INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MYOSIN IS THE MAJOR MOTOR PROTEIN in the contractile apparatus of all muscle types and cyclically interacts with the thin (actin) filament to generate force (see reviews, Refs. 33 and 35). In the heart, myosin cross-bridge action represents an important process in determining cardiac systolic and diastolic function. Cardiac myosin consists of two heavy chains (MYHC), - and -MYHC, and each heavy chain is associated with two types of light chains (MLC), the regulatory myosin light chain (RLC) and essential (ELC) myosin light chain. X-ray crystallographic analyses demonstrated that both ELC and RLC are associated with the neck region of MYHC (30, 31), and removing MLC from the chicken skeletal muscle myosin reduces the velocity of actin filament movement by 90% without significant loss of the myosin ATPase activity in an in vitro motility assay (18). Furthermore, RLC removal had little effect on isometric force, whereas ELC removal reduced the isometric force by over 50% (41). Mutations in either light chain can lead to cardiovascular disease (8, 42). These data all suggest that the MLCs play an important role in normal cardiac function and in the manner in which force is generated during the myosin-actin cross-bridge cycle.

In the mammalian heart, atrial (a)- and ventricular (v)-specific isoforms exist for both ELC and RLC. ELC1v and RCL2v are ventricular-specific light chains that are also expressed in adult slow skeletal muscle (35). Unique functional roles for the cardiac compartment specific expression of MLC isoforms have not been fully described, but in hyperthyroid rats, the cross-bridge kinetics of atrial fibers are faster than that of ventricular fibers, despite having the same MYHC composition (2). Recently, via transgenesis, an ELC1vELC1a transition was effected and resulted in altered kinetics of force development as well as changes in whole organ function (9). Thus there is the possibility that the unique MLC isoform compositions of the different cardiac compartments play a role in modulating the cross-bridge kinetics of the ventricles and atria. Consistent with the idea that the MLCs play an important role are data obtained from human clinical populations. For example, in human congenital heart disease, such as tetralogy of Fallot, double outlet right ventricle, infundibular pulmonary stenosis, and dilated cardiomyopathy, partial ELC isoform transitions occur in the ventricle (23). On the other hand, an increase in RLC2v protein is observed in human atria tissue with various cardiomyopathies (5, 17), and these changes along with others, such as alterations in troponin T splicing or troponin-I phosphorylation (1, 44), could underlie functional parameters of the heart. The mechanistic pathways underlying these changes, as well as their functional significance, are not clear, but it is likely that this response is part of a direct compensatory or adaptive mechanism as the heart attempts to maintain normal cardiac function in the face of a primary pathology.

In an attempt to more directly establish the structure-function relationships that may underlie MLC compartment specific expression, we recently produced transgenic (TG) mice that overexpress RLC2v, ELC1v, and ELC1a in a cardiac-specific manner (9, 14, 27). Although large increases in TG mRNAs were observed in both atria and ventricles, no differences were observed in total MLC protein levels, indicating that contractile protein stoichiometry was rigorously conserved. High levels of ectopic expression of RLC2v or ELC1v in the atria resulted in essentially complete replacement of the respective atrial isoforms with the ventricular species. Similar results were observed in the ventricles of mice in which ELC1a was expressed (9). This approach gives one the ability to study the consequences of the primary isoform switch as well as its sequelae on cardiac structure and function.

In this study we tested the hypothesis that MLC isoform replacement leads to changes in fiber cross-bridge kinetics. A comprehensive survey at the fiber level was carried out in TG mice exhibiting ELC1v  ELC1a, ELC1a  ELC1v, and RLC2a  RLC2v isoform shifts. We were also interested in determining the long-term effects, if any, of MLC isoform replacement, particularly in terms of overt cardiac remodeling or development of hypertrophy over the animal's lifetime. Maximum shortening velocity, maximum relative power (W), and unloaded shortening velocity (v) of both atrial and ventricular skinned fibers were examined. The data show that fiber kinetics are not affected when TG-encoded protein is expressed in its endogenous compartment but that ectopic replacement invariably leads to changes in the fiber's cross-bridge kinetics.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transgenic mice. FVB/N mice with cardiac specific overexpression of RLC2v (27), ELC1v (14), and ELC1a (9) and nontransgenic (NTG) controls were used in this study. Except where specifically noted, all mice were studied between 10 and 13 wk of age, and an equal distribution of male and female mice were used in each experimental group. TG mice were identified by PCR analysis of genomic DNA isolated from tail clips.

