Contraction Accelerates Myosin Heavy Chain Synthesis Rates in Adult Cardiocytes by an Increase in the Rate of Translational Initiation

Abstract

The purpose of this study was to determine the mechanism by which contraction acutely accelerates the synthesis rate of the contractile protein myosin heavy chain (MHC). Laminin-adherent adult feline cardiocytes were maintained in a serum-free medium and induced to contract at 1 Hz via electrical field stimulation. Electrical stimulation of contraction accelerated rates of MHC synthesis 28%, p < 0.05 by 4 h as determined by incorporation of [ 3 H]phenylalanine into MHC. MHC mRNA expression as measured by RNase protection was unchanged after 4 h of electrical stimulation. MHC mRNA levels in messenger ribonucleoprotein complexes and translating polysomes were examined by sucrose gradient fractionation. The relative percentage of polysomebound MHC mRNA was equal at 47% in both electrically stimulated and control cardiocytes. However, electrical stimulation of contraction resulted in a reproducible shift of MHC mRNA from smaller polysomes into larger polysomes, indicating an increased rate of initiation. This shift resulted in significant increases in MHC mRNA levels in the fractions containing the larger polysomes of electrically stimulated cardiocytes as compared with nonstimulated controls. These data indicate that the rate of MHC synthesis is accelerated in contracting cardiocytes via an increase in translational efficiency.Hypertrophic growth occurs in terminally differentiated adult cardiocytes by an increase in cellular mass via a relatively coordinate increase in the proteins comprising each of the cellular components (1). This accumulation of cardiocyte proteins occurs by an increase in rates of protein synthesis relative to rates of protein degradation (2). The anabolic changes that occur during hypertrophy of the adult myocardium are generally considered to be a compensatory response to an increase in hemodynamic load (3). As demonstrated in studies employing isolated papillary muscle preparations, both the active tension and passive strain components of load have been identified as mechanical stimuli for accelerating protein synthesis rates in adult myocardium (4, 5). These findings have been extended to isolated adult cardiocytes in culture. Rates of protein synthesis were accelerated in response to electrically stimulated cardiocyte contraction (6, 7), in response to a basal load as defined by adherence of the cardiocytes to the culture dish and establishment of a resting length (8) and in response to passive stretch of cardiocytes adherent to a deformable membrane (9). Thus, the integrity of adult cardiocytes in primary culture is maintained with respect to the ability to transduce changes in mechanical load into an anabolic response such as accelerated protein synthesis rate.The specific mechanisms by which cardiocyte protein synthesis is regulated probably occur at many levels, including transcriptional, post-transcriptional, and translational processes (10). It is well established, for example, that transcriptional and post-transcriptional mechanisms regulate steady state mRNA levels and are respon...

Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds

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Connexin43 pseudogene in breast cancer cells offers a novel therapeutic target

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Selective translation of mRNAs in the left ventricular myocardium of the mouse in response to acute pressure overload

“…After a 15 min incubation on ice, the homogenate was centrifuged at 12,000×g to produce a post-mitochondrial supernatant. The supernatant was layered onto a 15-50% linear sucrose gradient in a buffer containing 200 mM Tris-HCl (pH 7.5), 2.5 M KCl, and 100 mM MgCl 2 , and centrifuged at 100,000×g for 100 min at 4° C. A density gradient fractionator (Teledyne Isco, Inc.) was used to isolate monosome fractions (messenger ribonucleoprotein particles and free ribosomes) and polysome fractions [15]. The RNA in each fraction was extracted using Trizol® reagent.…”