Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice
Wenrui Huang, Jingsheng Liang, Katarzyna Kazmierczak, Priya Muthu, Divya Duggal, Gerrie P Farman, Lars Sorensen, Iraklis Pozios, Theodore P Abraham, Jeffrey R Moore, Julian Borejdo, Danuta Szczesna-Cordary, Wenrui Huang, Jingsheng Liang, Katarzyna Kazmierczak, Priya Muthu, Divya Duggal, Gerrie P Farman, Lars Sorensen, Iraklis Pozios, Theodore P Abraham, Jeffrey R Moore, Julian Borejdo, Danuta Szczesna-Cordary
Abstract
We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype.
Keywords: Contractile force; Echocardiography; Hypertrophic cardiomyopathy (HCM); Mutation; Myosin regulatory light chain; Transgenic mice.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Figures
A. Amino acid sequence of the human ventricular myosin RLC (GenBank accession no. P10916) and the sites of HCM-associated mutations (underlined); also indicated the Ser15 phosphorylation site and the Ca2+-binding site (shadowed). B. RLC-Lys104Glu expression in transgenic mice. Atrial (upper panel) and ventricular (lower panel) myofibrils from Tg-MUT L2, L3 and L7 mice were probed for protein expression: 95±2% (n=12) (L2), 97±1% (n=12) (L3) and 99±0.5% (n=12) (L7) of Lys104Glu RLC mutant were incorporated into the hearts of Tg-MUT mice compared to 97.0±3% (n=12) of Tg-WT L2 generated previously [6]. The data are presented as average of n experiments performed on cardiac myofibrils and heart extracts ± SEM. No differences in protein expression level were observed between the preparations. Note that RLCventr. migrated faster than RLCatrial while no differential gel mobility was monitored between the mouse and human RLCventr.. Abbreviations: NTg, non-transgenic; L, mouse line; RLCventr., ELCventr., ventricular isoforms of RLC or ELC. C. Real-time PCR quantification of human MYL2 gene expression in the atria and ventricles of Tg-WT and Tg-MUT L2, L3 and L7 mice. The results were expressed as a ratio ofMYL2 to GAPDH (housekeeping gene). Note that similar levels of mRNA expression were observed between atria (A) and ventricles (V) in all tested transgenic lines.
A. Amino acid sequence of the human ventricular myosin RLC (GenBank accession no. P10916) and the sites of HCM-associated mutations (underlined); also indicated the Ser15 phosphorylation site and the Ca2+-binding site (shadowed). B. RLC-Lys104Glu expression in transgenic mice. Atrial (upper panel) and ventricular (lower panel) myofibrils from Tg-MUT L2, L3 and L7 mice were probed for protein expression: 95±2% (n=12) (L2), 97±1% (n=12) (L3) and 99±0.5% (n=12) (L7) of Lys104Glu RLC mutant were incorporated into the hearts of Tg-MUT mice compared to 97.0±3% (n=12) of Tg-WT L2 generated previously [6]. The data are presented as average of n experiments performed on cardiac myofibrils and heart extracts ± SEM. No differences in protein expression level were observed between the preparations. Note that RLCventr. migrated faster than RLCatrial while no differential gel mobility was monitored between the mouse and human RLCventr.. Abbreviations: NTg, non-transgenic; L, mouse line; RLCventr., ELCventr., ventricular isoforms of RLC or ELC. C. Real-time PCR quantification of human MYL2 gene expression in the atria and ventricles of Tg-WT and Tg-MUT L2, L3 and L7 mice. The results were expressed as a ratio ofMYL2 to GAPDH (housekeeping gene). Note that similar levels of mRNA expression were observed between atria (A) and ventricles (V) in all tested transgenic lines.