Fiber isolation and analyses. Each mouse was heparinized (500 IU/kg ip) and euthanized with CO₂. After a thoracotomy was performed, the heart was rapidly removed and placed in relaxing solution [5.37 mM ATP, 30 mM phosphocreatine, 5.0 mM EGTA, 20.0 mM N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (BES), 7.33 mM MgCl₂, 0.12 mM CaCl₂, 10 mM dithiothreitol, 10 µg/ml leupeptin, and 32 mM potassium methanesulfonate, pCa 8.0, pH 7.0, ionic strength 175 mM] at 4°C. The solution contained 30 mM of 2,3-butanedione monoxime (BDM) to protect the myocardial tissue from cutting injury (25, 38). Small sections of either left ventricular papillary muscles or left atrial trabeculae were dissected in the BDM-relaxing solution to yield 0.5-mm diameter and 2- to 3-mm long strips. The strips were skinned by incubation in 5.5 mM ATP, 5.0 mM EGTA, 20.0 mM BES, 6.13 mM MgCl₂, 0.11 mM CaCl₂, 10 mM dithiothreitol, 10 µg/ml leupeptin, 121.8 mM potassium methanesulfonate, (pCa 8.0, pH 7.0, and ionic strength 175 mM) and 50% glycerol containing 0.5 wt/vol% Triton-X 100 for 12 h at 4°C. Strips were transferred into the same solution but without 0.5 wt/vol% Triton-X 100 and stored at 20°C. The skinned fiber experiments were performed using a commercially available apparatus (Scientific Instruments, Heidelberg, Germany). Fibers were dissected into bundles of 125-175 µm diameter, 1.3- to 2.0-mm long, under a dissecting microscope and mounted isometrically between a force transducer (KG3, Scientific Instruments) and a length-step generator in relaxing solution. Sarcomere length at resting tension was always 2.1 µm as detected by laser diffraction analysis. The contraction solution had the same composition as the relaxing solution except that EGTA was substituted with 5 mM Ca-EGTA. The free Ca²⁺ concentration was obtained by mixing the relaxing and contraction solution in the appropriate proportions. Strip tension (mN/mm²) was calculated by dividing force by fiber cross-sectional area, calculated from widths measured at the major axis. Activating solution had the same ionic composition as relaxing solution. The solutions were formulated by solving a set of simultaneous equations describing the multiple equilibrium of ions in the solutions (10).

Force-velocity relationship and unloaded velocity. Force-velocity relationships were determined by isotonic quick release at pCa 5.0 at 23°C. Load clamping for isotonic shortening was achieved by changing the mode of operation from length control to force control during isotonic steady-state force. Force values were measured by averaging the records only from 10 to 25 ms after each step to an isotonic load. During this interval, although steady-state shortening is not achieved, force remains relatively constant. The shortening velocity at a given force was fit to a hyperbola of the following form: (F + a)(v + b) = b(F₀ + a), where F is the force during isotonic shortening, F₀ is isometric force, v is the isotonic shortening velocity, and a and b are constants. This relationship was fitted using a commercially available program (Scientific Instruments) and the curve relative power (W) was calculated for each fiber (W = v · F/F₀). Unloaded shortening velocity at pCa 5.0 at 23°C was obtained by the slack-test method (7, 24) with minor modifications. Release amplitudes introduced to one end of a fiber varied between 5% and 13% of initial muscle length (L). Release abolished tension completely. After the onset of tension recovery, the fiber was restretched to the initial muscle length. Slack time was defined as the time between the completion of the length step and the moment at which force began to redevelop. L was plotted versus slack time (t) and a linear regression fit to the relationship. The slope of the regression line provided the measure of unloaded shortening velocity, which could be compared with the maximum velocity estimated from the fit to the force-velocity relationship.

