A large series of hydroxytyrosyl esters of C2-C18 fatty acids with increasing lipophilicity was prepared by a new highly efficient method based on acylation of methylorthoformate-protected hydroxytyrosol. All products were tested for relative antioxidant effect using ABTS assays in ethanolic medium and DCF assays in L6 cells. No linear correlation between lipophilicity and antioxidant effect was found. ABTS assays showed a growing antioxidant capacity, with respect to hydroxytyrosol, only for medium-sized ester chains (C4-C10) and a nearly constant capacity for the higher homologues. This has been rationalized by molecular dynamics experiments in terms of partial shielding of the catecholic hydroxyls by long-chain esters. A similar and dose-dependent pattern was observed in DCF assays in L6 cells, but a sharp antioxidant activity drop resulted for long-chain esters, probably due to membrane entrapment.

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A large panel of novel catecholic antioxidants and their fatty acid or methyl carbonate esters has been synthesized in satisfactory to good yields through a 2-iodoxybenzoic acid (IBX)-mediated aromatic hydroxylation as the key step. The new catechols are structural analogues of naturally occurring hydroxytyrosol (3,4-DHE). To evaluate structure/activity relationships, the antioxidant properties of all catecholic compounds were evaluated in vitro by ABTS assay and on whole cells by DCF fluorometric assay and compared with that of the corresponding already known hydroxytyrosyl derivatives. Results outline that all of the new catechols show antioxidant capacity in vitro higher than that of the corresponding hydroxytyrosyl derivatives. Less evident positive effects have been detected in whole cells experiments. Cytotoxicity experiments, using MTT assay, on a representative set of compounds evidenced no influence in cell survival.

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to β-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.