Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, FL 33136, USA
* Author for correspondence (e-mail: dszczesna{at}med.miami.edu)
Accepted 13 May 2005
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Summary |
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Key words: Hypertrophic cardiomyopathy, Myosin regulatory light-chain, Muscles, Physiology
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Introduction |
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In this report, we have examined the physiological consequences of the E22K mutation in skinned papillary muscle fibers isolated from transgenic mice expressing the E22K mutation of human cardiac RLC. The current study is a continuation of our previous work where we showed that the bacterially expressed E22K mutant of human cardiac RLC had dramatically altered Ca2+ binding- and phosphorylation-properties compared with the wild type (WT) RLC (Szczesna et al., 2001). Specifically, we demonstrated that the E22K mutation, which is localized in the proximity of the RLC phosphorylation site (Ser15) and its Ca2+ binding site (residues 37-48), prevented phosphorylation of RLC and decreased its affinity for Ca2+ by about 20-fold (Szczesna et al., 2001
). Moreover, our recent study utilizing E22K-reconstituted skinned porcine muscle preparations showed a slight increase in the Ca2+ sensitivity of force and myofibrillar ATPase activity compared to WT-reconstituted preparations (Szczesna-Cordary et al., 2004
). The current work supports our previous results and demonstrates that this E22K mutation when overexpressed in mouse cardiac muscle, increases Ca2+ sensitivity of myofibrillar ATPase activity and steady-state force by about
pCa50
0.1. Previous and current results suggest that the FHC-associated perturbations of the RLC Ca2+ binding site that lead to alterations in the Ca2+-dependent ATPase/force could be responsible for the E22K-linked pathogenesis of FHC. This supports our hypothesis that an intact Ca2+ binding site in RLC is important in the regulation of cardiac-muscle contraction in the normal and diseased state of the heart (Szczesna, 2003
). Moreover, we have examined the gross morphology of the transgenic (Tg)-E22K hearts that seemed to recapitulate the human phenotype of FHC associated with this RLC-E22K mutation (Kabaeva et al., 2002
; Poetter et al., 1996
). Longitudinal sections of hematoxylin and eosin stained whole hearts of 13-month-old Tg-E22K animals showed enlarged inter-ventricular septa and papillary muscles compared with Tg-WT and/or Non-Tg littermates. However, no cardiac hypertrophy was found by echocardiography examination or judging by the heart weight to body weight ratios.
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Materials and Methods |
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Analysis of protein expression
About 10 mg of left ventricle tissue from transgenic myc-E22K, myc-WT and Non-Tg were minced in a solution of 1% SDS, 1% ß-mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 1 µl/ml protease inhibitor cocktail (Sigma), homogenized, clarified by centrifugation 18,000 g for 10 minutes and quantitated by Pierce Coomassie-Plus Assay (Fig. 1A). The extracts were loaded at 5-20 µg per lane and run on 15% SDS-PAGE for Coomassie staining, while about 0.5-10 µg per lane was run for western blotting. Alternatively, cardiac myofibrils (CMF) prepared from left and right ventricular walls, septa and papillary muscles of transgenic mice were used in protein quantitation assays. The transgenic protein was quantitated with polyclonal RLC CT-1 antibodies produced in this laboratory (raised against 15 residues from the C-terminus of human cardiac RLC). For standard western blots the secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (Fig. 1A). We have also used fluorescent secondary antibodies that were conjugated with fluorescent dye Cy 5.5 to monitor transgenic and endogenous RLC (Fig. 1B). As shown in Fig. 1A, the transgenic myc-RLC (WT or E22K) migrates slower than the endogenous RLC due to the myc sequence attached to the WT protein. The percent transgenic protein was expressed as: transgenic myc-protein ÷ (endogenous protein + transgenic myc-protein) x 100. The transgenic protein was also visualized using monoclonal myc-antibodies (clone 9E10) raised to a peptide from the human MYC protein (sequence EQKLISEEDL) (Roche) (Fig. 1A). Quantitation of western blots was done using Scion Image Software (standard western blots recorded with UVP Imager) or the Odyssey Infrared Imaging System (LI-COR Inc., for fluorescent western blots and Coomassie stained gels) (Fig. 1C).
