Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin

Abstract

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Keywords: Cardiac myosin; Cardiomyopathy mutations; Force–velocity curves.

Fig. 1.

Sliding velocity as a function of lever arm length. The Dictyosteium discoideum myosin II gene was genetically engineered to produce four forms of S1 with different lever arm lengths (shown on the right), expressed in D. discoideum, and purified as described previously (Uyeda et al., 1996). The normal D. discoideum myosin S1 has two light chains. Constructs containing zero light chains and one light chain were expressed, as well as one containing three light chains. The latter was created by inserting an additional essential light chain (magenta) binding domain into the myosin II heavy chain. The fulcrum was inferred from the intercept of the straight line with the x-axis. v_o, unloaded sliding velocity; d, stroke size; t_s, strongly bound state time. Data are from Uyeda et al. (1996).

Fig. 2.

Schematic drawing to scale of essential components of the sarcomere. Tropomyosin (Tm) and the three subunits of troponin (Tn complex) decorate the actin filament. The domains of myosin are the S1 head (only one of two are shown for clarity), the coiled-coil S2, and the light meromyosin that forms the thick filament (bottom grey platform). The N-terminal domains of myosin binding protein-C (MyBP-C) bind to the thin actin filament and the C-terminal domains bind to the thick myosin filament. Titin (not shown) traverses in between the thin and thick filaments, closely associated with the thick filament backbone. Interactions among titin, MyBP-C, S1, and S2 are possible and perhaps regulatory, but have not been fully explored.

Fig. 3.

Schematic plot of a force–velocity (F–V) curve for heart muscle. The power curve is generated by multiplying the force times the velocity at each point along the F–V curve. The grey zone is the region of the power curve thought to dominate systolic contraction.

Fig. 4.

Schematic figure of the key steps of the actin-activated myosin chemomechanical cycle. The total cycle involves five key steps. The pre-stroke states of S1 are shown on the right and the post-stroke states on the left. Step 1: The pre-stroke S1 with bound ADP (D) and phosphate (P_i) undergoes a weak to strong transition in binding to actin. This is the rate-limiting step in the cycle and determines the total cycle time (t_c), which is 1/k_cat, where k_cat is the turnover number, or the number of substrate molecules each enzyme site converts to product per unit time. Step 2: While tightly bound to actin, the lever arm swings about its fulcrum point (black circle) to the right to its post-stroke position (black arrow), moving the actin filament to the left (bold blue arrow) with respect to the myosin thick filament. Step 3: ADP release frees the active site for binding of ATP (T). This step is related to the velocity of the actin filament sliding as the filament cannot move any faster than the myosin heads can let go. Step 4: ATP binding weakens the interaction of the S1 to actin. Although the ATP-bound heads still maintain a weak affinity for the actin, this state is typically drawn as dissociated from the actin filament. Step 5: ATP hydrolysis results in cocking of the head into the pre-stroke state (Shih et al., 2000).

Fig. 5.

Cartoon of the loaded in vitro motility assay and data obtained for human α- and β-cardiac sS1. (A) Myosin sS1 (red) and utrophin (green) are anchored to a microscope cover slip on the surface via the same attachment system involving binding of a C-terminal eight-residue peptide that specifically binds to SNAP-PDZ18 (light blue), which coats the surface. The utrophin puts a load on the actin filament and slows its gliding velocity. (B) Loaded in vitro motility percent time mobile data for α- (red) and β- (blue) human cardiac sS1. Solid lines show the best stop-model fit (Aksel et al., 2015) to the percent time mobile data. Dashed lines mark values determined from the fit that reflect the relative force generation by the two motors; a lower x-intercept means weaker force. Data are from Aksel et al., (2015).

Fig. 6.

Effects of TnI mutations on Ca²⁺ regulation of actin-activated myosin S1 ATPase. Percent of the maximal wild-type (WT) ATPase activity (100%) is plotted as a function of the negative logarithm of the Ca²⁺ concentration. WT (black) is compared with R21C (blue), S166F (green) and ΔK177 (red). Maximum WT activity was set to 100% and all other activities were normalized to this. Mean activities±standard error are plotted. Myosin S1 was prepared by proteolytic cleavage of bovine cardiac myosin.

Fig. 7.

Locations of 17 hypertrophic cardiomyopathy mutations in the myosin catalytic domain. (A) Cartoon showing two groups of HCM mutations. Four mutations are in the converter domain (dark green), and 13 are on or very near the myosin mesa (bright green). Sixteen of the HCM mutations were chosen because they have been documented to be the cause of HCM in families carrying these mutations (Alfares et al., 2015). The 17th is M531R, which, while only documented in one family, is a left ventricular non-compaction mutant myosin that appears to be hypercontractile in our studies. (B) The top view of the mesa showing 13 mutations on or near the mesa surface. The residues labeled in blue denote the positively charged amino acids. (C) The same view as in B except the surface charge distribution is shown. I263T, N444S, and M531R are slightly below the surface in acidic pockets, while the remainder of the mutations are on or very near the surface and most are arginine residues, seven of which form a particularly large domain of positive charge on the right half of the mesa as viewed.