Stretching Cardiac Myocytes Stimulates Protooncogene Expression*

Recently cellular protooncogenes have been found to be induced as an early response to pressure overload in cardiac hypertrophy. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocyte stretching stimulated expression of the protooncogene, c-fos, in a stretch length-dependent manner, followed by an increase in amino acid incorporation into proteins. c-fos mRNA levels were enhanced within 15 min by cardiocyte stretching, peaked at 30 min, and declined to undetectable levels by 240 min. In the presence of cycloheximide, a greater increase in c-fos mRNA was seen by stretching. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene including its promoter was also activated by stretching cardiac myocytes. These results suggest that mechanical loading directly regulates gene transcription without the participation of humoral factors in cardiocytes.

Recently cellular protooncogenes have been found to be induced as an early response to pressure overload in cardiac hypertrophy.
To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocyte stretching stimulated expression of the protooncogene, c-fos, in a stretch length-dependent manner, followed by an increase in amino acid incorporation into proteins. Since cardiac myocytes cannot divide later in life, they respond to the demand for increased mass by an increase in cell size (hypertrophy), not by an increase in cell number (proliferation) (1). In cardiac hypertrophy, the expression of specific genes, such as protooncogenes and fetal-type genes of contractile proteins, was induced as well as an increase in protein synthesis (2)(3)(4). Many factors, including hemodynamic overload and humoral factors, have been reported to induce cardiac hypertrophy (5,6). Recently, norepinephrine has been elucidated to induce cardiac hypertrophy and specific gene expression in cultured cardiac myocytes possibly by the activation of protein kinase C (7,8 dynamic overload directly induces specific gene expression without humoral factors remains unknown. We have reported that the expression of protooncogenes such as c-myc and c-fos genes was markedly stimulated in rat hearts by aortic stenosis (4). But, since the previous study was performed on hearts in uiuo, we could not rule out the participation of humoral factors. In the present study, to examine whether mechanical stimuli are directly coupled to specific gene expression, we cultured neonatal rat cardiocytes in elastic silicone dishes with defined serum-free medium, imposed mechanical stimuli in vitro by stretching adherent cardiocytes, and examined protooncogene expression.

RESULTS
As shown in Fig. lA, passive stretching of cardiocytes stimulated amino acid incorporation into proteins. The amino acid incorporation was significantly increased 2 h after stretching, and the stimulation was maintained for 8 h. Northern blot analysis using a c-fos-specific DNA revealed that myocyte stretching reproducibly induced the accumulation of c-fos mRNA, although there was no signal at the anticipated 2.2-kilobase transcripts in the control (Fig. 1B). Accumulation of c-fos mRNA was detected as early as 15 min after the passive stretching of cardiocytes and reached a maximal level at 30 min, followed by a decline to undetectable levels ( Fig.   2A).
To examine whether the increased mRNA levels of c-fos gene by stretching were regulated at the transcriptional level or post-transcriptional level, we transfected plasmids containing chimeric c-fos gene into primary cultures of neonatal cardiac myocytes and measured CAT activity. As shown in Fig. 4, the pSVOCAT plasmid showed very little CAT activity in either the absence or presence of stretch stimulation. By contrast, when pSVOfosCAT, which contained the 5' c-fos sequence, was introduced into the system, myocardial stretching reproducibly caused more than a 'i-fold increase in CAT activity, but there was little activity without stretching (relative acetylated chloramphenicol levels: stretch, 100% uersus control, 12 + 3%. n = 7). When the pSV2CAT vector, which A, construction of recombinant plasmid, pSVOfosCAT, containing the c-fos upstream and promoter region linked to the CAT coding sequence..A 1.4-kb NaeI fragment was cloned into the uniaue Hind111 site in the DSVOCAT vector. B. cultured cardiocytes were stretched by 10% 48 h after transfection with the plasmids pSVOCAT (a, b), pSVOfosCAT (c, d), and pSV2CAT (e, f) into primary l-day-old rat heart myocytes. Autoradiograms of thin layer chromatograms using the assay mixture after 1 h of incubation are demonstrated. a, c, e, control cells; b, d, f, stretched cells.
contained SV4O enhancer and early promoter sequences, was introduced into the cells, a large amount of CAT activity was observed. In this case, however, stretching did not cause additional activity.

DISCUSSION
There have been many reports that pressure or volume overload induces specific gene expression in cardiac hypertrophy (3,4,15,16). In the present study, mechanical stimuli directly induced specific gene expression as well as protein synthesis without the participation of humoral factors. Since cycloheximide did not inhibit but additively stimulated the induction of c-fos gene by stretching, this gene expression was not stimulated by newly synthesized proteins, and the mechanism of the c-fos mRNA accumulation by stretching might differ from that by cycloheximide.
Moreover, increased CAT activity of the transfected c-fos fusion gene indicated that the specific c-fos gene expression by stretching was regulated at least partially at the transcriptional level and that the stretchresponse element was located in the 5'-flanking DNA of the c-fos gene.
When the heart was exposed to hemodynamic overload, specific genes such as protooncogenes and hsp70 gene were induced and fetal type genes of contractile proteins were reexpressed (3,4,16). Recently, many reports have demonstrated that humoral factors such as thyroid hormone and norepinephrine induce gene expression in cultured cardiocytes (2,8,17). But because of difficulties in imposing work overload on cardiocytes in vitro, it is unknown whether mechanical stimuli directly cause these biochemical responses. In the present study, we imposed mechanical stimuli by stretching rat neonatal cardiocytes cultured in elastic dishes under defined serum-free medium and demonstrated that mechanical loading in cardiocytes induced specific gene expression as well as cell hypertrophy.
We have reported that the expression of the c-fos gene was stimulated by aortic stenosis in in uiuo heart studies (4). In the present study, the expression of c-fos protooncogene was markedly induced by mechanical loading without the participation of humoral factors. These results suggest that hemodynamic overload itself is one of the main factors to stimulate the expression of the c-fos gene in the heart in Go. The protein which it encodes, Fos, has been localized to the nucleus and is believed to play an important role in regulating the subsequent expression of other genes (18). Quite recently, Fos was elucidated to bind to the 12-O-tetradecanoylphorbol-13-acetate-responsive elements of some genes and to activate their gene transcription in cooperation with the transcriptional factor AP-1 (19, 20). Cardiocytes respond to hemodynamic overload by reinduction of fetal type contractile proteins as well as cell hypertrophy.
Bishopric et al. (8) suggested that the activation of protein kinase C may stimulate early developmental gene re-expression in cardiac hypertrophy. These results and observations lead us to speculate that some early responsive gene products like Fos may stimulate other subsequent gene expressions in the heart under conditions of hemodynamic overload. Further work is needed to elucidate the physiological roles of protooncogenes in cardiac hypertropk.