INTRODUCTION

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CONTRACTION OF CARDIAC muscle results from the cyclic interaction of actin and myosin under the influence of regulatory thick and thin filament proteins (28). Variable expression of contractile and regulatory protein isoforms in heart has been associated with differences in contractile properties, including Ca²⁺ sensitivity of tension (20, 29), sensitivity to acidosis (14, 16), and velocity of unloaded shortening (5, 24). Velocity of unloaded shortening is closely related to the actin-activated myosin ATPase rate (1, 5) and is thought to reflect the detachment rate of negatively strained cross bridges, i.e., cross bridges bound after the power stroke (10). The elaboration of the crystalline structure of myosin S1 suggests an influence of myosin light chains on the mechanical stability of the myosin head during force development, because the light chains are located at the hinge region between the myosin rod and the globular myosin head (23). In skeletal muscle, velocity of unloaded shortening has been related to alkali light chain isoform expression (7), and phosphorylation of regulatory light chain has been shown to influence the kinetics of tension development (15, 26). In the present study, we employed a transgenic approach to determine the effect of regulatory light chain isoform expression on kinetics of cardiac contraction, specifically on velocity of unloaded shortening in isolated atrial myocytes. A preliminary report of this work was presented in abstract form (4).

	MATERIALS AND METHODS

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Production of transgenic mice. A 700-bp fragment containing the complete coding region of murine ventricular regulatory light chain (MLC2v) cDNA and its 3'-untranslated sequence was ligated to the Sal I site of the cardiac-specific -myosin heavy chain promoter (8, 21). A 240-bp Not I-Hind III fragment containing the SV40 poly(A) signal was also ligated to the 3'-end of the -myosin heavy chain promoter/MLC2v construct to ensure correct 3' processing of the transgene. DNA used in the microinjection was released from the transgene vector by digestion with Sac I-Hind III to generate the linear 6.4-kb fragment, which was isolated and purified as described (8, 21). Purified DNA was microinjected into the pronuclei of single cell embryos derived from superovulated FVB/N females; surviving embryos were then implanted in pseudopregnant foster mothers. Founder mice were identified using PCR as described previously (8, 21). Stable transgenic lines were raised by breeding the founder transgenic mice with nontransgenic cohorts. The copy number of the transgene in each line was then determined by quantitative nucleic acid analysis.

Myocyte isolation. Hearts were excised from transgenic mice and littermate controls after euthanasia with inhaled methoxyflurane in accordance with institutional guidelines. The hearts were placed in ice-cold relaxing solution containing (in mmol/l) 1 free Mg²⁺, 100 KCl, 2 EGTA, 4 ATP, and 10 imidazole (pH 7.0). Atria were separated from ventricles and were then mechanically disrupted using a Polytron homogenizer (Kinematica). The resulting suspensions of cells and cell fragments were centrifuged at 165 g for 120 s, and pellets were then resuspended in 0.3% Triton X-100 for 6 min to permeabilize sarcolemmal, mitochondrial, and sarcoplasmic reticulum membranes. After being washed in fresh relaxing solution, the skinned myocytes were resuspended in relaxing solution and kept at 4°C until use.

Velocity of unloaded shortening in myocytes. On the stage of an inverted microscope, atrial myocytes were attached with silicone adhesive (Dow Corning) to stainless steel pins fastened to the active elements of a force transducer and motor (Cambridge) or piezoelectric translator (Physik Instrumente). After curing of the silicone attachments, atrial myocytes were transferred to relaxing solution, and sarcomere length was adjusted to 2.3 µm using on-line continuous videomicroscopy. Velocity of unloaded shortening was determined as described previously (25) at 15°C in maximally activating solution (pCa 4.5) containing in (mmol/l) 7 EGTA, 1 free Mg²⁺, 4 MgATP, 14.5 creatine phosphate, and 20 imidazole (ionic strength 180 mmol/l, pH 7.0) using the slack-test method. After steady tension was reached in maximally activating Ca²⁺ solution, the preparation was rapidly slackened; the time required to take up the imposed slack was measured as the interval between the beginning of the imposed slack length step and the onset of tension redevelopment. Slack step lengths were restricted to the range of sarcomere lengths in which velocity has been shown to be constant in isolated cardiac myocytes (3, 25, 27). Plots of slack length versus duration of unloaded shortening were included if well fit to a straight line (r  0.95).

