Benjamin L. Prosser, Ph.D. - US grants

DESCRIPTION (provided by applicant): Stretch-dependent X-ROS signaling: implications for cardiomyopathy PI: Benjamin L. Prosser A novel signaling pathway in heart cells, termed X-ROS signaling, links the lengthening of a heart cell during diastole to the generation of reactive oxygen species (ROS) and subcellular Ca2+ signaling (Prosser et al., Science 2011). Discovered and characterized by the PI, X-ROS signaling regulates the release of Ca2+ from sarcoplasmic reticulum stores in heart cells. While X-ROS signaling is a normal physiological mechanism, it is upregulated in disease processes (e.g. Duchenne muscular dystrophy) and can trigger Ca2+-dependent arrhythmias. Using innovative techniques that combine the control of cell length with patch-clamp electrophysiology, the PI will characterize unknown critical biophysical features of this physiological generation of ROS, and investigate the signaling cascade that results. This biophysical characterization in single heart cells will be performed under the guidance of Dr. W.J. Lederer (mentor, University of Maryland), a world leader in cardiac cellular electrophysiology and calcium imaging. Through the adoption of new techniques and training, the PI will next move X-ROS signaling from the cellular level to that of the whole heart. The PI will image ROS and calcium signaling in intact, Langendorff-perfused hearts where diastolic length is modulated by a change in pre-load pressure. These examinations of length- dependent signaling will be conducted in both healthy and diseased hearts, as our recent evidence suggests that hyper-active X-ROS may be linked to calcium-dependent arrhythmia in disease. These studies will be guided by Dr. David Kass (co-mentor, Johns Hopkins School of Medicine), a translational cardiologist who specializes in the role of ROS signaling in cardiomyopathy. As a clinician scientist, Dr. Kass will broaden the perspective and skill set of the PI, which will allo the PI to take a more integrative approach to his work. The PI has assembled an excellent research advisory committee who will provide the training and support to facilitate the proposed studies and the growth of the PI. The PI will remain based at Maryland, but will work under the guidance of both Drs. Lederer and Kass during the entire period of work. Additionally, the PI has access to state-of-the-art equipment, as well as excellent resources for career development at the University of Maryland and at Johns Hopkins. The proposed work and development plan will enable the PI to characterize the physiological and pathophysiological actions of X-ROS signaling from the subcellular to the whole heart level, and will thus enable him to carve his niche as an independent faculty member. The proposed program is part of the PI's long term goal to investigate molecular mechanisms of cardiac function, with a particular focus on ROS and Ca2+ signaling, and to apply this knowledge to clinically relevant conditions.

Project Summary: ?Detyrosinated microtubules in cardiomyocyte mechanics? The microtubule cytoskeleton performs a number of cellular functions including cargo transport and structural support. In certain forms of heart disease there is an extensive proliferation and post-translational modification of the microtubule network that correlates with declining contractility. It has previously been suggested that the increased density of microtubules may provide an intrinsic mechanical resistance to cardiac contraction, and therefore that targeting microtubules may restore contractility in heart disease. However, a detailed mechanistic understanding of how microtubules provide resistance is lacking, and this line of research has stalled. The PI and colleagues have made two important advances to move this field forward. First, we have developed imaging and labeling tools to observe and characterize microtubule behavior in beating heart cells. We observe that microtubules function like springs in the beating heart, a challenge to the conventional view. These spring like microtubules provide a mechanical resistance to heart cell contraction and stretch. Second, we have identified a novel element that appears to regulate the mechanical properties of the cytoskeleton. In new published (Kerr et al. Nature Communications, 2015) and preliminary evidence we show that detyrosination, a post-translational modification of tubulin, regulates the compression-resistance of the cytoskeleton and alters the spring-like behavior of microtubules. Importantly, reducing detyrosination decreases mechanical resistance and increases myocyte contractility, suggesting a potential therapeutic benefit in heart disease. In this proposal we will thus test the hypothesis that detyrosination increases cytoskeletal compression resistance, and that specifically reducing detyrosination can improve contractility in heart failure. We have 3 major goals of our proposal: 1) to determine if increasing detyrosination is sufficient to impair myocyte mechanics; 2) to determine the molecular mechanism by which detyrosination influences cytoskeletal mechanics; 3) to determine if increased detyrosination impairs myocyte function in human heart disease. Our team of cardiomyocyte physiologists, cytoskeletal biologists, and a cardiac physician scientist are ideally suited to achieving these goals. The successful completion of this work will reveal mechanistic insight into how detyrosination alters cytoskeletal mechanics, a finding with broad relevance to cell biology and with specific translational implications for human cardiac disease.

