St. Boniface Hospital Research

Ian M.C. Dixon
Principal Investigator, Molecular Cardiology
Institute of Cardiovascular Sciences

Research Focus

The work carried out by the members of Dr. Dixon’s group deals with investigation of the signaling pathways responsible for the occurrence of cardiac fibrosis in the development of pathological cardiac hypertrophy and overt congestive heart failure. In particular we are interested in myofibroblast contractility, migration and protein synthesis as they all play roles in wound healing and remodelling of extracellular matrix in diseased hearts.

Cardiac fibrosis is the abnormal expansion of the cardiac extracellular matrix and occurs in most types of heart disease. Fibrosis is the end-point of the production of too much collagen and other extracellular matrix component proteins. The extracellular matrix includes proteins that occupy the space between cells in an organ like the heart. The component proteins include fibrillar collagen types, proteoglycans, fibronectin and others. A key feature of the maintenance of the cardiac extracellular matrix is being exclusively deposited and removed exclusively by fibroblasts and contractile myofibroblasts.

The mechanisms involved in stimulation of cardiac fibrosis are not fully understood. In most cases, the marginal attenuation of cardiac fibrosis as a result of a given therapy is a beneficial side effect that may be coupled with other primary changes to other cells, especially cardiac myocytes. Very few drugs or agents are known to preferentially affect the behaviour of fibroblasts alone. As little information about the exclusive production of collagen is available, or even about the biology of cardiac fibroblasts themselves, the members of our lab are focused on these questions. In particular, our efforts are directed on those signaling proteins that either stimulate or repress fibroblast and myofibroblast activity in the heart. We are known for our work to investigate endogenous inhibitors of cardiac fibrosis.

Why is this work important?

Remodeling of the cardiac extracellular matrix has become a well-known modifier of cardiac performance and on-going or chronic wound healing is closely tied to heart failure. Our lab is interested in determining why natural inhibitors of wound healing undergo drastically reduced expression in injured or failing hearts. Our laboratory is known for its work on two such proteins, Smad7 and c-Ski – as well, we have addressed the anti-fibrotic properties of cardiotrophin-1, a cytokine of the IL-6 superfamily. The expression of these naturally occurring fibrosis-inhibiting proteins are tightly regulated in cardiac wound healing, especially after myocardial infarction. Adapting their function with the development of a new drug and applying it to failing hearts, may allow us to alter the course of the progression of heart disease. Thus if we are able to slow down the progression of fibrosis present in the vast majority of cardiac disease, we will then be able to preserve cardiac function after the disease has begun to affect the function of heart.

What techniques and equipment are used in this laboratory?

The majority of our work make use of primary myofibroblast culture (2-D). We have used viral contructs to induce and ablate specific gene expression and observe the effects on several end-points including collagen synthesis and secretion, myofibroblast migration, and myofibroblast contraction. The latter is carried out using both anchored and floating gel contraction assays. We have developed retroviral constructs for both pro- and anti-fibrotic agents, and have completed the first phase of investigation on the role of NCX1 (plasmalemmal Na-Ca exchange) in myofibroblast function. This complements other work wherein we have addressed the role of specific non-specific cation channels in delivery of Ca to the myofibroblast upon ligand-based excitation.

Thus we combine techniques in molecular biology with those of cell biology and microscopy to accomplish our research goals. We are also well versed in whole animal physiology, and are able to carry out combined in vivo and in vitro work using models of myocardial infarction. In the case of the latter model, we have almost two decades of research experience.

In addition to collaborations with investigators in the CIHR group, Dr. Dixon has developed a number of other collaborative efforts. Our work has led to the discovery of novel means of assessing cardiac collagen in cardiomyopathic hearts via FTIR microspectroscopy. Our lab is also collaborating with Drs. Wayne Giles and Lisa Chilton (University of Calgary) and investigating issues of CT-1’s role in myofibroblast function. This collaboration is focused on the electrical excitability of myofibroblasts in culture conditions, and how intracellular Ca movements are impacted.

Assistance from Drs. Anita Roberts and M. Fujii (NIH, Bethesda, MD), Dr. Francesco Ramirez (Mount Sinai, New York), Miyazono (Tokyo) and Paul Benya (UCLA) have allowed us to move into the area of recombinant adenovirus work (Ad Smad7 and Ad Smad 3) and also enabled us to directly assess the induction or inhibition of various factors on the response elements in fibrillar collagen promotor regions.