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Echocardiography examination
Mice were anesthetized with a mixture of ketamine (42.8 mg/kg), xylazine (8.6 mg/kg) and acepromazine (1.4 mg/kg) by intraperitoneal injection. Echocardiography was performed using Toshiba PowerVision 7000 equipped with real-time digital acquisition (Toshiba Medical Systems) (Yang et al., 1999). M-mode measurements of left ventricular chamber dimensions in diastole (LVIDD) and in systole (LVIDS), inter-ventricular septum thickness in diastole (IVSTD) and in systole (IVSTS), and posterior wall thickness (LVPWS) were performed as described (Yang et al., 1999
). Left ventricular shortening fraction (FS) was calculated from the formula: FS (in %) = [(LVIDD-LVIDS) ÷ LVIDD] x 100. The ejection fraction (EF) was calculated using the equation: EF (100%)=[(LVIDD)3(LVIDS)3 ÷ (LVIDD)3] x 100 (Yang et al., 1999
).
Myofibrillar ATPase activity
Cardiac myofibrils (CMF) were prepared from left and right ventricular walls, septa and papillary muscles of transgenic mice according to Solaro et al. (Solaro et al., 1971). Assays for myofibrillar ATPase activity of Non-Tg, Tg-WT and Tg-E22K CMF were performed in a solution of 20 mM MOPS pH 7.0, 40 mM KCl, 2.5 mM MgCl2, 2 mM EGTA and increasing concentrations of Ca2+ from pCa 9 to pCa 4.5. After a 5-minute incubation at 30°C, the reaction was initiated with 2.5 mM ATP and terminated after 10 minutes with 5% trichloroacetic acid. Released inorganic phosphate was measured according to Fiske and Subbarow (Fiske and Subbarow, 1925
).
Skinned fibers
Following euthanasia, hearts were quickly removed and rinsed free of blood in ice-cold saline (0.9% NaCl). Muscle strips 150-200 µm wide and 2-3 mm long were then quickly dissected from left ventricular papillary muscle, immersed in cold relaxing-solution and transferred into detergent-containing solution of 50% glycerol and 50% pCa 8 solution (108 M Ca2+, 1 mM Mg2+, 7 mM EGTA, 5 mM MgATP2+, 20 mM imidazole, pH 7.0, 15 mM creatinine phosphate, ionic strength adjusted to 150 mM with KPr), containing 1% Triton X-100 for 24 hours at 4°C. Owing to the small size of these muscle strips they were not attached to support during glycerination. After a 24-hour skinning process with 1% Triton X-100, the fibers were transferred to the same glycerol containing solution without Triton X-100 and stored at 20°C until tested.
Steady-state force development
A bundle of approximately 3 to 5 fibers isolated from a batch of glycerinated mouse papillary fibers was attached by tweezer clips to a force transducer, placed in a 1 ml cuvette and bathed in pCa 8 solution. The fibers were then tested for steady-state force development in pCa 4 solution (composition is identical to pCa 8 solution except that the Ca2+ concentration is 104 M) and relaxed in pCa 8 solution. Steady-state force development was monitored for the control, Non-Tg fibers as well as for Tg-WT and Tg-E22K fibers.
Ca2+ dependence of force development
After the initial steady-state force was determined, the fibers were relaxed in pCa 8 solution and then exposed to solutions of increasing Ca2+ concentrations (from pCa 8 to pCa 4). The maximal force was measured in each "pCa" solution followed by a short relaxation of the fibers in pCa 8 solution. Data were analyzed using the following equations:
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Rate of force activation and relaxation
For kinetic measurements the fibers were exposed to either DM-nitrophen or Diazo-2 to measure the rates of activation or relaxation, respectively, as described in Miller et al. (Miller et al., 2001).
