Male but not female mice carrying a single R403Q missense allele for cardiac α-myosin heavy chain (M-αMHCR403Q/+ and F-αMHCR403Q/+, respectively) develop significant hypertrophic cardiomyopathy (HCM) compared with male and female wild-type mice (M-αMHC+/+ and F-αMHC+/+, respectively) after ∼30 wk of age. We tested the hypothesis that myofilament mechanical performance differs between M-αMHCR403Q/+ and F-αMHCR403Q/+ at younger ages (10–20 wk) and could account for sex differences in HCM development. The sensitivity of chemically skinned myocardial strips to Ca2+ activation (pCa50) was significantly (P < 0.05) enhanced in male mice independent of genotype (M-αMHCR403Q/+: 5.70 ± 0.06, M-αMHC+/+: 5.63 ± 0.05, F-αMHCR403Q/+: 5.57 ± 0.03, F-αMHC+/+: 5.54 ± 0.04) by two-way ANOVA, whereas maximum developed tension was significantly enhanced in α-MHCR403Q/+ independent of sex (M-αMHCR403Q/+: 29.3 ± 2.3, M-αMHC+/+: 26.0 ± 1.4, F-αMHCR403Q/+: 30.2 ± 2.1, F-αMHC+/+: 26.2 ± 1.2 mN/mm2). The frequency of maximum work generated by sinusoidal length perturbation was significantly higher in αMHCR403Q/+ mice than in sex-matched controls (M-αMHCR403Q/+: 2.26 ± 0.47, M-αMHC+/+: 1.29 ± 0.18, F-αMHCR403Q/+: 3.21 ± 0.33, F-αMHC+/+: 2.52 ± 0.36 Hz). Unloaded shortening velocity was significantly enhanced in αMHCR403Q/+ and in female mice (M-αMHCR403Q/+: 2.26 ± 0.47, M-αMHC+/+: 1.29 ± 0.18, F-αMHCR403Q/+: 3.21 ± 0.33, F-αMHC+/+: 2.52 ± 0.36 muscle lengths/s), and normalized mechanical power, calculated from the tension-velocity relationship, was significantly enhanced in αMHCR403Q/+ independent of sex (M-αMHCR403Q/+: 60 ± 2 10−3, M-αMHC+/+: 37 ± 3 10−3, F-αMHCR403Q/+: 57 ± 3 10−3, F-αMHC+/+ 25 ± 3 10−3 muscle lengths/s × normalized tension). We did not find a statistically significant sex × mutation interaction for any measure of myofilament performance. Therefore, sarcomeric incorporation of the R403Q myosin similarly enhanced left ventricular myofilament mechanical performance in both male and female mice. The sex-dependent development of HCM due to the R403Q myosin may then be inhibited by female sex hormones, which may additionally underlie the observed sex differences for pCa50 and unloaded shortening velocity.
- hypertrophic cardiomyopathy
- myosin heavy chain
- isometric tension
- calcium sensitivity
hypertrophic cardiomyopathy (HCM) in the absence of recognizable hemodynamic risk factors can arise naturally as a consequence of a mutant allele for myosin heavy chain (MHC) (41). Assessing the functional consequences of HCM-associated mutations, such as the R403Q point mutation in cardiac α-MHC or β-MHC, has played an important role in the identification of modifications in myofilament performance that may lead to the HCM phenotype (9, 19, 36, 51). However, functional data from some models of the R403Q-mutated MHC have been indefinite. For example, myosin isolated from myocardium of HCM patients carrying an allele for the R403Q mutation reportedly reduce (5) or enhance (33) actin filament velocity (Vactin) in the myosin motility assay. Additionally, actin-activated myosin ATPase and Vactin of R403Q mutant MHC expressed by baculovirus/insect cell systems are reportedly reduced for rat cardiac α-MHC (44) and yet enhanced in chicken gizzard smooth muscle MHC (53).
