Genetics and Molecular Determinants of Cardiovascular Disease

Diagram showing the determinants of coronary artery disease

Coronary artery disease (CAD) continues to have an enormous impact on society despite marked progress in prevention and management. Our research program is exploring molecular and genetic aspects of the development of atherosclerosis and one of its major consequences, congestive heart failure.

Focus 

Consequences of CAD include sudden death, angina or myocardial infarction (requiring angioplasty, coronary artery bypass graft (CABG) surgery, or both) and loss of myocardium leading to congestive heart failure (CHF). The ultimate goal of our researchers is the prevention of CAD and CAD-related CHF. Studies are being done in both animals and humans to gain insights into gene-environment interaction for cardiovascular disease, early stage pathophysiology of the abnormal artery and diseased heart, and new approaches for prevention, stabilization, and perhaps reversal of disease in arteries and the heart.

Our research program has three main objectives:

  • To identify cellular and molecular pathways that translate a Western diet into CAD progression and contribute to the CHF phenotype post MI.
  • To determine genetic variants contributing to hypertension in humans and rats, early-onset CAD in humans and development of CHF in humans post MI.
  • To generate and examine animal model systems to assess function of genes and their variants for hypertension, CAD and CHF post MI.
Projects 

Tissue RAAS and Cardiac Remodeling and Dysfunction Post MI

Principal Investigator: Dr. Frans Leenen

Specific blockade of the brain RAAS, and general (i.e., both central and peripheral) blockade by an ARB both prevent sympathetic hyperactivity in rats post MI. However, effects on cardiac remodeling and dysfunction differ: The LV dilation is more inhibited by central blockade than by general blockade, and specific central blockade prevents (partially) the decrease in LV systolic function post MI, but general blockade by ARB leads to no change or further decreases. We hypothesize that peripheral blockade adversely affects the heart through both functional and structural mechanisms.

Central AT1-receptor blockade vs. general AT1-receptor blockade will start 2-3 days post MI and continue for 2-3 weeks (early phase) or 2-3 months (late phase). Assessments at the end include: LV function by Millar catheter, components of the circulatory and cardiac RAAS, Cardiac sympathetic activity as NE turnover rates in the non-infarcted part of the LV and in the RV, and post mortem parameters of cardiac remodeling including fibrosis, cardiomyocyte size and apoptosis. These experiments will explore a provocative new concept “adverse cardiac effects of AT1-receptor blockade.”

Genetic Basis of Salt-sensitive Hypertension in Rats

Principal Investigator: Dr. Frans Leenen

In genetically salt-sensitive rat-strains, Dahl S and SHR, CSF [Na+] increases on high salt intake and sympatho-excitatory and hypertensive responses to CSF [Na+] ↑ are increased. Both appear to depend on enhanced Na+-transport across the blood- and CSF-brain-barrier, involving amiloride/benzamil-sensitive sodium channels, presumably epithelial sodium channels (ENaC), and Na+,K+-ATPase. We proposed as specific hypothesis that “gain-of-function” of benzamil blockable sodium channels, i.e. ENaC, in the brain of salt-sensitive rat-strains leads to an increase in CSF [Na+] on high salt intake, and to enhanced sympathoexcitation and BP increase for a given CSF [Na+]. Increased plasma membrane activity of ENaC in genetically salt-sensitive rats is most likely due to functional mutations in genes encoding for proteins involved in post-transcriptional regulation of plasma membrane ENaC activity. We recently demonstrated that “Liddle”-like mutations in the coding regions of one or more of the ENaC genes, or functional mutations in the promoter regions of one or more of the ENaC genes unlikely play a role (Shehata et al., 2007).

Two lines of research are being pursued: expression and genotyping of ENaC genes and of ENaC activity regulating genes, and in vivo and in vitro functional Aof ENaC and Rof CSF and Brain [Na+].

Genetic Analysis and Expression of SLMAP in Human Heart Disease

Principal Investigators: Frederique Tesson and Balwant Tuana

Genetic programming involved in normal cardiac growth and development may be re-introduced in adult myocardium post MI and HF. The molecular basis for this reactivation is of much interest as its understanding may lead to new therapeutic approaches. We have isolated two genes, one of which (SLMAP) encodes proteins involved in excitation-contraction (e-c) coupling while the other (E2F6) is involved in regulating the cell cycle. As both the e-c coupling and cell cycle are involved in remodeling and cardiac growth we have been investigating the role of these genes in normal and diseased myocardium.

