Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we design an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modelling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on numerical results we design a simple device combining an insert with substrates containing pores with 0.4 um and 1.0 um diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with bulk electroporation, but with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability.

Serelaxin (RLX), the recombinant form of human Relaxin-2 hormone, protects the heart from the ischemia/reperfusion (I/R)-induced damage due to its anti-inflammatory, anti-apoptotic and antioxidant properties. RLX exerts its actions by binding to its specific RXFP1 receptor whereby it regulates multiple cellular transduction pathways. In this in vitro study we offer first evidence for the involvement of AMP kinase/Sirtuin1 (AMPK/SIRT1) pathway in the protection afforded by RLX against H/R-induced damage in H9c2 cells. Treatment of the H/R-exposed cells with RLX (17 nmol L−1) enhanced SIRT1 expression and activity. Inhibition of SIRT1 signaling with EX527 (10 µmol L-1) reduced the beneficial effect of the hormone on mitochondrial efficiency and cell apoptosis. Moreover, RLX upregulated AMPK pathway, as shown by the increase in the expression of phospho-AMPK active protein. Finally, AMPK pathway inhibition by Compound C (10 and 20 μmol L−1) abrogated the increase in SIRT1 expression induced by RLX, thus suggesting the involvement of AMPK pathway in this effect of RLX. The results of this study strengthen the concept that RLX exerts its cardioprotective effects against H/R-induced injury by multiple pathways which also include AMPK/SIRT1.

Abscisic acid (ABA) is a conserved “stress hormone” in unicellular organisms, plants and animals. In mammals, ABA and its receptors LANCL1 and LANCL2 stimulate insulin-independent cell glucose uptake and oxidative metabolism: overexpression of LANCL1/2 increases, and their si-lencing conversely reduces, mitochondrial number, respiration and proton gradient dissipation in muscle cells and in brown adipocytes. We hypothesized that the ABA/LANCL hor-mone/receptors system could be involved in thermogenesis. Heat production by LANCL1/2-overexpressing vs. double-silenced cells was compared in rat H9c2 cardiomyocytes with two different methods: differential temperature measurements using sensitive thermistor probes, and differential isothermal calorimetry. Results indicate that overexpressing cells generate an approx. double amount of thermal power compared with double-silenced cells, and that addition of ABA further doubles heat production in overexpressing cells. With the temperature probes, we find a time-scale of approx. 4 min for thermogenesis to “turn on” after nutrient addition. These results identify the ABA/LANCL hormone receptors system as a hitherto unknown regu-lator of cell thermogenesis. Pharmacologic means of increasing metabolic energy dissipation are considered a promising strategy to reduce oxidative stress and lipid accumulation at a cellular and organismic level: from this study, LANCL1-2 proteins emerge as targetable controllers of ther-mogenesis through their natural ligand ABA.

Arjunolic acid (AA) is a triterpenoid saponin majorly found in the Terminalia arjuna and is claimed to exert the cardiovascular protective effects as a phytomedicine. However, it is unclear how AA exerts the effects at the molecular level. Hence, this study used an in vitro model using lipopolysaccharide (LPS)-stimulated H9C2 and C2C12 myotubes to investigate the cardioprotective effects of arjunolic acid (AA) via MyD88-dependant TLR4 downstream signaling markers expression. The myotubes were developed by differentiating rat H9C2 and mouse C2C12 myoblast cells. The MTT viability assay was used to assess the cytotoxicity of AA. LPS induced in vitro cardiovascular disease model was developed in H9C2 and C2C12 myotubes. The treatment groups were designed such as control (untreated), LPS control, positive control (LPS+ pyrrolidine dithiocarbamate (PDTC)-25 µM), and treatment groups were co-treated with LPS and three doses of AA (50, 75, and 100 µM). The changes in the expression of TLR4 downstream signaling markers were evaluated through High Content Screening (HCS) and Western Blot (WB) analysis. The outcomes demonstrated that the expression of MyD88, MAPK, JNK, and NFκB markers were significantly upregulated in the LPS-treated groups compared to the untreated control. Evidently, the HCS analysis revealed that MyD88, NF-κB, p38, and JNK were significantly downregulated in the H9C2 myotube in the AA treated groups (50, 75, and 100 µM). For, the C2C12 myotube, the expression of NFκB was downregulated. TLR4 marker expression in H9C2 and C2C12 myotubes was subsequently decreased by AA treatment, suggesting possible cardioprotective effects of AA.