Hung Cao, Ph.D. - US grants

PI: Cao, Hung
Proposal #: 1652818

Heart diseases are the leading cause of death in the developed world due to failure to adequately replace lost ventricular myocardium (heart wall) damaged by loss of blood flow due to blood clots or other forms of obstruction. This failure is attributed to the limited ability of adult mammalian ventricular cardiomyocytes (CMs = heart muscle cells) to divide and regenerate the lost myocardium. In contrast, zebrafish hearts fully regenerate after 20% ventricular removal and thus provide a genetically tractable model system for heart regeneration investigations. While the zebrafish heart has been extensively assessed using immunohistochemistry and DNA and protein analyses to study the roles of different signaling pathways in cardiac development and regeneration, which is thought to have relevance for developing mammalian regeneration therapies, current approaches cannot elucidate the progress of the process (i.e., regeneration) of the same samples over time. The objective of this project, which builds on the PI's demonstrated ability to obtain quality (favorable high signal to noise ratio) electrocardiogram (ECG) signals in sedated animals, is to provide revolutionary polymer-based wireless devices for long-term acquisition of intrinsic ECG in freely swimming zebrafish models of heart regeneration. Broader society interests include the promise 1) to enable novel cardiac therapy; 2) to reduce cost and time of drug screening; and 3) to pave the way for numerous comfortable patch-based healthcare wearables for both patients and healthy populations. Integration of research with education activities are designed to help close the gap of engineering education and biomedical research, including efforts to 1) inspire and train students from different disciplinary and levels to follow higher education and a bioengineering career; 2) to nurture K-12 students directly and indirectly through their teachers by broadening knowledge in state-of-the-art hi-technology applied in bioengineering and medical research; 3) to translate cutting-edge technologies used in rigorous research to real world devices directly supporting society; 4) to engage multidisciplinary personnel from engineering, science and medicine to tackle unmet bioengineering challenge; and 5) to establish international collaborations to exchange innovations as well as to address global issues such as water quality.

Unlike mammalian hearts, which have limited ability to regenerate myocardium after ischemia induced infarct due to the limited capacity of adult cardiomyocytes (CMs) to divide and proliferate, zebrafish (Danio rerio) hearts fully regenerate after 20% ventricular resection. The central theme of this project lies in the applications of flexible and stretchable microelectronics and wireless power transfer to carry out heart regeneration studies in freely swimming zebrafish models. The project involves three milestones: 1) Development of flexible and stretchable micro-electrode array (MEA) membranes for long-term recording of electrocardiogram (ECG) in zebrafish models of heart regeneration: Four working electrodes approximately 200 um in diameter will be placed near the heart and a reference electrode will reside on the body. 2) Design and implementation of a wireless system (an "ECG Jacket") including wireless powering (inductive coupling or ultrasound) and data communication for remote and continuous electrocardiac monitoring of freely swimming fish (multiple fish monitored simultaneously) in a circular tank under biological investigations. 3) Deploying the system to elucidate the roles of specific genes towards myocardium regeneration in zebrafish (multiple mutant-like models with injuries induced by either amputation or a cryogenic approach) with potential translations to humans and to demonstrate the translational potentials for i) drug screening (zebrafish will be treated with well-known drugs that affect cardiac activity, e.g. amiodarone and verapamiland) and ii) physiological monitoring in humans with comfortable (flexible and stretchable) and unobtrusive patch-based devices (e.g., ECG, EEG, EOG, EMG, and blood pressure) in the home setting. The technologies developed will enable long-term (up to 2 months) ECG recordings of specific myocardial sites and thus uniquely determine the overall functionality of the area under investigation without effects of sedation. The conventional view of mammalian hearts as having virtually no regenerative capacity is now questioned by recent animal and human studies, in which new CMs may arise from existing CMs and progenitor or stem cells. The discovery of specific genes' roles towards heart regeneration in zebrafish studies would suggest methods to activate limited regenerative capacity in the human heart, garnering optimism about potential cardiac therapies.

Abstract
Non-technical:
This Major Research Instrumentation grant co funded by ECCS and CMMI of the Engineering directorate awarded to the University of Washington at Bothell provides funding for the acquisition of a variable-pressure scanning electron microscope with an Energy Dispersive X-Ray analysis. This high resolution imaging microscope fosters innovative research and facilitates multidisciplinary research collaborations at UW Bothell, a primarily undergraduate institution, with a large pool of talented students who have been traditionally underrepresented in the fields of Science, Technology, Engineering, and Mathematics (STEM). While the primary use of the SEM and EDX will be in support of research, it will also be integrated into research training and course curricula. The SEM will provide undergraduate students and graduate students with valuable hands-on experience in operating state-of-the-art instrumentation, an essential skill for students interested in future graduate studies and careers in STEM fields. Moreover, the instrument's impact on students with diverse background will be substantial, as forty nine percent of incoming first year students at UW Bothell are the first in their families to earn a four-year degree and seventy percent of first year students are from diverse backgrounds.

Technical:
The enhanced nanometer scale imaging resolution and elemental analysis capabilities will significantly enhance a large number of ongoing projects in science and engineering, including: (1) Development of highly efficient light trapping structures for organic solar cells; (2) Investigation of neural probes for neurotransmitter sensing; (3) Examining the impact of mutualistic coevolution on microbial phenotypes; (4) Characterization of novel bearing surfaces for total joint replacements as well as for the microstructure of electrically conducting polymer hydrogels; and, (5) Investigation of failure in polyurethane transformer structures. In addition, the instrument provides other capabilities such as energy dispersive X-ray spectroscopy microanalysis system, which can be used to determine the elemental composition of a specimen.