Ca²⁺ sensitivity of isometric tension in myocytes. Developed isometric tension was measured at sarcomere length 2.3 µm (15°C) in maximally activating Ca²⁺ solution (pCa 4.5) and submaximal Ca²⁺ solutions (pCa 5.7  pCa 5.1) as described previously (25). Isometric tensions measured at submaximal pCa (P) were expressed as a fraction of maximal (P_o); i.e., P_rel = P/P_o, and were then plotted versus pCa and analyzed by least squares regression using the Hill equation: log[P_rel/(1  P_rel)] = n(log[Ca²⁺]) + k, where n is the Hill coefficient, k is the intercept of the fitted line with the x-axis in pCa units, and [Ca²⁺] is Ca²⁺ concentration. Lines were fit to the tension-pCa curves by inserting constants derived from the above analysis into the following equation: P_rel = [Ca²⁺]ⁿ/(kⁿ + [Ca²⁺]ⁿ), where k denotes the pCa at which relative tension is half-maximal, i.e., pCa₅₀. Data were discarded if maximal tension declined by >15% during the experiment or if mean sarcomere length changed >0.2 µm between isometric relaxed and maximally activating conditions.

	RESULTS

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Expression of the MLC2v transgene. Neither transgenic mice nor their progeny demonstrated gross phenotypic abnormalities nor evidence of increased fetal or neonatal morbidity or mortality when compared with nontransgenic littermates. Expression of the myosin heavy chain promoter/MLC2v gene construct was measured in hearts of transgenic and nontransgenic mice using S1 nuclease and Northern dot-blot analyses. In hearts of transgenic mice, there was a four- to eightfold increase in total myosin light chain 2 (MLC2) RNA compared with nontransgenic littermate controls. Moreover, in atria of transgenic mice, the ratio of MLC2v to atrial regulatory light chain (MLC2a) RNA was ~2:1, whereas in atria of nontransgenic mice, the MLC2v transcript could not be detected. To examine MLC2 protein expression in hearts of transgenic mice, atrial myocyte samples from transgenic and wild-type mice were subjected to 15% PAGE followed by silver staining. As shown in Fig. 1, in atria of transgenic mice, native MLC2a protein expression was replaced by MLC2v protein expression. By densitometric analysis, the ratio of actin to MLC2v in pooled transgenic atrial myocytes was equivalent to the ratio of actin to MLC2a in wild-type myocytes. No significant differences in expression of other contractile or regulatory proteins were evident by comparison of transgenic and wild-type atrial myocyte proteins by gel electrophoresis.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Silver-stained SDS-PAGE (15% acrylamide) of electrophoretically separated cardiac myocyte proteins. Lane 1 (left): nontransgenic wild-type atrial myocytes; lane 2 (right): transgenic atrial myocytes. MHC, myosin heavy chain; TM, tropomyosin; TnI, troponin I; MLC1a, atrial essential light chain; MLC2a, atrial regulatory light chain; MLC2v, ventricular regulatory light chain.

Velocity of unloaded shortening and Ca²⁺ sensitivity of isometric tension in myocytes. The attachment procedure consistently provided low-compliance attachments of atrial myocytes in which sarcomere length could be monitored while relaxed and during maximal activation (Fig. 2). The slack-test procedure yielded reproducible linear plots of length step amplitude versus duration of unloaded shortening for step sizes between 16 and 22% of myocyte length (Fig. 3). As shown in Table 1, the velocity of unloaded shortening of MLC2v-expressing transgenic atrial myocyte preparations was greater than wild-type myocytes, 3.88 ± 1.19 (mean ± SD) versus 2.51 ± 1.08 muscle lengths/s, respectively (P < 0.05 by unpaired Student's t-test). No differences of peak isometric tension or Ca²⁺ sensitivity of tension were evident in comparisons of transgenic and wild-type myocytes (Fig. 4); the pCa₅₀ of transgenic (n = 8) and wild-type (n = 8) atrial myocytes was 5.40 ± 0.12 and 5.35 ± 0.07, respectively; Hill coefficients of transgenic and wild-type atrial myocytes were 3.56 ± 1.01 and 3.50 ± 0.51, respectively, and peak isometric tension of transgenic and wild-type atrial myocytes was 4.07 ± 1.43 and 3.52 ± 1.64 kN/m², respectively.

