Robert W. Neumar - US grants

DESCRIPTION (adapted from applicant's abstract): Brain ischemia caused by cardiac arrest and stroke kills 300,000 people and disables another 150,000 each year in the United States. The general goal of my research effort is to characterize the molecular events that cause postischemic neuronal death and develop clinically effective therapies to reduce brain damage after cardiac arrest and stroke. This proposal focuses on the causal role of calpain-mediated proteolysis. Calpains are a family of Ca2+-dependent cytosolic proteases. Brain calpain activity is increased by focal and global ischemia, and calpain inhibitors are neuroprotective. However, the mechanism by which calpains contribute to post-ischemic neuronal death has not been determined. Several Ca2+ regulatory proteins are known to be calpain substrates. These include plasma membrane Ca2+-ATPase, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, the ryanodine receptor Ca2+ channel, and the IP3 receptor Ca2+ channel. The hypothesis is that calpain-mediated proteolysis of Ca2+ regulatory proteins disrupts Ca2+ homeostasis in post-ischemic neurons. The result is a sustained elevation in cytosolic Ca2+ and persistent calpain activation in a positive feedback pathway that is potentially irreversible and ultimately leads to delayed neuronal death. This hypothesis will be tested using an established in vivo model of transient forebrain ischemia in rats. Specific Aim 1 will determine if post-ischemic calpain inhibition prevents delayed death of hippocampal CA1 pyramidal neurons. Specific Aim 2 will characterize calpain-mediated cleavage of Ca2+ regulatory proteins in the postischemic hippocampus by Western blot. Specific Aim 3 will localize calpain-cleaved Ca2+ regulatory proteins by immunohistochemistry using cleavage site-specific antibodies. Specific Aim 4 will analyze the functional consequences of Ca2+ regulatory protein cleavage in microsomes and synaptosomes after 1) calpain-mediated proteolysis in vitro or 2) transient ischemia in vivo. In addition, plasma membrane Ca2+-ATPase dysfunction in the post-ischemic hippocampus will be localized by in situ histochemistry. These studies will provide important insights into the causal mechanisms of 1) calpain-mediated neuronal injury, 2) disrupted neuronal Ca2+ homeostasis, and 3) delayed neuronal death. Elucidating these mechanisms is essential for the development of effective therapies for patients suffering from cardiac arrest and stroke.

DESCRIPTION (provided by applicant): Brain ischemia caused by cardiac arrest and stroke is a significant source of human morbidity and mortality. This proposal focuses on the role of calpains, a family of Ca2+-dependent cytosolic proteases, in delayed necrosis of post-ischemic neurons. Brain calpain activity is pathologically increased after brain ischemia, and calpain inhibitors are neuroprotective in preclinical models. However, the mechanism of calpain-mediated injury is unknown, and the relative roles of the two ubiquitous isoforms, u-calpain and m-calpain, have not been elucidated. Pathologic calpain activity requires sustained cytosolic Ca2+ elevation. Calpain-mediated cleavage of IP3 receptors (IP3R) and ryanodine receptors (RYR) generates stable dysregulated channels that have increased Ca2+ conductance. These observations support the hypothesis that calpains are not only activated by elevated cytosolic Ca2+, but under pathologic conditions contribute to sustained cytosolic Ca2+ overload in a potentially irreversible feed-forward pathway that ultimately causes neuronal necrosis. Specific Aim 1 will use in vivo adeno-associated viral (AAV) vector-mediated overexpression of the specific endogenous calpain inhibitor, calpastatin, to elucidate the causal relationship between post-ischemic calpain activity, cytosolic Ca2+ overload, electrophysiological dysfunction, and delayed necrosis of hippocampal CA1 pyramidal neurons in a rat model of transient forebrain ischemia. In Specific Aim 2, the relative role of u-calpain and m-calpain will be examined using AAV vector-mediated RNA interference in the same model. Specific Aim 3 will examine the role of calpain-cleaved IP3R1. Generation of calpain-cleaved IP3R1 in post ischemic neurons will be immunohistochemically characterized. A truncated IP3R1 mutant corresponding to the stable calpain-derived fragment will be expressed in Xenopus oocytes for nuclear patch clamp analysis of channel function and expressed in CA1 pyramidal neurons in vivo to determine if it causes calpain activity and delayed necrosis. Specific Aim 4 will utilize a similar approach to evaluate the role of calpain-cleaved RYR2. The proposed studies overcome significant obstacles limiting the mechanistic evaluation of calpain's role in in vivo ischemic brain injury. The results will provide fundamental insights into the mechanism of delayed post-ischemic neuronal necrosis, and facilitate the development of effective therapies for patients suffering from cardiac arrest and stroke.

