Abstract Body (Do not enter title and authors here): Background: Dynamin-related protein (DRP1) is hailed as the “master regulator” of mitochondrial morphosis events and is understood to orchestrate fission and membrane permeabilization: events largely regarded as the apoptotic “point of no return”. Historically, DRP1 has been targeted through genetic downregulation and small-molecule inhibition, thereby mitigating apoptosis and lethal ischemia-reperfusion (IR). Aim: Our goal was to establish whether knockdown of cellular DRP1 has a protective effect in HL1 cardiomyocytes subjected to lethal IR. Methods and Results: To test this hypothesis, HL1 cells were transfected with siRNA against DRP1 (DRP1 KD group), scrambled siRNA, or vehicle and subjected to a standard IR insult or a time-matched normoxic period. Transfection with DRP1 siRNA achieved ~80% reduction in cellular DRP1. Transfected HL1 cells and matched controls underwent IR and viability analysis with the MTT assay . Our data show that in normoxic controls, DRP1 knockdown was associated with ~35% reduction in viability (Figure). In the IR group, IR produced a ~55% reduction in viability. However, contrary to our hypothesis, DRP1 knockdown modestly exacerbated rather than attenuated - cell death (Figure). To provide a mechanistic explanation for these findings, immunoblotting for markers of mitochondrial injury (i.e., cytochrome c, cleaved caspase 3, cleaved OPA1) were assessed following DRP1 knockdown. Knockdown of DRP1 associated with increases in cytosolic cytochrome c, cleaved caspase 3 and processing of OPA1. Conclusion: Prior studies have reported cardioprotection when DRP1 is either silenced or inhibited. Our data; however, suggests that DRP1 knockdown may promote mitochondrial injury pathways and worsen viability.