Data from mouse models of the R403Q mutation in α-MHC, on the other hand, have provided more consistent findings. Myosin isolated from myocardium of α-MHCR403Q/R403Q homozygous mice demonstrate a marked enhancement of actin-activated ATPase, Vactin, and stall force of Vactin by α-actinin (7, 47). Similarly, cardiac myosin isolated from αMHCR403Q/+ heterozygous mice shows enhanced ATPase (2, 31, 47). Data from ATPase and Vactin assays generally correspond to each other and reflect actomyosin cross-bridge kinetics (48), whose enhancement in the R403Q myosin has been corroborated by a shorter cross-bridge time-on measured in the laser trap (47).
Left ventricular (LV) function in the αMHCR403Q/+ heterozygous mouse, which mimics the human genotype leading to HCM, is characterized in part by an earlier and higher peak LV pressure and an elevated free wall fractional shortening compared with these measures in the wild-type mouse, αMHC+/+ (11, 12, 24, 30, 43). Furthermore, cardiac myocytes isolated from αMHCR403Q/+ mice demonstrated an enhanced unloaded shortening velocity, suggesting that myofilament mechanical performance may be enhanced (16). These data from functionally intact myocardium imply a greater mechanical performance of the myocardial myofilaments containing the R403Q mutant α-MHC. Interestingly, only male (M) αMHCR403Q/+ mice develop HCM after 30 wk of age (11, 12, 24, 30, 43), a condition that appears to underlie the greater likelihood for M mice to develop heart failure compared with that shown in female (F) mice (17, 18, 20, 24, 30, 42, 45). Men carrying mutant alleles for sarcomeric proteins are likewise more likely than women to develop HCM (8, 22, 28, 49). These findings suggest that the sarcomeric incorporation of R403Q mutant myosin affects myofilament performance significantly enough in M to initiate the development of HCM but is relatively inconsequential to myofilament performance in F. Myofilament tension development and Ca2+ sensitivity of tension development, however, were not found to be significantly altered in the M-αMHCR403Q/+ group (2, 31). Other important physiological measures of myofilament mechanical performance, namely shortening velocity and power production, have not yet been reported in the αMHCR403Q/+ of either sex.
It has already been observed that cardiac contractile function and contractile reserve are enhanced in male compared with female animals (34, 38, 39). Gonadectomy in rats of either sex leads to a significantly reduced myosin ATPase consistent with the concomitant ∼30–40% reduction in α-MHC (3, 38, 39, 52). Testosterone recovers cardiac function in gonectomized rats of either sex (39), whereas estrogen reduces myofilament sensitivity to Ca2+ activation (4, 46, 52). Collectively, these observations imply that the sarcomeric incorporation of a differently performing myosin, such as the R403Q mutant αMHC, would modify myofilament performance and, in addition, in a sex-dependent manner.
In the present study, we sought to determine the fundamental myofilament basis for sex differences in the hypertrophic response due to the R403Q mutant myosin. We directly tested the hypothesis that several indexes of myofilament mechanical performance (namely, tension development, Ca2+ sensitivity, frequency of oscillatory work production, shortening velocity, and power production) would be different between M- and F-αMHCR403Q/+ myocardium. We found that myofilament performance in both M-αMHCR403Q/+ and F-αMHCR403Q/+ mice was significantly enhanced compared with sex-matched αMHC+/+ mice. However, there were no sex differences in αMHCR403Q/+ myofilament performance, which could account for the sex differences in the development of HCM.
All procedures were similar to those described previously (2, 31) and were reviewed and approved by the Harvard University and University of Vermont Institutional Animal Care and Use Committees. All reagents were purchased from Sigma (St. Louis, MO), except where noted.
Mice undergoing echocardiography at 40 wk.
Heterozygous αMHCR403Q/+ mice and αMHC+/+ littermate controls of 129SvEv background (40 wk old) underwent transthoracic echocardiography as described previously (24) using a Sonos 5500 ultrasonograph (Hewlett-Packard, Andover, MA) and a 12-MHz focused transducer. Mice received 2.5% Avertin and were warmed with a heating pad during the procedure. Standard measures of LV wall and chamber dimensions and fractional shortening were obtained as described previously (10, 24).
Solutions for skinned myocardial strips.
