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Peripheral arterial disease (PAD) affects 8 to 12 million people in the United States. Intermittent claudication (exercise-induced leg pain and severe walking limitation) is a hallmark of PAD which severely limits exercise capacity and deceases quality of life. Clinical studies demonstrate that the exercise pressor reflex (EPR), a neural reflex originating in skeletal muscle which contributes to the regulation of cardiovascular function during physical activity, is exaggerated in patients with PAD. Furthermore, the exaggerated EPR during exercise in PAD patients is seen before the onset of claudication. We hypothesize that in PAD, exercise-induced activation of muscle afferents (i.e. the EPR), which is initially below the threshold for pain , initiates an exaggerated vasoconstriction and worsens the ischemia of the exercising PAD muscle, which may further activate muscle afferents above the nociceptive threshold level and produce the familiar exertional pain symptoms of claudication. In Aim 1, We will examine the time-course of EPR function including heart rate, blood pressure and local muscle blood flow during static and dynamic exercise in a rat model of chronic PAD and in sham animals. We will determine the potential correlation between EPR function, muscle ischemia and pain behavior scores in both sham and PAD rats. Finally, we will determine if selective ablation of the EPR with the potent TRPV1 receptor agonist, resiniferatoxin (RTX), improves muscle perfusion during exercise and pain indices in PAD rats. Assessment of RTX efficacy will be carried out using a novel tissue clearing method in a TRPV1-tdTomato reporter mouse model. Furthermore, our pilot study found that neural inflammation indicated by macrophage infiltration occurs in lumbar DRGs post PAD. In parallel, we observed upregulated protein expression of nuclear factor erythroid 2-related factor (Nrf2, a transcription factor that mediates the cellular response to oxidative stress/inflammation) in lumbar DRGs post PAD. We believe that the purpose of upregulated Nrf2 protein is to counteract the influence of neural inflammation in DRG neurons post PAD. However, this self-limited compensatory response by an intrinsic DRG system may not be potent enough in DRG neurons to antagonize exogenous neural inflammation evoked by PAD. Therefore, in Aim 2, We hypothesize that selective upregulation of muscle afferent Nrf2 via Keap1-Knock-out (KO) will mitigate enhanced muscle afferent neuronal excitability and pain sensation in a mouse model of chronic PAD. We also hypothesize that local epidural delivery of hydrogel-based drug system with encapsulation of Nrf2 activator (i.e., Curcumin) will mitigate the exaggerated EPR and pain sensation in a rat model of chronic PAD. Finally, we propose to examine the potential influence of Nrf2 signaling onto DRG ion channel function such as potassium channels in muscle afferent neurons in PAD. To date, the underlying molecular/cellular mechanisms of the exaggerated EPR in chronic PAD has not been examined in any chronic PAD animal model. The long-term goal of this application is to discover the neural mechanisms operating in the exaggerated EPR and claudication and to provide a novel treatment for this disorder.

Excessive oxidative stress has been shown to be a potent pathophysiological mediator in multiple disease states. Cardiac and peripheral sympathetic nerve activity is increased in chronic heart failure, in part, due to augmented neural inflammation and oxidative stress. Importantly, several major antioxidant enzymes and molecules are decreased in the cytosol and mitochondria of pre-sympathetic neurons in chronic heart failure. A major regulator of these antioxidant molecules is the transcription factor, Nrf2. Previous studies from our laboratories have shown a reduction in Nrf2 in heart failure. Genetic knock down of Nrf2 in the rostral ventrolateral medulla of normal mice results in significant sympatho-excitation and hypertension. On the other hand, overexpression of Nrf2 results in sympatho-inhibition in mice with chronic heart failure along with a reduction in oxidative stress and an increase in several antioxidant enzymes. In order to determine if this mechanism plays a role in cardiac arrhythmogenesis in the post myocardial infarction (MI) chronic heart failure model we propose here to evaluate the role of Nrf2 in the stellate ganglia, a source of major sympathetic outflow to the heart. Three specific aims are proposed. Specific Aim 1: We will determine the time-course changes in neural inflammation-Oxidative stress-Nrf2 signaling in the SG post MI. We will evaluate message and protein for the key molecules involved in maintaining redox homeostasis in the SG, namely the Nrf2/Keap1 system and its target antioxidant enzymes, including NQO1, HO1, Catalase, SOD and glutathione peroxidase. We will also assess neural inflammation in the SG by measuring macrophage infiltration and pro-inflammatory cytokines. Correlations will be made between levels of Nrf2 and cardiac sympathetic tone across a wide range of cardiac function. Specific Aim 2: We will determine if selective knockdown and overexpression of Nrf2 in the SG alters cardiac sympathetic tone in normal and CHF animals by altering ganglionic neuronal excitability. Using Nrf2 and Keap1 floxed mice we will evaluate antioxidant enzyme expression, redox homeostasis, inflammation and arrhythmia incidence. These molecular alterations induced by Nrf2 will be evident in SG cell action potential generation. Specific Aim 3: We will determine if pharmaceutic manipulation of Nrf2 signaling in the SG by either systemic treatment with the well-known Nrf2 activators dimethyl fumarate (DMF) and sulforaphane114 or local SG delivery of micelle conjugated dynamic hydrogels with encapsulation of a Nrf2 activator reduces arrhythmogenesis and improves cardiac function in the post-MI rats. We hypothesize that the redox status and electrophysiological activity of SG neurons in CHF rats will be partially restored following treatment with Nrf2 activators. Furthermore, these effects will be abolished or attenuated in mice with stellate ganglia Nrf2 deficiency. We believe that this proposed research will lay a solid scientific foundation for developing a new therapy for the patients with CHF.