Regulation of mitochondria in myocardial ischemic preconditioning and ischemic/reperfusion injury.
This NIH funded Program Project focuses in on the mitochondria and its key role in cell survival and recovery of function following this type of insult. There is growing evidence linking triggers of cardiac preconditioning (PC) to changes in mitochondrial function, yet our knowledge is still incomplete as how the overall protein profile is altered by short versus long ischemic (I) episodes and how these changes are linked to either self-protective or detrimental effects.
With ischemic PC (IPC), a series of short sublethal ischemic events trigger a powerful cellular defense response that ultimately limits infarct size from a subsequent prolonged ischemia. Over the last several years, our lab undertook a broad-based proteomics analysis of hearts exposed to preconditioning stimuli and ischemia and showed that the effects converge on the mitochondrial proteome, manifested as changes in protein quantity, posttranslational modification (PTMs), and higher order supercomplex organization.
By carrying out a systematic in-depth proteomic investigation of the mitochondrial subproteome, including careful identification and quantification of specific post-translational modified amino acid residues of individual proteins and protein complexes, coupled with a plan to assess the functional effects of such changes, we will be able to understand how manipulating the mitochondrial proteome can result in either damage or cardioprotection, with the eventual goal of developing strategies to reduce the damage associated with acute myocardial infarction.
Proteomic alterations in Cardiac Dyssynchrony and Resynchronization
This NIH-funded Program Project has undertaken the first detailed assessment of how the failing heart is impacted by dyssynchronous contraction and the subsequent restoration of synchrony from bi-ventricular pacing.
Cardiac resynchronzation therapy (CRT) is to date the only treatment that improves systolic function of the heart and enhance survival. Our group (Drs. Kass and O'Rourke) uses a canine model of heart failure with dyssynchrony and then resynchronization. Our latest data shows that DHF and CRT affect the mitochondrial subproteome by specifically altering proteins in cellular redox control and oxidative phosphorylation (OxPhos) pathways, manifested as changes in both protein quantity and post-translational modifications (PTMs) within the mitochondria.
From a basic science perspective, the potential for intra-mitochondrial regulation by phosphorylation and other PTMs represents a paradigm shift in the field of bioenergetics, hence, we are only just beginning to catalog all of the changes and determine their functional effects. Our underlying hypothesis is that CRT converts the mitochondrial subproteome to a protected phenotype, reversing detrimental DHF?induced protein alterations that regulate key functions to improve ATP production and redox balance.
Post translational regulation of embryonic and cardiac stem cells
Transplanting stem cells into damaged myocardium is emerging as a novel means for acute repair and as a means to treat end-stage heart failure. The critical elements in therapeutic success will lie in being able to identify and manipulate proliferating stem cells to differentiate specifically into cardiac muscle upon demand. We have two funded projects that use synergistic and sophisticated proteomic technologies to address biological and clinical questions with respect to stem cells.
Two different funded grants will allow us to analyze several distinctive subproteomes and couple this finding to functional analysis in order to develop new tools and provide insight into the regulation of ESC. This work includes investigating the biologically important cell surface proteins, in particular the N-linked glycoproteome to create specific biomarker panel for the identification and monitoring of various stages of differentiation. Furthermore, we will ink cell surface receptor identification with identification of secreted regulatory factors (e.g. paracrine and autocrine factors) and how these alter stem cell and cardiac myocyte function and biology.
Biomarker development for clinical utility
Our lab had created a novel biomarker development pipeline that is based on de novo and targeted discovery coupled to validation for serum, plasma and urine. The underlying premise is that identification of specific protein isoforms and/or disease-associated PTMs will increase biomarker specificity. This stems from the fact, that most genes are the same throughout the body and thus, unique aspects of the cell specific proteome arise due to either difference in the protein quantity, cell specific isoforms or the specific regulation PTMs.
Our lab has worked to develop tools and robust processes for de novo discovery of disease-associated PTMs, and reliable method to quantify specific isoforms and PTMs. In the last few years, the CTSA/ICTR Biomarker Development Center was created and is housed within the Van Eyk laboratory with the mandate to investigators within JHU to develop and validate new biomarkers. Examples of biomarker projects are listed below.
Development of blood-based biomarkers for myocardial ischemia
In patients presenting to the emergency room with chest pain less than 15% will be ultimately be diagnosed as having ischemia or acute myocardial infarction (MI). The current serum based cardiac biomarkers available to clinicians can identify patients with even small amounts of myocardial necrosis based on detection of cardiac specific cardiac troponin I (cTnI) or cTnT. However, the diagnosis of cardiac ischemia in the absence of necrosis is difficult as there is no serum/plasma biomarker available for clinical use. Our group has undertaken in-depth proteomic analysis for discovery and validation of such a biomarker in a set of unique patient cohorts.
Development of urine and blood-based biomarkers for chronic kidney disease
This project is funded by NIDDK's RFA, "Chronic Kidney Disease Biomarker Discovery and Validation Consortium" we (Dr. Joe Coresh and colleagues) propose a comprehensive proteomic approach to discovery, verification and validation of biomarkers for kidney function and kidney damage.
Development of blood-based biomarker for brain ischemic infarction in children
In our pilot study driven by Drs. Allen Everett and James Casella in collaboration with the CTSA/ICTR Biomarker Development Center we have been using a non-biased proteomic analysis of plasma samples from the ongoing Pediatric Silent Infarct Transfusion (SIT) Trial from patients with or without sickle cell disease.
Novel Biomarkers of Aortic Aneurysms and Acute Aortic Dissections
One major step towards the understanding of aortic diseases was the discovery of the molecular mechanisms underlying Marfans syndrome, a common inherited connective tissue disorders in which patients are susceptible to life threatening aortic dissection. In collaboration with Dr. Hal Dietz, we have been funded through a Challenge grant to look for a biomarker that (1) would enable us to identify the patient at risk for a dissection among patients with known aortic aneurysms and (2) facilitate diagnosis of aortic dissection in the patient with acute chest pain.