A. Amino acid sequence of the human ventricular myosin RLC (GenBank accession no. P10916) and the sites of HCM-associated mutations (underlined); also indicated the Ser15 phosphorylation site and the Ca2+-binding site (shadowed). B. RLC-Lys104Glu expression in transgenic mice. Atrial (upper panel) and ventricular (lower panel) myofibrils from Tg-MUT L2, L3 and L7 mice were probed for protein expression: 95±2% (n=12) (L2), 97±1% (n=12) (L3) and 99±0.5% (n=12) (L7) of Lys104Glu RLC mutant were incorporated into the hearts of Tg-MUT mice compared to 97.0±3% (n=12) of Tg-WT L2 generated previously [6]. The data are presented as average of n experiments performed on cardiac myofibrils and heart extracts ± SEM. No differences in protein expression level were observed between the preparations. Note that RLCventr. migrated faster than RLCatrial while no differential gel mobility was monitored between the mouse and human RLCventr.. Abbreviations: NTg, non-transgenic; L, mouse line; RLCventr., ELCventr., ventricular isoforms of RLC or ELC. C. Real-time PCR quantification of human MYL2 gene expression in the atria and ventricles of Tg-WT and Tg-MUT L2, L3 and L7 mice. The results were expressed as a ratio ofMYL2 to GAPDH (housekeeping gene). Note that similar levels of mRNA expression were observed between atria (A) and ventricles (V) in all tested transgenic lines.
Notice a reduced E/A ratio in Tg-MUT compared with Tg-WT.
A. Hematoxylin and eosin (H&E), and B.Masson’s trichrome stained LV sections from 4.6 (young), 8.6 (intermediate) and 15.8 (old) month-old Tg-WT and 3.9 (young), 8 (intermediate) and 14.6 (old) month-old Tg-MUT mice. Note the progression of fibrosis in Tg-MUT hearts in mice of 8 to 15 months of age.
A. Hematoxylin and eosin (H&E), and B.Masson’s trichrome stained LV sections from 4.6 (young), 8.6 (intermediate) and 15.8 (old) month-old Tg-WT and 3.9 (young), 8 (intermediate) and 14.6 (old) month-old Tg-MUT mice. Note the progression of fibrosis in Tg-MUT hearts in mice of 8 to 15 months of age.
A. Transmission electron micrographs showing sarcomeric ultrastructure of LV tissue from 6 month-old transgenic mice. The magnification is 10,500 X. Bar = 1 μm. Black arrows depict the Z-discs and white arrows the mitochondrial structures. Note, more mitochondria are recruited in the mutated hearts compared to WT controls. B. Assessment of mitochondrial content in Tg-MUT vs. Tg-WT mice. Upper panel: SDS-PAGE of heart extracts probed for VDAC (porin) protein and normalized for the myosin ELC, used as a loading control. Lanes 1 and 3, Tg-WT 4 and 14 month-old, respectively; lanes 2 and 4, Tg-MUT, 4 and 14 month-old, respectively. Lower panel: Band intensity ratios derived from gel samples run for young and old Tg-WT and Tg-MUT extracts. 14 myocardial extracts were used for each group of mice. Note that the mitochondrial content was significantly (P<0.05) higher in young and old Tg-MUT vs. Tg-WT hearts. C. Analysis of RLC phosphorylation. The level of RLC phosphorylation was determined with phospho-specific RLC antibodies and compared to the total RLC content assessed with a rabbit polyclonal RLC CT-1 antibody recognizing total RLC protein. Myofibrillar preparations from left ventricles of Tg-MUT (L2 and L3) and Tg-WT L2 mice were used. Upper panel, representative Western blot demonstrating lower RLC phosphorylation in Tg-MUT compared with Tg-WT mice. Lane 1, Tg-WT (3 month-old); lane 2, Tg-MUT (3 month-old); lane 3, Tg-WT (18 month-old); lane 4, Tg-MUT (17 month-old); lane 5: −P RLC standard protein; lane 6, +P RLC standard protein. Lower panel, quantification of RLC phosphorylation in Tg-MUT vs. Tg-WT mice. The data were collected from 13 independent Western blots. Note that young and old Tg-MUT mice showed a significantly lower level of endogenous RLC phosphorylation compared to age matched Tg-WT (P<0.05). Abbreviations: VDAC, Voltage-dependent anion channel; −P, unphosphorylated RLC; +P, phosphorylated RLC; L, mouse line.
