Familial Hypertrophic Cardiomyopathy Mutations in the Regulatory Light Chains of Myosin Affect Their Structure, Ca2+Binding, and Phosphorylation*

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca2+ binding site, had dramatically altered Ca2+ binding properties. The K Cavalue for E22K was decreased by ∼17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca2+. Interestingly, Ca2+ binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the α-helical content of phosphorylated R58Q greatly increased with Ca2+ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lowerK Ca than wild-type light chain, whereas phosphorylation of this mutant increased the Ca2+ affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca2+ affinity, the opposite was true for A13T. The α-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca2+ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

There is substantial evidence that myosin regulatory light chains (RLC) 1 play a primary regulatory role in scallop and smooth muscle contraction, but their functional role in mammalian striated (skeletal and cardiac) muscle contraction is unclear. RLC, together with the essential light chain, stabilizes the 8.5-nm-long ␣-helical neck of the myosin head, with the N terminus of RLC wrapped around the heavy chain (1). Smooth muscle contraction is initiated by RLC phosphorylation with a Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) (2,3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role (4). It was shown that RLC phosphorylation increased the Ca 2ϩ sensitivity of force in skinned skeletal (5)(6)(7) and cardiac (8) muscle fibers. In the human heart, several RLC isoforms are expressed (9,10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11,12). FHC is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (13), myosin-binding protein C (14), ventricular RLC and essential light chain (11,12,15), troponin T (16), troponin I (17), ␣-tropomyosin (18), actin (19), and titin (20). Depending on the affected gene, and the site of the mutation, FHC has variable presentation with regard to its degree and severity and the extent of myocardial disarray. The clinical manifestations of FHC range from benign to severe heart failure and to sudden cardiac death. The best characterized clinical cases include patients with ␤-myosin heavy chain mutations who have a high level of cardiac hypertrophy and those with troponin T mutations who have less hypertrophy, but a higher incidence of sudden cardiac death in young adults. The first three identified mutations in the RLC (A13T, E22K, and P95A) were shown to be associated with a particular subtype of cardiac hypertrophy defined by mid-left ventricular obstruction (11). Two other RLC mutations (F18L and R58Q), identified by Flavigny et al. (12), were associated with a typical form of hypertrophic cardiomyopathy, which causes increased left ventricular wall thickness and abnormal electrocardiograph findings with no mid-cavity obliteration.
The three-dimensional structure of the RLC demonstrates the close proximity of FHC mutations to either the phosphorylation site of RLC (Ser-15) or the Ca 2ϩ binding site (amino acids 37-48). Because of this distinctive arrangement of the FHC mutations within the RLC, it was of interest to study their effect on the phosphorylation properties of human cardiac RLC (HCRLC) as well as their effect on Ca 2ϩ binding. We have also investigated how these FHC mutations influence the secondary structure of the HCRLC as well as the combined effects of phosphorylation and Ca 2ϩ binding on their structure. We demonstrate that both processes, phosphorylation and Ca 2ϩ bind-ing, are significantly altered by the FHC mutations and their effect depends upon the specific location of the missense mutation. The alterations in contractility that would result from these mutations are not known at present, and it is therefore not possible to know precisely how they might trigger the hypertrophic process. It is likely, however, that such a response would be part of a direct compensatory and/or adaptive mechanism of the heart to maintain normal cardiac function. Assessing the mechanism by which FHC mutations alter RLC function will lead to a better understanding of the physiological role of RLC in the regulation of cardiac muscle contraction.

MATERIALS AND METHODS
Mutation, Expression, and Purification of Wild-type HCRLC and the FHC Mutants-The cDNA for wild-type HCRLC was cloned by reverse transcription-polymerase chain reaction using primers based on the published cDNA sequence (GenBank accession no. AF020768) and standard methods (21). The FHC RLC mutants: A13T, F18L, E22K, R58Q, and P95A, were generated using overlapping sequential polymerase chain reaction (21). Wild-type and mutant cDNAs were constructed with an NcoI site at the N-terminal ATG and a BamHI site following the stop codon to facilitate ligation into the NcoI-BamHI cloning site of the pET-3d (Novagen) plasmid vector and transformation into DH5␣ cloning host bacteria for expression of the cDNAs of the wild-type HCRLC and the FHC mutants. The cDNAs of the proteins were transformed into BL21 expression host cells and protein expressed in large (16 liters) cultures.
