At the core of the heart muscle are contractile proteins. We have sought to understand some of the mechanisms of heart failure from the perspective of the cardiac actin protein. While changes to interactions between some ACTC variants and specific proteins have been studied, very little is known about the biophysical properties of naturally occurring ACTC mutants related to heart disease themselves.
When the ACTC mutants were ranked within each assay, two main groups were observed: those ACTC mutants with changes in actin protein stability and those with changes in actin protein polymerization. However, it should be noted that the magnitudes of any changes in these ACTC mutant proteins were subtle. These subtle changes might reflect the late development of some HCM or DCM cases in humans.
We observed a strong correlation between Tm values and DNase-I IC50 values for five of the ACTC proteins examined. However, for E99K and A331P ACTC, intermonomer interactions may bury some of the D-loops of ACTC, resulting in disproportionate changes in the two parameters. This is most likely the case for E99K ACTC, where modification with TMR reduced an elevated Tm reminiscent of F-actin to that of monomeric WT ACTC. It is not clear if such interactions would impact the heart, since actin filaments form for both E99K and A331P ACTC proteins and a host of regulatory proteins surround the actin filament in situ to encourage proper filament structure.
Our rankings suggested a pattern where intrinsic deficiencies occur with mutations in subdomain 3 of actin (, R312H, A331P and Y166C) while mutations in subdomains 1 and 4 do not affect the intrinsic properties of the actin, but more likely affect protein-protein interactions. The Y166C ACTC mutant exhibited polymerization deficiency through an increased Cc, abnormal filament morphology, and lowered Pi
release. This mutation is located in the hydrophobic cleft of actin which is known to be involved in F-actin contacts 
. Similarly, studies on yeast actin show that mutations within the W-loop (a.a. 165–172) of yeast actin also impact actin polymerization. 
Correlation of location and effects of ACTC mutations.
The R312H ACTC mutant possessed reduced protein stability accompanied with higher polymerization Cc, and increased nucleotide and Pi release rates. The fast production of inorganic phosphate during polymerization of R312H ACTC is likely due to breakage of filaments, resulting in rapid treadmilling and ATP hydrolysis, as seen with other unstable mutant actin proteins 
. These observations are confirmed by previous work showing the effect of the R312H mutation on actin protein stability 
. Altered contractile protein interactions were observed with reconstituted regulated R312H ACTC filaments leading to increased calcium sensitivity 
. It is not clear how instability of the R312H ACTC variant is related to changes in protein interactions; however, the inclusion of the filament stabilizing toxin phalloidin to the assays may have influenced the behaviour of the variant ACTC protein.
The A331P ACTC mutant also follows a pattern of altered protein stability, but with less severity. Ala-331 is located in subdomain 3 of actin, beside Pro-332 and Pro-333 in the hinge region between the large and small domains 
. The addition of another Pro residue results in faster nucleotide release rates, supporting the hypothesis that movement of this region is critical for nucleotide binding 
. A331P ACTC protein also displayed a relatively high F-actin Cc and a slow inorganic phosphate release rate, suggesting a slight defect in polymerization., something that was seen with A331P yeast actin 
. The yeast actin A331P protein Tm
of 51.6°C as measured by CD 
also agrees with the hypothesis that A331P ACTC is structurally unstable. However, the A331P ACTC protein showed a Tm
higher than WT ACTC protein. Intermonomer interactions similar to those likely occurring E99K ACTC may also occur with A331P ACTC to affect the Tm
and DNase-I IC50
Aside from potential intermolecular interactions of E99K ACTC protein under monomeric conditions, ACTC mutations in subdomain 1 of actin (E99K and E361G) did not otherwise adversely affect the intrinsic properties of the actin molecule itself and more likely affect protein interactions. Previous work has shown that the E99K mutation affects myosin and tropomyosin binding 
, while the E361G mutation affects α-actinin binding 
and the regulation of actin filaments by troponin I 
The locations of both A230V and M305L mutations in subdomain 4 are not thought to be involved in important F-actin contacts or actomyosin interactions. The increase in Pi release rate of M305L ACTC protein may reflect the location of the mutation within the nucleotide-binding cleft 
. The A230V ACTC mutation is believed to be in the tropomyosin-binding site 
Based on the pattern emerging from our data, we predict that the remaining ACTC mutations located in subdomain 3 (P164A, S271F, A295S, and R312C) affect protein stability or actin polymerization, while mutations in subdomain 1 (H88Y, F90del, R95C) impact protein interactions with other sarcomere proteins. We can now examine the interaction of the ACTC proteins with important actin binding proteins, including myosin, thin filament regulatory proteins, and myosin binding protein-C to test our model and further illuminate the molecular alterations that occur with changes in the ACTC protein that are related to the development of cardiomyopathies.