A. Transmission electron micrographs showing sarcomeric ultrastructure of LV tissue from 6 month-old transgenic mice. The magnification is 10,500 X. Bar = 1 μm. Black arrows depict the Z-discs and white arrows the mitochondrial structures. Note, more mitochondria are recruited in the mutated hearts compared to WT controls. B. Assessment of mitochondrial content in Tg-MUT vs. Tg-WT mice. Upper panel: SDS-PAGE of heart extracts probed for VDAC (porin) protein and normalized for the myosin ELC, used as a loading control. Lanes 1 and 3, Tg-WT 4 and 14 month-old, respectively; lanes 2 and 4, Tg-MUT, 4 and 14 month-old, respectively. Lower panel: Band intensity ratios derived from gel samples run for young and old Tg-WT and Tg-MUT extracts. 14 myocardial extracts were used for each group of mice. Note that the mitochondrial content was significantly (P<0.05) higher in young and old Tg-MUT vs. Tg-WT hearts. C. Analysis of RLC phosphorylation. The level of RLC phosphorylation was determined with phospho-specific RLC antibodies and compared to the total RLC content assessed with a rabbit polyclonal RLC CT-1 antibody recognizing total RLC protein. Myofibrillar preparations from left ventricles of Tg-MUT (L2 and L3) and Tg-WT L2 mice were used. Upper panel, representative Western blot demonstrating lower RLC phosphorylation in Tg-MUT compared with Tg-WT mice. Lane 1, Tg-WT (3 month-old); lane 2, Tg-MUT (3 month-old); lane 3, Tg-WT (18 month-old); lane 4, Tg-MUT (17 month-old); lane 5: −P RLC standard protein; lane 6, +P RLC standard protein. Lower panel, quantification of RLC phosphorylation in Tg-MUT vs. Tg-WT mice. The data were collected from 13 independent Western blots. Note that young and old Tg-MUT mice showed a significantly lower level of endogenous RLC phosphorylation compared to age matched Tg-WT (P<0.05). Abbreviations: VDAC, Voltage-dependent anion channel; −P, unphosphorylated RLC; +P, phosphorylated RLC; L, mouse line.
A. Transmission electron micrographs showing sarcomeric ultrastructure of LV tissue from 6 month-old transgenic mice. The magnification is 10,500 X. Bar = 1 μm. Black arrows depict the Z-discs and white arrows the mitochondrial structures. Note, more mitochondria are recruited in the mutated hearts compared to WT controls. B. Assessment of mitochondrial content in Tg-MUT vs. Tg-WT mice. Upper panel: SDS-PAGE of heart extracts probed for VDAC (porin) protein and normalized for the myosin ELC, used as a loading control. Lanes 1 and 3, Tg-WT 4 and 14 month-old, respectively; lanes 2 and 4, Tg-MUT, 4 and 14 month-old, respectively. Lower panel: Band intensity ratios derived from gel samples run for young and old Tg-WT and Tg-MUT extracts. 14 myocardial extracts were used for each group of mice. Note that the mitochondrial content was significantly (P<0.05) higher in young and old Tg-MUT vs. Tg-WT hearts. C. Analysis of RLC phosphorylation. The level of RLC phosphorylation was determined with phospho-specific RLC antibodies and compared to the total RLC content assessed with a rabbit polyclonal RLC CT-1 antibody recognizing total RLC protein. Myofibrillar preparations from left ventricles of Tg-MUT (L2 and L3) and Tg-WT L2 mice were used. Upper panel, representative Western blot demonstrating lower RLC phosphorylation in Tg-MUT compared with Tg-WT mice. Lane 1, Tg-WT (3 month-old); lane 2, Tg-MUT (3 month-old); lane 3, Tg-WT (18 month-old); lane 4, Tg-MUT (17 month-old); lane 5: −P RLC standard protein; lane 6, +P RLC standard protein. Lower panel, quantification of RLC phosphorylation in Tg-MUT vs. Tg-WT mice. The data were collected from 13 independent Western blots. Note that young and old Tg-MUT mice showed a significantly lower level of endogenous RLC phosphorylation compared to age matched Tg-WT (P<0.05). Abbreviations: VDAC, Voltage-dependent anion channel; −P, unphosphorylated RLC; +P, phosphorylated RLC; L, mouse line.