About Ian M.C. Dixon

Dr. Dixon has worked as a Professor of Physiology at the Institute of Cardiovascular Sciences since 1992. He obtained his PhD at the University of Manitoba in 1990, and then completed a post-doctoral fellowship at the University of Toronto with Drs. Mike Sole and C.C. Liew. During his career, he has published 75 full-length research papers and 14 book chapters along with 91 abstracts. Dr. Dixon is a past MRC (CIHR) scholar, MHRC scholar, and has held studentships and post-doctoral fellowships from the Heart and Stroke Foundation as well as the Medical Research Council of Canada. He is a past and current winner of the Heart and Stroke Foundation of Manitoba Robert E. Beamish Memorial Award for excellence in cardiovascular research (2002 and 2007), and also is a current recipient of the Myles Robinson Heart Health Scholarship. He currently serves CIHR as the scientific officer of Cardiovascular Sciences committee C, and has reviewed for Wellcome Trust, Alberta Heritage Foundation, and the Michael Smith Foundation.

Dr. Dixon has supervised the research activities of 14 graduate students (graduated) and is currently supervising three students. He is a reviewer for various cardiovascular journals including Cardiovascular Research, Circulation, Circulation Research, American Journal of Physiology, J Cell Science, J Cell Physiology and others. He is married to Kelly and they have two children.

For more information, please contact:

Ian M.C. Dixon
[t] 204 235 3419
[f] 204 233 6723
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In Detail

Research Update: Roll of calcium in fibrosis

Through his research, Dr. Dixon hopes to find a way to slow or stop the occurrence of cardiac fibrosis. Fibrosis is important as a disease modifier and can be viewed as the “straw breaking the camels’ back” in the progression of heart failure. If he can pinpoint why fibroblast cells become active, and then try to find ways to inhibit their tendency to create runaway wound healing eg, where its not needed, then he may be able to find a way to stop inappropriate cardiac scarring.

Fibroblasts play a role in almost every kind of heart disease, they are the common thread. Fibrosis is the end result in many forms of heart disease. If one can control the fibroblasts and their differentiated relatives (myofibroblasts), then it may be possible to slow or stop fibrosis.

One of Dr. Dixon’s recent studies looks at calcium’s role in fibroblast function. Calcium (Ca), which is found throughout the body, is very important for muscle contraction – especially in a beating heart. Even in cells that reside in the matrix surrounding the muscle, (like fibroblasts), calcium is very important. Fibroblasts use calcium for all of their functions, including their control of proper outputs of collagen.

Having found that NCX1 blocking drugs can be beneficial in suppressing fibroblast activity, and thus demonstrate “proof of concept” that this protein is worth targeting, they will now try to find out how to control NCX1 expression in fibroblasts and myofibroblasts. They discovered last year that NCXI operates in fibroblasts (controlling the activity of the fibroblasts – thus controlling the release of collagen). They published a paper on this about one year ago.

One of Dr. Dixon’s graduating Masters students carried out the above study within the context of her thesis. They believe that NCX1 manipulation could be the key to preventing or stopping fibrosis. Dr. Dixon and his team is the first research group to investigate NCX1 in the context of cardiac fibroblast and myofibroblast function.

Cardiac fibrosis is the abnormal expansion of the cardiac extracellular matrix and occurs in most types of heart disease. Fibrosis is the end-point of the production of too much collagen and other extracellular matrix component proteins. The extracellular matrix includes proteins that occupy the space between cells in an organ like the heart. The component proteins include fibrillar collagen types, proteoglycans, fibronectin and others. A key feature of the maintenance of the cardiac extracellular matrix is being exclusively deposited and removed exclusively by fibroblasts and contractile myofibroblasts.