Expressed proteins were purified using a Q-Sepharose column followed by a DE-52 column, both equilibrated with 2 M urea, 25 mM Tris-HCl, 0.1 mM PMSF, 1 mM dithiothreitol, 0.001% NaN 3 , pH 7.5. The proteins were eluted with a 1000-ml salt gradient of 0 -300 mM KCl. The elution profiles from both the Q-Sepharose and the DE-52 columns for wild-type (WT) HCRLC and A13T occurred in the range of 160 -200 mM KCl, for F18L, R58Q, and P95A between 210 -250 mM KCl, and for E22K between 230 and 280 mM KCl. The final purity of the proteins was tested using 15% SDS-PAGE.
Phosphorylation of Wild-type HCRLC and Its FHC Mutants-The proteins were phosphorylated with Ca 2ϩ -calmodulin activated myosin light chain kinase (MLCK) in a solution containing: 50 M protein (in 20 mM imidazole, 50 mM NaCl, 0.1 mM PMSF, pH 7.5), 0.5 M MLCK, 5.0 M calmodulin, 0.1 mM CaCl 2 , 12 mM MgCl 2 and 5 mM ATP. A catalytically active truncated fragment of the rabbit skeletal muscle MLCK was used in this study (22). The MLCK, missing the first 256 amino acids, was expressed in Sf9 cells infected by a recombinant virus (23). The phosphorylation reaction was carried out for 2 h at room temperature or overnight on ice. After phosphorylation the proteins were purified using a Q-Sepharose column (conditions as described above). The level of phosphorylation was measured using 8% urea-PAGE (24).
Flow Dialysis-Flow dialysis was performed in a solution of 100 mM KCl, 20 mM imidazole, pH 7.0 (22°C). All proteins were equilibrated in this buffer prior to the measurements. The flow dialysis experiments were performed according to Colowick et al. (25) with modifications. Briefly, the upper chamber of the apparatus containing the protein and the labeled substrate ( 45 Ca 2ϩ ) was separated by a membrane from the lower chamber. The buffer was pumped through the lower chamber at a constant rate of 1.5 ml/30 s. The upper chamber was first equilibrated with 0.4 ml of buffer for 15 min, followed by the protein (0.4 ml) for 5 min. After adding 45 Ca 2ϩ , an equilibrium was attained by flowing buffer through the lower chamber for 5 min. After steady state was reached, unlabeled substrate (Ca 2ϩ ) was added at regular intervals and in varying concentrations. Fractions were collected every 30 s, and the effluent was sampled for measurement of radioactivity. The specific radioactivity of 45 Ca 2ϩ used in the experiment was 12-16 mCi/mg (from PerkinElmer Life Sciences), and 2 Ci of 45 Ca 2ϩ per experiment gave sufficient radioactivity in the dialysate for accurate measurements. Data were presented using Scatchard analysis (26, 27).
C Ca-bound and C Ca-free represent the concentration of the bound and free metal, respectively, C p is the concentration of the protein, n is the total number of Ca 2ϩ binding sites, and K Ca is the Ca 2ϩ binding affinity. CD Measurements-Far-UV circular dichroism spectra (CD) were obtained using a 1-mm path quartz cell in a Jasco J-720 spectropolarimeter. Spectra were recorded at 195-250 nm with a bandwidth of 1 nm at a speed of 50 nm/min and a resolution of 0.2 nm. Analysis and processing of data were done using the Jasco system software (Windows Standard Analysis, version 1.20). Ten scans were averaged, base lines subtracted, and no numerical smoothing applied. Mean residue ellipticity ([] MRE , in degrees⅐cm 2 /dmol) for the spectra were calculated (utilizing the same Jasco system software) using the following equation (28 -30).
[] is the measured ellipticity in millidegrees, Cr is the mean residue molar concentration, and l is the path length in cm. The optical activity of the buffer with or without Ca 2ϩ was subtracted from relevant protein spectra. The ␣-helical content for each protein was calculated using the standard equation for [] at 222 nm (31).
f H is the fraction of ␣-helical content (f H ϫ 100, expressed in %). The measurements were performed at 22°C, in 10 mM phosphate buffer, 60 mM NaF, and 1 mM EGTA or 0.1 mM CaCl 2 at pH 7.0. NaF was used to avoid the strong absorption of chloride ions in the far ultraviolet (32). The protein samples were equilibrated with respective Ca 2ϩ buffers before the measurements. Spectra are presented as the mean residue ellipticity.

RESULTS
To study the effects of FHC mutations on the phosphorylation, Ca 2ϩ binding and the secondary structure of HCRLC, we have cloned and expressed the WT and five FHC mutants of HCRLC: A13T, F18L, E22K, R58Q, and P95A. Fig. 1A presents the amino acid sequence of human ventricular RLC and all the known specific missense mutations that have been associated with FHC. The Ca 2ϩ binding (residues 37-48) and the phosphorylation sites (Ser-15) are also illustrated. Fig. 1B demonstrates the three-dimensional representation of the HCRLC derived from the crystal structure of chicken skeletal myosin S1 (1). The N-terminal region of HCRLC containing the phosphorylation site and two of the FHC mutations, A13T and F18L, is not shown since this region of the RLC was unresolved in the reported crystal structure (1). The three-dimensional representation of the HCRLC (Fig. 1 B) suggests that the mutations A13T, F18L, E22K, and P95A are located in close proximity to the phosphorylation site of RLC (Ser-15), whereas the E22K and R58Q mutations occur in the immediate extension of the helices flanking the Ca 2ϩ binding loop.
Ca 2ϩ Binding to HCRLC and Its FHC Mutants-HCRLC-WT and the FHC mutants were tested for Ca 2ϩ binding using the flow dialysis method. As shown in Fig. 2 and Table I Table I (Table I, n ϭ 3, p Ͻ 0.003). However, two other FHC mutants, E22K and R58Q, dramatically changed the RLC's Ca 2ϩ binding properties. The K Ca value decreased by ϳ17-fold for the E22K mutant ( Fig. 2 and Table I, n ϭ 3, p Ͻ 0.001), whereas the R58Q mutation completely impaired Ca 2ϩ binding ( Table I).
Effect of Phosphorylation-Phosphorylation of HCRLC-WT with Ca 2ϩ -calmodulin activated MLCK decreased its Ca 2ϩ binding affinity by 7.4-fold (K Ca ϭ 0.90 Ϯ 0.06 ϫ 10 5 M Ϫ1 , Fig.  3A and Table I, n ϭ 3, p Ͻ 0.001). However, phosphorylation of the FHC mutants affected their Ca 2ϩ binding properties in various ways (Figs. 3 and 4). The most dramatic effect was observed for the E22K mutant. This substitution prevented the E22K mutant from becoming phosphorylated. Even a 20-fold increase of the enzyme/substrate ratio and longer incubation time did not result in phosphorylated E22K (Fig. 4, lane 5). As shown in Fig. 4, the gel migration of the E22K mutant was slower than HCRLC-WT, F18L, and R58Q mutants, due to the Glu 3 Lys replacement that resulted in an additional positive charge of the E22K protein. The E22K mutation changes the isoelectric point of HCRLC from pI ϭ 4.92 to pI ϭ 5.10; therefore, the nonphosphorylated E22K migrates slower than the nonphosphorylated wild-type HCRLC. Accordingly, the phosphorylated forms of HCRLC-WT and its FHC mutants migrated faster than the nonphosphorylated ones due to the acidic phosphate group attached to Ser-15 of the phosphorylated proteins (Fig. 4). A large effect of phosphorylation was also observed for the A13T mutation. As shown in Fig. 1, this mutation is located next to Ser-15, the phosphorylation site of HCRLC. Phosphorylation of A13T resulted in a large increase (K Ca value increased from 2.06 Ϯ 0.23 ϫ 10 5 M Ϫ1 to 1.33 Ϯ 0.02 ϫ 10 6 M Ϫ1 ) in its Ca 2ϩ binding affinity compared with nonphosphorylated A13T (Fig. 3B and Table I, n ϭ 3, p ϭ 0.016). Interestingly, phosphorylated A13T demonstrated a 15fold greater affinity for Ca 2ϩ than phosphorylated HCRLC-WT, whereas nonphosphorylated A13T bound Ca 2ϩ with a 3-fold lower affinity than nonphosphorylated-WT. No effect of phosphorylation on Ca 2ϩ binding to F18L was observed (Table I, n ϭ 3). However, there was an interesting effect of phosphorylation on Ca 2ϩ binding to the R58Q mutant. This mutant did not bind Ca 2ϩ in the nonphosphorylated state but did bind Ca 2ϩ when phosphorylated (K Ca ϭ 3.04 Ϯ 1.02 ϫ 10 5 M Ϫ1 ) ( Table I).