A. Maximal tension assessment at saturating (pCa 4) calcium concentrations. Note Tg-MUT skinned papillary muscle strips developed a 1.4-fold lower maximal force (per cross-sectional area of muscle strip) compared with Tg-WT (PB. Calcium sensitivity of force in skinned papillary muscle strips from Tg-WT vs. Tg-MUT mice. No significant (P>0.05) changes in the Ca2+ sensitivity of force or Hill coefficient values were observed between the strips from Tg-MUT animals compared with Tg-WT. Error bars represent SEM. C. Muscle relaxation dependence in Tg-MUT and Tg-WT papillary muscle strips. A significant reduction in relaxation rates was observed in Tg-MUT vs. Tg-WT muscle strips (P<0.01). D. Assessment of passive tension (passive stiffness) under relaxation conditions (pCa 8). Error bars represent SEM. Note significantly increased levels of passive tension in Tg-MUT compared to Tg-WT mice (P<0.001, as determined by ANOVA for repeated measurements).
A. Maximal tension assessment at saturating (pCa 4) calcium concentrations. Note Tg-MUT skinned papillary muscle strips developed a 1.4-fold lower maximal force (per cross-sectional area of muscle strip) compared with Tg-WT (PB. Calcium sensitivity of force in skinned papillary muscle strips from Tg-WT vs. Tg-MUT mice. No significant (P>0.05) changes in the Ca2+ sensitivity of force or Hill coefficient values were observed between the strips from Tg-MUT animals compared with Tg-WT. Error bars represent SEM. C. Muscle relaxation dependence in Tg-MUT and Tg-WT papillary muscle strips. A significant reduction in relaxation rates was observed in Tg-MUT vs. Tg-WT muscle strips (P<0.01). D. Assessment of passive tension (passive stiffness) under relaxation conditions (pCa 8). Error bars represent SEM. Note significantly increased levels of passive tension in Tg-MUT compared to Tg-WT mice (P<0.001, as determined by ANOVA for repeated measurements).
A. Maximal tension assessment at saturating (pCa 4) calcium concentrations. Note Tg-MUT skinned papillary muscle strips developed a 1.4-fold lower maximal force (per cross-sectional area of muscle strip) compared with Tg-WT (PB. Calcium sensitivity of force in skinned papillary muscle strips from Tg-WT vs. Tg-MUT mice. No significant (P>0.05) changes in the Ca2+ sensitivity of force or Hill coefficient values were observed between the strips from Tg-MUT animals compared with Tg-WT. Error bars represent SEM. C. Muscle relaxation dependence in Tg-MUT and Tg-WT papillary muscle strips. A significant reduction in relaxation rates was observed in Tg-MUT vs. Tg-WT muscle strips (P<0.01). D. Assessment of passive tension (passive stiffness) under relaxation conditions (pCa 8). Error bars represent SEM. Note significantly increased levels of passive tension in Tg-MUT compared to Tg-WT mice (P<0.001, as determined by ANOVA for repeated measurements).
A. Maximal tension assessment at saturating (pCa 4) calcium concentrations. Note Tg-MUT skinned papillary muscle strips developed a 1.4-fold lower maximal force (per cross-sectional area of muscle strip) compared with Tg-WT (PB. Calcium sensitivity of force in skinned papillary muscle strips from Tg-WT vs. Tg-MUT mice. No significant (P>0.05) changes in the Ca2+ sensitivity of force or Hill coefficient values were observed between the strips from Tg-MUT animals compared with Tg-WT. Error bars represent SEM. C. Muscle relaxation dependence in Tg-MUT and Tg-WT papillary muscle strips. A significant reduction in relaxation rates was observed in Tg-MUT vs. Tg-WT muscle strips (P<0.01). D. Assessment of passive tension (passive stiffness) under relaxation conditions (pCa 8). Error bars represent SEM. Note significantly increased levels of passive tension in Tg-MUT compared to Tg-WT mice (P<0.001, as determined by ANOVA for repeated measurements).
A. Native myosin ELC was exchanged with 10 nM SeTau-ELC in myofibrils as described in Materials and Methods. In the image, the gray circle represents a projection of the confocal aperture on the sample plane (diameter 0.5 μm). The black and white scale is from 0 (black) to 168 (white). Scale bar = 5 μm, sarcomere length = 2.1 μm. Images were acquired on a PicoQuant Micro Time 200 confocal lifetime microscope. The sample was excited with a 640 nm pulsed laser and observed through a LP 650 nm filter.B. The anisotropy values associated with different cross-bridge states are 0 for MT MDP, intermediate for AMDP and maximum for AM, AMD and AMT* (rigor, strongly bound or near rigor) states. Autocorrelation function (ACF ) of such a process is shown schematically as the dashed line. Abbreviations used in the 3 state model of muscle contraction: M, Myosin; D, ADP; P, inorganic phosphate; A, F-actin; T, ATP.
