Brown 2015 Abstract MiPschool Cape Town 2015
|Protection of post-ischemic mitochondrial bioenergetics by targeting cardiolipin-dependent membrane fluidity.|
Event: MiPschool Cape Town 2015
In this study we determined the efficacy of the cardiolipin-targeting peptide MTP-131 (Bendavia) on mitochondrial function in isolated hearts, permeabilized fibers, and isolated mitochondria. We hypothesized that this peptide would improve mitochondrial membrane fluidity during ischemia-reperfusion (I/R). Fluidity is a biophysical parameter of the membrane that influences the assembly of mitochondrial respiratory supercomplexes. MTP-131 is a cell-permeable peptide known to confer cardioprotection in pre-clinical models and is currently being investigated in Phase 2 clinical trials for cardiovascular disease1-4.
Mitochondria from rat ventricle were isolated and placed in the chamber of a modified Oroboros Instruments O2k high-resolution respirometer. Seeking to mimic physiological state 3 respiration in heart, we clamped ADP levels at 75uM using a hexokinase/2-deoxyglucose clamp. Mitochondria were treated with either saline or 1uM MTP-131 prior to hypoxia. In this protocol, mitochondria make themselves hypoxic by consuming the oxygen in the chamber in about 10 minutes. Hypoxia lasted for 25 minutes, followed by reoxygenation. Mitochondrial membrane fluidity was monitored continuously throughout the protocol using the fluidity-dependent fluorophore MC540. Membrane fluidity decreased sharply at the onset of reoxygenation (32,335 ± 3948 AU). Mitochondria treated with MTP-131 displayed higher membrane fluidity in early reperfusion (43,322 ± 6862 AU). These data were corroborated in intact heart studies, where both lipid head-group and acyl side-chain fluidity were restored with MTP-131. The improvement in fluidity was associated with greater density of mitochondrial respiratory supercomplexes and decreased degradation of respiratory protein complexes assessed with blue-native gel electrophoresis.
Mitochondrial respiration in permeabilized ventricular fibers was significantly impaired after I/R. Complex I-dependent respiration after reperfusion was 208±19 v 42±9 pmol O2 mg-1∙s-1 in control v I/R, respectively; p<0.05. Complex II-dependent respiration was also lower (753±41 v 168±13 pmol∙mg-1∙s-1 in control versus I/R; p<0.05). Perfusion with MTP-131 during reperfusion significantly increased Complexes I- (100±13 pmol O2 mg-1∙s-1) and II-dependent (334±63 pmol O2 mg-1∙s-1) respiration (p<0.05 versus untreated for both). Finally, this recovery of bioenergetics was associated with a significant reduction in infarct size in MTP-131 treated animals.
In summary, our results provide evidence that post-ischemic mitochondrial function can be rescued by targeting the cardiolipin-dependent assembly of mitochondrial supercomplexes.
Labels: MiParea: Respiration, mt-Membrane Pathology: Cardiovascular Stress:Ischemia-reperfusion Organism: Rat Tissue;cell: Heart Preparation: Intact organism, Permeabilized tissue, Isolated mitochondria Enzyme: Supercomplex
Coupling state: OXPHOS Pathway: N, S, NS HRR: Oxygraph-2k
School of Medicine, East Carolina Univ, NC, USA. - [email protected]