Myofibrillar Ca²+-stimulated Mg²⁺-ATPase activity. Purified myofibrils (20) were obtained from appropriate amounts of atrial and ventricular tissue (~50 mg protein corresponding to 1 ventricle or 10 atria). Myofibrillar Mg²⁺-ATPase activity was determined in incubation medium (in mM: 66 KCl, 60 imidazole, 6 MgCl₂, 5 EGTA, and 5.33 ATP at pH 7.0). The 5-min reaction was initiated with the addition of the sample and stopped with 15% trichloroacetic acid. The reaction mixture was centrifuged, and P_i produced by ATP hydrolysis was measured. Ca²⁺-stimulated Mg²⁺-ATPase activity was determined by subtracting the activity at pCa 5.0 from the activity at pCa 8.0.

Statistics. All values are expressed as means ± SE. The significance of the differences between TG and NTG mice was determined by Student's t-test (P < 0.05).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All cDNAs used in these studies were placed into the -MYHC promoter cassette, which contains the full-length -MYHC transcriptional apparatus upstream and a human growth hormone or SV40 polyadenylation signal downstream of the insertion site of cDNA (Fig. 1). In the present study, we examined a single line from each of three different TG constructs: RLC2v (line 97) (27), ELC1v (line 66) (15), and ELC1a (line 34) (9). An additional construct, consisting of a full-length -MYHC cDNA, was also made and is described more fully below. In each of these lines, no gross phenotypic abnormalities could be discerned, and we have noted no evidence of increased fetal, neonatal, or adult morbidity or mortality when compared with NTG littermates. In the lines carrying the light chain transgenes, SDS-PAGE analyses indicate that normal protein stoichiometry was conserved in both atria and ventricles but that substitution of the endogenous isoform with the relevant TG-encoded protein is essentially complete (9, 15, 27). Additionally, all muscle strips used for the fiber studies had no apparent histological changes or fibrosis.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the constructs used to generate the transgenic (TG) animals. MyHC, myosin heavy chain; Essential MyHC (ELC)1a, ELC1v, regulatory MyHC (RLC)2v, and -MYHC cDNAs were each linked to the 5.5-kbp -MYHC promoter containing the first three noncoding exons (grey boxes) of the -MYHC transcript. Immediately after the inserted cDNAs, polyadenylation signals are derived from either the human growth hormone (hGH) gene or SV40. The relative lengths of the DNAs used are not drawn to scale. The lines and the degree of substitution with the transgenically encoded protein have been described previously (9, 15, 27). a, Atria; v, ventricular.

Because the purpose of this study was to determine the primary effect of isoform substitution on the mechanics and kinetics of the fibers in the absence of any secondary compensatory effects due to hypertrophy, animals were used as soon as isoform substitutions had reached steady state (10-13 wk). Normalized adult heart weights showed that no hypertrophy was present in either the atria or ventricles of any of the lines with the exception of very mild atrial hypertrophy in the hemagglutinin (HA)-tagged construct (data not shown). No activation of the fetal gene program, a sensitive marker for the hypertrophic response, was detected (data not shown).

Fiber preparations from TG and NTG ventricles and atria. The studies below depended on isolation of fibers with reproducible geometries, both within single preparations of atria and ventricles, as well as between multiple mice. Careful but rapid dissection of the respective tissue under low magnification using a dissecting microscope, as well as precise trimming such that reproducible fiber geometries were obtained, resulted in acceptable levels of variation between experimental trials and excellent conservation of internal structure. Sarcomere striations were easily visible in these skinned preparations and appeared uniform throughout the fiber (data not shown). We wanted to compare data obtained from the murine fibers with previous data obtained from other rodent preparations, and a comprehensive set of analyses was carried out using NTG hearts. Sets of tracings obtained from left ventricular skinned fibers for the force-velocity relationship and slack test (data not shown) demonstrated values that were well within published parameters (13, 21, 32). As described in MATERIALS AND METHODS, although steady-state shortening was not achieved, during the short time interval (10-25 ms), force remained relatively constant. The mechanical analyses, carried out on skinned fiber strips isolated from NTG left atrial trabeculae and left ventricular papillary muscles, underscore the unique characteristics of the different fibers (Fig. 2). Although atrial isometric force is lower, the maximum and unloaded shortening velocities of the atrial fiber were faster without noticeable differences in ATPase activity (Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Mechanical analyses of non-TG (NTG) skinned fibers from left atria and ventricles, and mechanical analysis of skinned fiber strips from left atrial trabecula and left ventricular papillary muscles in NTG mice. A: force-velocity relationship; B: force-relative power relationship; C: slack test. Parameters of the atrial fibers were as follows: maximum shortening velocity 3.2 ± 0.1 m · l · s1, maximum relative power 0.57 ± 0.05 m · l · s1, and unloaded shortening velocity 4.4 ± 0.2 m · l · s1. Parameters of the ventricular fibers were as follows: maximum shortening velocity 2.3 ± 0.2 m · l · s1, maximum relative power 0.44 ± 0.03 m · l · s1, and unloaded shortening velocity 3.1 ± 0.3 m · l · s1. Significant differences between the atrial and ventricular fibers are present in the maximum shortening velocity, maximum relative power, and unloaded shortening velocity (P < 0.05).

