Raw data (Excel file) for figures from American Heart Association 19TPA34910011.

Cardiovascular disease is the most common cause of death among the elderly and the prevalence of heart failure increases dramatically with age. Cardiac aging is associated with significant alterations in cardiac structure and function, including hypertrophy and contractile dysfunction. Mitochondria are important organelles for energy supply and are also recognized as gatekeepers in the control of cell survival. There is a progressive accumulation of damaged mitochondria with age, and this is considered to be a critical contributor to the aging process. Selective elimination of damaged mitochondria by autophagy (mitophagy) plays a central role in preservation of mitochondrial quality. However, mitophagy declines with age and the molecular mechanisms responsible for these defects have been elusive and pathways that could be targeted to improve the mitochondrial quality in the aging heart have not been defined. Chronic low grade inflammation is another hallmark of aging (inflammaging) which has been causatively linked to aging-related disease. The NLRP3 inflammasome, a major contributor to maladaptive inflammation, is activated in response to various stimuli including mitochondrial damage to produce pro-inflammatory cytokines. RhoA is a small G-protein that serves as a proximal downstream effector of numerous GPCRs and is also responsive to oxidative stress, functioning as a protective signaling molecule in the heart. The effect of RhoA on cardiac aging has not, however, been determined. We propose based on findings in cardiac specific RhoA knockout (KO) mice that RhoA-mediated signaling limits the progression of cardiac aging. We hypothesize that this occurs through stimulation of mitophagy and suppression of NLRP3 inflammasome activity.<b>Aim 1: Does RhoA regulate mitophagy to delay the aging process?</b>The goal of this Aim is to demonstrate that RhoA signaling regulates mitophagy to preserve mitochondrial quality and thus delays the myocardial aging. Proposed studies will use cardiac specific RhoA KO mice to determine the role of RhoA signaling in cardiac aging. Conditional cardiac specific RhoA deletion in the adult heart will also be used. The effect of activation of RhoA on mitophagy and cardiac aging will be directly tested using adeno-associated virus serotype 9 (AAV9)-mediated gene delivery of active RhoA. We will determine whether RhoA signaling induces mitophagy by stabilizing PINK1 protein through protein kinase D (PKD) mediated phosphorylation. This hypothesis will be examined using PINK1 KO and PDK1 KO mice.<b>Aim 2: Does RhoA attenuate inflammasome activation in the aging heart?</b>In this Aim, we will determine whether the NLRP3 inflammasome is activated in the aging heart and whether it is negatively regulated by RhoA. NLRP3 inflammasome activity in the aging heart will be assessed by several different methods including visualization of caspase-1 activity in cardiac sections and the effect of RhoA expression (AAV9-RhoA) or RhoA deletion (KO) on the NLRP3 inflammasome determined. The significance of inhibition of the NLRP3 inflammasome in RhoA-mediated protection against aging will be examined using NLRP3 KO mice. We will determine if mitophagy regulated by RhoA signaling leads to attenuation of NLRP3 inflammasome activation in the aging heart. The possibility that RhoA-mediated signaling directly inhibits the assembly and activation of the NLRP3 molecular complex will also be examined.