Expression of constructs of WT-α\alpha-cardiac actin and its mutants in different cell lines and primary rat cardiac myocytes

Das Ziel dieser Arbeit war Untersuchung von Mutationen des kardialen $\alpha$-Aktins, die in manchen Fällen der hypertrophen Kardiomyopathie (HCM) gefunden wurden. Mit Konstrukten, die N-terminal einen GFP- (grün fluoreszierte Protein) oder einen Hemagglutinin tag erzeugten, wurden Wildtyp (WT) und die zwei Mutanten - Y166C und M305L - des kardialen $\alpha$-Aktins zunächst fibroblastische NIH3T3, Kardiomyozyten ähnliche HL-1 und humane A431 (epidermoide Tumor-) Zellen transfiziert. Die Ergebnisse dieser Untersuchungen waren widersprüchlich. Die Y166C-Mutante mit GFP konnte intrazellulär nicht polymerisieren, jedoch mit dem HA-tag war diese Mutante fähig zu polymerisieren. Um primäre Cardiomyocyten zu transfizieren wurden zwei rekombinante adenovirale Konstrukte (WT und Y166C) hergestellt. In Herzmuskelzellen konnten WT und die Y166C-Mutante sehr gut polymerisieren und sich in sarkomerische Strukturen einbauen. Jedoch erzeugte die Y166C Mutante kürzere Sarkomere im Vergleich zu WT-kardialem $\alpha$-Aktin

Integration of cardiac actin mutants causing hypertrophic (p.A295S) and dilated cardiomyopathy (p.R312H and p.E361G) into cellular structures

The human mutant cardiac $\alpha$-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the $\textit {baculovirus/Sf21}$ insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in $Ca^{2+}$-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends