Developmental regulation of cardiac MAP4 protein expression.

It has been shown that the level of expression of microtubule-associated protein 4 (MAP4) mRNAs changes throughout neonatal heart development [Chapin SJ, et al. 1995. Biochemistry 34:2289]. In the present study, both immunofluorescence and western blotting methods were used to monitor MAP4 protein expression levels in the developing heart. By both methods, it was shown that the levels of total MAP4 protein were maximal during the first postnatal week, and then declined progressively to adulthood. In addition, four major electrophoretic species that reacted with MAP4-specific antibodies (called bands 1-4) were observed in all heart tissue samples. Three of the four bands decreased in abundance throughout postnatal development, but at different rates. The fourth band remained relatively constant in abundance with increasing postnatal age. To determine if phosphorylation events might contribute to this heterogeneity, western blotting experiments using phospho-specific antibodies and phosphatase digestion of extract samples were performed. No phosphorylation-specific antibody staining was observed and no significant changes were demonstrated in the bands after phosphatase treatment, implying that the observed complexity was due mainly to alternative start site or differential isoform expression. Finally, it was discovered that cardiomyocyte MAP4 associated with drug- and cold-stable microtubules in early neonatal myocytes. Thus, the complex regulation of MAP4 protein expression may play a key role in the functional differentiation of myocyte microtubules during heart development. Copyright 2006 Wiley-Liss, Inc.