This study examines any genetic linkage of SLMAP to dilated cardiomyopathy (DCM) or HF in humans. Mutations in SLMAP gene may lead to alterations in its structure and function which impact cardiac dysfunction in human heart disease. We have examined the human SLMAP gene sequence from 200 patients with DCM/HF with a view to determine whether variations in the genomic sequence of the Human SLMAP are involved in the development of DCM and HF. Using DHPLC, sequence analyses and qPCR we have defined several polymorphisms in the SLMAP gene and that its level of expression may be altered in HF. We propose to further analyze SLMAP using Sequencing, DHPLC and q PCR to determine any genetic variations and changes in expression of SLMAP in human HF.

SLMAP Alterations in Post MI and Animal

Principal Investigators: Balwant Tuana and Dr. Frans Leenen

These studies will determine the functional significance of SLMAP isoforms in the myocardium. Distinct isoforms of SLMAP may be expressed in developing myocardium and serve diverse roles in cardiac growth and function. We have been examining the expression of SLMAP in normal myocardium and post MI. A transgenic mouse model over expressing SLMAP in myocardium has been established. Functional studies using echocardiography and ECG analysis as well as studies in isolated cardiomyocytes will be conducted to assess the role of SLMAP. Genotyping/phenotyping of the Tg model with a view to define the role for SLMAP in normal development as well as cardiac remodeling Post-MI will be investigated.

Role of E2F Pathway in Normal and Post MI Myocardium

Principal Investigators: Balwant Tuana and Dr. Frans Leenen

These studies will define the role of E2F transcriptional control in cardiac growth and remodelling by regulating it through the repressor E2F6. We propose that E2F6 serves to regulate cardiac growth and remodeling by controling the E2F responsive gene program.

We have been examining the E2F pathway with a focus on E2F6 in animal models of HF. The E2F6 repressor serves to regulate the cell cycle and apoptosis by repressing genes that are controlled by the transcription factor E2F1. A mouse model with cardiac targeted expression of E2F6 has been generated and studies on the phenotype particularly with respect to cardiac growth/death and remodeling are underway. These studies will utilize in vivo functional techniques in Tg mice as well as analysis of isolated cardiomyocytes to determine changes in cardiac cell function, gene expression, remodeling, cell growth, death and differentiation.

The Role of Cathepsin G in the Development and Progression of Atherosclerosis

Principal Investigators: Dr. Frans H. H. Leenen

This study examines the role of Cathepsin G during the development and progression of atherosclerosis by mediating the local conversion of angiotensinogen and Ang I to Ang II at the site of lesion formation, resulting in an increase in lesion formation via enhanced Ang II-mediated foam cell development.

To date, we established the necessary parameters to measure by QRT-PCR cathepsin G, angiotensinogen, angiotensin receptor and β-actin in non-diseased and atherosclerotic moue tissue extracted by laser capture microdissection. We are currently in the process of crossing cathepsin G null mice with atherosclerosissusceptible apolipoprotein E null mice (apoe-/-) in order to perform in vivo atherosclerosis study that will allow us to determine the extent of both early and late stage lesion development between cathepsin G competent versus deficient apoe-/- mice. Within the next two years we also plan to use bone-marrow transplantation to examine macrophage-specific expression of cathepsin G during lesion progression in atherosclerosis-prone mice.

The Role of Innate Immunity in the Process of Cardiac Remodeling Following a Myocardial Infarct

Principal Investigators: Dr. Frans Leenen, Dr. John Veinot, and Alexandre Stewart

These studies will examine the role of various innate immune cells, namely Natural Killer (NK) cells and Natural Killer T (NKT) cells in the process of myocardial remodeling following occlusion of the left anterior descending coronary artery (LAD) in mice. Immediate participation of the innate immune system may lead to greater damage of the myocardium and a slowing of repair in the affected area.

Using two well-known mouse models that carry specific deficiency in either NK cells (Ly49a transgenic mice) or NKT cells (J alpha 18 null mice), we plan to induce a MI in these mice by ligation of the LAD. The process of myocardial damage and repair post MI will be followed in both immune competent or deficient mice by various histological tools, as well as LV function by echo and Millar catheter.

