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Mar Drugs
2018 Jun 02;166:. doi: 10.3390/md16060192.
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A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.
Kim HK
,
Cho SW
,
Heo HJ
,
Jeong SH
,
Kim M
,
Ko KS
,
Rhee BD
,
Mishchenko NP
,
Vasileva EA
,
Fedoreyev SA
,
Stonik VA
,
Han J
.
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Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca2+. To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low KD of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.
Figure 1. The chemical structure and cell viability effect of echinochrome A (Ech A). (A) The chemical structure of Ech A (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone; FW = 266.2). (B) The cell viability according to the dose of Ech A. Each group, n = 3–5.
Figure 2. Ech A enhances cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). (A) The protocol for the differentiation of cardiomyocytes from the mESCs using the hanging drop method to produce differentiated embryoid bodies (EBs). (B) The beating number per minute (BPM) of EBs during differentiation. Each group, n = 3. * p < 0.05 (C) Representative FACS analyses and the percentage of mESC-derived αMHC+ cardiomyocyte-like cells according to the dose of Ech A. Each group, n = 3. (D) Relative mRNA expression levels of cardiomyocyte specific genes in nontreated control (Con), Ech A treatment (Ech), heart tissue from neonatal (7 day, Neo) or adult mice (10 weeks, Adult). Each group, n = 3. * p < 0.05 vs. Con, †p < 0.05 vs. Ech A. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (E) Western blot analyses for the expression of cardiomyocyte specific proteins incubated with the control vehicle (Con) or 50 µM of Ech A. (F) Images displaying αMHC-GFP+, α-actinin+, cTnT+, and Cx43+ cells incubated with Con or 50 µM of Ech A (scale bars, 50 μm). SCC: side scatter cell, cTnI: cardiac troponin I, cTNT: cardiac troponin T, Cx43: connexin 43, Mef2c: Myocyte Enhancer Factor 2C, Mlc2: Myosin light chain 2.
Figure 5. Computer simulated Ech A-PKCι docking model. (A) A three-dimensional (3D) model of Ech A-PKCι (PDB:3A8W) docking simulation was generated by using the online DockingServer site (https://www.dockingserver.com). (B) Magnified 3D structure of the Ech A-PKCι binding site.
Figure 6. In vitro surface plasmon resonance binding assay for PKCι and Ech A. The PKCι binding response to Ech A was measured in the range of 0–40 nM of Ech A. The flow rate was 30 μL/minute. The association and dissociation times were 3 min each. The calculated binding constants are shown at the bottom. ka: Association rate, kd: Dissociation rate, KD (Affinity) = kd/ka (n = 3).
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