The overall hypothesis for this project is that increased mitochondrial generation of superoxide causes a disruption of vascular smooth muscle heme biosynthesis by ferrochelatase (FECH), resulting in a loss of the beneficial vascular regulatory effects of soluble guanylate cyclase (sGC). It is known that oxidation of the sGC heme promotes loss of regulation by nitric oxide (NO) and sGC degradation by proteolysis. While the mitochondrial heme generating enzyme FECH has an iron-sulfur cluster essential for its stability, which is a potential target for disruption by superoxide, the literature appears to ack evidence for the consequences of FECH dysfunction on the heme-dependence of sGC regulation in any biological system. Due to the potential importance of angiotensin II (AngII) in promoting human vascular dysfunction in multiple diseases and previous evidence for mechanisms associated with increased superoxide generation in subcellular sites including mitochondria, and disruption of NO and its associated heme-dependent regulation of sGC/cGMP-mediated vasodilation, we hypothesized and found evidence for both FECH activity being disrupted by AngII and for FECH controlling the expression and NO-mediated activation of sGC. Studies in Aim 1 focus on showing how AngII regulation of increases in mitochondrial superoxide cause a disruption in heme biosynthesis by FECH that impairs vascular regulation by sGC in isolated mouse and bovine coronary arteries treated with AngII and siRNA or mechanistic probes modulating subcellular sources of superoxide and FECH. Arteries from mice deficient in AngII type-1 receptor (AT1R), Cu, Zn-SOD (SOD1), mitochondrial matrix SOD2, FECH, Nox1 and Nox2 oxidase will be used in these studies to define how AngII regulation of cytosolic and mitochondrial superoxide influences regulation by the sGC and FECH systems.
Aim 2 investigates new preliminary observations suggesting how promoting availability of protoporphyrin IX (PpIX, an activator of sGC) and heme from ?-aminolevulinic acid (ALA) appears to protect ferrochelatase and sGC regulation potentially through a cGMP-mediated inhibition of mitochondrial superoxide that may involve cGMP preventing the depletion of SOD2. Studies in Aim 3 focus in defining the in vivo importance of the disruption of ferrochelatase and heme- associated sGC regulation of vascular function and protection provided by ALA, using a mouse model of osmotic minipump delivery of AngII. Radiotelemetry monitoring of blood pressure, echocardiography, and evaluation of changes in isolated arteries and in vivo arteriolar function in the skeletal muscle microcirculation will be evaluated. The role of subcellular sources of superoxide in the disruption of ferrochelatase will also be assessed in AngII-infused mice deficient in AT1R, SOD1, SOD2, Nox1 and Nox2, together with a therapy specifically targeting mitochondrial superoxide. These studies are expected to document that mitochondrial superoxide disruption of ferrochelatase has an important role in sGC-associated vascular dysfunction, which can be targeted with a beneficial ALA therapy regulating the activity, expression and NO-stimulation of sGC.
This project investigates the novel observation that vascular diseases associated with increased angiotensin II could promote the loss of a key protective vasodilator system (guanylate cyclase activation by nitric oxide) as a result of disrupting the production of a needed heme cofactor made by mitochondria. It also investigates a potential novel therapeutic approach based on treatment with an amino acid normally used to produce heme, because it appears to have disease reversing beneficial effects.
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