Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle.

Abstract

Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (Ip) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.