂- and COOH-terminal sequences of TM determine its actin affinity and cooperativity (25, 34). The basis for the slight but significant increase in heart rate present in the TPM 3 TG mice is unclear. The role that TM plays in the conduction system is obscure; TPM 3 is a protein that is normally found only in skeletal muscles and may substitute for -TM in the specialized cells of the conduction system. Heart rate differences could be observed in the TPM 3 mice relative to control groups even with increased concentrations of isoproterenol. It is unclear whether these increased heart rates were a primary or secondary effect of the transgene being expressed in the generalized cardiomyocyte population. Interestingly, alterations in heart rate have been reported with cardiac-specific overexpression of another contractile protein, myosin binding protein C (40).

Surprisingly, the TPM 3 physiological phenotype was opposite of what we anticipated. The TM isoforms are highly homologous at the amino acid level: -TM to -TM: 86%; -TM to TPM 3: 87%; and TPM 3 to -TM: 91%. -TM and TPM 3 are predominantly found in slow-twitch muscles, whereas -TM is more prevalent in all types of striated muscle. From the amino acid homologies and expression profiles, we predicted that TPM 3-overexpressing mice would exhibit a physiological profile similar to -TM TG mice but less pronounced due to the higher homology with -TM. However, in the working heart preparations and closed-chest preparations of TPM 3 mice, +dP/dt, and dP/dt are increased, and TPP and the RT_1/2 are decreased. In the -TM mice, there is a decreased dP/dt and an increased RT_1/2. Also, in the -TM mice, there are no changes in contraction parameters with the working-heart preparations, but there is a decrease in maximum shortening velocity of isolated myocytes (39). TPM 3 force/pCa analysis shows a rightward shift in the calcium sensitivity (decreased sensitivity) that was directly opposite the results of the -TM phenotype (which exhibit a leftward shift or increased calcium sensitivity) (29). The opposite results in Ca²⁺ sensitivity correlate with an increase in dP/dt. The apparent steeper slope in the pCa-maximum force curve for the TPM 3 at the 2.3-µm sarcomeric length may account for the increased +dP/dt even though there was a decreased sensitivity to Ca²⁺. To determine whether sarco(endo)plasmic reticulum Ca²⁺ ATPase and phospholamban contributed to this change in calcium response, we measured their protein levels by Western blot analysis. There are no significant differences in these protein levels between NTG and TG mice (data not shown). Although we cannot rule out that isoform changes in contractile or calcium regulatory proteins occurred in the TG mice, no gross quantitative alterations in the levels of the proteins were detected.

In summary, our data show that compared with controls, hearts of mice expressing TPM 3 demonstrate increased contractile and relaxation parameters, decreased myofilament Ca²⁺ sensitivity, and an attenuation of myofilament length-dependent Ca²⁺ activation. These unexpected physiological differences associated with TPM 3 expression may be due to unique properties of this isoform. It is possible that changing the hydrophobicity of amino acid residues alters the secondary structure of the TM, allowing for faster transitions from the "on" state to the "off" state of the myofilament structure, leading to increased basal myocardial function of the TPM 3 TG mice. As seen with -TM point mutations associated with familial hypertrophic cardiomyopathy and dilated cardiomyopathy, it is possible that a few amino acid differences among the TM isoforms can drastically alter various functions (i.e., cooperativity, steric hindrance, blockage of myosin interactions, TM dimer formation, head-to-tail interactions, and interactions with actin and TnT) (16, 21). An important finding of this investigation is that the TG fiber bundles demonstrate an attenuated length-dependent activation. This result indicates an important role for TM structure relations in the variability of near-neighbor interactions along the thin filament of various muscle types (3). Our result also indicates that the mechanism of length-dependent activation involves structure-function relations of TM in addition to current theories that are based on changes in interfilament spacing (6, 7, 15, 22).