Solutions were formulated by solving equations describing ionic equilibria (14). Relaxing solution (pCa 8) contained 5 mM EGTA, 5 mM ATP, 1 mM Mg2+, 240 U/ml creatine kinase, 40 mM phosphocreatine, and 190 meq ionic strength (pH 7.0). Activation solution was same as relaxing solution but with pCa 4. Rigor solution was same as activating but without ATP, creatine kinase, and phosphocreatine. Storage solution was same as relaxing but with 10 μg/ml leupeptin and 50% wt/vol glycerol. Skinning solution was same as storage but with 1% vol/vol Triton X-100.
Analyses for cardiac sarcomere performance in mice at 10–20 wk.
αMHCR403Q/+ and αMHC+/+ mice aged 10–20 wk were acquired from the laboratory of Dr. J. G. Seidman. Mice were killed by cervical dislocation, and hearts were rapidly excised and placed in 95% O2-5% CO2 bubbled Krebs solution containing 30 mM 2,3-butanedione monoxime (26). Right ventricles were trimmed away, and LV papillary muscles were removed. Papillary muscles were dissected to yield at least four thin strips (∼120–160 μm diameter, ∼800 μm length) with longitudinally oriented parallel fibers as described previously (26
At least two strips from each heart were Ca2+ activated from pCa 8.0 to pCa 4.5. At each pCa point, strips underwent sinusoidal length perturbations of amplitude 0.125% strip length and over frequencies ranging between 0.125 and 250 Hz as previously described (2, 31). Individual recordings of normalized isometric tension (T/Tmax) were fit to the Hill equation: T/Tmax = [Ca2+]n/([Ca2+]50n + [Ca2+]n), where [Ca2+]50 = Ca2+ concentration at half activation, pCa50 = −log [Ca2+]50, and n = Hill coefficient using a two-parameter nonlinear least squares algorithm (Sigma Plot 11.0, SPSS, Chicago, IL).
At maximum Ca2+ activation, pCa 4.5, strips underwent one of two different additional protocols: 1) temperature was reduced to 27°C and slack test was applied to determine unloaded shortening velocity Vslack (32), or 2) temperature was reduced to 17°C and a force-clamp protocol was executed to acquire the force-velocity relationship. These reductions in temperature slowed kinetics sufficiently to affect accurate recording of respective measures.
Individual recordings of force-velocity relationship were fit to the hyperbolic Hill equation: (V + b)/b = (1 + a/Tmax)/(T/Tmax + a/Tmax), where a and b are fitted parameters, Tmax = measured maximum activated tension, T/Tmax = prescribed tension for force clamp as fraction of Tmax, and V = recorded velocity at T/Tmax (15). Characteristics of mechanical power (P) were calculated as follows: P = V × T/Tmax, extrapolated maximum velocity (Vmax) = b(Tmax/a) [in muscle lengths (ML)/s], velocity at maximum power (Vopt) = b[(Tmax/a) + 1]1/2 − b (in ML/s), tension at maximum power as a fraction of Tmax (Topt/Tmax) =[(a/Tmax)2 + a/Tmax]1/2 − a/Tmax (no units), and maximum power (Pmax) = VoptTopt/Tmax (in ML/s × T/Tmax) (15).
Cardiac myosin isoform distribution.
Approximately 10 mg of ventricular tissue were homogenized in buffer containing (in mM) 300 KCl, 150 KH2PO4, 20 NaP2O7, 10 MgCl, 5 DTT, and 2 ATP (pH 6.8). Myosin was extracted in the above buffer for 1 h on ice with gentle agitation and then clarified with centrifugation (14,0000 g × 1 min). The protein concentration of the resulting supernatant was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). Myosin isoform separation was achieved by the method of Reiser and Kline (27, 35). In brief, isolated cardiac myosin (2–5 μg) was run on separating gels (contain 8% acrylamide and 5% glycerol) for 30 h at 200 V and 8°C. After silver staining, relative isoform content was determined by densitometric analysis with the Fluormax-2 imaging analysis system (Bio-Rad). Separation of α-MHC and β-MHC was verified with bovine cardiac myosin.
Cardiac quantitative real-time PCR analysis.
