Familial hypertrophic cardiomyopathy: Functional effects of myosin mutation R723G in cardiomyocytes

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the β-cardiac myosin heavy chain (β-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy.

Hypertrophic Cardiomyopathy

“…Thin filament mutations: In contrast to HCM mutations in the thick filaments, mutations in the thin filaments enhance Ca ++ sensitivity of myofibrillar ATPase activity, and hence of force generation 110, 111, 114, 116 . These changes appear to be the direct effects of the mutations, as they precede the development of cardiac hypertrophy, at least in the murine models 98 .…”

“…These changes appear to be the direct effects of the mutations, as they precede the development of cardiac hypertrophy, at least in the murine models 98 . Changes occurring after the development of cardiac hypertrophy, whether in the human or murine heart, are subject to secondary modifications of the sarcomere proteins, including phosphorylation of TNNI3 and MYBPC3, post-translational modifications that are known to affect sarcomere functions 116, 117 .…”

Cardiomyopathies and Related Changes in Contractility of Human Heart Muscle

“…It may be the case that idiopathic DCM hearts with found mutations may have normal phosphorylation of TnI, whereas hearts without mutation rather have a reduced phosphorylation level. Alongside with TnI, MyBP-C and myosin light chain 2 (or regulatory) phosphorylation also decreased by 1.5–2.5 folds in hearts with cardiomyopathies [67,73,96,97,98]. Phosphorylation of MyBP-C may be responsible for about half of the change in EC 50 at low sarcomere length, but not at high sarcomere length, and is an important factor in length-dependent activation [12].…”

“…Several studies indicate that uncoupling maybe not an intrinsic property of human HCM [67,73] (Figure 4A). Treatment with PKA decreased Ca 2+ -sensitivity of human cardiomyocytes with HCM mutations in MYBPC3 and MYH7 , as well as with mutations in thin filament proteins: TNNI R145W and TNNT2 K280N.…”

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

“…This has complicated the interpretation of the experimental data because mutations in α-cardiac myosin have different effects than mutations in β-cardiac myosin (Lowey et al, 2008; Palmer et al, 2008; Witjas-Paalberends et al, 2014; Nag et al, 2015). Other studies have examined human muscle fibers purified from skeletal muscle biopsies or from ventricular samples obtained from patients who had cardiac surgeries (Köhler et al, 2002; Seebohm et al, 2009; Brenner et al, 2012; Kraft et al, 2013; Witjas-Paalberends et al, 2014). Measurements on human recombinant β-cardiac myosin are just beginning to be reported and are promising for examining large numbers of different mutations to establish structure-function relationships.…”