The Role of MLIP (Muscle Lamin Interacting Protein) Expression in Both Early and Late Stage Atherosclerotic Lesion Formation

Principal Investigators: Patrick Burgon

The MLIP protein has been detected in both early and late-stage lesions from apoe-/- mice. Expression of MLIP may affect lesion progression in a temporal manner, such that MLIP expression early in the disease process would be considered proatherogenic, whereas expression in more advanced lesions would be considered anti-atherogenic.

We plan to carry out detailed atherosclerosis studies in apoe-/- mice to examine the role of MLP during lesion progression. These studies will involve crossing apoe-/- mice with mice that are either deficient in MLIP (mlip-/- mice) or carry a MLIP transgene (mlip transgenic mice) leading to moderate over-expression of MLIP.

Genetic Determinants Contributing towards the Development of Heart Failure following MI or Dilated Cardiomyopathy

Principal Investigators: Frederique Tesson, Dr. Frans Leenen, Balwant Tuana, and Dr. John Veinot

The central hypothesis of this project is that the progression to HF in patients at risk is genetically determined. The objectives are to study the contribution of candidate genes related to two key pathways, the RAAS and the phosphorylation system of calcium handling proteins, in the development of HF, as well as the involvement of the SLMAP.

The activity of the RAAS is increased in patients with heart failure secondary to myocardial infarction (MI) or dilated cardiomyopathy (DCM). We are determining whether multiple common DNA polymorphisms in the RAAS related genes collectively contribute to the development of HF in patients at risk. Foremost among biochemical features that remain common to the failing myocardium regardless of its aetiology are alterations in cellular calcium handling. We are establishing whether a genetically determined imbalance in the expression of genes from the phosphorylation system of calcium handling proteins predicts progression to HF in patients at risk. We are also screening for genetic variation the coding sequence and the 5’-flanking region of the SLMAP gene of 200 patients with DCM/HF and comparing the mRNA levels, protein expression and location of SLMAP in biopsies from control and DCM/HF individuals.

Interactions of the Nucleoskeleton Proteins and Their Role in the Onset of Dilated Cardiomyopathy and its Progression to Heart Failure

Principal Investigators: Frederique Tesson, Balwant Tuana, Patrick Burgon, and Dr. John Veinot

We are studying the interactions of the nucleoskeleton proteins and their role in the onset of dilated cardiomyopathy and its progression to heart failure. Our hypotheses are that 1) The nuclear envelope integrity is impaired in some DCM patients because of genetic variations in lamin A/C gene or in a lamin partner gene located at the inner nuclear membrane which may represent a first deleterious event for the development of HF; and 2) the functional consequence of LNMA and interacting protein mutations is mediated, at least in part, by the disruption of SUMO1 functions.

Gene-environment Interactions in Complex Genetic Diseases

Principal Investigators: Frederique Tesson, Dr. Frans Leenen, and Alexandre Stewart

We are studying gene-environment interaction in complex genetic diseases that are strong predictors of cardiac disorders, focusing on three clinical diseases and their most significant environmental factors as well as primary interventions: type 2 diabetes and exercise, obesity and diet, and hypertension and salt. Our experimental approach is to go from candidate gene strategy to genome wide association (GWA) studies.

Establishment and Utilization of Adenoviral and Lentiviral Technology to Investigate the Molecular Role of Novel Cardiovascular Genes in Primary Cardiomyocyte Cultures and Transgenic Mouse Models

Principal Investigators: Patrick Burgon, Alexandre Stewart, and Balwant Tuana

The adult mammalian heart is incapable of regenerating injured muscle. During the onset of pathological hypertrophic or dilated cardiomyopathies a number of the fetal heart genes are reactivated, yet there is no evidence of cardiomyocyte re-entry into the cell cycle. Understanding how this transition is controlled may provide new insight that will result in the development of therapies that regenerate injured heart muscle. Initial studies have established the use of lentivirus and adenovirus as effective delivery systems for gene over-expression and gene knock-down (shRNAi technology) in cultured cells. Future studies will involve further validation and refinement of the techniques in in vitro systems, followed by the translation of the technology into in vivo models. Specifically we will directly address the role of novel genes such as MLIP, Vestigial-like transcription cofactors, and E2F6 in heart development/function, and extend the research programs of all group members in a synergistic manner.

Role of MLIP in Idiopathic Human Dilated Cardiomyopathy and Heart Failure

Principal Investigators: Patrick Burgon and Frederique Tesson

The goal of this project is to identify and study the potential genetic linkage of MLIP in idiopathic DCM in the human population. Preliminary evidence demonstrates that MLIP over-expression in the mouse heart leads to bradycardia with a prolonged P-R interval. In addition, MLIP expression is dramatically down regulated in a mouse model that develops DCM shortly after birth. A collaborative effort will identify MLIP mutations in a human population with idiopathic DCM and then to characterize these mutations through structure-function analysis of the identified mutants.