Heterozygous α-MHCR403/+ (2 M and 2 F) and αMHC+/+ (7 M and 7 F) mice at 7–18 wk of age were killed by cervical dislocation. Cardiac ventricles were immediately harvested. The total RNA isolation was prepared from the ventricular tissue of each mouse separately using TRIzol reagent (Invitrogen, Carlsbad, CA).
Reverse transcription of total RNA (2 μg) was carried out by random hexamer primers and MultiScribe reverse transcriptase from high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). The cDNA from tissue samples of different mice was used as a template for each replicate in each experiment. The specific oligonucleotide primers were designed using Primer3 (http://frodo.wi.mit.edu/) or retrieved from Primer Bank (http://pga.mgh.harvard.edu/primerbank/) (Table 1) (37, 50). The 15-μl PCR reaction performed by 7500 fast real-time PCR system (Applied Biosystems) contained Power SYBR green PCR master mix (Applied Biosystems), 7.5 pmol of each primer, and 1 μl of cDNA template. The relative levels of mRNA expression normalized to control hypoxanthine guanine phosphoribosyl transferase 1 were calculated according to the ΔΔCT method. Dissociation curve analysis was performed after each complete PCR to confirm an expected DNA product.
Western blot analysis.
Tissue samples were also separately homogenized with tissue homogenizer in lysis buffer (50 mM Tris·HCl, 150 mM NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, 1 mM EDTA, pH 7.4, and 0.1% SDS) containing protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (Pierce, Rockford, IL). Protein concentration was determined using bicinchoninic acid protein assay (Pierce).
Protein extracts were subjected to reducing SDS-PAGE and transferred to polyvinylidene difluoride membrane. Mouse monoclonal cardiac troponin T antibody (1:1,000; ab8295, Abcam, Cambridge, MA) and rabbit anti-troponin I polyclonal antibody (1:1,000; no. 4002; Cell Signaling, Danvers, MA) were used as primary antibody to detect 20 μg of cardiac troponin T and cardiac troponin I, respectively. Rabbit anti-phospho-troponin I polyclonal antibody (1:1,000; no. 4004, Cell Signaling) was used to detect 60–80 μg of phosphorylated cardiac troponin I. All secondary antibodies (1:5,000; Santa Cruz, Santa Cruz, CA) were horseradish peroxidase conjugated. Rabbit anti-GAPDH polyclonal antibody (1:10,000; ab36840, Abcam) was used as internal control to normalize the amount of proteins among Western blots. The detection of horseradish peroxidase was performed using SuperSignal West Pico chemiluminescent substrate (Pierce). Autoradiograph was developed using BioMax light film (Kodak, Rochester, NY). The band intensities of protein levels were quantified by densitometry with National Institutes of Health Image J software (http://rsb.info.nih.gov/ij/) (1).
All data are presented as means ± SE. Multiple measures from a single heart were averaged together to provide a single value for that heart. Two-way ANOVA was performed using sex (M, F) and R403Q mutation (αMHC+/+, αMHCR403Q/+) as group identifiers. To detect dependencies on Pi, a repeated-measures ANOVA was performed with values taken at the various Pi concentrations as the repeated measures. For data describing gene expression and protein characteristics, post hoc analyses was performed for each sex between αMHC+/+ and αMHCR403Q/+. For data describing myofilament performance characteristics, post hoc analyses was performed using Duncan's test to detect differences among all four groups: M-αMHC+/+, M-αMHCR403Q/+, F-αMHC+/+, and F-αMHCR403Q/+. Analysis was performed using SPSS 10.0. Statistical significances are reported at the P < 0.05 and P < 0.01 levels.
Hypertrophic response and gene expression.
The development of HCM, as indicated by thicker LV posterior wall and narrower LV chamber dimensions (Table 2), was significant in the M-αMHCR403Q/+ but not in the F-αMHCR403Q/+ mice at age 40 wk. Fractional shortening was also significantly elevated in the M-αMHCR403Q/+ but not in the F-αMHCR403Q/+ mice (Table 2). These data are consistent with previous findings of enhanced LV performance and thicker LV walls in the M-αMHCR403Q/+ and relatively normal LV performance and wall thickness in F-αMHCR403Q/+ mice (13, 29).