Statistical analysis
Data are expressed as the average of n experiments ± s.e.m. (standard error of the mean). For experiments where only a few repetitions were available for the statistical analysis, the standard deviation (s.d.) was shown. The statistically significant difference between the pCa50 values in the Ca2+ sensitivity of myofibrillar ATPase activity and force development between the Tg-WT and the Tg-E22K mutant was determined utilizing an unpaired Student's t-test (Sigma Plot 8.0), with significance defined as P<0.05.
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Results |
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The gross heart morphology of Non-Tg, Tg-WT and and Tg-E22K mice is presented in Fig. 2A,B. Fig. 2A shows the longitudinal sections of the whole hearts from representative 13-month-old Non-Tg, Tg-E22K and Tg-WT animals. Fig. 2B shows higher magnification views of the respective heart slides shown in Fig. 2A. As indicated by arrows, enlarged inter-ventricular septa and papillary muscles (encircled) were observed in Tg-E22K mice compared with Tg-WT or Non-Tg littermates (Fig. 2A). The microscopic views of the ventricular and septal sections show no difference between the groups and no myofibrillar disarray was observed (Fig. 2B). Surprisingly, the echocardiography examination did not show any significant differences between the groups. The left ventricular chamber dimensions in systole and diastole, ejection or shortening fractions were relatively similar in all animals (Table 1). This result was in accord with the heart weight to body weight ratios (data not shown) that did not indicate cardiac hypertrophy in Tg-E22K vs control animals. Therefore, the human phenotype of septal and papillary muscles hypertrophy observed in patients harboring this mutation (Kabaeva et al., 2002; Poetter et al., 1996
) could only be seen qualitatively in the slides of the whole hearts of Tg-E22K vs Tg-WT vs Non-Tg mice.
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Myofibrils from mouse ventricular, septal and papillary muscles of all groups were examined for their Ca2+ sensitivity of ATPase activity. In the control assays, transgenic wild-type (Tg-WT) mice expressing human isoform of RLC were compared to Non-Tg mice to test whether the Ca2+ sensitivity was RLC-isoform-dependent. As expected, the midpoints of Ca2+ sensitivity for these two curves were not different (Fig. 3) and pCa50=6.27±0.04, n=7 (Tg-WT) vs pCa50=6.23±0.03, n=10 (Non-Tg) (P>0.05). Statistically significant differences in the pCa50 values of the Ca2+ sensitivity of myofibrillar ATPase activity were observed between Non-Tg or Tg-WT and each line of mutant mice: Tg-E22K, line 2 (L2) (pCa50=6.40±0.03, n=8) and line 4 (L4) (pCa50=6.41±0.03, n=7) (P0.01). Both mutant lines increased the Ca2+ sensitivity of ATPase by about
pCa50
0.14 compared with Tg-WT myofibrils (P
0.01) (Fig. 3B). Analogous effects of the E22K-dependent increase in Ca2+ sensitivity of force were observed in skinned papillary muscle fibers which, in addition, showed a slight gene dose effect. As demonstrated in Fig. 4, significant differences in the pCa50 values of the Ca2+ sensitivity of steady-state force were observed between Non-Tg (pCa50=5.54±0.01, n=14) or Tg-WT (pCa50=5.53±0.01, n=10) and each of the mutant lines, Tg-E22K, L2 (pCa50=5.62±0.02, n=7) and L4 (pCa50=5.65±0.01, n=12) (P
0.001). Similar to the myofibrillar ATPase data, there was no difference in the midpoint of steady-state force-pCa dependence of Tg-WT and Non-Tg mice showing no variation in the force-pCa relationship between the murine and human isoforms of RLC. Interestingly, there was a slight gene-dose effect in the force development observed in the mutant fibers, i.e. between Tg-E22K, line 4 (87% transgene) and Tg-E22K, line 2 (65%) (Fig. 4A,B). The Ca2+ sensitivity of force in Tg-E22K L4 was slightly higher than in Tg-E22K L2 (pCa50=5.65 vs 5.62) indicating a correlation between the increase in the pCa50 value with the increase of the transgenic protein expression level but the difference was not statistically significant (Fig. 4). The largest statistically significant increase in force development was caused by Tg-E22K L4, and compared with Tg-WT, the difference in
pCa50 was
0.12 (P<0.001). Moreover, the steepness (nH) of the force-pCa dependences of the mutant fibers were lower than those of Non-Tg or Tg-WT fibers with the respective nH values of 2.23±0.06, 2.44±0.08, 1.95±0.09 and 2.07±0.06 for Non-Tg, Tg-WT, Tg-E22K L2 and Tg-E22K L4 fibers (Fig. 4).
