Publication Date

2008-11-24

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2008-10-23

First Committee Member

Sandra K. Lemmon - Committee Chair

Second Committee Member

Si M. Pham - Committee Member

Third Committee Member

James D. Potter - Committee Member

Fourth Committee Member

Keith A. Webster - Mentor

Fifth Committee Member

Rakesh C. Kukreja - Outside Committee Member

Abstract

Bnip3 is a BH3-only member of the Bcl-2 family of apoptotic proteins. Our laboratory has previously shown that Bnip3 induces a unique pathway of cardiac myocyte cell death, characterized by mitochondrial dysfunction, cytochrome c release and DNA fragmentation. Bnip3 is induced by hypoxia and the death pathway is activated by concurrent acidosis. We have shown that hypoxia-acidosis creates an environment that is permissive to calpain but not caspase activation and is characterized by enhanced DNase(s) activity as evidenced by genomic DNA fragmentation. This dissertation describes the nuclear consequences of Bnip3 activation by hypoxia-acidosis. Chapter 3 presents my evidence that hypoxia with progressive acidosis in cardiac myocytes results in a biphasic activation of DNases. In phase 1, [pH]o 6.9-6.7, apoptosis-inducing factor (AIF) is released from the mitochondria and translocates to the nucleus. AIF release coincided with the loss of mitochondrial membrane potential and with the release of cytochrome c from the mitochondria. In Phase II, [pH]o 6.3-6.0, DNase II translocates from the cytoplasm to the nuclear compartment. Nuclear localization of DNase II was associated with the collapse of endosomal pH gradients, indicated by diffuse Lysotracker Red staining and with single strand DNA nicks. Both phases of DNase release were independent of Bnip3, the mPTP and calpains. Neither phase involved activation of caspase-dependent DNases. Chapter 4 describes a unique role for Bnip3 in the modulation of histone acetylation. I found that hypoxia with acidosis in cardiac myocytes but not hypoxia alone stimulated a global increase in the acetylation of histones H3 and H4. Acetylation was initiated at [pH]o ~ 6.8 and increased as the pH declined. Histone hyperacetylation was associated with an increase in histone acetyltransferase (HAT) activity but no change in deacetylase (HDAC) activity. Knockdown of Bnip3 protein expression with siRNA dramatically reduced both histone H3 and H4 acetylation levels and HAT activity indicating an essential role for Bnip3 in this process. Components of the hypoxia-acidosis death pathway including the mPTP and calpains are not required for Bnip3-mediated histone hyperacetylation. These results reveal a novel role for Bnip3 in regulating HAT activity and histone acetylation which may lead to altered cardiac gene expression.

Keywords

Bnip3; Hypoxia; Cardiac Myocytes; Apoptosis; Gene Regulation; Histone Acetylation; DNA Fragmentation; Apoptosis Inducing Factor; Dnase II; Caspase Activted DNase

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