Molecular Relationship of Cardiac Fetal Gene Reactivation and Heart Failure

Principal Investigators: Patrick Burgon, Dr. Frans Leenen, and Alexandre Stewart

Little is known about the molecular basis for the transition from hyperplastic to hypertrophic-based myocardial growth. Endogenous MLIP is localized to both nuclei in neonatal myocytes and is expressed in a transient biphasic manner during the critical phase of perinatal heart development. The promoter of MLIP has not been characterized, yet MLIP expression is striated muscle-specific and developmentally regulated. Our goal is to identify the transcription factors that drive MLIP expression to shed light on the mechanism of cardiac muscle differentiation and withdrawal from the cell cycle. We will further expand these studies into rodent MI models to define the role MLIP in heart failure.

Characterization in Mice of Novel Human Genes Associated with Hypertension and Coronary Artery Disease Risk Identified in Ongoing Genome-wide Association Studies using High Density SNP Microarrays

Principal Investigators: Alexandre Stewart, Dr. Frans Leenen, Frederique Tesson

The goal of this project is to generate and characterize transgenic mice that express newly identified gene variants associated with the risk of coronary artery disease or salt-sensitive hypertension to determine how these variants affect the progression of disease in well characterized mouse models. We are coordinating a large genome-wide association study that has identified novel genes associated with the risk of coronary artery disease. As an example of this strategy, we have identified a functional risk polymorphism in the metalloproteinase ADAM-TS5 that we are characterizing at the biochemical level in cultured cells and for which a transgenic mouse expressing the mouse wild type ADAM-TS5 and ADAMTS5 bearing the polymorphic mutation are being generated. These ADAM-TS5 transgenic mice will be crossed to mice prone to atherosclerosis to determine whether the polymorphism accelerates the onset of disease.

Transcription Regulation in the Transition to Heart Failure Following Myocardial Infarction

Principal Investigators: Alexandre Stewart, Patrick Burgon, and Balwant Tuana

The goal of this project is to identify and elucidate the mechanisms of altered gene expression following MI that promote the transition to heart failure in cultured cardiac myocytes and in animal models. The myocardium reactivates fetal genes following myocardial infarction through a stereotypical program controlled by a network of transcription factors. We have identified the TEAD4 transcription factor (a member of the TEAD family of transcription factors formerly called RTEF-1) as a critical component of this program. TEAD4 is induced by hypoxia in cardiac myocytes and its elevated expression activates a protein phosphatase that targets the cardiac gap junction protein connexin and impedes cardiac conduction leading to atrial tachyarrhythmias. In addition, transgenic mice with cardiac myocyte-specific over-expression of TEAD4 develop diastolic dysfunction, as measured by conduction catheters.

We have also identified a novel family of TEAD-interacting cofactors related to a Drosophila protein called vestigial that controls flight muscle development in the fly. We named these cofactors Vestigial-like (Vgll1-4). We showed that Vgll2 is specifically expressed in skeletal muscle and is required for muscle differentiation. Vgll2 is transiently activated at the onset of hypertrophic cardiomyopathy. In addition, we showed that the cardiac-enriched Vgll4 protein participates in the hypertrophic response with TEAD factors. Studies are underway to understand how Vgll2 and Vgll4 participate in transcription regulation. In collaborative projects, we have screened skeletal and cardiac muscle yeast two-hybrid libraries and identified novel transcription partners of Vgll2 and Vgll4 that further elucidate the transcription factor network.

Staff 

Investigators

  • Dr. Frans Leenen, MD, PhD, FRCPC
  • Balwant Tuana, PhD
  • Frederique Tesson, PhD (University of Ottawa)
  • Patrick Burgon, BAppSc, PhD
  • Alexandre Stewart, BScH, MSc, PhD

Trainees 

Postdoctoral Research Fellows

  • T. Ramsamy
  • A. Al-Madhoun
  • N. Sylvius
  • E. El-Shahat
  • M. Nader
  • B. Westendorp

PhD Graduate Students

  • M. Shehata
  • S. Amin
  • N. Rafatian
  • S. Labib

Other Students

  • J. Chan
  • K. Shaw
  • M. Lee Choon

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