[unreadable] DESCRIPTION (provided by applicant): Approximately 150,000 Americans are treated for out-of-hospital cardiac arrest every year, but less than 5% survive with good neurological outcome. Induced hypothermia is the only therapy clinically proven to increase survival and improve neurological outcome of patients resuscitated from cardiac arrest. Although the optimal temperature range (32 to 34 [unreadable]C) is well established, critical gaps in our fundamental knowledge regarding the optimal time of onset, duration, and rate of rewarming have left clinicians guessing how to best apply this clinically effective intervention. Unfortunately, comprehensive optimization in clinical trials is profoundly limited by feasibility, time, and cost. To address these issues, we propose using a rat model of cardiac arrest to systematically evaluate two critical variables in the application of therapeutic hypothermia: optimal time of onset, and optimal duration of therapy. We hypothesize that therapeutic hypothermia after cardiac arrest is 1) equally effective when initiated between 1 and 8 hours after cardiac arrest, and 2) most effective when maintained for at least 48 hours. Our proposed Specific Aim will test these hypotheses using a 3 x 3 factorial study design that will compare therapeutic hypothermia (33.0 +/-1.0 [unreadable]C) initiated 1, 4, or 8 hours after resuscitation from cardiac arrest, maintained for 24, 48 or 72 hours, and rewarmed at a fixed rate of 0.25 [unreadable]C per hour. This study design will allow us to identify the optimal onset and duration of therapeutic hypothermia as well as potential interactions between the two parameters. Primary outcome parameters will include survival and Morris water maze behavioral function, and secondary outcome will be survival with good neurological function. Our structural surrogate outcome will be total brain atrophy based on morphometric quantification of total brain volume. A subset of rats will be instrumented for serial blood sampling to be used for future biomarker analysis. This study will, for the first time, systematically optimize the only proven therapy for patients resuscitated from cardiac arrest. The results will provide the fundamental knowledge that is urgently needed to design feasible, focused, and definitive clinical trials. In addition, this translational paradigm will be available to efficiently evaluate and optimize therapies that might be compared to or used in combination with therapeutic hypothermia. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Ischemic brain injury due to cardiac arrest or stroke represents a major cause of mortality and disability. Although the precise mechanisms of delayed post-ischemic neurodegeneration remain incompletely understood, disruption of Ca2+ homeostasis appears to play a major role. One potential cause of disrupted calcium homeostasis in post-ischemic neurons is proteolytic modification of Ca2+ regulatory proteins. This proposal focuses on caspase 3- and calpain-mediated cleavage of the inositol (1,4,5)-trisphosphate receptor (IP3R), a Ca2+ release channel located on the endoplasmic reticulum (ER). Both published evidence and our own preliminary data suggest that caspase 3- and calpain-mediated cleavage of the type 1 IP3R (IP3R1) generates a constitutively open channel that allows Ca2+ to leak from the ER and impairs the ER capacity to buffer cytosolic calcium overload. The aims of this proposal will test the hypothesis that caspase 3- or calpain- mediated cleavage of IP3R1 generates a constitutively open channel that irreversibly disrupts intracellular Ca2+ homeostasis and contributes to neurodegeneration after excitotoxic and ischemic injury. Specific Aim 1 will measure the channel properties of caspase 3- and calpain-cleaved of IP3R1 and their effect on intracellular calcium homeostasis. Specific Aim 2 will investigate the effect of caspase 3- and calpain-cleaved IP3R1 in primary neuron culture under baseline conditions and after excitotoxic injury. Specific Aim 3 will investigate the effect of caspase 3- and calpain-cleaved IP3R1 on neurons in vivo under baseline and post-ischemic conditions. Overall, the results of these experiments with provide critical insight into the mechanism by which pathologic proteases cause acute neurodegeneration through disruption of intracellular calcium homeostasis. In addition, blocking the caspase- and calpain-cleaved forms of IP3R1 could be a novel therapeutic target for neuroprotection after ischemic brain injury. PUBLIC HEALTH RELEVANCE: A growing body of evidence suggests that sustained disruption of neuronal calcium homeostasis plays a causal role in neuronal death after brain ischemia. This proposal tests the hypothesis that pathologic proteases, caspase-3 and calpains, disrupt neuronal calcium homeostasis through cleavage of the inositol (1,4,5)-trisphosphate receptor, a Ca2+ channel located on the endoplasmic reticulum. The results of these experiments with provide fundamental insight into the mechanism of post-ischemic neurodegeneration and potentially identify a novel therapeutic target for neuroprotection after ischemic brain injury.