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In summary, although there was an increase in the Ca2+ sensitivity of force in E22K mutant skinned papillary muscle fibers, there was no difference in the kinetics of Ca2+-dependent activation and Ca2+-dependent relaxation in all examined transgenic lines.
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Discussion |
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To elucidate the mechanism by which the E22K mutation in RLC results in ventricular and/or septal hypertrophy and alters cardiac-muscle contraction in humans, we have generated transgenic mice overexpressing human cardiac E22K-RLC in murine heart. The expression of two lines of myc-WT and two lines of myc-E22K has been made under the control of the murine -myosin heavy-chain promoter. The gross morphology of hematoxylin and eosin stained longitudinal sections of Tg-E22K mouse hearts demonstrated that their inter-ventricular septa as well as their papillary muscles were larger than those of Tg-WT or Non-Tg littermates. These experiments were performed utilizing hearts from 9-month-old mice (data not shown) and 13-month-old mice (Fig. 2). In both cases, the hearts of Tg-E22K mice demonstrated visibly enlarged inter-ventricular septa as well as papillary muscles (Fig. 2). These results suggested that the human phenotype of the E22K-mutated hearts (Kabaeva et al., 2002
; Poetter et al., 1996
) could be recapitulated in transgenic mice. As was shown by Kabaeva (Z. T. Kabaeva, PhD thesis, 2002), older individuals of a three-generation family carrying the mutation showed moderate hypertrophy of the entire septum, whereas younger patients had only mild or basal septal hypertrophy. Surprisingly, echocardiography abnormalities seen in most of the human patients with the E22K mutation have not been detected in our transgenic mice. The echocardiography examinations (Table 1) showed no significant differences in chamber dimensions in systole and diastole between Tg-E22K, Tg-WT and Non-Tg mice. The ejection and shortening fractions were also similar between the animals. At present, we have no obvious explanation why physiological differences between the mutant and the control lines as well as the different appearance in the histopathology images could not be supported by echocardiography results. Perhaps, more measurements and the use of newer echocardiogram technology developed for small animals could solve this discrepancy in the future. In vivo analysis of transgenic mice expressing the E22K mutation in the murine RLC isoform was recently reported by Sanbe and colleagues (Sanbe et al., 2000
). Unlike the murine model carrying the human RLC gene-locus, mice with the mutated murine RLC cDNA failed to exhibit either overt hypertrophy or mid-ventricular cavity obstruction. Since the major focus of their study was the essential light-chain of myosin, no functional examination was performed or reported on their E22K-RLC transgenic mice (Sanbe et al., 2000
).
The first investigated and reported transgenic animal models to study FHC-associated changes in the Ca2+ sensitivity of cardiac-muscle contraction were those expressing the thin filament regulatory proteins such as tropomyosin and troponin (James et al., 2000; Miller et al., 2001
; Muthuchamy et al., 1999
; Wolska et al., 1999
). Our report presents similar Ca2+-dependent alterations in contraction caused by a thick, and not thin, filament-containing protein, namely the myosin RLC. Moreover, this work is the first to investigate the functional consequences of the E22K mutation in human ventricular RLC in transgenic mice. Our findings that the E22K mutation increased Ca2+ sensitivity of myofibrillar ATPase activity and steady-state force development in transgenic skinned-cardiac-muscle preparations follow our earlier results in solution and in the reconstituted porcine fiber system. We showed that this mutation decreased Ca2+-binding to isolated RLC (Szczesna et al., 2001
) and slightly increased the Ca2+ sensitivity of force in the E22K-reconstituted porcine cardiac-muscle preparations (Szczesna et al., 2001
). Our current studies with the E22K animal model support our earlier findings and therefore reveal the usefulness of transgenesis in the physiological characterization of the human disease in mice. Interestingly, the effect of the E22K-mediated increase in the Ca2+ sensitivity of force was shown by Levine and colleagues in biopsied slow skeletal muscle fibers from an E22K-diseased human patient (Levine et al., 1999
; Levine et al., 1998
). Consistently with our work, they showed an increase in the Ca2+ sensitivity of force development. Since the same gene expresses the ventricular- and slow-twitch skeletal muscle RLC (Kubalak et al., 1994
; Sarkar et al., 1971
), the comparison of their results from slow skeletal fibers with the ventricular fibers used in our work seems justified.
