John Auchampach - US grants

DESCRIPTION (provided by applicant): The goal of this research is to define the mechanisms by which A3 adenosine receptors (ARs) protect against myocardial ischemia/reperfusion injury. We will test the hypothesis that A3ARs are protective by multiple mechanisms including preservation of mitochondrial function, reduction of apoptosis, and attenuation of inflammation. We predict that the A3AR signals via kinase signaling pathways that ultimately results in protection by activation of mitochondrial KATP channels. A key feature of our hypothesis is that we predict that A3ARs are expressed in cardiac myocytes. We will test these hypotheses by examining the effect of selective A3AR agonists in an isolated mouse heart model of global ischemia/reperfusion and a clinically relevant in vivo dog model of infarction. The isolated mouse heart model will be used to correlate post-ischemic contractile function (left ventricular pressure) and cell necrosis (LDH released) with metabolic state (high-energy phosphates), mitochondrial integrity (mitochondrial membrane potential, respiration, and rate of ATP synthesis), apoptosis (DNA fragmentation, TUNEL staining, caspase activation), and pro-inflammatory cytokine signaling (NF kappa B activation, TNF alpha production). The in vivo dog model of infarction will be used to compare infarct size with markers of inflammation (neutrophil infiltration). The mitochondrial specific KATP channel blocker sodium 5-hydroxydecanoate will be used to delineate the role of mitochondrial KATP channels in preserving mitochondrial and cardiac function. Experiments will be performed to determine whether A3ARs signal via specific protein kinases (PKC-epsilon or delta; PI-3/akt kinase) and/or generation of reactive oxygen species. Information obtained from these studies will advance the understanding of the fundamental questions involved in cellular protection. The results of theses studies should also provide new insights into the therapeutic utility of targeting A3ARs in patients with ischemic heart disease.

DESCRIPTION (provided by applicant): Current evidence suggests that A3 adenosine receptors (A3ARs) are expressed in cardiac myocytes, where they regulate responses to ischemic stress. In addition, A3ARs are expressed in cells involved in the inflammatory response including neutrophils, macrophages, and endothelial cells. In these cells, they mediate anti-inflammatory actions. In preliminary studies, we have shown that newly developed A3AR agonists provide profound cardiac protection against tissue damage induced by myocardial ischemia/reperfusion (I/R) injury in mice, rabbits, and dogs whether they are given prior to ischemia or at the time of reperfusion. The goal of this research proposal is to understand the mechanisms responsible for this tissue protection. Our general hypothesis is that A3AR agonists act dually on cardiac myocytes to reduce myocardial injury during ischemia, and act on bone marrow-derived cells to reduce inflammation during reperfusion. The studies involve the use of genetic approaches to: 1) produce a congenic line of mice with the A3AR gene selectively deleted from cardiomyocytes using the Cre/LoxP strategy, and 2) to produce from congenic A3AR gene "knock-out" mice, chimeric mice lacking or expressing the A3AR in bone marrow-derived cells using standard transplantation techniques. An in vivo mouse model of infarction and an isolated mouse heart model of global ischemia and reperfusion will be used to determine the relative importance of A3ARs expressed in the myocardium versus inflammatory cells in regulating I/R injury. Additional studies are proposed to study cellular signaling responses of A3ARs in specific populations of inflammatory cells from "wild-type", conventional A2AAR gene "knock-out", and conventional A3AR gene knock-out" mice. Collectively, these studies will combine several state-of-the-art techniques to provide new definitive information on the cell-specific mechanisms by which A3AR activation provides protection from I/R injury.

