Myofilament incorporation determines the stoichiometry of troponin I in transgenic expression and the rescue of a null mutation

The highly organized contractile machinery in skeletal and cardiac Muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (Tnl) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental Tnl isoform switching in normal hearts, the postnatal presence of slow skeletal muscle Tnl lasted significantly longer in the hearts of cardiac Tnl (cTnl) knockout (cTnl-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental Tnl. Transgenic postnatal expression of an N-terminal truncated cTnl (cTnl-ND) using alpha-myosin heavy chain promoter effectively rescued the lethality of cTnl-KO mice and shortened the postnatal presence of slow Tnl in cardiac muscle. cTnl-KO mice rescued with different levels of cTnl-ND over-expression exhibited similar levels of myocardial Tnl comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase Tnl stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnl in heterozygote cTnl-KO mice was sufficient to sustain the normal level of myocardial cTnl, indicating that cTnl is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells. (C) 2009 Elsevier Inc. All rights reserved.