Logo image
Back
Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P
Journal article   Peer reviewed

Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P

Fan Bai, Hannah M Caster, Peter A Rubenstein, John F Dawson and Masataka Kawai
Journal of molecular and cellular cardiology, Vol.74, pp.64-75
09/2014
DOI: 10.1016/j.yjmcc.2014.04.014
PMCID: PMC4264970
PMID: 24793351
url
http://doi.org/10.1016/j.yjmcc.2014.04.014View
Open Access

Abstract

Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca2+], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is the tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca2+ sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0.06 T0 and 0.85±0.06 T0, respectively, N=11). A331P also showed decreased pCa50 (5.57±0.03, N=13) compared to that of WT (5.69±0.02, N=11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype. •Sf-21 cell expressed actins were polymerized in myocardium to form the thin filament.•WT actin reconstituted myocardium behaved exactly the same as the native myocardium.•A331P decreased the tension and pCa50 of reconstituted fibers compared to WT actin.•Cross-bridge kinetics of reconstituted fibers were similar between A331P and WT actin.•Diminished contractility with A331P actin must cause the characteristic HCM phenotype.
 Hypertrophic cardiomyopathy Cross-bridge kinetics A331P Actin-myosin interaction Actin Ca2 + sensitivity

Details

Metrics

18 Record Views
19 Times Cited - Web of Science
Logo image