Secondary Structure of HCRLC and Its FHC Mutants-Far-UV CD spectroscopy was used to detect changes in the secondary structure of the HCRLC-WT and its FHC mutants in response to phosphorylation and Ca 2ϩ binding. The CD spectra for nonmetal bound (apo) proteins are presented in Fig. 5A. The effect of the FHC mutation on the HCRLC-WT secondary structure was quantified from changes in the mean residue ellipticity at 222 nm. This wavelength has been shown to be sensitive to changes in the ␣-helical content of the protein (31, 33). Table  II presents Table  I. Flow dialysis was performed in the solution of 100 mM KCl, 20 mM imidazole, pH 7.0, and 2 Ci of 45 Ca 2ϩ /experiment. phosphorylation on HCRLC-WT and its FHC mutants. Data are the average of 2-4 experiments (n), each consisting of 10 scans. The variation in the ␣-helical content of the HCRLC-WT and its FHC mutants was below 2% for the individual experiments. As shown in Fig. 5A and Table II, the ␣-helical content of HCRLC-WT and its FHC mutants ranged from 18% to 29%, with the highest for A13T. Replacement of the alanine residue with threonine (A13T) increased the ␣-helical content of HCRLC from 18% (n ϭ 4) to 29% (n ϭ 2). This significant effect of the FHC mutation on the secondary structure of the HCRLC (p Ͻ 0.01) was quite surprising since alanine has a high potential to form ␣-helix. The second significant change was brought about by the E22K mutation, which resulted in an increase in the ␣-helical content of HCRLC from 18% to 24% (n ϭ 2, p Ͻ 0.01). All other FHC mutations of HCRLC did not alter its ␣-helical content in the apo-state ( Fig. 5A and Table II).
Effect of Ca 2ϩ Binding- Fig. 5B and Table II show the effect of Ca 2ϩ binding on the far-UV CD spectrum of HCRLC-WT. Similar to other EF-hand Ca 2ϩ -binding proteins, the binding of Ca 2ϩ to HCRLC-WT increased its ␣-helical content from 18 to 23%. Mean residue ellipticity at 222 nm for HCRLC-WT (Ϫ7681) was similar to previous work by Huang et al. (30) (Ϫ7060) and Wu and Yang (33) (Ϫ7500) for rabbit skeletal muscle RLC. Using the equation utilized in this study to calculate the ␣-helical content, these values of the mean residue ellipticity at 222 nm from Huang et al. (30) and Wu and Yang (33) would yield 15.6% and 17%, respectively compared with our 18% for HCRLC-WT (Table II). Similar to HCRLC-WT, the P95A mutant also had an increase in ␣-helical content upon Ca 2ϩ binding (Table II). Interestingly, the binding of Ca 2ϩ to the A13T mutant, which had the highest ␣-helical content among all FHC mutants in the apo-state, caused a decrease (not increase) in its ␣-helical content from 29% to 25% (Table II, n ϭ 2, p Ͻ 0.05). However, the amount of ␣-helical content of the Ca 2ϩ -bound A13T was the same as for the other Ca 2ϩbound mutants. The binding of Ca 2ϩ to the F18L mutant produced very little change in its ␣-helical content (Table II, n ϭ 3, p Ͼ 0.1). The R58Q mutant did not bind Ca 2ϩ in its nonphosphorylated form, and Ca 2ϩ did not significantly affect its ␣-helical content. However, the phosphorylation restored its Ca 2ϩ binding and the amount of ␣-helical content greatly increased on binding of Ca 2ϩ to phosphorylated R58Q (28% , Table II, n ϭ 2).
Effect of Phosphorylation-Phosphorylation of HCRLC-WT did not change its ␣-helical content or the binding of Ca 2ϩ to the phosphorylated form (Table II, n ϭ 4). Only Ca 2ϩ binding to nonphosphorylated HCRLC-WT had increased ␣-helical content (23% , Table II, Fig. 5B). No change in ␣-helix was observed for the phosphorylated F18L and R58Q mutations in the apostate. However, a dramatic effect of phosphorylation was observed for the A13T mutant, whose ␣-helical content was decreased from 29% to 19% (n ϭ 2, p Ͻ 0.01, Table II, Fig. 5C). The phosphorylated A13T mutant had the same ␣-helical content as HCRLC-WT in both phosphorylated and nonphosphorylated states (Table II). Interestingly, the effect introduced by the FHC mutation (replacement of the alanine with threonine) was reversed by the phosphorylation of the mutant (decrease in ␣-helix from 29% to 19%). As mentioned above, the R58Q mutation had the same ␣-helical content before and after phos-  Table  I and Fig. 2). Data were analyzed using Scatchard plot: C Ca-bound /C Cafree/C p ϭ ϪK Ca ⅐C Ca-bound /C p ϩ n⅐K Ca , where C Ca-bound and C Ca-free represent the concentration of the bound and free Ca 2ϩ , respectively; n is the number of the total Ca 2ϩ binding sites of the protein; and K Ca is the Ca 2ϩ binding affinity constant. phorylation; however, Ca 2ϩ binding only occurred with the phosphorylated form and this increased its ␣-helical content from 20% to 28% (n ϭ 2, p Ͻ 0.01, Table II). DISCUSSION This study investigates the effects of FHC mutations in myosin RLC on Ca 2ϩ binding, phosphorylation, and secondary structural properties.
Since the ventricular myosin RLC belongs to the superfamily of EF-hand Ca 2ϩ binding proteins, it was of interest to investigate the effect of the FHC mutations on its Ca 2ϩ binding properties. Unlike other EF-hands, RLC contains only one Ca 2ϩ binding EF-hand domain, between amino acids 37 and 48. We have studied all known FHC RLC mutants with special attention to two mutations, E22K and R58Q, located in the immediate extension of the helices flanking the HCRLC Ca 2ϩ binding site. Indeed, whereas A13T, F18L, and P95A mutants decreased the Ca 2ϩ affinity of RLC by 3-fold, the E22K and R58Q mutants had even greater reduction in Ca 2ϩ affinity compared with HCRLC-WT. The K Ca value for E22K was decreased by 17-fold, and the R58Q mutant did not bind Ca 2ϩ at all. Interestingly, Ca 2ϩ binding to the R58Q mutant was restored upon phosphorylation while the E22K mutant could not be phosphorylated. Even a 20-fold increase of the MLCK-calmodulin concentration did not result in phosphorylation of the E22K mutant (Fig. 4).