Fiber mechanical analyses from TG hearts. RLC2v expression in both atria and ventricles of adult hearts leads to no noticeable perturbation at the protein level in the ventricle where RLC2v is normally expressed. However, replacement of the endogenous atrial isoform with the TG-encoded protein was essentially complete with normal stoichiometric relationships being conserved (27). We wanted to determine the effects of overexpression in both the atria and ventricles. In the ventricle, it is formally possible that by merely driving high levels of TG transcription [RNA levels of RLC2v are eightfold higher in this particular line (27)], phenotypic consequences could present. Mechanical analyses were first carried out using fibers derived from NTG littermates: left atrial trabeculae or left ventricular papillary muscle were used. The isometric force of the atrial fibers was 5.9 ± 0.3 versus 9.9 ± 0.6 mN/mm² for the ventricular fibers (P < 0.05). Although atrial isometric force was lower, maximum shortening velocity, maximum relative power, and the unloaded shortening velocity were all higher compared with the ventricular fiber (Fig. 3). These data, along with data from the other two TG mice analyzed in this study, are summarized in Table 2.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Mechanical analyses of skinned fiber strips from left atrial trabeculae (A-C), RLC2v TG left ventricular papillary muscles (D-F; closed circles and squares), and NTG mice (open circles and squares). A, D: force-velocity relationship; B, E: force-relative power relationship; C, F: slack test. Parameters of the atrial fibers in the RLC2v mice were the following: maximum shortening velocity 2.4 ± 0.1 m · l · s1, maximum relative power 0.49 ± 0.03 m · l · s1, and unloaded shortening velocity 2.7 ± 0.2 m · l · s1. Parameters of the NTG atrial fibers were the following: maximum shortening velocity 3.2 ± 0.1 m · l · s1, maximum relative power 0.57 ± 0.05 m · l · s1, and unloaded shortening velocity 4.4 ± 0.2 m · l · s1. Significant differences (P < 0.05) presented in the maximum shortening velocity and unloaded shortening velocity between TG and NTG fibers in the (ectopic) atrial compartment. Parameters of the ventricular fibers in the RLC2v mice were the following: maximum shortening velocity 2.4 ± 0.1 m · l · s1, maximum relative power 0.48 ± 0.02 m · l · s1, and unloaded shortening velocity 3.3 ± 0.2 m · l · s1. Parameters of the ventricular fibers in NTG mice were the following: maximum shortening velocity 2.3 ± 0.1 m · l · s1, maximum relative power 0.44 ± 0.03 m · l · s1, and unloaded shortening velocity 3.1 ± 0.3 m · l · s1. No significant differences presented between TG and NTG ventricular fibers.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Mechanical analysis of skinned fiber strips from left atrial trabeculae (A-C), left ventricular papillary muscles (D-F) in ELC1v (closed circles and squares), and NTG mice (open circles and squares). A, D: force-velocity relationship; B, E, force-relative power relationship; C, F: slack test. Parameters of the atrial fibers in the ELC1v mice were the following: maximum shortening velocity 2.4 ± 0.1 m · l · s1, maximum relative power 0.50 ± 0.03 m · l · s1, and unloaded shortening velocity 2.7 ± 0.3 m · l · s1. Parameters of the atrial fibers in NTG mice were the following: maximum shortening velocity 2.9 ± 0.1 m · l · s1, maximum relative power 0.62 ± 0.02 m · l · s1, and unloaded shortening velocity 3.8 ± 0.2 m · l · s1. In the atrial compartment, maximum shortening velocity, maximum relative power, and unloaded shortening velocity differed significantly (P < 0.05) between the TG and NTG fibers. Parameters of the TG ventricular fibers were the following: maximum shortening velocity 2.4 ± 0.1 m · l · s1, maximum relative power 0.49 ± 0.02 m · l · s1, and unloaded shortening velocity 3.1 ± 0.2 m · l · s1. Parameters of the NTG ventricular fibers were the following: maximum shortening velocity 2.4 ± 0.1 m · l · s1, maximum relative power 0.47 ± 0.02 m · l · s1, and unloaded shortening velocity 3.1 ± 0.1 m · l · s1. No significant differences presented.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Mechanical analysis of skinned fiber strips from left atrial trabeculae (A-C), left ventricular papillary muscles (D-F) in ELC1a (closed circles and squares), and NTG mice (open circles and squares). A, D: force-velocity relationship; B, E: force-relative power relationship; C, F; slack test. Parameters of the TG atrial fibers were the following: maximum shortening velocity 2.8 ± 0. m · l · s1, maximum relative power 0.61 ± 0.03 m · l · s1, and unloaded shortening velocity 3.8 ± 0.1 m · l · s1. Parameters of the NTG atrial fibers were the following: maximum shortening velocity 2.7 ± 0.1 m · l · s1, maximum relative power 0.60 ± 0.03 m · l · s1, and unloaded shortening velocity 3.9 ± 0.2 m · l · s1. In the atria, no significant differences were present. Parameters of the TG ventricular fibers were the following: maximum shortening velocity 2.6 ± 0.1 m · l · s1, maximum relative power 0.53 ± 0.01 m · l · s1, and unloaded shortening velocity 3.8 ± 0.1 m · l · s1. Parameters of the NTG ventricular fibers were the following: maximum shortening velocity 2.3 ± 0.1 m · l · s1, maximum relative power 0.45 ± 0.02 m · l · s1, and unloaded shortening velocity 3.0 ± 0.1 m · l · s1. In the ventricular compartment, maximum shortening velocity, maximum relative power, and unloaded shortening velocity differed significantly (P < 0.05) for the TG and NTG fibers.