Gene expression of natriuretic polypeptide A measured by RT-PCR was elevated in the hearts of both M- and F-αMHCR403Q/+ mice at 7–18 wk compared with sex-matched controls (Fig. 1). Gene expression levels of natriuretic polypeptide B, skeletal actin, cardiac troponin T, and cardiac troponin I were not significantly affected by the R403Q allele (Fig. 1). The similar gene expression profiles observed between the male and female mice indicated a similar hypertrophic response of the cardiac tissue initiated in both males and females. Nevertheless, sex differences in LV performance and HCM development in the αMHCR403Q/+ genotype may indicate sex differences in the mechanical performance of the R403Q myosin incorporated into the myofilaments. We therefore tested whether myofilament protein characteristics and performance in the αMHCR403Q/+ mice were differentiated between M and F animals at an earlier age and accordingly could be responsible for the sex differences in the development of HCM.
Myofilament protein characteristics.
Cardiac myosin in all four groups at age 10–20 wk was composed of α-MHC and did not include any detectable portion of β-MHC (Fig. 2A). Therefore, measures of myofilament mechanical performance cannot be due to the incorporation of β-MHC. It has been reported previously that ∼50% of the myosin is the R403Q mutant (12, 24).
The phosphorylation status of troponin I was reduced in both F- and M-αMHCR403Q/+ mice (Fig. 2, B and C). Any differences in sarcomere performance observed between αMHCR403Q/+ and αMHC+/+ could therefore not be attributed to troponin I phosphorylation.
Isometric tension-pCa and tension-Pi relationships at 10–20 wk.
The Ca2+ sensitivity of myofilament activation was not different between M-αMHCR403Q/+ and M-αMHC+/+ mice (Fig. 3A) or between F-αMHCR403Q/+ and F-αMHC+/+ mice (Fig. 3B). However, myofilaments from M mice of either genotype were significantly more sensitive to Ca2+ activation than those of F mice (sex main effect P < 0.05; Table 3). This result highlights a sex difference in this particularly important physiological attribute of myofilament activation and relaxation, which may contribute to the sex differences in the development of the HCM phenotype. There was a statistically significant elevation in Tmax in the αMHCR403Q/+ mice by ∼10% compared with αMHC+/+, as indicated by a significant mutation main effect (P < 0.05; Fig. 3C). Marginal trends for an elevation in Tmax were observed previously for the αMHCR403Q/+ mice but were without statistical significance (2, 31). There was also a significant mutation × Pi interaction for Tmax (P < 0.05), indicating that αMHCR403Q/+ group is more sensitive to the reduction with tension as occurs with Pi exposure. This result can be visualized in Fig. 3D, where the recorded tension in both the M- and F-αMHCR403Q/+ populations was diminished by Pi with a greater sensitivity than in the sex-matched αMHCR403Q/+ mice.
Sinusoidal length perturbations and oscillatory work.
Sinusoidal length perturbations were normalized by the muscle length providing a strain on the activated myofilaments. An example of a single cycle of sinusoidal strain is illustrated as the dotted line in Fig. 4A. The resultant stress, which is the sinusoidal change from steady-state tension, will lag behind the strain, illustrated by the example at 9 Hz in Fig. 4A, or will lead the strain, illustrated by the example at 15 Hz in Fig. 4A.
The work loops presented in the stress-strain relationships of Fig. 4B indicate the amount of work that is generated by the myofilaments (i.e., when loop is counterclockwise, work is positive) or is absorbed by the myofilaments (i.e., when loop is clockwise, work is negative). At maximum Ca2+ activation, work was generated at a higher frequency range in M-αMHCR403Q/+ mice than in M-αMHC+/+ mice (Fig. 4C) and also for F-αMHCR403Q/+ mice vs. F-αMHC+/+ mice (Fig. 4D).
The maximum work produced during sinusoidal length perturbation of activated myofilaments was higher in the αMHCR403Q/+ mice than in sex-matched αMHC+/+ mice (Fig. 5, A and B) when myofilaments were exposed to pCa 7 or pCa 8 and therefore not Ca2+ activated. These data indicate that there must have been an enhanced Ca2+-independent activation of the thin filament in the αMHCR403Q/+ mice compared with sex-matched αMHC+/+ mice. However, when the thin filament was Ca2+ activated, work production rose with the degree of Ca2+ activation but also similarly between αMHCR403Q/+ and sex-matched αMHC+/+ mice (Fig. 5, A and B).