View larger version (159K): [in this window] [in a new window]   Fig. 2.   Photomicrograph of an MLC2v-expressing transgenic atrial myocyte preparation while relaxed in pCa 9.0 solution (A) and during maximal activation in pCa 4.5 solution (B). Scale bar is 25 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Method of determining unloaded shortening velocity. A: expanded force vs. time records obtained during slack releases of 17, 20, and 22% of myocyte length. Duration of unloaded shortening is represented by horizontal line from the beginning of the imposed slack release to the onset of tension redevelopment (arrows). B: corresponding superimposed length vs. time records of slack releases of 17, 20, and 22% of myocyte length. C: duration of unloaded shortening vs. size of all slack releases for a single atrial myocyte preparation; in this preparation, unloaded shortening velocity was 1.95 muscle lengths/s (r = 0.98).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Ca2+ sensitivity of isometric tension of MLC2v-expressing transgenic atrial myocytes () and nontransgenic controls (). No differences in pCa50 (5.40 ± 0.12 vs. 5.35 ± 0.07) or Hill coefficient (3.56 ± 1.01 and 3.50 ± 0.51) were evident.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, the influence of regulatory light chain isoform expression on velocity of unloaded shortening was determined in atrial myocytes. Using a transgenic approach to express ventricular light chain in atrium resulted in increased velocity of unloaded shortening of transgenic atrial myocytes compared with wild-type atrial myocytes. Previous studies have demonstrated that regulatory light chain influences kinetics of contraction in skeletal muscle. Myosins devoid of regulatory light chain move actin filaments at only 35% the velocity of native myosin in an in vitro motility assay (13). Furthermore, in skinned skeletal muscle preparations, partial extraction of regulatory light chain reduces unloaded shortening velocity (9, 19). Thus the results of the present study extend to cardiac muscle the important conclusion that the regulatory light chains influence the kinetics of contraction.

Expression of MLC2a and MLC2v is normally chamber specific beyond day 12 of embryonic development in the mouse (11, 12). The amino acid sequences of MLC2a and MLC2v, shown in Fig. 5, demonstrate 58% identity and 77% homology between the two isoforms. The regions of greatest differences between the aligned sequences of the isoforms are found at residues 62-74 and 109-121 of MLC2v corresponding to residues 58-70 and 105-117 of MLC2a, located at the junctions of helices C and D and helices E and F, respectively (23, 30). Considering the location of regulatory light chain at the hinge region of the myosin backbone and head (23), regional charge or secondary structural differences between regulatory light chain isoforms may influence cross-bridge interactions by affecting movement of cross-bridge heads away from the thick filament backbone. Such a mechanism has been proposed to account for the increased extent and rate of force development observed in skeletal muscle upon phosphorylation of regulatory light chain (15, 26), although no effects of phosphorylation on velocity of unloaded shortening have been observed. Recent data suggest regulatory light chain phosphorylation may influence cross-bridge kinetics in cardiac muscle as well; Morano et al. (17) reported a correlation between regulatory light chain phosphorylation and the rate of tension development in porcine ventricular fibers after photolytic release of ATP. The results of the present study indicate that regulatory light chain isoforms influence the turnover kinetics of cross bridges in myocardium. The regions of isoform sequence divergence responsible for the observed differences in kinetics may be further investigated using techniques of site-directed mutagenesis.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Amino acid sequence comparison of MLC2v and MLC2a (see Ref. 11). Dashes designate identical amino acids; asterisks indicate gaps in sequences for alignment for maximal homology.

The greater shortening velocity of transgenic atrial myocytes expressing MLC2v is consistent with greater power-generating requirements of ventricular compared with atrial tissue. Because power is the product of force and velocity, the observed greater velocity of transgenic atrial myocytes suggests that the MLC2v isoform may contribute to the greater power-generating capabilities of ventricular tissue. The results of the present study differ somewhat with the previous observation that maximum shortening velocity of atrial trabeculae of hyperthyroid rats is greater than that of ventricular trabeculae (2). Because both the regulatory and essential light chains differ between atrium and ventricle, the observations in hyperthyroid rats may reflect a dominating effect of atrial essential light chain to increase velocity. Consistent with this observation, recent reports have described increased maximal velocity of shortening of human ventricular tissue from patients with pressure-overloaded right ventricles expressing atrial essential light chain (18) and increased velocity of actin translocation by ventricular myosin from patients with hypertrophic cardiomyopathy containing mutations of the essential light chain (22). By avoiding potentially confounding metabolic influences of hyperthyroidism and hemodynamic influences of pressure overload and hypertrophy in previous studies, the present study has directly shown that the isoform of regulatory light chain expressed in myocardium is an independent determinant of the kinetics of cardiac contraction.