Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

<div><p>Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.</p></div

Differential protein expression by undifferentiated ADSCs and ADSCs-derived cells in the absence or presence of Y-27632.

<p>Expression of cMHC (A), cTnI (B), Connexin 45 (C) and RyR2/DHPR (D) in the ADSCs (day 3) and ADSCs-derived cells (day 16, in the absence or presence of Y-27632) by flow cytometer analyses. Right column figures represent results from 3 independent experiments. * <i>P</i><0.05.</p

Changes of ROCK related signaling molecules before or after the differentiation of ADSCs into cardiomyocytes.

<p>(A) Phosphorylation levels of JNK. (<b>B</b>) Phosphorylation level of Akt. Lane 1 represents undifferentiated ADSCs (day 3). Lanes 2 and 3 representbeating cells (day 16) in the absence or presence of Y-27632, respectively. Phosphorylation level  =  phosphorylation/total. * <i>P</i><0.05.</p

Immunofluorescence staining of contracting clone for cardiac- and muscle-proteins.

<p>Beating cells were with anti-cMHC (red), anti-α-actin (green), anti-cTnI (red) and anti-Nkx2.5 (red) antibodies. No specific staining was obtained with anti-MyoD (red) and anti-α-SMA (red) antibodies. Nuclei (blue) were stained with Hoechst 33342.</p

Morphology and expression of cardiac markers before and after differentiation of ADSCs into cardiomyocytes.

<p>(A) Morphological change of ADSCs into cardiomyocytes in the absence (left panels) or presence (right panels) of ROCK-specific inhibitor Y-27632. After 6 days of culture, spindle shaped cells (black arrow), small round cells (gray arrow) and small tube cells (white arrow) appeared in the top panel, with no beating. After 12 days, single contractive cells (white arrow) were shown in the center panel; After 18dyas, myotube-like structure formed a cohesive network in the bottom panels and they began to contract synchronously. Scale bars  = 100 µm. (B) Expression of cardiac genes was determined by RT-PCR before (lane 1 for undifferentiated ADSCs) and after (lane 2 for beating cells) differentiation of ADSCs into cardiomyocytes. Rat heart tissue was used as a positive control (lane 4), with no cDNA as a negative control (lane 3). Expression of α-cardiac actin, α-MHC, β-MHC, ANP, cTnT, GATA4 and MEF-2C mRNA was higher in ADSCs-derived beating cells than in undifferentiated ADSCs.</p

Expression of cardiac proteins, excitation-contraction coupling proteins and signaling proteins in beating cardiomyocytes.

<p>Expression of MLC-2v(A), cTnT(B) and RyR2/DHPR (C) in the SCR siRNA and ROCK siRNA groups by flow cytometer analyses. (D) Change of JNK and PI3K/Akt from Western Blot analyses. Lane 1 represents undifferentiated ADSCs (day 3), while lanes 2 to 5represent SCR siRNA, ROCKI siRNA, ROCKII siRNA and Both siRNA groups, respectively (day 16). Phosphorylation level  =  phosphorylation/total. * <i>P</i><0.05.</p

Primers for reverse transcription PCR and real time PCR and sequences for RNA interference.

<p>Primers for reverse transcription PCR and real time PCR and sequences for RNA interference.</p

Morphological change of actin cytoskeleton during the differentiation of ADSCs.

<p>ADSCs and ADSCs-derived cardiomyocytes were stained with phalloidin for F-actin (green). The white arrows point to stained stress fibers. After 3 days of culture in the absence of Y-27632 (top left panel), stress fibers in cells weres parse. After 7 days of culture in the absence of Y-27632 (top right panel), stress fibersshifted from sparse to dense distribution. The long stress fibers fully occupied the cytoplasm of contractive cells after 16 days of culture in the absence of Y-27632 (middle left and bottom left panels). The contractive cells in the presence of Y-27632 showed much less compact stress fibers in the cytoplasm, in comparison with un-treated cells (after 16 days of culture, middle rightand bottom right panels). The white arrow indicates stress fibers. Nuclei (blue) were stained with Hoechst 33342, Scale bar  = 100 µm.</p

Dirhodium(II)-Catalyzed Sulfide Oxygenations: Catalyst Removal by Coprecipitation with Sulfoxides

The dirhodium­(II) carboxylate complex Rh₂(esp)₂ (esp = α,α,α′,α′-tetramethyl-1,3-benzenedipropanoate) was shown to catalyze the sulfoxidation of organic sulfides using <i>tert</i>-butyl hydroperoxide as the oxidant. Due to the unique structure of Rh₂(esp)₂ and its stable Rh₂(II,II) catalyst resting state, the rhodium catalyst is able to precipitate as a Rh₂(esp)₂–sulfoxide complex following the reaction which makes separation of the catalyst from the products very convenient. The precipitated Rh₂(esp)₂–sulfoxide complexes could be reused to catalyze sulfide oxygenation reactions without considerable loss of activity. Mechanistic studies suggest that the axial ligands fine-tune the redox potential of the dirhodium­(II,II) compounds and determine the predominant catalyst species in the oxidation reaction

TiO₂‑Catalyzed <i>n</i>‑Valeraldehyde Self-Condensation to 2‑Propyl-2-Heptenal: Acid Catalysis or Base Catalysis?

Several TiO₂ samples with different morphologies and structures, nano/microcomposite of TiO₂ (anatase), TiO₂ whisker (anatase), and nano-TiO₂ (anatase), were prepared and characterized by means of N₂ adsorption–desorption, CO₂-TPD, and NH₃-TPD, and their catalytic performance for <i>n</i>-valeraldehyde self-condensation was investigated. The results indicated that the conversion of <i>n</i>-valeraldehyde was correlated with their acid amount while the selectivity of 2-propyl-2-heptenal was associated with their base amount. Since the catalytic performance of nano-TiO₂ (anatase) was the best, its preparation process was further studied and the suitable preparation conditions were obtained. Then the effect of reaction conditions on the catalytic performance of nano-TiO₂ (anatase) for <i>n</i>-valeraldehyde self-condensation was investigated and the suitable reaction conditions were obtained as follows: a weight percentage of TiO₂ catalyst of 15 wt %, a reaction temperature of 190 °C, and a reaction time of 10 h. Under the above reaction conditions, the conversion of <i>n</i>-valeraldehyde, 2-propyl-2-heptenal yield, and selectivity were 94.6%, 93.7%, and 99.1%, respectively. The TiO₂ catalyst could be reused four times without a significant loss in its catalytic performance, which was different from most of the literature. The catalytic stability of TiO₂ catalyst was associated with the properties of the active sites, especially acid–base property. Not as some of the literature claimed that their TiO₂-catalyzed reactions were base-catalyzed reactions, the TiO₂ catalyst used in this work possessed much greater acid amount than base amount. To assess the role of acidic and basic sites in <i>n</i>-valeraldehyde self-condensation, ammonia and carbon dioxide were separately used as a probe molecule for poisoning the corresponding active sites. The results confirmed the key role of acid sites in <i>n</i>-valeraldehyde self-condensation. Therefore, we were convinced that the TiO₂-catalyzed <i>n</i>-valeraldehyde self-condensation was mainly an acid-catalyzed reaction