Point mutations in the contractile proteins of the myocardium are linked to both hypertrophic (HCM) and dilated (DCM) cardiomyopathies, pathologies characterized by distinctly different patterns of ventricular dysfunction and remodeling, where remodeling is the change in cardiac chamber morphology associated with a pathophysiologic state. Specifically, HCM (a leading cause of sudden death in people under 35 years old [1
]) is characterized by a thickening of the ventricular walls with maintained or even enhanced contractility [2
]. DCM, in contrast, is characterized by an increase in left ventricular chamber dimensions, a thinning of ventricular walls, and a depression of myocardial contractility, ultimately progressing to clinically overt heart failure [3
]. Although the pathway from sarcomeric point mutation to pathology is unclear, this differential remodeling response presents a unique opportunity to characterize the primary insult to the contractile protein machinery associated with these mutations.
In contrast to the better characterized effects of human cardiomyopathy associated mutations in myosin and the muscle regulatory proteins, troponin (Tn) and tropomyosin (Tm), much less is known about the functional significance of the 9 mutations identified in actin, 7 of which cause HCM and 2 of which cause DCM [4
]. Given the highly conserved nature of actin and its integral role in muscle contraction, a mutation in a key functional domain of actin could specifically affect the mechanics, kinetics and/or regulation of the actomyosin interaction. A recent study with expressed human actin revealed that the HCM-causing mutation, E99K, significantly affected the dynamics of the actomyosin interaction [8
]. Specifically, both a decrease in the binding affinity of the myosin S1-subfragment to E99K actin, and decreased actin filament velocity in an in vitro
motility assay were observed. This amino acid residue is located in subdomain 1 of actin and in the absence of Ca++
is proximate to Tm [9
]. As such this mutation could affect the regulatory properties of Tn and Tm, particularly at low calcium levels.
The DCM associated mutation (R312H) may also affect thin filament regulation. This mutation is located in subdomain 3, a region of actin that is postulated to make important electrostatic contacts with Tm. The neighboring residue (D311) forms one of the strongest interactions between Tm and actin [10
]. Thus a mutation that results in a charge change, such as is seen with this mutation, could perturb interactions with Tm and dynamically affect the azimuthal movement of Tm as a function of thin filament activation.
Recent advances in optical trapping methods have provided a unique opportunity to study and quantify the regulatory process of muscle activation at the molecular level. Our laboratory [11
] has shown that this assay can be used to quantify the degree to which Tn and Tm inhibit the actomyosin interaction and to determine the relative effects of cooperative activation by myosin strong-binding. Given the different subdomain locations of these two actin mutations and their potential to differentially affect thin filament activation, we have chosen to study the regulatory effects of these two actin mutations. As the thin filament is activated by both calcium and myosin strong-binding, the in vitro
motility and the laser trap assay are used to investigate these facets of thin filament activation. Thus, by directly comparing the molecular regulatory properties of the HCM mutation (E99K) with the DCM mutation (R312H), we may gain insight in the molecular triggers that initiate the divergent patterns of pathological ventricular remodeling associated with these two distinct clinical phenotypes.