Superoxide produced by mitochondrial site IQ inactivates cardiac succinate dehydrogenase and induces hepatic steatosis in Sod2 knockout mice

Abstract

Superoxide produced by mitochondria has been implicated in numerous physiologies and pathologies. Eleven different mitochondrial sites that can produce superoxide and/or hydrogen peroxide (O₂^.-/H₂O₂) have been identified in vitro, but little is known about their contributions in vivo. We introduce novel variants of S1QELs and S3QELs (small molecules that suppress O₂^.-/H₂O₂ production specifically from mitochondrial sites I_Q and III_Qo, respectively, without compromising bioenergetics), that are suitable for use in vivo. When administered by intraperitoneal injection, they achieve total tissue concentrations exceeding those that are effective in vitro. We use them to study the engagement of sites I_Q and III_Qo in mice lacking functional manganese-superoxide dismutase (SOD2). Lack of SOD2 is expected to elevate superoxide levels in the mitochondrial matrix, and leads to severe pathologies and death about 8 days after birth. Compared to littermate wild-type mice, 6-day-old Sod2^-/- mice had significantly lower body weight, lower heart succinate dehydrogenase activity, and greater hepatic lipid accumulation. These pathologies were ameliorated by treatment with a SOD/catalase mimetic, EUK189, confirming previous observations. A 3-day treatment with S1QEL352 decreased the inactivation of cardiac succinate dehydrogenase and hepatic steatosis in Sod2^-/- mice. S1QEL712, which has a distinct chemical structure, also decreased hepatic steatosis, confirming that O₂^.- derived specifically from mitochondrial site I_Q is a significant driver of hepatic steatosis in Sod2^-/- mice. These findings also demonstrate the ability of these new S1QELs to suppress O₂^.- production in the mitochondrial matrix in vivo. In contrast, suppressing site III_Qo using S3QEL941 did not protect, suggesting that site III_Qo does not contribute significantly to mitochondrial O₂^.- production in the hearts or livers of Sod2^-/- mice. We conclude that the novel S1QELs are effective in vivo, and that site I_Q runs in vivo and is a significant driver of pathology in Sod2^-/- mice.