## 3 Results and Discussion
### In Vitro Findings
Conversely, in the ISO + compound 10 (10 nM) treated group, cell size was significantly decreased compared to those treated with ISO alone. Compound 10 was unable to counteract in a significant manner the effects of ISO at higher doses (25 to 100 nM). These findings indicate that, similar to compound 8, compound 10 was able to counteract ISO-dependent effects on H9c2 cells. However, the first effective dose of compound 10 was lower than that of compound 8 (10 nM vs. 25 nM). Figure 6 also indicates that compound 10 alone did not exert significant effects on cell size at 10 nM, while this parameter was significantly increased when administered at higher doses (i.e., 50 nM and 75 nM). Figure 4: Effects of compounds 8 and 10 on cell viability in H9c2 cardiomyocytes. H9c2 cells were treated with vehicle (Control) or increasing concentrations of (**A**) compound **8** (1 nM-100 nM) or (**B**) compound **10** (1 nM-100 nM) for 24 h. Cell viability was determined using the MTT assay and was expressed as the percentage of control cells exposed only to vehicle (indicated as Control). Results are represented as mean \(\pm\) SEM (\(n=6\) per group). Significant differences were detected by one-way ANOVA followed by [PERSON]’s test, \(p<0.01\) (**) vs. Control group. Figure 5: Effects of compound 8 on cardiomyocytes hypertrophy induced by isoproterenol (ISO) in vitro. (**A**) Morphological staining of H9c2 cells exposed to vehicle (indicated as Control), ISO (100 \(\upmu\)M), ISO + compound 8 (from 10 nM to 100 nM) and compound 8 (from 10 nM to 100 nM) for 24 h. Scale bars: 25 \(\upmu\)m. (**B**) Cell surface area (%) of H9c2 cells exposed to vehicle (indicated as Control), ISO (100 \(\upmu\)M), ISO + compound 8 (from 10 nM to 100 nM) and compound 8 (from 10 nM to 100 nM) for 24 h. Data are expressed as mean \(\pm\) SEM derived from two independent measurements for each group. \(p<0.05\) (*); \(p<0.001\) (***) vs. Control group; \(p<0.05\) (#); \(p<0.01\) (###); \(p<0.001\) (###) vs. ISO group (one-way ANOVA and Newman-Keuls multiple comparison test). Our data are of interest in the view of \(\beta\)-blocker monitoring and dosing, to avoid or minimize their side effects [54]. \(\beta\)-blockers reach their therapeutic goal mainly by inducing negative chronotropic effects that prolong heart diastole and rest. In this context the choice of an appropriate dose is vital to avoid concomitant adverse negative inotropic effects. Accordingly, we used the \(\beta\)-adrenergic receptor antagonist propranolol as a positive control. As depicted in Figure 7, both morphological staining and cell surface area indicated that in H9c2 cotreated with propranolol (1 \(\upmu\)M), ISO failed to increase cardiomyocyte size. Figure 6: Effects of compound 10 on cardiomyocytes hypertrophy induced by isoproterenol (ISO) in vitro. (**A**) Morphological staining of H9c2 cells exposed to vehicle (indicated as Control), ISO (100 \(\upmu\)M), ISO + compound 10 (from 10 nM to 100 nM) and compound 10 (from 10 nM to 100 nM) for 24 h. Scale bars: 25 \(\upmu\)m. (**B**) Cell surface area (%) of H9c2 cells exposed to vehicle (indicated as Control), ISO (100 \(\upmu\)M), ISO + compound 10 (from 10 nM to 100 nM) and compound 10 (from 10 nM to 100 nM) for 24 h. Data are expressed as mean \(\pm\) SEM derived from two independent measurements for each group. \(p<0.05\) (*); \(p<0.01\) (*); \(p<0.001\) (***) vs. Control group; \(p<0.05\) (#); \(p<0.01\) (##) vs. ISO group (one-way ANOVA and Newman-Keuls multiple comparison test). Our compounds were effective at lower doses (nM range) with respect to the classic \(\beta\)-blocker propranolol (uM range), according to data that support beneficial effects of low dosage especially in patients suffering from ventricular hypertrophy [55]. Clinical evidence indicates that, in practice, \(\beta\)-blockers are often used at half or lower doses compared to manufacturers' recommendations [56]. For some drugs, lower doses even improved the disease outcomes [57].