There are a few possible mechanisms for the E22K-mutated myocardium to initiate changes in the Ca2+ sensitivity of force/ATPase that could potentially trigger ventricular and/or septal hypertrophy as seen in human patients harboring this mutation. As a subunit of myosin, the RLC is known to stabilize the neck region of the myosin head, also called the lever-arm (Szczesna, 2003). This region of the myosin head has been postulated to undergo conformational changes that are important for the power stroke (Rayment et al., 1993a
; Uyeda et al., 1996
). As pictured in Fig. 5, the E22K mutation occurs at the N-terminus of RLC, which is in close proximity to the C-terminal RLC-binding sequence of myosin heavy-chain in the head portion of myosin. This region in the crystal structure (NCBI numbers 1WDC and 1QVI) makes a sharp bend and connects the myosin head with the myosin rod (Gourinath et al., 2003
; Houdusse et al., 1997
; Rayment et al., 1993b
). One can predict that any structural alterations in this pivotal region of myosin may affect the function of the lever arm and cause alterations in Ca2+-dependent force generation in muscle. Lack of RLC phosphorylation caused by this E22K mutation, as shown by Szczesna and colleagues (Szczesna et al., 2001
), could contribute to these myosin-dependent perturbations in force generation. However, the RLC is a member of the EF-hand Ca2+-binding-protein family, like troponin C (TnC) and calmodulin. Ca2+-binding to the RLC, like to other EF-hand Ca2+-binding-proteins, could be a part of the Ca2+-handling machinery in the muscle cell. The E22K mutation is localized in the helix preceding the Ca2+-binding-loop of RLC and, as we have shown previously, replacement of the negatively charged glutamic acid with the positively charged lysine decreased binding of Ca2+ to RLC (KCa) 20-fold (Szczesna et al., 2001
). One can speculate that, if binding of Ca2+ to the RLC plays a role in the overall Ca2+ homeostasis, this E22K amino acid change could directly interfere with this process because the RLC would no longer bind Ca2+ with the affinity of the wild-type protein. The observed increase in Ca2+ sensitivity of force/ATPase in Tg-E22K fibers could be a consequence of the E22K-mediated decrease in RLC Ca2+-binding leading to an increase in overall Ca2+ concentrations that could be utilized by TnC, the key player of the Ca2+ regulation of muscle contraction. Similar cellular concentrations of both of these Ca2+ buffers, the RLC and the TnC, support this line of thinking. Another possibility is that the Ca2+ affinity and/or dissociation of TnC could be directly affected by this RLC mutation, leading to TnC-mediated Ca2+ alterations during contraction. Further experiments are needed to test the latter hypothesis. Interestingly, the kinetics of muscle contraction were not altered by this FHC RLC mutation and no significant changes in activation and relaxation rates among all three groups of animals were found. These results are in accord with in vitro motility assays by Poetter and colleagues, where the E22K-mutated myosin isolated from cardiac biopsies of affected individuals showed normal actin-filament translocation compared with control samples (Poetter et al., 1996
).
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In conclusion, our results suggest that E22K-mediated structural perturbations in the RLC-affecting Ca2+-binding-properties of the mutated thick filaments are responsible for triggering the abnormal function of the filaments that in turn may initiate a hypertrophic process and lead to heart failure.
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Acknowledgments |
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References |
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