Construction of a TG mouse with significantly reduced cardiac power output. Changes in cardiac mechanics, kinetics, and/or power are often invoked as playing a primary role in triggering an initial hypertrophic response. However, despite significant modulation of these parameters in the TG lines, no hypertrophy occurred. We wanted to determine whether a more significant deficit in motor kinetics and fiber mechanics would necessarily result in hypertrophy. To this end, an HA epitope tag (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) was inserted after the methionine initiator codon in a full-length -MYHC cDNA. The cDNA was placed in the promoter construct (Fig. 1) and used to produce multiple lines of TG mice. A line was chosen in which ~50% of the total -MYHC protein consisted of the TG-encoded polypeptide. As was the case for the MLC overexpressors, no perturbation in myofibril stoichiometry was detected despite a twofold increase in -MYHC transcript (data not shown). We hypothesized that this tag would sterically interact with ELC, slow velocity, and decrease power. The data show that this indeed is the case (Fig. 6). The HA tag impacted significantly on the normal shortening velocity of the fiber, force-relative power relationships, and ability to translocate actin filaments, as measured using the in vitro motility assay (11, 26). A similar construct lacking the HA tag was also made and used to produce multiple lines of TG mice. Despite levels of expression similar to the HA-tagged construct, no effects on either fiber kinetics or mechanics could be detected. In addition, despite these deficits, no changes in cardiac function, as measured using the isolated working heart preparation, were observed when 7-mo-old mice were analyzed (data not shown). Even after 1 yr, no statistically significant ventricular hypertrophy (e.g., Fig. 7) or changes in Mg²⁺-ATPase activity (Table 1) could be detected.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Hemagglutinin (HA)-tagged -MYHC. Mechanical analysis of skinned fiber strips from TG and NTG left ventricular papillary muscles (closed squares and open squares, respectively). A: force-velocity relationship, B: force-relative power relationship, C: slack test; D: in vitro myosin motility assay. The bar graph indicates the velocities of actin filaments (µm/s). Parameters of the NTG ventricular fibers were the following: maximum shortening velocity 2.4 ± 0.1 m · l · s1, maximum relative power 0.50 ± 0.02 m · l · s1, unloaded shortening velocity 4.0 ± 0.2 m · l · s1, and filament velocity 5.97 ± 0.13 µm/s. Parameters of the TG ventricular fibers were the following: maximum shortening velocity 2.1 ± 0.1 m · l · s1, maximum relative power 0.31 ± 0.03 m · l · s1, unloaded shortening velocity 2.9 ± 0.3 m · l · s1, and filament velocities 4.92 ± 0.12 µm/s. Maximum shortening velocity, maximum relative power, unloaded shortening velocity, and myosin motility all differed significantly (P < 0.05) between the TG and NTG fibers.

