Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilative Cardiomyopathy (p.R312H and p.E361G)

The human mutant cardiac α-actins p.A295S or p.R312H (plus p.R312K) and p.E361G correlated with hypertrophic or dilative cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. After purification their biochemical and cell biological properties were analysed and compared to wild type (wt) cardiac actin identically obtained or conventionally isolated from bovine hearts. DNase I inhibition and their polymerization behaviour indicated that all c-α-actins had maintained their native state. Cardiomyopathy type specific differences were observed except for the p.R312K mutant, which behaved like wt c-α-actin. The extent of myosin-S1 ATPase stimulation by the c-actin variants and its Ca2+-sensitivity after decoration with tropomyosin (cTm) and troponin complex (cTn) varied being highest for the HCM p.A295S and lower for both DCM mutants. Similar Ca2+-sensitivity differences were observed by recording the fluorescence increase of pyrene-cTm in the absence or presence of myosin-S1 and/or the actin-binding N-terminal fragment of cardiac myosin binding protein C (N-cMyBP-C). Transfection experiments showed the incorporation of the c-actin variants into existing cytoskeletal elements of non-muscle cells. Wt and p.A295S c-α-actin preferably incorporated into the microfilament system and p.R312H and p.E361G into the submembranous actin network of MDCK cells. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding for HA-tagged c-α-actins showed their incorporation into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferably incorporated at the minus ends. Our data indicate functional differences of the c-α-actins that may be causative for the different cardiomyopathy phenotypes.

Hypertrophic cardiomyopathy mutations at the folded-back sequestered β-cardiac myosin S1-S2 and S1-S1 interfaces release sequestered heads and increase myosin enzymatic activity

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, thereby leading to hypercontractility at the molecular level.

Stepwise C-Terminal Truncation of Cardiac Troponin T Alters Function at Low and Saturating Ca2+

Activation of striated muscle contraction occurs in response to Ca2+ binding to troponin C (TnC). The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin catalyzed ATP hydrolysis. It now appears that the levels of activity at both low and saturating Ca2+ are modulated by the C-terminal 14 amino acids of cardiac troponin T (TnT). We made a series of mutants of human cardiac troponin T, isoform 2, with deletions from the C-terminal end: Δ4, Δ6, Δ8, Δ10 and Δ14. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+, the change in acrylodan tropomyosin fluorescence at low Ca2+, and the degree of Ca2+ stimulation of the rate of binding of rigor myosin S1 to pyrene-labeled actin-tropomyosin-troponin. Together, these measurements define the distribution of actin-tropomyosin-troponin among the 3 regulatory states. Results from rates of rigor S1 binding deviated from other measurements when > 8 residues of TnT were deleted. That deviation was due to increased rates of binding of rigor S1 to pyrene-labeled actin with truncated TnT at saturating Ca2+. Such behavior violated a key assumption in the determination of the B state by this method. Nevertheless, all methods show that as residues were removed from the C-terminus of TnT there was approximately a proportional loss of the inactive B state at low Ca2+ and an increase in the active M state at saturating Ca2+. Most of the C-terminal 14 residues of human cardiac troponin T are essential for forming the inactive B state at low Ca2+ and for limiting the formation of the active M state at saturating Ca2+.

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

Do cardiac actin mutations lead to altered actomyosin interactions?

It is currently hypothesized that increased heart muscle contractility leads to hypertrophic cardiomyopathy (HCM), and reduced contractility leads to dilated cardiomyopathy (DCM). To determine if changes in the core interaction between actin and myosin occur due to mutations in the cardiac actin gene (ACTC), we measured the interactions between myosin and 8 ACTC mutant proteins found in patients with HCM or DCM. R312H showed a decreased actin-activated myosin S1 ATPase rate (13.1 ± 0.63 μmol/L/min) compared to WT (15.3 ± 1.6 μmol/L/min), whereas the rate with E99K was significantly higher (20.1 ± 1.5 μmol/L/min). In vitro motility assays with varying ATP concentrations showed that the KM for E99K remains unchanged with a significantly decreased Vmax (1.90 ± 0.37 μm/sec) compared to WT (3.33 ± 0.46 μm/sec). Based on a 5 nm myosin step size, we calculated a duty ratio of approximately 0.04 for WT and the majority of mutant actins; however, the duty ratio for E99K was twice as high. Based on our analysis of 8 ACTC mutants, we infer that mutations in ACTC lead to disease through various molecular mechanisms. While changes in actomyosin interactions with the E99K mutation might cause increased ATP usage and tension leading to HCM, measurable changes in the basic interaction between actin and myosin do not appear to be involved in the mechanisms of disease development for the other ACTC mutants tested.