Cardioprotection by isosteviol derivate JC105: A unique drug property to activate ERK1/2 only when cells are exposed to hypoxia‐reoxygenation

Abstract In the present study, we have investigated potential cardioprotective properties of Isosteviol analogue we recently synthesized and named JC105. Treatment of heart embryonic H9c2 cells with JC105 (10 μM) significantly increased survival of cells exposed to hypoxia‐reoxygenation. JC105 (10 μM) activated ERK1/2, DRP1 and increased levels of cardioprotective SUR2A in hypoxia‐reoxygenation, but did not have any effects on ERK1/2, DRP1 and/or SUR2A in normoxia. U0126 (10 μM) inhibited JC105‐mediated phosphorylation of ERK1/2 and DRP1 without affecting AKT or AMPK, which were also not regulated by JC105. Seahorse bioenergetic analysis demonstrated that JC105 (10 μM) did not affect mitochondria at rest, but it counteracted all mitochondrial effects of hypoxia‐reoxygenation. Cytoprotection afforded by JC105 was inhibited by U0126 (10 μM). Taken all together, these demonstrate that (a) JC105 protects H9c2 cells against hypoxia‐reoxygenation and that (b) this effect is mediated via ERK1/2. The unique property of JC105 is that selectively activates ERK1/2 in cells exposed to stress, but not in cells under non‐stress conditions.


| INTRODUC TI ON
Cardioprotection can be defined as a property of cardiac muscle to withstand challenges by virtue of intracellular signalling pathways that, when activated, increase cellular resistance to metabolic challenges including ischaemia-reperfusion. It is a consensus view that development of clinically viable and safe therapeutic cardioprotective strategies is warranted. It is generally accepted that a cardioprotective-based strategy would be useful in the therapy of ischaemic heart disease and other cardiac diseases. Such therapy would not be mutually exclusive with traditional therapies aimed to reconstitute coronary blood flow or decrease myocardial metabolic demands, but would be rather complementary to those therapies. 1 Isosteviol (STV) is a glycan of stevioside, which has been widely used in Paraguay since 16th century, and is currently used as a food supplement in over 90 countries. 2 It has been suggested that STV is a pharmacologically active substance deserving to be tested as a potential therapy for diabetes mellitus, hypertension and heart failure. 2 We have recently synthesized two series of analogues from isosteviol with modifications at C-16, C-19 positions (in series one) and at C-15, C-16 positions (in series two). We have demonstrated that one of those analogues (compound 9), named JC105, protects zebrafish embryos against doxorubicin-induced cardiotoxicity in vivo. This particular finding suggests that this compound could have cardioprotective properties. 3 Therefore, we decided to test cardioprotection afforded by JC105. To do that, we have applied hypoxia-reoxygenation challenge on rat heart embryonic H9c2 cells, which is an experimental model used to study cardioprotection in quick and reliable manner. 1,4 We have found that JC105 protects H9c2 against hypoxia-reoxygenation by selectively activating ERK1/2 during hypoxia-reoxygenation without affecting ERK1/2 signalling pathway under normoxic conditions. This is the first account ever of a compound having such properties. naphthalene-4-carboxylic acid) was synthesized from isosteviol (ent-16-ketobeyeran-19-oic acid) as described with details in our recent work (compound 9 in Ref. [3]). The effect of concentration of 10 μM of JC105 was selected to be studied based on our preliminary data (see also Ref. [3]).

| H9c2 cells and treatment
The H9c2 cardiomyocytes were obtained from the Cell Bank of

| Western blot analysis
For Western blotting, H9c2 cells were lysed using RIPA buffer containing protease inhibitors (1×) (Biosharp). The cell lysates were collected, all antibodies. All the blots were incubated in TBST with 5% skimmed milk, and either phospho or total protein was determined using horseradish peroxidase conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG) and enhanced chemiluminescence reagent. The Western blot band intensities were analysed using Quantiscan software. All the Western blots were performed in triplicates. For OCR analysis, 1 mmol/L sodium pyruvate, 2 mmol/L l-glutamine and 10 mmol/L glucose were added into the XF assay medium and pH was adjusted to 7.4. After measuring basal respiration, oligomycin (1 μmol/L), carbonyl cyanide (trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μmol/L) and rotenone/antimycin A (R/A) (1 μmol/L) were injected in sequence from port A, B and C respectively. In the ECAR assay, 2 mmol/L l-glutamine was added to XF assay medium and pH was adjusted to 7.4. Glycolytic flux (glycolytic reserve and glycolytic capacity) was recorded by sequence addition of glucose (10 mmol/L), oligomycin (1 μmol/L) and 2-deoxyglucose (50 mmol/L) from Port A,