ROLE ON Cal. Acad. Sum. INST.BASE NAME

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the number one cause of death in the United States and ischemic heart disease is the leader in mortality among patients with CVD. Most individuals with ischemic heart disease have blocked coronary arteries and cardiac tissue has a lack of perfusion (ischemia), resulting in myocardial damage and further injury following reperfusion. The novel CYP-epoxygenase metabolites of arachidonic acid (AA), 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) are increased during ischemia and particularly following reperfusion. Endogenously produced EETs and exogenously administered EETs produce marked cardioprotective effects in dog, rat and mouse hearts; however, the mechanisms responsible for these beneficial effects remain unclear. Based on intriguing preliminary data, opioid receptors and nitric oxide (NO) release may be 2 major players in EET-mediated cardioprotection. This study will utilize rats and genetic knockout mice of nitric oxide synthase (NOS) isoforms as well as cellular models of cardiomyocytes and cardiac fibroblasts to elucidate in depth, the key contributing factors responsible for EET-induced cardioprotection. The hypothesis to be tested is that upon ischemia/reperfusion, EETs released from cardiomyocytes and cardiac fibroblasts are potent cardioprotective agents that induce the further release of endogenous NO to produce their beneficial effects. Specifically, we will (1) determine that nitric oxide (NO) is a mediator of EET-induced cardioprotection in intact rat and mouse hearts and cardiomyocytes. Both rats and NOS knockout mice and cardiomyocytes (H9c2 cells) will be used to demonstrate that EET-induced cardioprotection is mediated via a NO signaling pathway. NOS isoforms (eNOS, nNOS or iNOS) and their signaling pathways activated by the EETs will be identified. (2) Determine that cardiac fibroblasts play a role in releasing EETs and possibly NO as regulatory factor(s) to protect cardiomyocytes from hypoxia/reoxygenation injury. (3) Determine if cross-talk occurs between the two major cardioprotective factors, EETs and opioids, in cardioprotection. Importantly, EETs are readily hydrolyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) which have no effect on myocardial infarct size. This finding suggests that novel synthetic EET analogs with superior pharmacokinetics may represent better therapeutic targets than sEH inhibitors and labile EETs. The newly synthesized longer-acting EET analogs will be used to elucidate signaling pathways of the EETs and to reduce infarct size. As leaders in the this field, identification of novel stable analogs of endogenous EETs will be a major goal of this project as well as demonstrating their powerful anti-ischemic and anti-inflammatory actions in cellular and whole animal models of cardiac injury. The long term goal is to obtain better characterization of a novel endogenous system with multiple therapeutic targets that may suggest combined therapy for better treatment of ischemia/reperfusion injury.

ABSTRACT The interstitial concentration of adenosine remains elevated following MI continuing well after the injured tissue has healed and a scar has formed. However, the impact of adenosine signaling during post-MI remodeling and chronic ischemic heart failure remains unknown. In other organs, the novel concept has emerged that adenosine becomes damaging if it remains persistently elevated in tissues by activating pathways that promote fibrosis. This has been observed in experimental animal models of chronic lung, liver, kidney, and skin diseas- es. While the mechanisms by which adenosine becomes damaging vary, it generally acts to promote exces- sive tissue repair via activation of the A2BAR. The A2BAR is a unique member of the AR family abundantly ex- pressed in macrophages linked to cellular activation and production of inflammatory/fibrogenic cytokines. To determine the contribution of adenosine signaling during post-MI remodeling, we examined the effect of genet- ic deletion or blockade of the A2BAR (ATL-801) in a mouse model of permanent coronary artery ligation. Our preliminary data show a marked reduction in fibrosis with loss of A2BAR signaling at 8 weeks post-MI, which was associated with reduced inflammatory/fibrogenic activity in heart tissue, less dysfunction, and improved compliance. Central hypothesis: Augmented production of adenosine persists following MI, which contributes to fibrosis, pathological remodeling, and heart failure via activation of the A2BAR subtype. Aim #1 will define the role of A2BAR signaling during post-MI remodeling. We will test whether genetic deletion or inhibition of the A2BAR will slow the rate of fibrosis that develops in surviving myocardium following MI resulting in less dysfunc- tion and ultimately prolonged survival. This aim will be accomplished using ATL-801, Adora2b-/- mice, as well as a new line of Adora2b mutant rats created by our lab. Aim #2: To delineate the mechanisms by which A2BAR signaling contributes to adverse post-MI remodeling. Utilizing a conditionally targeted mouse line lack- ing expression of the A2BAR in myeloid-derived cells, assessments of macrophage infiltration/activation, and molecular analyses of post-MI heart tissue, we will test the hypothesis that persistent exposure to adenosine following MI stimulates the production of inflammatory mediators from macrophages that causes chronic in- flammation, fibroblast activation, and fibrosis. Aim #3: To delineate the magnitude and consequences of en- hanced adenosine production during post-MI remodeling and ischemic heart failure. We will assess changes in the interstitial concentration of adenosine in the surviving myocardium during the course of post-MI remodel- ing. We hypothesize that the interstitial concentration of adenosine will increase progressively during late re- modeling as the heart fails and that its enzymatic removal with pegylated-adenosine deaminase therapy will be protective. Completion of this work is expected to identify adenosine as an important fibrogenic mediator in the heart that contributes to pathological remodeling following MI through its interaction with the A2BAR subtype, and potentially identify a novel target for therapeutic intervention for patients with ischemic heart disease.

