Quantitative Assay of Skeletal Muscle alpha-actin Expression In Normal and Pathological Human and Mouse Hearts

Abstract

We have developed a polyclonal antibody specific to skeletal muscle actin (ACTA1) in the presence of cardiac actin (ACTC) and have used it to quantify the skeletal actin content of human and mouse cardiac muscle. Heart muscle myofibrils were separated by SDS-PAGE and Western blotted. The membrane was stained first with MemCode total protein stain and then probed in with the anti-skeletal muscle actin antibody, visualised by ECL. The ECL band signal was normalised to MemCode-stained actin band. Skeletal muscle myofibrils were used as a 100% skeletal actin standard. For the negative control we used myofibrils from skeletal muscle of a skeletal actin knockout mouse crossed with a transgenic mouse over-expressing cardiac actin in skeletal muscle. There was no detectable signal from the skeletal actin antibody in the pure cardiac actin control.Human non-failing donor heart muscle contained 21±2% skeletal actin (n=9). This is comparable to previous estimates using N-terminal sequencing or Mass spectroscopy. In both end-stage failing heart muscle and in myectomy samples from HCM muscle the skeletal actin content was much higher (58±5% in both cases, n=12, 11). The increase in skeletal actin content of myopathic muscle was highly significant (p< 0.001). Mouse heart muscle (C57BL/6 strain) contains 26±3% skeletal actin (n=7). This is similar to human heart. ACTC DCM mutation E361G expressed at 50% in mouse heart has 16±3% (n=8) skeletal actin but ACTC HCM mutation E99K expressed at 50% is not significantly different from NTG 24±2% (n=5). We conclude that in human heart, acquired heart failure or failure secondary to HCM is associated with an increased content of skeletal muscle actin. In contrast, in mouse genetic models of HCM and DCM skeletal muscle actin content may be lower than normal.