At or near maximum Ca2+ activation, the frequency of maximum work production was significantly higher in the αMHCR403Q/+ mice than in sex-matched αMHC+/+ mice (Fig. 5, C and D). The frequency of maximum work production also rose with Pi concentration (Fig. 5, E and F) as indicated by a significant Pi main effect (P < 0.05). Additionally, there was a significant mutation main effect and mutation × Pi interaction (P < 0.05). These latter results can be visualized in Fig. 5, E and F, as a higher frequency of maximum work and a greater Pi sensitivity of the frequency of maximum work in the αMHCR403Q/+ compared with sex-matched αMHC+/+ mice. There were no mutation × sex or mutation × sex × Pi interactions.
Vslack and force-velocity relationships.
Maximum Vslack, measured by the slack test, was elevated in F compared with M mice (sex main effect P < 0.05) and in αMHCR403Q/+ compared with αMHC+/+ mice (genotype main effect P < 0.05) (Fig. 6, A and B). By post hoc analysis, Vslack in M-αMHC+/+ mice was significantly lower than that shown in the other groups (Fig. 6, C and D). The higher Vslack due to the R403Q mutation is consistent with the finding of higher actin velocities in motility assays using R403Q myosin isolated from homozygous or heterozygous mice (47).
Figure 7, A and B, demonstrates the force-clamp method used to assay the tension-velocity relationships. Figure 7, C and D, illustrates the resulting tension-velocity relationships. Velocity of shortening at all loads was elevated in the αMHCR403Q/+ muscle strips compared with sex-matched αMHC+/+ mice. Correspondingly, mechanical power output at all loads was higher in the αMHCR403Q/+ muscle strips of both M and F animals (Fig. 7, C and D).
Table 4 provides values for hyperbolic Hill characteristics of the tension-velocity and tension-power relationships for the four mouse populations. The curvature of the tension-velocity relationships was suppressed in the αMHCR403Q/+ mice, as indicated by the elevated values for a/Tmax (Table 4) and as can be seen in Fig. 7, C and D (15). The extrapolated value of Vmax was not significantly different due to sex or R403Q mutation. However, the directly measured loaded shortening under all loaded conditions, including the lowest load of only 4% Tmax, was significantly higher in M- and F-αMHCR403Q/+ than in M- and F-αMHC+/+ (Fig. 7, C and D). Vopt is the velocity of shortening at Pmax output and was significantly higher in the αMHCR403Q/+ mice, independent of sex (genotype main effect P < 0.05; Table 4).
The relative tension at Pmax output (Topt/Tmax) was higher in M- than in F-αMHCR403Q/+ mice (Table 4). This is an important result because it suggests that M sex and the R403Q mutation independently promote myofilament performance to achieve higher power output at higher mechanical loads than in αMHC+/+. Pmax was likewise higher in M and in αMHCR403Q/+ mice. Interestingly, Topt/Tmax and Pmax were similar between M-αMHCR403Q/+ and F-αMHCR403Q/+ mice, indicating that sex played no role in determining these measures of enhanced myofilament performance due to R403Q mutation.
Sex hormones and HCM development.
To explore the influence of sex hormones on HCM development and LV function, we removed the gonads of a small number of mice and measured LV dimensions and function via echocardiography. Interestingly, thickening of the LV wall was diminished or nonexistent in the castrated M-αMHCR403Q/+ compared with M-αMHC+/+ mice, but LV wall was thicker in the oopherectomized F-αMHCR403Q/+ than in F-αMHC+/+ mice (Table 5). In addition, LV fractional shortening was similarly enhanced in M-αMHCR403Q/+ and F-αMHCR403Q/+ mice compared with sex-matched αMHC+/+ mice (Table 5), in contrast to the lack of enhanced fractional shortening observed in F-αMHCR403Q/+ mice with intact sex hormones (Table 2). We did not at this time undertake the analysis of myofilament performance in the gonadectomized populations.
