Narine Sarvazyan - US grants

Doxorubicin and its derivatives are among the most potent anticancer drugs known. Unfortunately their therapeutic efficacy is severely restricted by a dose-accumulated cardiotoxicity. Our preliminary results using adult rat cardiomyocytes as a working model, have shown evidence for the mechanism which could explain how reactive oxygen species generated by doxorubicin provoke myofibrillar degeneration while not causing severe oxidative damage. Specifically, 1) we visualized directly doxorubicin accumulation in cardiac mitochondria and subsequent increase in intracellular oxidation in living cardiomyocytes, 2) observed that doxorubicin administration is associated with redistribution of the protein kinase C epsilon isoform from cytosol to myofibrils, and 3) detected disruption of the periodicity of actin staining after repetitive exposure of myocytes to clinically relevant drug concentrations. Based on these data we propose the following sequence of events: doxorubicin rapidly accumulates in cardiac mitochondria; it then initiates lipid peroxidation via formation of superoxide and drug complexes with transition metals; and although the degree of lipid peroxidation is small and no significant membrane damage occurs, it leads to the activation of phospholipases; lipase thereupon release several second messengers, including arachidonic acid, activating a specific protein kinase C isoform; the kinase then initiates myofibrillar degeneration. To support the above hypothesis we aim to 1) establish a causal relationship between observed doxorubicin-induced kinase translocation and increased free radical formation; 2) to determine whether doxorubicin-induced free radicals and/or protein kinase C translocation are prerequisites for changes in myocyte myofibrillar organization and cell contractility; 3) to uncover role of phospholipase A2 in doxorubicin-induced activation of kinase and ensuing changes in myofibrillar organization and contractility. The proposed experiments aim to reveal explicit pathways through which reactive oxygen species are involved in anthracycline- induced cardiomyopathy. The studies will also provide new information about kinases involvement in myofilament degeneration and interaction between reactive oxygen species and signal transduction pathways.

[unreadable] DESCRIPTION (provided by applicant): Cardiac arrhythmias arise from abnormalities of either impulse propagation (reentry-based) or impulse initiation (focal or ectopic). The development of reentry arrhythmias, which involves rotation of an excitation wave around an anatomical or functional block, was observed both in vitro and in vivo and conceptually is well understood. In contrast, our comprehension of ectopic (non-reentrant) arrhythmias has a major gap. To gain initial insights into this process we propose to use a range of available models of cardiac tissue (both experimental and theoretical) in which infarct-like area will be created. Our preliminary studies have revealed that development of ectopic arrhythmias proceeds via an essential step, which we named an ectopic nexus (EN) It refers to a functional state of an injured cardiac tissue in which multiple poorly-coupled ectopic sources form a transient "breeding" microenvironment in which ectopic activity develops from individual cells into slowly propagating ectopic waves confined to the area of injury. The waves of excitation from surrounding healthy tissue fail to invade the EN, allowing slow ectopic waves to co-exist side-by-side with normal propagation pattern. Subsequent relief of EN conditions results in an escape of the ectopic waves leading to an arrhythmia. The EN is a novel concept, which, if it does occur in vivo, has important implications for both understanding and clinical treatment of arrhythmias and ventricular fibrillation. However, experimental and theoretical models employed in our previous studies had several limitations and the relevance of the EN concept to in vivo arrhythmias needs to be further established. Specifically, one needs to know whether EN is limited to 2D cultures of cardiac cells or to a specific set of experimental conditions, how electrical activity match data obtained using calcium transients, whether the EN occurs in a 3D environment, and many other questions. The goal of this application is to provide answers to these questions in order to establish a pathophysiological significance of EN. [unreadable] [unreadable]

1231549/ Sarvazyan

INTELLECTUAL MERIT. The proposed studies are at the intersection of different fields, including tissue bioengineering, cardiology, immunology, and tissue transplantation. The researchers propose to use an innovative approach to reduce rejection of embryonic stem cell (ESC)-derivatives by non-syngeneic hosts. The studies will use the latest stem cell differentiation techniques, molecular biology methods, and decellularized scaffold engineering. Projects will be executed by an interdisciplinary team of researchers with published expertise in the aforementioned fields. The creative and novel strategy of diminishing the expression of Major Histocompatibilty Complex Class I molecules and up-regulating Fas ligand expression is a practical and well-conceived plan that will help advance knowledge in developing universally immunocompatible tissues. The facilities and all required resources are fully available to the PI and her research team at the George Washington University to succeed in these studies.

BROADER IMPACT. The researchers aim to develop new methods by which graft rejection can be suppressed. Their studies will contribute to the general understanding of how immunogenic molecules on the surface of undifferentiated and differentiated ESC can influence tissue transplantation. As a result of these studies minimally immunogenic ESC will be developed. The use of such cells in tissue engineering can help to reduce the need for aggressive immunosuppressive treatments. In the long term, this strategy can help to decrease the rates of morbidity and mortality associated with many degenerative diseases.

