CIMED has awarded five Pilot and Feasibility grants for 2014

Joseph C. Corbo, MD, PhD
Associate Professor, Pathology and Immunology

“High-throughput functional analysis of non-coding regions related to arrhythmias”

Genome-wide association studies (GWAS) are identifying large numbers of genetic variants associated with cardiac arrhythmias and related ECG traits. The majority of these GWAS hits fall within non-coding regions. We hypothesize that many of these hits tag causal sequence variants that perturb the activity of cis-regulatory elements (CREs; i.e., enhancers/promoters) that regulate cardiac genes. Currently, no high-throughput technology exists for assaying the function of non-coding cis-regulatory regions and their variants in cardiac disease. Yet, the advent of whole-genome sequencing will soon require the development of tools for interrogating the functional significance of non-coding variants on a massive scale. To address this challenge, we propose to apply a novel technology called CRE-seq (Cis-Regulatory Element analysis by sequencing) to the analysis of human non-coding cis-regulatory regions relevant to cardiac arrhythmias. CRE-seq leverages next-generation sequencing of barcoded reporter libraries to quantify the activity of thousands of cis-regulatory elements in a single experiment. We plan to implement CRE-seq in the mouse heart to functionally evaluate cardiac cis-regulomes, focusing on CREs around 12 GWAS loci implicated in variation in the electrocardiographic QT interval. In the future, this approach will permit us to directly measure the effects of cis-regulatory variants in patient-derived, disease-relevant CREs on an unprecedented scale, thereby laying the groundwork for a personalized functional genomics of non-coding variation as it relates to cardiac disease.

************************************

Jianmin Cui, PhD
Professor, Biomedical Engineering

“ATP binding to the cytosolic domain of KCNQ1 channel proteins”

Genome-wide association studies (GWAS) are identifying large numbers of genetic variants associated with cardiac arrhythmias and related ECG traits.  The majority of these GWAS hits fall within non-coding regions. We hypothesize that many of these hits tag causal sequence variants that perturb the activity of cis-regulatory elements (CREs;  i.e., enhancers/promoters) that regulate cardiac genes.  Currently, no high-throughput technology exists for assaying the function of non-coding cis-regulatory regions and their variants in cardiac disease.  Yet, the advent of whole-genome sequencing will soon require the development of tools for interrogating the functional significance of non-coding variants on a massive scale.  To address this challenge, we propose to apply a novel technology called CRE-seq (Cis-Regulatory Element analysis by sequencing) to the analysis of human non-coding cis-regulatory regions relevant to cardiac arrhythmias.  CRE-seq leverages next-generation sequencing of barcoded reporter libraries to quantify the activity of thousands of cis-regulatory elements in a single experiment.  We plan to implement CRE-seq in the mouse heart to functionally evaluate cardiac cis-regulomes, focusing on CREs around 12 GWAS loci implicated in variation in the electrocardiographic QT interval.  In the future, this approach will permit us to directly measure the effects of cis-regulatory variants in patient-derived, disease-relevant CREs on an unprecedented scale, thereby laying the groundwork for a personalized functional genomics of non-coding variation as it relates to cardiac disease.

************************************

Beth Kozel, MD, PhD
Assistant Professor, Pediatrics and Genetics

Dorothy K. Grange, MD
Professor, Pediatrics

“Cardiovascular characterization and drug screening in the K_ATP channel disorder, Cantu Syndrome”

Cantu syndrome is a rare multisystem disorder, caused by activating mutations in ABCC9 and KCNJ8, genes that encode the accessory and pore-forming subunits of the K_ATP channel respectively.  K_ATP channels are expressed in multiple human tissues and mutant forms have the potential to cause a range of disease phenotypes depending on the tissue distribution of the affected subunit.  Preliminary studies in humans with ABCC9 gene mutations and Cantu syndrome have identified cardiovascular abnormalities attributable to the channel defect, including conduction abnormalities, lower than average blood pressure, decreased vascular stiffness and abnormally dilated and thickened hearts.  To continue these studies and to move toward external funding, the immediate goals of this multi-investigator collaborative venture are to further develop these preliminary advances with more detailed clinical characterization of the cardiovascular abnormalities in individuals with Cantu syndrome and to develop cellular assays that will allow us to ask mechanistic questions about the function of K_ATP channels in affected individuals.