We report here that sex differences in the development of HCM due to the incorporation of the R403Q mutant myosin are not due to sex differences in the underlying myofilament performance. Relative to sex-matched αMHC+/+ mice, we found that cardiac myofilaments of the αMHCR403Q/+ mice had a higher Tmax (Fig. 3C), a higher frequency of maximum work (Fig. 5, C and D), a higher sensitivity of tension and frequency of maximum work to Pi (Figs. 3D and 5, E and F), a higher Vslack (Fig. 6D), a higher shortening velocity at nearly all mechanical loads (Fig. 7C), and a higher mechanical power output at nearly all mechanical loads (Fig. 7D and Table 4). These attributes, measured at the level of the skinned myocardial strip, strongly suggest an elevation in myofilament kinetic and mechanical performance due to the R403Q mutant myosin (7, 33, 47).
Interestingly, these measures of the enhanced myofilament performance due to the R403Q mutation were independent of sex, although sex played a separate and significant role in affecting some measures of myofilament performance. Specifically, F sex was associated with a lower myofilament sensitivity to Ca2+ activation (Fig. 3A), consistent with previous reports of enhanced myofilament Ca2+ sensitivity in overectomized F rats (4, 46, 52). F sex was also associated with a higher Vslack, as measured by the slack test (Fig. 6D). The underlying mechanisms, however, are not clear, although our data (Fig. 2) suggest that myosin isoform profile and troponin I phosphorylation were not contributing factors. Myofilament kinetics, characterized here by the frequency of oscillatory work, were not significantly elevated in the F-αMHC+/+ compared with M-αMHC+/+ mice and were not more sensitive to Pi and therefore could not explain the higher unloaded velocity in the F-αMHC+/+ mice.
Measures of myofilament velocity and power underloaded conditions, however, were significantly more sensitive to load in the F-αMHC+/+ mice, as indicated by the lowest measures of the parameter a/Tmax (Table 4) (15). Additionally, Pmax production occurred at lower tensions in the F-αMHC+/+ animals (e.g., Topt in Table 4). Thus we speculate that those molecular mechanisms responsible for the load sensitivity of myofilament shortening velocity and power production underlie the greater Vslack and lower Topt results in the F-αMHC+/+ mice. At this time, it is not clear what these molecular mechanisms may be, although an upregulation of the atrial myosin essential light-chain isoform and/or an elevated level regulatory light-chain phosphorylation represent two possible mechanisms at the myofilament level (6, 25). M sex was associated with a higher shortening velocity (Fig. 7C) and power production (Fig. 7D and Table 4) under all loaded conditions. These results imply that myofilament performance contributes substantially to the enhanced cardiac contractile function in the LV of males compared with females (38).
The higher frequency of maximum work, force production, Vopt, and Pmax in the αMHCR403Q/+ mice at the myofilament level mimic and likely underlie the enhanced systolic elastance (13), rate of pressure development (29), and circumferential fractional shortening (24, 42) observed for LV function in the M-αMHCR403Q/+ mice (Table 2). One detrimental effect of the enhanced power generation by the R403Q mutant α-MHC in the M-αMHCR403Q/+ mice could be the mechanical disruption of intersarcomeric and intermyocyte connections, as indicated by the loss of Z-line registration and by the development of myocyte disarray and fibrosis (24, 40, 47).
As stated above, the enhanced characteristics of myofilament performance measured here were independent of sex, whereas enhanced LV systolic performance was not observed in the F-αMHCR403Q/+ mice (Table 2). These observations suggest that F sex hormones neutralize the intrinsically enhanced myofilament performance of the F-αMHCR403Q/+ mice in vivo. Indeed, we found that gonadectomized αMHCR403Q/+ mice exhibit similarly narrowed chamber dimensions and enhanced fractional shortening in both sexes (Table 5). These data would imply that the development of HCM due to a R403Q mutant allele may be accelerated by M sex hormones, as suggested by others (21, 23), and/or by the absence of F sex hormones (3, 21, 46).
This study was funded by National Heart, Lung, and Blood Institute Grant HL-59408.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 by the American Physiological Society