Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart.

The vertebrate heart contains two myosin heavy chain isoforms, alpha and beta, which are differentially expressed. To establish a murine model for gene-targeting experiments, we defined the precise temporal expression of the myosin isoforms during cardiogenesis and obtained quantitative measurements of cardiac performance. The relative levels of the alpha- and beta-cardiac transcripts were determined by isolating the RNA from the hearts of CD-1 mice during development and hybridizing the preparations to probes that detect specifically the alpha- or beta-cardiac myosin heavy chain mRNAs. The data indicate that, although both isoforms are present from the onset of cardiogenesis, the beta-isoform predominates during embryogenesis and fetal development. This relation is reversed after the first day of life with a significant drop in the absolute transcript levels during the switch; and alpha/beta ratio of 16:1 is maintained in the neonate, and the relatively high levels of the alpha-transcript remain throughout the adult stages. To be able to make functional comparisons between normal and transgenic mice, we obtained indexes of myocardial function in isolated retrogradely perfused and in work-performing heart preparations in normal and hypodynamic mouse hearts. We found that the physiology of the mouse heart is similar to the rat heart in that we observed a positive staircase in the force-frequency relation of the mouse Langendorff preparation. We also saw contractile responses of more than twice control induced by paired stimulation and persistent postextrasystolic potentiation. As is the case for the rat, in the work-performing mouse heart, afterload (Starling resistance, pressure) changes produced a steeper Starling function curve than did changes in preload (volume, venous return).

Muscle isoactin expression during in vitro differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from mouse blastocysts. ES cells can differentiate into complex embryoid bodies (EBs) which exhibit many of the characteristics of 4-10-d embryos, including areas which rhythmically contract. The expression of the four muscle isoactins was examined in EBs by using transcript-specific probes for each of the muscle actin mRNAs and selectively reactive MAbs to muscle actins. Northern blot analyses from undifferentiated ES cells and EBs after 5, 10, 15, and 20 d in suspension culture demonstrated that no muscle actin transcripts could be detected in the undifferentiated cells, whereas during differentiation, the vascular and enteric smooth muscle isoactin mRNAs were easily detected. To further define the pattern of expression polymerase chain reaction analyses were carried out on RNA isolated from individual EBs. The data indicated that all four muscle-specific actin genes are transcribed. We also demonstrated the presence of muscle actins in at least two distinct cell populations within the EBs using selectively reactive MAbs. Fibroblast-like cells exhibit significant levels of the two smooth muscle actins (vascular and enteric) localized to stress fibers. In addition, one or both of the striated muscle actins (cardiac and skeletal) are expressed in cardiomyocyte-like cells. As is the case in embryonic heart, alpha-smooth muscle actin and the striated muscle actin(s) are incorporated into well organized sarcomeres in these cardiomyocyte-like cells. Thus, differentiating EBs provide an in vitro system to study both striated and smooth muscle cell gene expression.