************************************

Stacey Rentschler, MD, PhD
Assistant Professor, Medicine and Developmental Biology

“Regulation of Cellular Electrophysiological Phenotype by Notch and Wnt Signaling”

Wolff-Parkinson-White (WPW) syndrome affects 3 in 1000 individuals and is characterized by ventricular preexcitation, palpitations, and rarely sudden cardiac death.  We previously described a mouse model that recapitulates many aspects of WPW syndrome in which Notch signaling is activated within a subset of the myocardium during development.  In addition to morphologic abnormalities, Notch activation in the myocardium leads to cell autonomous electrophysiological changes consistent with reprogramming of ventricular cardiomyocytes to specialized Purkinje-like cells.  We are seeking to delineate the molecular pathways downstream of Notch that mediate these distinct phenotypic effects in an attempt to understand the developmental basis for WPW, which may also shed light on arrhythmia syndromes more broadly.  Interestingly, Notch pathway mutations have recently been associated with Brugada syndrome, where electrophysiological changes occur without significant structural abnormalities.  Surprisingly, in our WPW model, in contrast to the Notch-mediated morphologic abnormalities, Notch-mediated electrophysiological changes were not rescued by β-catenin protein stabilization.  In fact, the combined increase in Notch and Wnt signaling has a more dramatic effect on cellular electrophysiology, resulting in a more robust Purkinje-like phenotype.  Aim 1 tests the hypothesis that Notch and/or Wnt globally remodel the electrical phenotype of cardiomyocytes including K+, Ca2+ and Na+ currents, and that the effect is different in the right versus left ventricles.  These intrinsic differences may have profound implications for human diseases including arrhythmogenic right ventricular cardiomyopathy, which preferentially affects the right ventricle and may be associated with altered Wnt activity.  Aim 2 tests the hypothesis that genes encoding voltage-gated K+ channel subunits are direct transcriptional targets of Notch and/or Wnt using chromatin immunoprecipitation (ChIP) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq).  Completion of these aims will provide the preliminary data to directly test the mechanism for how transcription factor signaling networks regulate the cellular electrophysiological phenotype in future studies.

************************************

Jonathan R. Silva, PhD
Assistant Professor, Biomedical Engineering

“Fluorescent Unnatural Amino Acid Tracking of Membrane Protein Conformation”

The primary aims of this proposal are 1) to synthesize an environmentally sensitive un-natural amino acid (UAA) 2) to incorporate the UAA into an ion channel, and 3) observe changes in conformation, including intracellular changes, in a population of ion channels that are expressed in mammalian cells.

In the decade following the combination of spectroscopy and voltage clamp, known as voltage clamp fluorometery (VCF), it has been widely applied to study the dynamics of extracellular changes in conformation that are caused by the movement of positive charges within the voltage sensing domain of voltage-gated ion channels.  However, there are two major drawbacks to the technique in its current form.  First, the method uses cystein labeling, which is non-specific labeling and causes a large increase in the background signal.  Further, only extracellular positions can be labeled due to the prevalence of intracellular cysteine residues.  The second drawback is that the technique is currently limited to Xenopus oocytes, whose cytoskeletal interaction with many channels severely alters their phenotype.  The use of un-natural amino acids in mammalian HEK cells overcomes both of these limitations by allowing residues to be introduced at extracellular, intracellular and membrane-spanning locations.

Two very recent advances lead us to believe that mammalian VCF is currently realizable. In May of this year, Kalstrup and Blunck demonstrated the ability to track intracellular Shaker K+ channel conformations using the UAA 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid, Anap. Then, in August, the Schultz group published a vector that can be used to introduce Anap into mammalian cell proteins.  We have obtained this vector, pAnap, from the Schultz group and have built a patch-clamp fluorometry rig that is suitable for making these measurements.  The final barrier is availability of Anap, of which large quantities are needed, is not sold commercially, and is difficult to synthesize.  This proposal will support the our effort to create an abundant Anap supply and adapt our patch-fluorometry rig for use with UAAs.