Heart failure is an epidemic syndrome affecting 5.7 million Americans in which the weakened heart is unable to supply sufficient blood flow to the body. Approximately 10% of these patients will fail to respond to medical therapy and progressively worsen to develop advanced heart failure, for which the only definitive therapy is cardiac transplantation. As the supply of suitable donor hearts is limited to ~2000 per year in the US, the current and future care of advanced heart failure patients requires therapeutic alternatives. For some patients, mechanical circulatory support in the form of an implantable pump device can provide short- or long-term support, and in 1-2% of recipients, the heart improves to the point where the pump can be removed, termed myocardial recovery. Currently, it is not possible to predict which heart failure patients will respond to medical therapy alone, which will benefit from device support, and which have no potential for recovery. For device recipients, quantifying myocardial recovery to inform if, and when, to explant the device requires invasive testing. Thus, our long range goals are to develop novel strategies to identify patients with potential for recovery and develop non-invasive methods to monitor disease progression and recovery more precisely. In the short-term, our focus is on the development of novel cell surface markers to address these goals. Our previous studies of human cells in culture have successfully applied a mass spectrometric approach to capture and identify extracellular domains of cell surface glycoproteins, termed Cell Surface Capture (CSC). The proposed work extends these efforts and focuses on the implementation of innovative approaches to improve the CSC to enable identification and quantitation of cell surface proteins from live patient cells, as for many human diseases and syndromes like advanced heart failure, it is not possible to model the disease phenotype in vitro, and access to primary cells is severely limited. The current CSC requires 100 million cells. The studies outlined in this application are designed to develop a microscale-CSC (CSC) to reduce the amount of starting material required by 10-fold. The specific goals of this application are to use new bioconjugation reagents and an innovative processing pipeline to develop CSC and to expand its scope to capture a broader class of cell surface proteins with greater sequence coverage for improved quantitation (Aim 1); apply CSC during in vitro cardiomyocyte differentiation from pluripotent stem cells and on cells isolated from human hearts (Aim 2); and apply CSC to primary heart cells from patients with varying degrees of myocardial recovery (Aim 3). The identification of new markers and generation of reagents for identifying human cardiomyocytes with distinct developmental phenotypes will enable future mechanistic studies to determine the stages and molecular characteristics of cells amenable to repair. Once validated on primary heart cells derived from clinical samples, the technological advances resulting from the proposed studies will be widely applicable to researchers across a wide range of fields, from stem cell biology to cancer and patient diagnostics.

Public Health Relevance

This proposal will develop a new strategy to selectively quantify cell surface proteins on live cells derived from patient samples. Once developed, this strategy will enable the analysis of human cell types and diseases that cannot be adequately modeled in cell culture systems, including complex disorders such as advanced heart failure. The proposed studies will identify new markers for assessing patients with advanced heart failure and for sorting stem cell-derived cardiomyocytes at specific stages of maturation, which is highly relevant to regenerative medicine, disease modeling, and drug development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL134010-02
Application #
9246573
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Lundberg, Martha
Project Start
2016-04-01
Project End
2021-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
2
Fiscal Year
2017
Total Cost
$385,000
Indirect Cost
$135,000
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Fujinaka, Chelsea M; Waas, Matthew; Gundry, Rebekah L (2018) Mass Spectrometry-Based Identification of Extracellular Domains of Cell Surface N-Glycoproteins: Defining the Accessible Surfaceome for Immunophenotyping Stem Cells and Their Derivatives. Methods Mol Biol 1722:57-78
Moehring, Francie; Waas, Matthew; Keppel, Theodore R et al. (2018) Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin. J Proteome Res 17:2635-2648
Haverland, Nicole A; Waas, Matthew; Ntai, Ioanna et al. (2017) Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes. Proteomics 17:
Mallanna, Sunil K; Waas, Matthew; Duncan, Stephen A et al. (2017) N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm. Proteomics 17:
Boheler, Kenneth R; Gundry, Rebekah L (2017) Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets. Stem Cells Transl Med 6:131-138
Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk et al. (2016) Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes. Stem Cell Reports 7:543-556
Boheler, Kenneth R; Gundry, Rebekah L (2016) Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets. Stem Cells Transl Med :