SVCT2-Dependent plasma and mitochondrial membrane transport of ascorbic acid in differentiating myoblasts

• Published in: Pharmacological research Vol. 159; p. 105042
• Main Authors: Fiorani, Mara, Scotti, Maddalena, Guidarelli, Andrea, Burattini, Sabrina, Falcieri, Elisabetta, Cantoni, Orazio
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01-09-2020
• Subjects: C2C12 cells; L-ascorbic acid; Mitochondrial SVCT2; Myoblasts; Myotubes; Plasma membrane SVCT2; Plasma membrane SVCT2; Mp; IP3R; DTT; Mt; C2C12 cells; BSA; FBS; RT-PCR; IB; EB; AA; PBS; Mitochondrial SVCT2; L-ascorbic acid; Myotubes; CK; ER; PVDF; Myoblasts; SVCT; Mb; Mc; ROS; HB; DHA; MRB; GAPDH; PM; TEM
• ISSN: 1043-6618; 1096-1186
• DOI: 10.1016/j.phrs.2020.105042

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•C2C12 myoblasts and myotubes express high affinity plasma membrane SVCT2.•C2C12 myoblasts express low-affinity mitochondrial SVCT2.•C2C12 myotubes present progressively reduced mitochondrial SVCT2 expression.
The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.