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First published online 2 August 2005
doi: 10.1242/jcs.02492

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The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice

Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, FL 33136, USA

View larger version (61K): [in a new window]   Fig. 1. Protein expression in transgenic mouse lines. Approximately 10 mg of left ventricle tissue from transgenic myc-E22K, line 2 (L2) and line 4 (L4), myc-WT, line 1 (L1) and line 2 (L2) as well as Non-Tg mice were processed (see Materials and Methods) and quantitated on 15% SDS-PAGE using either Coomassie gel staining or polyclonal RLC CT-1 antibodies produced in this laboratory (raised against 15 residues from the C-terminus of human cardiac RLC). (A) SDS-PAGE stained with Coomassie and western blots of Non-Tg, Tg-WT (L1 and L2) and Tg-E22K (L2 and L4) expressed in mouse left ventricular tissue. Cardiac myofibrils prepared from left and right ventricular walls, septa and papillary muscles of transgenic mice were loaded at 10-20 µg per lane, run on 15% SDS-PAGE for Coomassie staining, while approximately 0.1-10 µg of the left-ventricular (LV) muscle extracts were loaded per lane for western blotting. The blots were calibrated with purified recombinant myc-WT that showed a linear dependence in the range of 10-75 ng. The highest loading concentration of 75 ng is shown in the right lane of each blot. The endogenous RLC and transgenic myc-WT, and myc-E22K proteins were all quantitated using polyclonal RLC CT-1 antibodies. Note that transgenic myc-WT and myc-E22K proteins migrate slower than the endogenous RLC because of the myc sequence attached to their N-termini. This was true for the Coomassie-stained gel (left upper panel) and also for RLC-antibody-stained western blots (right panels). Alternatively, the amount of transgene expression was quantitated using monoclonal myc-antibodies (clone 9E10) raised to a peptide from the human MYC protein (left lower panel). (B) Visualization of western blots with fluorescence. Western blots treated with polyclonal RLC CT-1 antibodies were further processed with fluorescent secondary antibodies conjugated with fluorescent dye Cy 5.5. Like in Fig. 1A, myc-RLC migrates slower than endogenous RLC because of the additional myc sequence attached to the N-terminal region of Tg-E22K or Tg-WT proteins. Note that no background fluorescence was found in the blot, indicating that no nonspecific antibody binding or ectopic transgene expression occurred. (C) Multi-determination of transgenic protein expression. Coomassie-stained gels as well as standard and fluorescent western blots were quantitated. Percentages of protein expression were determined by using Coomassie staining (black bars), western blots `standard' (gray bars) and western blots `fluorescence' (striped bars) were as follows: Tg-WT L1, 8.9±5.0 (n=12), 11.9±5.2 (n=6) and 7.4±2.6 (n=8); Tg-WT L2, 24.5±3.8 (n=12), 29.2±8.8 (n=3) and 23.1±3.1 (n=9); Tg-E22K L2, 69.8±2.9 (n=12), 60.4±6.0 (n=6) and 65.4±7.1 (n=8) and Tg-E22K L4, 86.8±4.3 (n=12), 92.3±2.9 (n=12) and 80.7±3.0 (n=4), respectively. Data are expressed as the average of n measurements ± s.d.

View larger version (119K): [in a new window]   Fig. 2. Histology of transgenic mouse hearts. (A) Longitudinal sections of whole mouse hearts of Tg-E22K vs Tg-WT or Non-Tg mice. After euthanasia, the hearts of 13-month-old mice from Non-Tg, Tg-WT and Tg-E22K mice were excised and immersed in 10% buffered formalin. Longitudinal sections of the mouse hearts were stained with hematoxylin and eosin for overall morphology and hypertrophy. Note that the inter-ventricular septum (IVS) of Tg-E22K mice appears more massive than septa of Tg-WT or Non-Tg mice. Also, the papillary muscles of the left ventricles in Tg-E22K hearts appear more substantial than in Tg-WT or Non-Tg mice. (B) Microscopic views of the inter-ventricular septa (upper panels) and left ventricles (lower panels) of Tg-E22K vs Tg-WT or Non-Tg mouse hearts. As indicated, no myofilament disarray was observed in any presented heart tissue. Additionally there was no variation between different mouse lines. LVW, left ventricular wall; IVS, inter-ventricular septum.