| Statistical analysis
Data were analysed with a one-way analysis of variance (ANOVA) using SigmaPlot 12.5 (Jandel Scientific). All the results were expressed as means ± SEM and the 'n' represents number of independent experiments. P < .05 was considered statistically significant.
F I G U R E 1 JC105 protects H9c2 cells against hypoxiareoxygenation. Bar graph depicting cell survival under normoxic conditions and under hypoxia-reoxygenation (H/R) in the absence (control or H/R respectively) and presence of 10 μM JC105 (JC105 or H/R + JC105 respectively). Each bar represents mean ± SEM (n = 6-10). *P < .001 when compared to hypoxia-reoxygenation alone (H/R)

| JC105 treatment alone under normoxic condition does not activate known cardioprotective signalling pathway
When H9c2 cells were treated with JC105 (10 μM) alone under normoxic condition for 24 hours, it did not affect the phosphorylation levels of ERK, AKT and AMPK. Furthermore, it also did not affect the levels of SUR2A and GAPDH ( Figure S1).

| JC105 improved (and U0126 inhibited JC105 induced recovery) mitochondrial function and glycolytic capacity of H9c2 cells in H/R injury
To test the effects of JC105 on cellular bioenergetics, we assessed mitochondrial respiration and glycolytic activity of H9c2

F I G U R E 3 U0126 inhibits JC105induced ERK1/2 phosphorylation. Original
Western blots and corresponding graphs for P-ERK1/P-ERK2 and T-ERK1/T-ERK2 under depicted conditions. *P < .001 when compared to the control. Each bar represents mean ± SEM (n = 3). Applied

| D ISCUSS I ON
JC105 is a novel derivative of isosteviol we recently synthesized. actor to be mitigated, depending on the pathophysiological context. 7 In addition, it has been suggested that ERK1/2 also mediates cardioprotection. 6 In the present study, JC105 did not affect ERK1/2 phosphorylation under normoxic conditions, which would imply that this compound has no effect on ERK1/2 phosphorylation. However, JC105 increased phosphorylation of ERK1/2 in is a regulatory subunit of sarcolemmal K ATP channels, which levels regulate myocardial resistance to metabolic stress. 9,10 If JC105 activates ERK1/2, it was logical to expect that it would also increase SUR2A levels. Indeed, our Western blotting experiments demonstrate that SUR2A followed ERK1/2 pattern, that is JC105 did not affect SUR2A levels in normoxia, but it increased SUR2A levels in hypoxia-reoxygenation. These findings are another evidence that JC105 activates ERK1/2 specifically during hypoxia-reoxygenation and it also explains cardioprotection afforded by JC105 as it is well established that increased levels of SUR2A mediate cardioprotection by regulating timing of K ATP channels opening during stress and preventing stress-induced decrease in subsarcolemmal levels of ATP. 11,12 However, ERK1/2 activation can have many other effects with potential cardioprotective outcome in addition to SUR2A regulation. As an example, it has been shown that ERK1/2 promotes cell survival by activating pro-survival BCL2 proteins (BCL-2, BCL-XL and MCL-1) and repressing pro-death proteins (BAD, BIM, BMF and PUMA). More recently, a link between ERK1/2 signalling, DRP1 and the mitochondrial fission machinery has been established. 6 We have recently published that isosteviol protects the myocardium against ischaemia-reperfusion by inhibiting DRP1 phosphorylation and mitochondrial fission. 13 Here, we have found that JC105 stimulates DRP1 phosphorylation and it has been previously shown that inhibition of DRP1 is cardioprotective. 13  should be pointed out that, so far, any compound that was cardioprotective at in vitro level was also cardioprotective in in vivo experimental models. 4 In conclusion, we have shown that JC105 harbours cardioprotective properties that are associated with ability of this compound to selectively activate ERK1/2 signalling in cells under stressful conditions.

CO N FLI C T O F I NTE R E S T
The authors confirm that no conflict of interest exists.

AUTH O R S ' CO NTR I B UTI O N S
KSMA, JR, N. F. and AJ: Contribution to the acquisition, analysis, interpretation of data and contribution to writing of the manuscript.
WT: Initiation, designing and supervision of the study, analysis of the data and drafting the manuscript. All authors: Reading and approving the final version of the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be available from the corresponding author (WT) upon reasonable request.