Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type


Degree Name




First Advisor

Karin Przyklenk


Mitochondrial fusion and fission, collectively termed mitochondrial dynamics, are among the core mechanisms responsible for maintaining mitochondrial health and functional integrity. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission. Recent studies suggest that i) mitochondrial dynamics, particularly, mitochondrial fission, serves as a mediator of cell fate in the setting of ischemia-reperfusion (IR) injury, and, ii) inhibition of DRP1 and mitochondrial fission provides cardioprotection against IR injury. However, the precise role of DRP1 translocation to mitochondria in the pathogenesis of myocardial ischemia-reperfusion injury has not been established.

Using an established model of hypoxia-reoxygenation (HR) in cultured HL-1 cardiomyocytes, we tested three hypotheses:

I. subcellular redistribution of DRP1 is i) triggered by HR, and ii) plays a mechanistic role in HR-induced cytochrome c release and cell apoptosis;

II. inhibition of DRP1 translocation prior to hypoxia is cardioprotective;

III. inhibition of DRP1 in a time-frame that is relevant as a therapeutic strategy (i.e., begun at reoxygenation) will also attenuate cardiomyocyte death, although possibly less robust than pretreatment.

In support of Hypothesis I, our results demonstrated that HR was associated with DRP1 translocation to mitochondria, cytochrome c release into cytosol, and caspase 3 cleavage (harbinger of apoptosis). Subsequently, and consistent with Hypothesis II, we established a cause-effect relationship between DRP1 translocation and cardiomyocyte injury in the setting of HR injury. Both pretreatment with Mdivi-1 (a specific inhibitor of DRP1; 50 µM) and knockdown of DRP1 expression by transfection with DRP1 siRNA significantly reduced DRP1 translocation to mitochondria, attenuated cytochrome c release, blunted caspase 3 cleavage and apopotic cell death, better-preserved mitochondrial morphology and improved cell viability. However, in contrast to Hypothesis III, Mdivi-1 given at reoxygenation, was not cardioprotective. Rather, we observed a paradoxical result: Mdivi-1, given at reoxygenation, attenuated apoptosis, but did not reduce total cell death and, in some cases (prolonged exposure at a dose of 50 µM) exacerbated cell death. This exacerbated cell death with delayed Mdivi-1 treatment was in part rescued by co-administration of Necrostatin-1, suggesting that necroptosis (programmed necrosis) may play a role in this phenomenon.

In conclusion, our results show that DRP1 translocation to mitochondria plays a mechanistic role in mediating cardiomyocyte injury in the context of hypoxia-reoxygenation injury, and reveal a complex temporal relationship between inhibition of mitochondrial fission and cardioprotection.