View larger version (92K): [in this window] [in a new window]   Fig. 7.   HA-TG and NTG hearts. Shown are hearts of a HA-tagged MYHC TG mouse, along with its NTG littermate. Despite the deficits in power production, no apparent hypertrophy or overt remodeling has occurred. LA; left atrium.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cardiac-specific overexpression of the various MLCs induced the desired isoform switches in the ectopic compartments. In previous studies, we did not see any fibrosis or sarcomere disarray in any of the lines used. The -MYHC promoter is highly active in both atria and ventricles after birth (34). However, cardiac contractile protein stoichiometry is highly regulated, and the contractile apparatus is unperturbed in the compartment containing the isoform that corresponds to the TG protein. Thus we were unable to detect any differences in either fiber mechanics or kinetics in the ventricles of the RLC2v or ELC1v mice or in the atria of the ELC1a transgenics. These results support the idea that the changes in cross-bridge kinetics are due to the ectopic MLC isoform switches and do not reflect secondary nonspecific effects. The heavy chain isoform is identical in mouse atria and ventricles, although the overall architectures differ. Atrial myocytes contain fewer myofibrils than ventricular myocytes (12), and this may be the structural basis for the lower tension observed in this compartment. In addition, myofibrillar and cellular organization are less ordered in the atria (3, 12). The ability of the atrial myocardium to shorten faster than that of the ventricular myocardium has been observed previously in several mammalian species, including dogs (40), pigs (37), humans (21), and rats (3). Our data are consistent with these results, indicating that limitations in preparing and analyzing the murine fibers were not a confounding factor. The data strongly support the hypothesis that an isoform of MLC can help regulate the actomyosin cross-bridge cycling rate, and the different MLC isoforms partially underlie the different contractile profiles of the atrium and ventricle.

The cross-bridge kinetics of the TG mice provide some potential insight into the role the MLCs play. In the atrial fibers of the RLC2v transgenics, slower maximum and unloaded shortening velocities were observed. In the atrial fibers of the ELC1v mice, both shortening velocity and relative power were reduced. On the other hand, faster maximum and unloaded shortening velocities, as well as an increase in relative power, were observed in ventricular fibers of ELC1a transgenics. The data suggest that, all things being equal, the ventricular MLC isoforms slow cross-bridge kinetics in atrial fibers, whereas atrial MLC isoforms speed the kinetics in ventricular fibers.

Lowey and co-workers (19), using reconstituted, in vitro systems, showed that MLC removal from skeletal muscle myosin reduced the velocity of actin filament movement by 90% without a significant loss in myosin ATPase activity. Consistent with these data, the TG experiments show that MLC isoform switching altered the cross-bridge kinetics without significant changes in ATPase activity. The myosin head consists of a motor domain and an extended long -helix region to which the MLCs bind and presumably help stabilize (29, 30). This neck region mainly serves as a lever, through which small displacements that occur in the motor domain are amplified. In striated muscle, the globular head region of myosin can function as an actin-activated ATPase and minimal motor, but the region containing the MLCs is important for the transduction of the energy that is generated by ATP hydrolysis into rapid movement (36). The different cross-bridge kinetics between the tissue-specific MLC isoforms may represent different efficiencies in the conversion of chemical energy into movement.