SUMMARY Over 200,000 people are treated for out-of-hospital cardiac arrest (OHCA) each year in the United States, and less than 1 in 10 survive with favorable neurologic function. Extracorporeal cardiopulmonary resuscitation (ECPR) using percutaneous veno-arterial extracorporeal circulation (VA-ECMO) is emerging as a potentially effective resuscitation strategy for OHCA patients who fail standard therapy. In published recent case series, survival rates range from 4-33%. Although promising, the efficacy of ECPR for OHCA has yet to be determined in a prospective randomized clinical trial. The overall goal of our research team is to design and perform the first prospective randomized clinical trial to evaluate the efficacy of ECPR for refractory OHCA. The goal of this NHLBI Clinical Trial Pilot Studies application is to generate the preliminary feasibility and efficacy data necessary and sufficient to design a full-scale clinical trial that will provide definitive guidance on the value of ECPR for refractory OHCA. Our first Aim is to demonstrate the efficacy of a training program designed to assure that emergency department providers can rapidly and safely initiate ECPR. Our second Aim is to evaluate the feasibility and performance of an EMS resuscitation strategy incorporating rapid identification and transport of patients with refractory OHCA to an ECPR-capable emergency department. Our third Aim is to evaluate the feasibility and performance of an ED protocol for initiating ECPR in patients with refractory OHCA. Our final Aim is to design and optimize an adaptive clinical trial that will maximize the performance of the planned Phase III multicenter study. The results of these aims will be used to prepare and submit a R01 application for a prospective randomized clinical trial to evaluate the efficacy of ECPR for Refractory Out-of- hospital Cardiac Arrest (EROCA Trial). The potential impact of such a trial is significant. Widespread implementation of ECPR for refractory OHCA could double overall survival rates, saving >10,000 lives each year in the United States. However, emergency medical systems, emergency departments and hospitals need evidence-based guidance regarding the value of implementing this promising but resource intensive therapy. The goal of our team is to provide the essential evidence needed to enable value-based implementation.

K12 Career Development Program in Emergency Critical Care Research University of Michigan PD/PIs: Robert Neumar, M.D.-Ph.D.; David J. Pinsky, M.D. The goal of the proposed program is to prepare a diverse group of early career clinician-scientists for leadership roles and independent research careers in emergency critical care research through a multidisciplinary, mentored career development program focused on developing innovative approaches to severe, acute, life-threatening illness and injury in emergency settings. This career development program for advanced learning in emergency critical care research will be led by Robert W. Neumar, M.D.-Ph.D., Chair of Emergency Medicine, and David J. Pinsky, Director of the Samuel Frankel Cardiovascular Center (CVC). Scholars may choose mentoring teams led by experienced and highly published senior clinician-scientists in Emergency Medicine, Pulmonary and Critical Care, Neurology, General and Trauma Surgery, Cardiovascular Disease, Biomedical Engineering, and Biostatistics. Career development will emphasize all phases of emergency critical care research and practice in a collaborative culture emphasizing the close interdependence of excellence in direct patient care and clinical research. Commitment of faculty, across not only the breadth of the University of Michigan Health System (UMHS), but also multiple schools at UM, represents a singular strength of this program. Mentors with active NIH funding will guide Scholars in designing individual development plans and achieving milestones including academic courses, professional development training, expert consultations, and research to produce preliminary data for research project funding. Scholars will be expected to transition to individual K or R01 funding by the end of the third year of K12 support. Immediate/Long-term Objectives: 1. Recruit 4 junior faculty Scholars with diverse backgrounds and demonstrated commitment to emergency critical care clinical research and innovative approaches to screening, diagnosis, and clinical management of patients manifesting cardiovascular/neurovascular/ pulmonary/ disease, sepsis, or trauma. 2. Pair each Scholar with a multidisciplinary mentoring team committed to the Scholar's development and achievement of milestones set forth in a customized individual career development plan for three years. 3. Provide comprehensive on-the-job/on-campus clinical research career development program leading to an M.S. in Clinical Research Design and Statistical Analysis (CRDSA) offered for clinician-scientists. 4. Advance Scholars toward independent research careers through additional didactic training in clinical trial design and oversight, data safety and monitoring, data management and security, scientific writing, grant writing, technology development, scientific presentations, responsible conduct of research, and leadership. 