A Subpopulation of Cardiomyocytes Expressing α-Skeletal Actin Is Identified by a Specific Polyclonal Antibody

Abstract

Abstract —The NH 2 -terminal decapeptide of α-skeletal actin that contains a primary sequence specific for this isoform was used to raise a polyclonal antibody in rabbits. Using sequential affinity chromatography, we recovered from serum antibodies reacting exclusively with α-skeletal actin when tested by immunoblotting and immunofluorescence. Epitope mapping by means of competition assays with synthetic peptides indicated that the acetyl group and the first 9 amino acids are essential for specificity. The monospecific antibody was then used to investigate the distribution of α-skeletal actin in the myocardium of newborn and normal or hypertensive (with or without fibrotic areas) adult rats. Immunostaining of normal heart revealed that α-skeletal actin is diffusely distributed within practically all myocardial fibers of the newborn rat, whereas it is restricted to a small proportion of adult rat cardiomyocytes, which appear intensely stained. A correlation, albeit not complete, was found between the distribution of α-skeletal actin and β-myosin heavy chain. During cardiac hypertrophy induced by aortic ligature between the renal arteries, the expressions of α-skeletal actin mRNA and protein were increased. The distribution of immunostaining had a focal pattern similar to that of normal adult rats, reactive fibers being more numerous and more intensely stained compared with normal myocardium. Positive fibers were particularly abundant at the periphery of fibrotic areas. Using this antibody, we have demonstrated for the first time the differential distribution of α-skeletal actin in heart tissues. Changes in the distribution of this isoform in hypertrophic heart provide new insight into the mechanisms by which the heart adapts to work overload. This antibody will prove useful in exploring the mechanisms of expression of α-skeletal actin and in defining its role in physiological and pathological situations. The full text of this article is available at http://www.circresaha.org.