Abstract Sensoriis, Inc is a company that, develops evidence-based sensing solutions to support biological investigations and address health care problems. The goal of this NIH STTR grant with University of Washington (UW) is to provide a flexible system to assess cardiac electrical activities in zebrafish models, supporting heart disease studies and drug screening. Unlike humans, zebrafish hearts can fully regenerate following cardiac injury, thereby providing a tractable model system to study endogenous heart regeneration. Zebrafish have also proven to be an ideal vertebrate model system for phenotype-based screening owing to their physiological similarity to mammals. Further, zebrafish model enables a forward genetic approach to reveal the genetic basis and underlying molecular mechanisms of numerous heart diseases. The conventional setup for cardiac phenotype acquisition in zebrafish (i.e. electrocardiogram ? ECG) involves sedation causing variation in functionality. To date, there is no system which can offer cardiac phenotype monitoring in freely-swimming zebrafish, not to mention for multiple fish simultaneously. In this context, we propose and develop 1) a wireless flexible ?jacket? to be worn by zebrafish for real-time assessment of electrical cardiac phenotypes, namely ECG; and 2) a simple-yet-novel apparatus to collect ECG of multiple awake fish. Our devices provide pivotal platforms for cardiac phenotype-related investigations. The obtained data will be processed by smart algorithms to detect aberrant ECG patterns in real time. The proposed systems will facilitate related studies using zebrafish models. Further, the success of this platform also paves the avenue for regenerative medicine and developmental biology studies as well as stem cell-based therapies for cardiac repair. In Phase I of this STTR grant, we will develop i) a polymer-based microelectrode array (MEA) jacket that could be comfortably worn by the zebrafish and provide wireless ECG acquisition; and ii) a 4-chamber apparatus for simultaneous recording of ECG in awake fish. Machine learning-based programs with embedded algorithms will be developed to distinguish ECG patterns such as heart rate, ST and QT intervals, thus can identify anomalies, such as arrhythmias or prolonged QTs. For proof of concept, the system will be validated and compared.

Early life sleep disruption (ELSD) has been shown to affect the development of complex social behaviors in animals, impairing social bonding between prairie voles in a manner reminiscent of autism in humans. In order to understand the underlying changes in brain development, there is a dire need to develop innovative tools that can measure neurotransmitter levels in real-time during complex social interactions among two or more animals. In this project, the research team will design, implement and test novel integrated biosensor microprobes that will simultaneously record two key brain neurotransmitters (L-glutamate, an excitatory neurotransmitter, and Gamma Aminobutyric Acid [GABA], an inhibitory neurotransmitter) in real time during complex social interactions using this vole model of autism. The biosensor system will be made wireless and will also be combined with electroencephalography (EEG), as a measure of brain electrical activity. This highly collaborative project advances neuroengineering by developing a much-needed method to measure Excitation:Inhibition (E:I) balance in the brain in real-time, which will inform fundamental questions about the brain control of social interactions in healthy and autistic individuals.

The specific tasks of this study are as follows: Task 1) Develop and test wired, enzyme-based flexible integrated dual-sensor probes to assess L-glutamate and GABA levels in prairie voles in order to examine E:I balance in the brain during complex social interactions. Task 2) Develop and test a wireless system with an integrated L-glutamate and GABA flexible dual-sensor probe to study changes in the E:I balance the brain during complex social interactions. Task 3) Develop and test a wireless dual sensor probe integrated with EEG as a way to investigate effects of ELSD on EEG gamma oscillations as a functional readout of E:I balance during complex social interactions. The microprobes and systems developed in this project can be generalized to other studies of neurological disorders using rodents or larger animal models. The outcomes would help reveal how neural processes may go awry in neurodevelopmental disorders, as well as enable the next generation of neural prostheses, therapeutics and brain machine interfaces.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Sensoriis, Inc is a company that, develops evidence-based sensing solutions to support biological investigations and address health care problems. The goal of this NIH SBIR Phase II grant with University of California Irvine is to provide novel systems to assess cardiac electrophysiology in zebrafish models, supporting heart disease studies and drug screening. Heart disease plagues the world as the leading cause of mortality. Cardiac arrhythmic diseases alone contributed about 350,000 deaths annually in the U.S. Although causative genes for some of them have been partially discovered, genetic basis for the majority remains poorly understood. The zebrafish (Dario rerio) model system is an important vertebrate experimental model owing to its small size, low-cost for maintenance, short generation time, amenable and conserved genetics, and optical transparency. Zebrafish have long been used as model system for understanding human cardiac development, disease, and regeneration. Further, zebrafish model enables a forward genetic approach to reveal the genetic basis and underlying molecular mechanisms of numerous heart diseases. Owing to the physiological similarities to humans?, zebrafish have also proven to be an ideal model system for drug screening. The conventional setup for cardiac phenotype acquisition in zebrafish (i.e. electrocardiogram ? ECG) involves anesthesia causing variation in functionality. To date, there is no system which can offer cardiac phenotype monitoring in freely-swimming zebrafish, not to mention for multiple fish simultaneously. Further, data processing and analysis have been done manually, making it impossible to conduct large-scale studies. In this context, we propose to establish a long-term roadmap using multidisciplinary approaches to enable i) novel devices and systems to provide reliable ECG data of multiple fish (both adult fish and larvae) over a long period of time; ii) cloud-based systems to effectively process and interpret as well as study large-scale data; and iii) a host of cardiac studies as well as drug investigations using the zebrafish models and our novel tools.