Familial Hypertrophic Cardiomyopathy – is radial
stiffness of human muscle fibers affected by
mutations in the myosin head domain?
Theresia Kraft, Ina Stehle, Birgit Piep, Faramarz Matinmehr, Thomas Mattei, Bernhard Brenner
Molecular and Cell Physiology, Hannover Medical School, D-30625 Hannover, Germany
Familial Hypertrophic Cardiomyopathy (FHC) is characterized by asymmetric hypertrophy of the
left ventricle and the interventricular septum (2). Clinically FHC can lead to diastolic or systolic
heart failure or sudden cardiac death particularly at young age. Cardiomyocyte and myofibrillar
disarray as well as interstitial fibrosis are hallmarks of the disease (3). Prevalence of hypertrophic
cardiomyopathy in a general population of young adults is 1:500 (2). Nearly all genotyped FHC
patients carry a mutation in a sarcomeric protein, of which mutations in the ß-myosin heavy chain
(ß-MyHC) account for more than 1/3 of the cases. Direct functional effects of these mutations as
well as disease pathogenesis are still poorly understood. We investigate effects of ß-MyHCmutations on sarcomere function and structure to characterize the role of specific myosin head
subdomains for the force generating mechanism of striated muscle (4, 5) and to identify initial
triggers of the disease at the cellular level.
Studying functional effects of different FHC-related missense mutations in the converter domain of
the ß-MyHC-head enabled us to address the question which structural element is mainly responsible
for elastic distortion of the acto-myosin complex during force generation. Such elastic distortion is a
key feature of cross-bridge function since it allows the myosin head domain to produce force during
isometric contraction when no sliding of the myofilaments occurs. We found that the two myosin
head domain mutations R719W and R723G, which are located in a small alpha-helix of the
converter domain, cause a significant increase in resistance to elastic distortion (stiffness) of the
muscle fibers from the patients (4, 5). We could trace the increased fiber stiffness to a higher
stiffness of the mutated myosin heads. This implies that the converter region, which became stiffer
with the mutations, is one of the main elastically distorted elements of the myosin head.
Figure 1: Crystal structure of
the myosin head domain (S1
structure from Rayment et al.
(1)),
which
highlights
mutations in the converter
region.
We
studied
Arg719Trp and Arg723Gly
to clarify whether these
mutations affect stiffness of
the myosin head not only in
parallel to the fiber axis but
also in perpendicular, i.e.,
radial direction.
In our last project at DORIS-Beamline A2 in 2012 we experimentally addressed the question
whether FHC-mutations R719 and R723 affect the stiffness of the myosin heads not just along the
muscle fibers axis but also in perpendicular, i.e., radial direction. For our experiments we used slow
skeletal muscle fibers since the ß-cardiac myosin isoform is the same in the myocardium and slow
skeletal muscle. M. soleus biopsies of patients with these mutations and control individuals were
chemically permeabilized and single fibers were isolated. From arrays of some 10-15 of fibers with
the different mutations, respectively, we recorded 2D-X-ray diffraction patterns under rigor
conditions (without nucleotide, i.e., all myosin heads are strongly attached to actin) and under
relaxing conditions. We exposed the fibers with increasing concentrations of high molecular weight
dextran (2%-8% of T500, MW 470kDa). These large molecules cannot penetrate into the fibers and
therefore exert increasing osmotic compression, i.e. a defined radial force on the muscle fibers. In
equatorial diffraction patterns of these fibers we analyze the distance of the [1,0] or [1,1] planes of
the equatorial (cross-sectional) lattice (d1,0 or d1,1). From these equatorial lattice parameters, we
determine the distance between the filaments without and with applied radial forces. Thus we can
characterize elastic properties of the mutated myosin heads in radial direction. Figure 2 shows
equatorial diffraction patterns of muscle fibers from a healthy control and from a patient with
mutation R719W without dextran compression and in the presence of 8% dextran, which
corresponds to an osmotic pressure of 123*102 Pa (6).
Fig.2: The equatorial reflections of muscle fibers from human M. soleus under relaxing conditions (ionic
strength 80mM), 2D-X-ray diffraction patterns recorded at beamline A2, HASYLAB. Left, no dextran;
Right, compressed with 8% dextran. Note the larger distance between the left and right [1,1] and [1,0]
reflection, respectively, with 8% dextran. This corresponds to reduced filament distance under dextran
compression. High dextran concentration causes a high background and thus reduced reflection intensities.
We are currently in the process of analyzing the diffraction patterns we recorded at A2 to determine
the filament distances without and with osmotic compression. This will show whether the myosin
heads with the mutations can resist the radial force of osmotic compression more strongly compared
to normal myosin heads. Since the effect of these mutations on stiffness of the myosin heads in
axial direction was quite significant (2-3fold increase; (4, 5)), we expect to see higher stiffness also
in radial direction. This is of particular interest also because the mutated residues are non-conserved
residues in different myosin isoforms, which exhibit different stiffness of the myosin heads. Thus,
this and further studies on different myosin isoforms will provide new insights into the molecular
basis of elastic deformation of the myosin head domain and of protein compliance in general.
Acknowledgements: We thank Dr. Sergio Funari for his constant support during our beamtimes at
beamline A2. We are also grateful for all the friendly support we received over the years from the
HASYLAB-administration and for travel grants for all co-workers from DESY/HASYLAB. This work was
also supported by a grant from the DFG (KR 1187/18-1, 2).
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