After 24 h, very small percentages of the cells treated with the extract

and chromatin-modifying agents reacted with α-actinin and myosin heavy chain (2.09% and 1.97%, respectively), while only 0.4% and 1.59% of the cells expressed cardiac troponin T and atrial natriuretic peptide (table 1).  Table 1 Percentages of the cells that showed positive reaction to various cardiomyocyte markers After 10 days, the percentages of the α-actinin and myosin-heavy-chain-positive cells treated with both extract and chromatin-modifying agents were higher than before so that 76% and 64.9% of the fibroblasts reacted with antibodies against these markers, respectively. Inhibitors,research,lifescience,medical However, just 7.3% and 1.3 % of the cells expressed cardiac troponin T and atrial natriuretic peptide (figure 2). In the cultures exposed to 5-aza-dC and TSA but

not to the cardiac extract, the fibroblasts also expressed myosin heavy chain and α-actinin, although the percentage of such cells was less than Inhibitors,research,lifescience,medical that of the cells treated with the extract (17.6% and 20.3%, respectively). In the cultures exposed to chromatin-modifying agents, 1.4% and 2.2% of the cells expressed Inhibitors,research,lifescience,medical atrial natriuretic peptide and cardiac troponin, respectively. Meanwhile, 1.4 % and 2.2% of the cells permeabilized in the presence of the cardiomyocyte extract expressed atrial natriuretic peptide and cardiac troponin, respectively (table 1). The antibodies did not react with the untreated cells. Figure 2 Extract and chromatin-modifying-agents-treated cells expressed myosin heavy chain and α-actinin but not atrial natriuretic peptide and cardiac troponin after 10 days. FITC (left), DAPI (middle), and Merged (right) Twenty-one days after Inhibitors,research,lifescience,medical the extract treatment, Inhibitors,research,lifescience,medical a higher percentage

of the cells expressed cardiac troponin and atrial natriuretic peptide (50% and 43.7%, respectively), while no Verteporfin change was observed in the percentage of α-actinin and myosin-heavy-chain-positive cells (67.9% and 75%, respectively) (figure 3). In the cultures only permeabilized in the presence of the cardiomyocyte extract, 23%, 18%, 9.3%, and 12.2% of the cells expressed α-actinin, myosin heavy chain, atrial natriuretic peptide, and cardiac troponin, respectively. Although the fibroblasts that were exposed to the chromatin-modifying agents were able to express myosin heavy chain and α-actinin after 21 days (20% and 35%, Phosphatidylinositol diacylglycerol-lyase respectively), the expression of the other markers was negligible. The expressed markers showed a parallel arrangement in most of the reacting cells. The untreated cells expressed negligible amounts of cardiomyocyte markers at 21 days after the beginning of the experiment as well as at the other period times (table 1). Figure 3 Extract and chromatin-modifying-agents-treated cells expressed all cardiomyocyte markers after 21 days.