Heart failure is often marked by stiffening of cardiac tissue that impairs the heart?s ability to relax. The microtubule network ? a part of the cardiomyocyte cytoskeleton ? provides an internal stiffness that can impede cardiomyocyte contraction and relaxation. We have recently found that cardiomyocyte microtubule network stiffness is tightly regulated by post-translational detyrosination and that microtubules, detyrosination, and cytoskeletal cross-linkers are consistently elevated in heart failure, with concomitant increases in cardiomyocyte stiffness. Our findings that reducing detyrosination lessens microtubule network density and contractile defects in cardiomyocytes from patients with advanced heart failure supports detyrosination as a therapeutic target. At the same time, these findings raise important questions about the processes driving remodeling of the microtubule network in heart failure and the consequences of sustained increases in the microtubule network over time. Accordingly, the proposed research will test the hypothesis that remodeling of the cardiac microtubule network is a reversible adaptation to altered mechanical stress, which when sustained, contributes to pathological hypertrophy and contractile dysfunction. Studies under three aims will address the multiple components of this hypothesis. Aim 1 experiments will determine if mechanical stress is sufficient to drive cell-autonomous remodeling of the microtubule network using a mechanobiology toolkit to isolate the contribution of three key mechanical stressors (pre-load, after-load, and matrix stiffness) on microtubule network remodeling. Aim 2 experiments will extend our mechanical manipulations to the tissue and organ level to characterize microtubule network remodeling under relevant in vivo contexts. Aim 3 studies will employ in vitro and in vivo genetic manipulations to determine whether sustained increases in detyrosination contribute to pathologic cardiac hypertrophy in the presence and absence of chronic pressure overload. Our overall study design uses novel and complementary experimental approaches to both exploit strengths of model systems and mitigate their shortcomings. This includes primary cardiomyocytes from human myocardium to complement findings from animal models and engineered tissue constructs. This cross-species, multi-scale approach balances the dual goals of reductionist rigor and integrative relevance that furthers ultimate clinical translation. Together this work will provide insight into the causes of microtubule network changes in heart failure and help determine if preventing or reversing these changes is therapeutically beneficial.

Project Summary Neuro-developmental disorders (NDD) are often caused by genetic mutations that lead to haploinsufficiency, or a loss-of-gene function. Mutations that cause haploinsufficiency of neurodevelopmental genes affect over 200,000 births each year, and commonly result in congenital malformations, intellectual disability, epilepsy and motor and behavioral impairments. While symptomatic treatments exist, for most conditions there are no treatments that directly correct the reduced levels of the haploinsufficient gene. Recent research by our group and others has revealed that many NDD-causing genes are normally under active repression by microRNAs, small, non-coding RNAs that function in gene silencing. This raises the possibility of removing this microRNA ?brake? to increase gene expression and help restore normal function in NDD haploinsufficiency. We hypothesize that the expression of downregulated genes can be restored through steric-blocking antisense oligonucleotides (ASO) that specifically disrupt gene repression by microRNAs. Our preliminary data support this hypothesis and demonstrate that ASOs that prevent a particular microRNA from binding to STXBP1 (a common NDD-causing gene) robustly upregulate gene and protein expression. We propose a novel pipeline to systematically develop ASOs to treat genetic NDD. First, we will use bioinformatic and wet lab experimental approaches to identify and validate microRNAs that potently repress leading NDD-causing genes. Next, we will use a high-throughput microRNA reporter assays to screen ASOs that can relieve this microRNA-mediated gene repression. Third, we will take lead ASOs identified from the high-throughput screen and test them on neurons derived from patients with genetically-defined NDD to determine if they can rescue gene expression and function. Together, this work will establish pre-clinical efficacy for an ASO-based therapy targeting microRNAs to treat neurodevelopmental disorders arising from haploinsufficiency, and lay the groundwork for first-in-human applications.