The production of a heart with significant deficits in force production (Table 2), but no ventricular hypertrophy, is illuminating, and the data indicate that a decrease in power does not constitute sufficiency for activation of a (the) hypertrophic signaling pathway(s). The lack of a hypertrophic response may be related to the relatively small changes we observed in fiber mechanics, with the magnitude of the deficits simply not being large enough to activate the requisite pathways. Inserting the HA epitope at the amino-terminal region of the MYHC protein produced a myosin that exhibited sizable reductions in velocity and force generation at the molecular and fiber levels (Fig. 6). X-ray studies indicate that the ELC is proximal to the hypervariable amino-terminal region of MYHC and that their interaction influences cross-bridge cycling events (6). During the power stroke phase, in which actin movement occurs, myosin undergoes a conformational bending, allowing ELC to interact with a distal site on the myosin head and thus entrapping the amino-terminus within the hinged region. According to this model, the size of the amino-terminal region dictates the extent to which bending occurs and any compromise in bending would ultimately result in slower motility and possibly force production.

The relatively hydrophilic HA epitope consists of a short peptide of nine amino acids that are believed to adopt a loop conformation. This may compromise cross-bridge cycling through steric hindrance of myosin bending during the power stroke. Alternatively, the effects on cross-bridge cycling could well be determined by the very nature of the ELC-amino-terminus interaction. ELC plays a critical role in myosin function and its removal results in a substantial reduction in actin motility (19, 39) and isometric force production (41). Should the amino-terminus stabilize the ELC, the presence of the HA epitope could interfere with this relationship, giving rise to the impaired kinetics. That no hypertrophy results, even when cardiac power is reduced, is interesting. Whereas unlikely based on the observations from other models (43), it is formally possible that the deficit is within tolerable limits and when the atria and ventricles are similarly affected, no hypertrophy results. For example, when the values for relative power are used to determine absolute power, the calculation shows that the TG ventricle produces 32% of its normal value. The atrium normally operates at 20% below the ventricular power value so that, if the atrium is used as a standard, the HA tag causes only a 17% deficit below normal atrial levels.

A subset of these data are at odds with previous data in which the rate of shortening and relengthening was measured in single cardiomyocytes derived from the atria and ventricles of the RLC2v TG mice (4, 28). Those data reported that the shortening velocity of TG RLC2v atrial myocytes was faster compared with that of NTG controls. The reason(s) for the different results between myocytes and skinned fibers in these mice is not readily apparent. Using skinned fibers, we can directly examine the contractile apparatus, excluding confounding factors such as alterations in calcium handling and protein phosphorylation (16, 22). We have also noted the rather arbitrary process involved in choosing cardiomyocytes for measurement from the general population of isolated cells: cells are selected on the basis of their overall morphology and clarity of the striations. It may be that this selection results in a nonrepresentative pool of cells, and the data generated do not accurately reflect the in vivo behavior of the myofibrils overall. Alternatively, changes in the external matrix could influence the kinetics of the fibers and mechanics, and whereas these changes would more accurately reflect the behavior of the fibers within the milieu of the heart, they would not be reflected in the kinetics of cardiomyocyte shortening and relengthening. Finally, it is possible that compensatory phenomena are being selected as the colony cycles through multiple generations. The cardiomyocyte studies were performed using mice that were bred during an initial expansion of the colony, approximately four years ago, whereas the fiber studies were done on mice bred within the last 6 mo. We have noticed that our colonies, even though they are carried on inbred strains, undergo subtle changes over the years. For example, in mice carrying a targeted cystic fibrosis mutation, survival rates for the colony have increased 10-fold over a 28-mo breeding period, and clearly, the severity of the disease, as measured by early mortality, is being attenuated (J. Whitsett, personal communication). In any case, the fiber data emphasize the importance of subjecting the contractile apparatus to multiple analytical modalities, at the molecular, biochemical, cellular, fiber, and whole heart levels.