A Measurements were performed on myosin isolated from left and right ventricles of Tg-MUT and Tg-WT mice (6-7 hearts per preparation). ATPase assays were run in triplicate with n=16 individual measurements for WT and n=13 for Tg-MUT. Note, significant differences in Vmax and no difference in Km between the groups. B. Actin sliding velocity as a function of α-actinin concentration. Tg-MUT myosin propelled actin at a higher unloaded sliding velocity. However, once α-actinin was applied to the flow cell the velocity dropped until reaching a plateau of ~0.25 μm/s. Conversely Tg-WT myosin had a slower unloaded velocity that dropped less steeply with α-actinin concentration until ~4 μg/ml α-actinin. At higher α-actinin concentrations velocity declined to a plateau similar to Tg-MUT. C. Fraction motile filaments as a function of applied α-actinin. The fraction of motile filaments for both Tg-WT and Tg-MUT illustrates a similar relationship as observed for velocity in B for the mutant myosin. However, WT myosin plateaus at a higher percent filaments motile than the mutant myosin, consistent with a reduction in force generation by Tg-MUT [41]. Error bars represent SEM.
A Measurements were performed on myosin isolated from left and right ventricles of Tg-MUT and Tg-WT mice (6-7 hearts per preparation). ATPase assays were run in triplicate with n=16 individual measurements for WT and n=13 for Tg-MUT. Note, significant differences in Vmax and no difference in Km between the groups. B. Actin sliding velocity as a function of α-actinin concentration. Tg-MUT myosin propelled actin at a higher unloaded sliding velocity. However, once α-actinin was applied to the flow cell the velocity dropped until reaching a plateau of ~0.25 μm/s. Conversely Tg-WT myosin had a slower unloaded velocity that dropped less steeply with α-actinin concentration until ~4 μg/ml α-actinin. At higher α-actinin concentrations velocity declined to a plateau similar to Tg-MUT. C. Fraction motile filaments as a function of applied α-actinin. The fraction of motile filaments for both Tg-WT and Tg-MUT illustrates a similar relationship as observed for velocity in B for the mutant myosin. However, WT myosin plateaus at a higher percent filaments motile than the mutant myosin, consistent with a reduction in force generation by Tg-MUT [41]. Error bars represent SEM.
A Measurements were performed on myosin isolated from left and right ventricles of Tg-MUT and Tg-WT mice (6-7 hearts per preparation). ATPase assays were run in triplicate with n=16 individual measurements for WT and n=13 for Tg-MUT. Note, significant differences in Vmax and no difference in Km between the groups. B. Actin sliding velocity as a function of α-actinin concentration. Tg-MUT myosin propelled actin at a higher unloaded sliding velocity. However, once α-actinin was applied to the flow cell the velocity dropped until reaching a plateau of ~0.25 μm/s. Conversely Tg-WT myosin had a slower unloaded velocity that dropped less steeply with α-actinin concentration until ~4 μg/ml α-actinin. At higher α-actinin concentrations velocity declined to a plateau similar to Tg-MUT. C. Fraction motile filaments as a function of applied α-actinin. The fraction of motile filaments for both Tg-WT and Tg-MUT illustrates a similar relationship as observed for velocity in B for the mutant myosin. However, WT myosin plateaus at a higher percent filaments motile than the mutant myosin, consistent with a reduction in force generation by Tg-MUT [41]. Error bars represent SEM.
At the sarcomere level, the Lys104Glu mutation leads to a decrease in contractile force, increased maximal ATPase activity, and a decrease in muscle relaxation rate. It also results in elevated passive tension. Disruption of myosin load-dependent mechanochemistry is most likely responsible for adverse changes in force production in Tg-MUT mice. These molecular events further trigger morphological and functional remodeling of the heart manifested by abundant mitochondria, development of fibrosis and altered diastolic indices. These changes are anticipated to result in diastolic disturbance in Tg-MUT mice, which together with inefficiency in energy use and diminished RLC phosphorylation may trigger the development of Lys104Glu-RLC induced heart disease.
Source: PubMed