Date Published: April 9, 2019
Publisher: Public Library of Science
Author(s): Rita Rezzani, Gaia Favero, Matteo Ferroni, Claudio Lonati, Mohammed H. Moghadasian, Partha Mukhopadhyay.
Interactive relationships among metabolism, mitochondrial dysfunction and inflammation at skeletal muscle level play a key role in the pathogenesis of disorders related to oxidative stress. Mitochondrial dysfunction and oxidative stress result in cellular energy deficiency, inflammation and cell death inducing a vicious cycle that promotes muscle wasting. The histidine-containing dipeptides, carnosine and anserine, are carbonyl scavengers whose cytoprotective contributions extend beyond the antioxidant defence, but the physiological meaning of these capacities is actually limited. In the present study, we compared and investigated the potential protective effects of three different histidine-containing dipeptides: carnosine, anserine and carnosinol, a carnosine-mimetic new compound, against oxidative stress induction in rat L6 skeletal muscle cells. The hydrogen peroxide induced-oxidative stress significantly altered cell morphology, induced apoptosis, oxidative stress and inflammation, decreased mitochondrial peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)/sirtuin3 pathway and the antioxidant system. Notably, all three investigated dipeptides in the present study, with a different extent and in a concentration-dependent manner, reduced myotube oxidative stress, apoptosis and inflammation. The present study underlined that carnosinol, maintaining the safety condition of carnosine and anserine, was the more efficient studied dipeptide in the preservation of mitochondrial environment mediated by PGC-1α and sirtuin3 expression and thereby in the reduction of oxidative stress-related alterations in this in vitro skeletal muscle model. Furthermore, we observed that carnosinol’s antioxidant effects are not blocked inhibiting sirtuin3, but are maintained with almost the same extend, indicating its multiple capacities of reactive carbonyl species-scavenging and of mitochondrial modulation through PGC-1α. In conclusion, carnosinol retained and surpassed the efficacy of the well-known investigated histidine-containing dipeptides improving oxidative stress, inflammation and also cell metabolism and so becoming a greatly promising therapeutic carnosine derivate.
Carnosine and anserine are versatile histidine-containing dipeptides (HCDs) identified in vertebrates, including horses, greyhounds, camels and humans . HCDs are stored in several tissues with the highest concentration occurring in skeletal muscle . These dipeptides have several important physiological properties and, in particular, carnosine plays many roles in maintaining health, including antioxidant activity . Anserine has similar effects, acting as an antioxidant and carbonyl scavenger  and affecting renal sympathetic nerve activity and blood pressure because, in kidney, it is expressed in concentration two times higher than those of carnosine .
The oxidative stress induced by incubation of L6 myotubes with H2O2, as previously observed also by , exhibited a typical morphological pattern of apoptosis: cells with small size and several membrane blebbing, while few cells were apparently preserved. In particular, numerous H2O2-treated myotubes looked rounded and detached (Fig 1A and 1B) respect to untreated control cells that appeared elongated and with a rod shape, except for few dead cells that showed a reduced size (Fig 1C). Cells incubated only with carnosinol, carnosine or anserine, independently of the concentration, showed the same normal morphological pattern (Fig 1D–1L) observed in untreated control myotubes. Interestingly, H2O2-reduction of cell viability and cell size were prevented, but to a different extent, by carnosinol, carnosine or anserine pre-incubation (Fig 1M–1U). The pre-incubation of H2O2-treated cells with 10 mM carnosinol (Fig 1M) or 10 mM carnosine (Fig 1N) showed prevalently scattered and round cells with membrane blebs; nevertheless, 10 mM carnosinol pre-incubation induced an increasing cell survival rate, not evident after pre-incubation of H2O2-incubated cells with carnosine 10 mM or anserine 10 mM and 20 mM (Fig 1O and 1R). These latter showed almost the same pattern of H2O2-incubated cells and just a weak preservation of myotubes morphology with a reduction of membrane blebs at the concentration of 30 mM (Fig 1U). Remarkably, the pre-incubation of H2O2-treated cells with carnosinol at the concentrations of 20 mM and 30 mM preserved, greatly and in a concentration-dependent manner, cell shape and size (Fig 1P and 1S) showing a cell morphology comparable to untreated control and preserving cell shape and size also respect to concentration-matched H2O2-treated cells pre-incubated with carnosine, which presented some round dead cells with blebs (Fig 1Q and 1T).
In the present study, we investigated the role of carnosinol, a new carnosine analogue, against oxidative stress and inflammation induced by H2O2-treatment of L6 skeletal muscle cells in vitro. Its effects have been evaluated in comparison to carnosine and anserine and the findings are very interesting demonstrating that it is more efficacy respect to the other well-known HCDs.