View larger version (25K): [in a new window]   Fig. 3. Myofibrillar ATPase activity in transgenic cardiac-muscle preparations. Myofibrils from ventricular and papillary muscles from transgenic mice were examined for myofibrillar ATPase activity in a solution (see Materials and Methods) and increasing concentrations of Ca2+ (pCa 9 to pCa 4.5). After a 5-minute incubation period 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). (A) ATPase-pCa relationship for Non-Tg, Tg-WT, Tg-E22K L2 and Tg-E22K L4 mouse muscle myofibrils. Statistically significant differences in the pCa50 values of the Ca2+ sensitivity of myofibrillar ATPase activity were observed between Non-Tg (n=10) or Tg-WT (n=7) and two lines of the mutant mice, Tg-E22K, line 2 (L2) (n=8) and line 4 (L4) (n=7) (P0.01). A difference of the pCa50=0.14 was observed between Tg-WT and Tg-E22K L4 myofibrils (P=0.01). Respective nH values of the ATPase-pCa relationships were: 1.54±0.17, 1.67±0.20, 1.38±0.14 and 1.45±0.11 for Non-Tg, Tg-WT, Tg-E22K L2 and Tg-E22K L4 myofibrils. (B) The pCa50 values for the ATPase-pCa dependences of Non-Tg, Tg-WT, Tg-E22K L2 and Tg-E22K L4 mouse muscle myofibrils. The Ca2+ sensitivity in Tg-E22K L2 (pCa50=6.40±0.03, n=8) and L4 (pCa50=6.41±0.03, n=7) myofibrils was increased compared with Non-Tg (pCa50=6.23±0.03, n=10) or Tg-WT (pCa50=6.27±0.04, n=7) myofibrils. As indicated, the differences were statistically significant (P0.01). Data are expressed as the average of n experiments ± s.e.m.

View larger version (19K): [in a new window]   Fig. 4. Ca2+ sensitivity of steady-state force development in transgenic papillary muscle fibers. A bundle of approximately three to five fibers isolated from glycerinated mouse papillary muscle fibers was attached by tweezer clips to a force transducer and placed in a 1 ml cuvette. After the initial steady-state force was measured in pCa 4 solution, the fiber bundles were exposed to solutions of increasing Ca2+ concentrations (from pCa 8 to pCa 4). Measurements were performed for control, Non-Tg fibers as well as for Tg-WT, Tg-E22K L2 and Tg-E22K L 4 fibers. (A) The force-pCa relationship for Non-Tg, Tg-WT, Tg-E22K L2 and Tg-E22K L4 mouse papillary muscle fibers. Significant differences in the pCa50 values of the Ca2+ sensitivity of steady-state force were observed between Non-Tg (n=14) or Tg-WT (n=10) and two lines of Tg-E22K, line 2 (L2) (n=7) and line 4 (L4) (n=12) (P0.001). The largest difference of the pCa50=0.12 was observed between Tg-WT and Tg-E22K L4 fibers (P<0.001). The steepness of the curves was lower for the mutant fibers and the respective nH values were: 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. (B) The pCa50 values for the force-pCa relationships of Non-Tg, Tg-WT, Tg-E22K L2 and Tg-E22K L4 mouse papillary muscle fibers. The Ca2+ sensitivity in Tg-E22K L2 (pCa50=5.62±0.02, n=7) and L4 (pCa50=5.65±0.01, n=12) fibers was increased compared with Non-Tg (pCa50=5.54±0.01, n=14) or Tg-WT (pCa50=5.53±0.01, n=10) fibers. As indicated, the differences were statistically significant (P0.001). Data are expressed as the average of n experiments ± s.e.m.

View larger version (39K): [in a new window]   Fig. 5. Schematic representation of the regulatory light-chain of myosin (RLC) and the RLC-binding sequence of myosin heavy-chain. Sequences of the RLC and myosin heavy-chain were derived from the following crystal structures: NCBI numbers 1QVI and 1WDC (Gourinath et al., 2003; Houdusse et al., 1997). The FHC RLC one-point missense mutations are labeled. The A13T and F18L mutations are in the region of RLC that has not been solved in any of the existing crystal structures. The D166V mutation of RLC, the last identified to date, is incorrectly indicated as D166L in the original paper by Richard et al. (Richard et al., 2003) (revised information of P. Charron).