This project assumes participation of junior faculty, undergraduate and graduate students, as well as high school students from the DC public school system. Throughout the years the PIs lab hosted and trained over 30 students. Their team was and continues to be very inclusive; they trained students of many faiths, different races and ethnical backgrounds, including many from underrepresented groups and minorities. Students involved in the proposed studies will be trained in the latest stem cell and molecular biology methods, as well as in scaffold-engineering protocols.

Regeneration of tissues is one of the hottest topics in modern science, which is of high interest to the general public. The researchers plan to promote knowledge about the current state of research in this rapidly developing field through professional seminars, publications, and public lectures.

DESCRIPTION: We propose a novel approach to treat insufficient venous flow. It relies on the use of minipumps made of engineered cardiac muscle. If successful, this approach has the potential to revolutionize the treatment of chronic venous disease and other causes of limited flow, including direct injury and paralysis of lower limb muscles. Specifically, our long term goal is use a patient's stem cells to create a rhythmically beating sheath of cardiac muscle cells that surrounds medium-sized veins. These 'Cardiomyocyte-based Venous Assist Devices or CMVAD will aid flow without requiring recreation of the heart's structural complexity. The main goal of this application is to obtain proof-of-the concept data for this novel approach using rat neonatal cardiomyocytes and human embryonic stem cell derived cardiomyocytes. Experiments will be structured along two specific aims. The first specific aim is to test the ability of CMVAD o create pressure within close-ended excised segments of canine, porcine or human saphenous veins and to examine how much the electric and mechanical stimulation of CMVAD improves its physical strength and force of contraction. The second specific aim is to a) compare different CMVAD designs for their ability to continuously propel fluid thru an excised vein segment with functional unidirectional valve and b) test feasibility of peristaltic fluid propulsion by electriclly stimulating a downstream end of an elongated CMVAD sleeve placed around vein segment without functional valve. To the best of our knowledge the proposed methodology would be one of the first examples of using tissue engineering protocols not just to repair damaged organs but to design entirely new ones - either outside the organ's original anatomical location or using the functionality of specialized cells from different tissues.

In spite of recent success in tissue engineering blood vessels and patches for repairing damaged regions of the heart, there has been little success in tissue engineering supportive pumping systems that can produce one-way flow, which often require valves that prevent flow reversal. Chronic venous insufficiency, which can affect 20-30% of the people over the age of 50, is an example of such an unmet need. In chronic venous insufficiency, blood pools in affected veins and doesn't return to the heart efficiently, which can lead to swelling, skin changes, varicose veins, ulcerations, and even loss of limbs. The goal of this project is to perform exploratory work to tissue engineer a biological valveless pump based on recently discovered novel physical principles governing the functioning of an embryonic heart, in which there is one-way flow without significant valve control. Pumps of different designs will be systematically tested for their ability to create one-way flow. Key parameters, including the pump's outer radius and length, the thickness and consistency of a gelatinous inner layer and the position and the contraction frequency of its active segment (a rhythmic cuff made of cardiac muscle cells), will be examined. If successfully developed, valveless pumps can be useful in multiple other projects. On a microscale, ongoing efforts to build organs-on-a-chip devices to study various diseases and their treatment using patient-derived cells can clearly benefit from a more physiologically relevant version of circulation that involves energy efficient valveless pumping. On a macroscale, implantable valveless pumps might be able to aid venous blood or lymphatic flow since they can be made suitable to different anatomical scales, including the peripheral circulation.

The goal of this project is to use tissue-engineered cardiac muscle cuffs to create effective directional pumping action in a valveless fashion. Theoretically, this can be achieved in the presence of a sectional difference in mechanical compliance along the cuff and by asymmetric stimulation with respect to such gradients. The theoretical basis and a man-made impedance pump of this kind was first proposed by Gerhart Liebau in the 1950s. Later it was suggested that this mechanism can play an important role in heart development and aortic flow. The main goal of this EAGER project is to recreate this fascinating mechanism of valveless pumping by combining novel tools of tissue engineering tools and 3D bioprinting and to test theoretical predictions that addition of a gelatinous inner layer to the classic Liebau impedance pump can significantly improve the ability of these pumps to generate unidirectional flow. The work will involve 3D printing of live tissue constructs containing defined amounts of cardiac myocytes and fibroblasts. It will also rely on a newly developed 3D bioprinting approach that uses agarose slurry as a support bath to sustain free-standing hydrogel structures in cell culture environments. Pacing of the contractile segment of the tissue engineered Liebau pumps will be achieved by external electrical field or by light-based stimulation of myocytes expressing channelorhodopsin. If successful, such biomimetic pumps can i) serve as energy- efficient flow generators in microdevices such as human-on-the-chip, ii) be used as experimental models for modeling function of embryonic heart during normal development or in diseased states, iii) be recreated from patient's own cell for the purpose of improving flow of biological fluids at specific locations.

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.