The R58Q mutation that had Ca 2ϩ binding completely eliminated, was quite surprising since other EF hand Ca 2ϩ -binding proteins, e.g. troponin C, calmodulin, contain the Gln residue (and not Arg) in the equivalent position in the helix C-terminal of the Ca 2ϩ binding site. The arginine residue of HCRLC, however, is very conserved across species and a wide spectrum of other RLCs contains the Arg residue in this position (Fig. 6). It would be interesting to determine whether substitution of Arg to Gln in these RLCs would also result in the inactivation of their Ca 2ϩ binding site in the nonphosphorylated state. Interestingly, phosphorylation of the R58Q mutant at Ser-15 restored the Ca 2ϩ binding site (Table I)  ner. This was also observed for the E22K mutation with a greatly reduced Ca 2ϩ affinity that could prevent phosphorylation. It is worth mentioning that the glutamic acid that follows the phosphorylation site of RLC and precedes its Ca 2ϩ binding site, is also conserved among species (Fig. 6). Moreover, the substitution of the positively charged Lys for the acidic Glu residue resulted in a significant increase in ␣-helical content of the E22K mutant compared with HCRLC-WT (Table II). All these changes induced by the E22K mutation are most likely affecting the interaction of the mutated light chain with the heavy chain of myosin. One could speculate that this mutation, which eliminates phosphorylation of the protein and reduces its affinity for Ca 2ϩ , alters working cross-bridges during contraction and may contribute to the development of hypertrophy in the human heart (34). As mentioned above, the three-dimensional representation of the HCRLC (Fig. 1B) suggests that mutations A13T, F18L, E22K, and P95A are located in close proximity to the phosphorylation site of HCRLC. Thus, any alterations affecting the structure and/or sequence near this site would be expected to influence the phosphorylation properties of the HCRLC and/or the relationship between phosphorylation and Ca 2ϩ binding. The first mutation located near the phosphorylation site (Ser-15), A13T, resulted in 3-fold decrease in the K Ca value, while phosphorylation of this mutant caused an additional 6-fold increase in Ca 2ϩ affinity. The K Ca value for phosphorylated A13T was 15 times larger than phosphorylated HCRLC-WT. Therefore, the consequences of the FHC mutation (A13T) were most profound in conditions where the protein became phosphorylated. Thus, these results clearly suggest a link between Ca 2ϩ binding and phosphorylation, as we have reported previously (35). Unpredictably, substitution of the Ala to Thr residue resulted in a large increase in the ␣-helical content (Table II, Fig. 5A). This was quite surprising since alanine is known to be predisposed to form ␣-helical structures. However, this residue (Ala-13) is located in the region of the RLC, which was not resolved in the crystal structure of chicken skeletal muscle myosin S1 (1) and it is hard to predict the precise effect of the FHC mutation on the secondary structure of HCRLC. The ␣-helical content of the A13T mutant returned to a normal level (that of HCRLC-WT) upon phosphorylation. Therefore, phosphorylation of A13T attenuated whatever sterical constraints were introduced by this FHC mutation. This result suggests that phosphorylation of HCRLC during contraction could act as a backup mechanism attenuating the physiological consequences of the FHC mutation in the working heart. This was also true with the R58Q mutation whose Ca 2ϩ binding properties were restored upon phosphorylation. Even in HCRLC-WT, phosphorylation reduced the Ca 2ϩ dependent elevated ␣-helical content (Table II).
Our results as well as others (36 -39) suggest that phosphorylation of the regulatory light chains of myosin could have an important physiological role in the regulation of cardiac muscle contraction. Moreover, the relationship between phosphorylation and Ca 2ϩ binding to RLC plays a key role in the working heart (35,39). One could speculate that both of these processes may operate as adaptive and/or protective mechanisms to either attenuate the effect of the FHC mutations and/or improve performance of the working muscle. Alterations introduced by the FHC mutations most likely interfere with the interaction of the RLC with the heavy chain of myosin (40) and affect the function of myosin cross-bridges during force generation (41). The region of the myosin heavy chain that contains the RLC has been postulated to undergo conformational changes that are important for working muscle (42,43). The motions of this region of the myosin head were predicted by crystallographic models (42,44) and studied further by fluorescence polarization spectroscopy (45). It was demonstrated that, during active contraction, the RLC binding domain of the myosin head undergoes repetitive conformational changes (tilt and twist) and therefore may play an active role during force generation in muscle. Therefore, alterations introduced by the FHC mutations could interfere with the physiological function of the RLC and contribute to malfunctioning of the human heart. Patients with the FHC RLC mutations have developed a phenotype of hypertrophic cardiomyopathy, without sudden death (11,12). Our results suggest that phosphorylation and Ca 2ϩ binding to HCRLC may simultaneously act to protect and attenuate the negative physiological consequences of the FHC mutations. Further work, in progress in our laboratory, is aimed at determining the physiological consequences of these mutations. FIG. 6. Amino acid sequence alignment of various isoforms of myosin regulatory light chains. Note that the residues Phe-18, Glu-22, Arg-58, and Pro-95, which are mutated in the human ventricular HCRLC of the FHC patients, are very conserved across the species.