Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes

Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.     

Elevation in heat shock protein 72 mRNA following contractions in isolated single skeletal muscle fibers

The purpose of the present study was 1) to develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n=26) or not stimulated to contract (REST; n=14). The relative mean developed tension of EX fibers decreased to 29+/-7% of initial peak tension at the stimulation end point. Following treatment, individual fibers were allowed to recover for 1 (n=9), 2 (n=8), or 4 h (n=9) prior to isolation of total cellular mRNA. HSP72, HSP60, and cardiac alpha-actin mRNA content were then assessed in individual fibers using quantitative PCR detection. Relative HSP72 mRNA content was significantly (P<0.05) elevated at the 2-h postcontraction time point relative to REST fibers when normalized to either HSP60 (18.5+/-7.5-fold) or cardiac alpha-actin (14.7+/-4.3-fold), although not at the 1- or 4-h time points. These data indicate that 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed, 2) HSP60 and cardiac alpha-actin are suitable endogenous normalizing genes in skeletal muscle following contractions, and 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 h after a single bout of fatiguing contractions, despite minimal temperature changes and without influence from extracellular sources.     

Beta-adrenergic regulation of a myocardial actin gene via a cyclic AMP-independent pathway.

The skeletal alpha-actin gene encodes a major component of the embryonic cardiac sarcomere that is strongly and selectively re-induced during beta-adrenoceptor-mediated hypertrophy in neonatal rat cardiac myocytes. We present evidence that beta-adrenergic induction of this gene is mediated, not by cAMP, but by a calcium-dependent pathway involving ryanodine-sensitive calcium stores. Nifedipine-induced blockade of the plasma membrane L-type calcium entry channel prevented induction of skeletal alpha-actin mRNA by isoproterenol. Activation of calcium entry by the dihydropyridine agonist Bay K8644 independently induced skeletal alpha-actin mRNA, as did cholera toxin-mediated activation of Gs. Induction of skeletal alpha-actin mRNA by compounds that directly elevate cAMP was weak relative to their effects on other cAMP-dependent phenomena and required calcium entry. In addition, selective inhibition of protein kinase A with KT5720 did not block beta-adrenergic induction of skeletal alpha-actin. Calcium ionophore A23187 did not induce skeletal actin, but prevented its induction by isoproterenol. Ryanodine had bimodal effects: 10(-10) M ryanodine induced skeletal alpha-actin mRNA, whereas 10(-6) M ryanodine prevented skeletal actin induction by beta-adrenergic stimuli. We postulate that beta-adrenergic stimulation of skeletal alpha-actin mRNA requires G-protein-coupled calcium channel activation and compartmentalized calcium release in a manner independent of the cAMP/protein kinase A signal pathway.     

Actin typing of rhabdomyosarcomas shows the presence of the fetal and adult forms of sarcomeric muscle actin

We analyzed actin expression in two human rhabdomyosarcomas as well as in three rhabdomyosarcomas induced in rats by the injection of nickel sulfide. All five tumors exhibited appreciable amounts of the sarcomeric alpha-actin types, in line with their myogenic differentiation. The level of these actins was particularly high in the rat tumors, which according to morphological criteria, all showed a higher degree of differentiation than the human tumors. Interestingly, in both human tumors and in two of the three rat tumors, the level of the cardiac alpha-actin type was significantly higher than that of adult skeletal muscle alpha-actin. Taken together with the results of recent reports indicating that the cardiac alpha-actin type is a marker of embryonic and fetal skeletal muscle, our findings indicate that rhabdomyosarcomas express the embryonic sarcomeric actin isoform.