5. Immerse each Scholar in the mentor's research program, a diverse campus network of collaborating critical care researchers, and stimulating scholarly discussion and ideation, and clinical patient care. 6. Monitor and improve program quality and effectiveness through a combination of metrics, active Scholar/mentor feedback, and post-program Scholar career tracking. This innovative program will be housed in the Michigan Center for Integrative Research in Critical Care (MCIRCC), a comprehensive research enterprise devoted to transforming critical care medicine by accelerating science from bench to bedside. MCIRCC, administratively anchored within the Department of Emergency Medicine, is a team science hub for innovative, interdisciplinary, collaborative, translational research on acute illnesses and injuries. Scholars will become affiliates of MCIRCC, the Cardiovascular Center Clinical Research (C3RG) Group, and other relevant multidisciplinary research centers at UM. These centers of excellence emphasize cutting-edge research, collaboration and networking, scholarly and research information exchange, clinical research training, and biomedical technology development that will transform not only emergency critical care research, but also future patient care. The proposed education program will provide: 1) a broad knowledge and training in modern clinical and translational science; 2) coursework to address gaps in scientific training; 3) didactic and experiential training in intellectual/philosophical approaches to modern investigation and technology development; 4) improved scientific and grant writing skills; 5) enhanced research mentoring and leadership skills. In addition to the mentored research and the M.S. in CRDSA, educational programming will leverage the well-established Michigan Center for Clinical Health Research (MICHR), an NIH CTSA offering very successful education, training, and research/academic mentoring programs to enhance and support dozens of training and career development programs for graduate students, postdoctoral fellows, and faculty throughout the Medical School.

Project Summary/Abstract Over 400,000 patients are treated for sudden cardiac arrest each year in the U.S., but fewer than 1 in 5 survive after in-hospital cardiac arrest (IHCA), and fewer than 1 in 10 survive after out-of-hospital cardiac arrest (OHCA). The overwhelming majority of deaths are caused by failure to achieve return of spontaneous circulation (ROSC) using standard cardiopulmonary resuscitation (CPR) and advanced cardiovascular life support (ACLS). Extracorporeal cardiopulmonary resuscitation (ECPR) using percutaneous veno-arterial extracorporeal membrane oxygenation (ECMO) is rapidly emerging as a feasible and effective resuscitation strategy for patients that fail standard resuscitation efforts. It is estimated that up to 10% of treated OHCA and IHCA patients are potential candidates for ECPR, and widespread implementation could save up to 10,000 lives each year in the U.S. Although return of a spontaneous heartbeat can be initially achieved in most patients with ECPR, less than one third survive with good neurologic outcomes. Therefore, more research is needed to maximize the potential of this life saving therapy. A fundamental barrier to the success of ECPR after prolonged cardiac arrest is the ?no- reflow phenomenon,? defined as inadequate vital organ reperfusion despite restoring normal cardiac output. This project's central hypothesis is that intravascular complications of total-body ischemia and reperfusion, including microvascular coagulation, leukocyte-adhesion, and neutrophil extracellular trap (NET) formation cause no- reflow and prevent recovery of heart and brain function when ECPR is used to treat prolonged cardiac arrest. Our proposed Specific Aims will test this hypothesis in a clinically relevant swine model of ECPR after prolonged cardiac arrest that is established in the UM investigator laboratories. Aim 1 will elucidate the impact of intravascular coagulation on recovery of heart and brain function after prolonged cardiac arrest treated with ECPR. Experiments will: 1) compare the effectiveness of indirect (heparin) and direct (argatroban) thrombin inhibition early during CPR and 2) evaluate the effectiveness of thrombolytic therapy (streptokinase) at initiation of ECPR. Aim 2 will examine the impact of leukocyte-mediated inflammation on recovery of heart and brain function after prolonged cardiac arrest treated with ECPR. Proposed experiments will compare the effectiveness of standard leukocyte filtration to our novel leukocyte modulation (L-MOD) device during ECPR. Aim 3 will determine the impact of therapies identified in Aim 1 and Aim 2 on 7-day survival, cardiovascular function, and neurologic function after prolonged cardiac arrest treated with ECPR. Overall, the results of these aims will advance the field by providing new fundamental knowledge about the mechanisms of no-reflow and proof-of- concept evidence that therapeutic strategies effectively targeting ?no-reflow? improve outcomes with ECPR after prolonged cardiac arrest. This study results will serve as the foundation for pre-clinical optimization studies and, ultimately, clinical trials that will bring new therapies to the field with the goal optimizing outcomes after prolonged cardiac arrest.