Off-campus WSU users: To download campus access dissertations, please use the following link to log into our proxy server with your WSU access ID and password, then click the "Off-campus Download" button below.

Non-WSU users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Access Type

WSU Access

Date of Award

January 2022

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Karin Przyklenk

Second Advisor

David Evans

Abstract

Cardiovascular disease (including coronary heart disease and myocardial infarction) is the leading global cause of death and disability. Myocardial infarction (MI) is defined by a cessation blood flow to a region of the heart, wherein oxygen and nutrient deprivation leads to cardiomyocyte injury and death. The only targeted therapy against MI is timely reperfusion. Reperfusion, while necessary to salvage ischemic myocardium, paradoxically introduces its own injury component – ischemia-reperfusion injury (IR). Mitochondria have been implicated at the nexus of IR injury, with our evolving understanding of mitochondrial quality control processes suggesting mitochondrial morphosis as a key determinant of cardiomyocyte fate. Mitochondrial fission has been extensively studied as a key event in IR injury progression; however, recent data suggest that altered mitochondrial fusion machinery, including the inner-membrane protein, optic atrophy protein (OPA1), may play a causal role in determining cardiomyocyte fate during IR. Using an in-vitro model of ischemia-reperfusion in an immortalized cardiomyocyte cell line (HL1), we tested three hypotheses:

i. Myocardial ischemia-reperfusion injury triggers the proteolytic processing of OPA1 through pathological activation of OMA1 protease early during the reperfusion period. These events coincide with pathological DRP1 translocation and precede cytochrome c release and activation of apoptosis.

ii. Cardioprotection through IPC and RIPC can be achieved in a pure cardiomyocyte model, and, these protective strategies will attenuate OPA1 proteolysis and DRP1 translocation, thereby blunting cytochrome c release and apoptosis.

iii. OPA1 plays a causal role in determining cardiomyocyte fate during IR, whereby depletion of OPA1 through siRNA knockdown will sensitize cardiomyocytes to IR injury, increase markers of apoptosis, and in IPC and RIPC cohorts, blunt the cardioprotective effect on viability. The functions of OPA1 and DRP1 are interdependent, where knockdown of one will alter the functionality of the other.

In support of hypothesis I, our results demonstrate that IR is associated with increased OMA1 activity within 30-minutes following reperfusion. The increased proteolytic activity of OMA1 explains the observed proteolytic processing of OPA1 at 30-minutes post-R, where we see significant loss of L-OPA1 and emergence of S-OPA1. Extending this timeline out to 120-minutes post-R, we have provided evidence that following OPA1 proteolysis, total OPA1 content is progressively lost from mitochondria fractions. In our HL1 model of IR, injury triggered the pathological translocation of DRP1 (detected in mitochondrial fractions), increased detection of cytochrome c within the cytosol, and ultimately, led to cleavage-activation of Caspase 3. Novel to this project, is the finding that following IR-induced processing of OPA1, S-OPA1 undergoes subcellular redistribution from mitochondrial to cytosolic compartments. Subsequently, contrary to our assertion in hypothesis II, cardioprotection achieved with IPC and RIPC did not attenuate the processing of mitochondrial OPA1, rather, IPC and RIPC attenuated the amount of S-OPA1 detected within the cytosol. However, the effect of IPC and RIPC on cytosolic OPA1 was disparate: i.e., IPC significantly attenuated cytosolic S-OPA1, while RIPC gave a modest and non-significant attenuation of cytosolic S-OPA1. This effect of cardioprotection on cytosolic S-OPA1 was mirrored by the effects of IPC and RIPC on pathological DRP1 translocation to the mitochondria. Despite these disparities between IPC and RIPC, both were significantly cardioprotective and attenuated markers of apoptosis. Given recent published data evidencing sensitization of cardiomyocytes to IR injury when OPA1 is depleted, we sought to provide evidence for a causal role of OPA1 in IR injury in hypothesis III. Despite our supporting evidence for pathological changes in OPA1 in response to IR, our evidence does not provide support for OPA1 as a causal determinant of cardiomyocyte fate, since siRNA depletion did not sensitize to IR nor did it blunt the protection achieved with IPC and RIPC. In conclusion, our results provide evidence that mitochondrial OPA1 content does not play a causal mechanistic role in ischemia-reperfusion injury, however, given the association between cardiomyocyte viability and S-OPA1 present in the cytosol, our results suggest that the subcellular redistribution of OPA1 may play a role in the pathogenesis of IR-induced apoptosis. Furthermore, our data reveal a complex relationship between OPA1 and DRP1, where modulation of one protein, leads to subcellular alteration in the other.

Download
Off-campus Download

Share

COinS