PROJECT SUMMARY Tip60 protein, also known as Tat-interactive protein 60 kD, has been associated with induction of apoptosis, the DNA damage response (DDR), and cell-cycle senescence -- functions that are not mutually exclusive -- in cell-lines and in cancer cells. We have shown that Tip60 is a vital protein in the early embryo, and that it undergoes a curious isoprotein shift as neonatal cardiomyocytes (CMs) enter replicative senescence, an event that was recently shown to be induced by activation of the DDR in these cells. Although the heart is enriched in Tip60 protein, its role in this organ remains unclear. A recent study using cultured neonatal CMs revealed that ischemia increases Tip60 content and consequent apoptosis, effects that were prevented in Tip60- depleted cells. This result is consistent with our previous finding in heterozygous Tip60+/- mice showing that modest reduction of Tip60 protein in the adult heart caused release of CMs from cell-cycle arrest while inhibiting apoptosis. Taken together, these findings suggest that reduction or inactivation of Tip60 should enhance cardioprotection during ischemia, by suppressing CM death and permitting CM regeneration. To test this possibility we have developed an animal model in which Tip60 can be conditionally depleted. Using these mice we are addressing Specific Aims to test the twofold hypothesis that Tip60 is recruited to the telomere during early neonatal stages to induce the DDR and replicative senescence in CMs (Aim 1), and that depletion or inactivation of Tip60 in the adult heart confers cardioprotection from ischemia by inhibiting apoptosis and permitting CM regeneration (Aim 2). Experimentally, the first Aim will determine whether Tip60 becomes associated with the CM telomere at early neonatal stages, and whether its depletion reduces telomere length and disrupts the DDR while extending the window of neonatal CM proliferation. The second Aim will determine whether knockdown or transient inactivation of Tip60 in the ischemic adult heart inhibits apoptosis while permitting CM regeneration. Fulfillment of this hypothesis will advance our understanding of how the post- mitotic differentiated state of CMs is attained, while establishing Tip60 as a cardioprotective target.  

Funds are requested to cover costs to upgrade an existing VisualSonics Vevo 770 high-frequency ultrasound imaging system to the technologically advanced Vevo 3100 model with components and analysis software specifically designed for cardiovascular and oncology applications. The specific technological enhancements with the Vevo 3100 model include improved transducer technology that provides higher resolution images and faster frame rates, expanded Doppler features, and improved image analysis software (strain analysis and 3D reconstruction software). The application is fiscally conservative by taking advantage of expiring trade-in offers from the manufacturer and by limiting purchase of accessories to only those that are required by users. The system will immediately aid 16 principal investigators from eight different MCW academic departments focused on research related directly to human health. Disease topics include ischemic heart disease, heart failure, atherosclerosis, coronary artery disease, hypertension, diabetes, kidney disease, and pancreatic, lung, and breast cancer. If funds are awarded, the new instrument will be housed in an existing small animal imaging shared facility, operated and maintained by the project PI and his staff. MCW Administration through the Department of Pharmacology & Toxicology will provide administrative and financial support. Enhancement in imaging technology is a key component of the planned growth of research at MCW. The addition of new cutting-edge imaging technology will support existing NIH-funded research, improve the quality of current research, increase competitiveness of our faculty for future grant support, and assist in our main goal to discover new treatments and cures for human diseases.