While clinicians use a number of drugs to partially alleviate or attenuate this process, the mechanisms involved in stimulation of cardiac fibrosis are not fully understood. In most cases, the marginal diminution of cardiac fibrosis as a result of a given therapy is a beneficial side effect that may be coupled with other primary changes to other cells, especially cardiac myocytes (muscle cells). Very few drugs or agents are known to preferentially affect the behaviour of fibroblasts alone. As little information about the exclusive production of collagen is available, or even about the biology of cardiac fibroblasts themselves, the members of Dixon’s lab are focused on these questions. In particular, Dr. Dixon’s efforts are focused on those signaling proteins that either stimulate or repress fibroblast and myofibroblast activity in the heart. His laboratory is known for its work on elucidating endogenous inhibitors of cardiac fibrosis.

“In fibrosed hearts, the matrix effectively wraps heart muscle cells in a molecular cement – collagen and other matrix proteins. There are many clinical problems which could spring from too much matrix deposition. Our work has helped to show that cardiac fibrosis contributes to heart failure.” He points out the remodeling of the cardiac extracellular matrix has become a well-known modifier of cardiac performance and on-going or chronic wound healing is closely tied to heart failure. He adds, “Our work will clarify the molecular mechanisms of cardiac fibrosis, and will aid in the development of effective and specific drugs to alleviate this problem.”

Dr. Dixon goes on to explain that fibrosis may result from inappropriate wound healing by cardiac fibroblasts and is interested in finding out why natural inhibitors of wound healing undergo drastically reduced expression in injured or failing hearts. His laboratory is known for its work on one such protein, Smad7. “The expression of these naturally occurring fibrosis-inhibiting proteins are tightly regulated in cardiac wound healing, especially after myocardial infarction.”, he explains. “We’re trying to find out why specific factors, like Smad7, inhibit the collagen output of myofibroblasts and whether other cardiac proteins share this property."

“If we can adapt their function with the development of a new drug and apply it to the issue of developing heart failure, we may be able to alter the course of disease progression.”

THE COLLAGEN CONNECTION

In pathological cardiac hypertrophy, a number of systems that aid in heart growth become abnormally activated, causing both the cardiac muscle cells and the extracellular matrix to increase in size. The expansion of the matrix impedes the electrical conductivity, which enables communication from cell to cell, and also hampers the transport of nutrients and removal of metabololic wastes. The cells can no longer contract or relax properly, which leads to a decline in the heart’s pumping performance and ultimately to heart failure. This progression of events commonly occurs following a myocardial infarction.

The major molecules of the extracellular matrix are collagens of various types. In healthy hearts, the synthesis and deposition of collagen are very tightly regulated by the fibroblast cells, which make up the connective tissue. After studying the various control points for this regulation, Dr. Dixon is now investigating the changes in cardiac collagen expression and the altered profile of cardiac collagen subtypes, after myocardial infarction. His laboratory has shown that cardiac collagen concentration is increased in cardiac tissue remote to the infarct, as well as in the infarct itself, but more importantly this occurs as a result of altered Smad signaling in the post-MI heart. With complementary in vitro experiments, his laboratory has also demonstrated the changes in collagen content are due to increased deposition, rather than a reduction in degradation.

The Dixon lab has also pinpointed other novel protein molecules, including cardiotrophin-1 (CT-1), which is a member of a group of cytokines important in inflammation. Dr. Dixon feels that unlike other closely related IL-6 superfamily members, CT-1 may serve a unique role, and may provide an early signal for wound healing in the heart, in the setting of acute post-MI.

CHARTING PATHWAYS TO POTENTIAL THERAPIES

While focusing on the target genes identified for further study, Dr. Dixon’s group will test their impact on the expression of matrix and the biology and behaviour of cardiac myofibroblasts. It is hoped that molecular pathways that trigger the onset of fibrosis can be charted and, ultimately, new therapies can be developed to correct the condition at its source.

Depicted on the left is a cardiac ventricular myofibroblast. We used stop-motion photography to illustrate its ability to contract. The myofibroblast in the field is plated on collagen type I matrix that was applied to a plastic culture dish (literally “painted on”) and then fixed to the plate with overnight UV light exposure. The following day, ventricular myofibroblasts were seeded onto the compressible collagen matrix at relatively low density. The actual time for the contraction duration for the depicted cell is ~ 30 seconds.

Myofibroblasts are muscle-like fibroblasts,and are major contributors to cardiac wound healing as well as eventual global ventricular fibrosis that may follow a particularly extensive damage, such as that occurring after a large heart attack. Note the compartmentalization of the contraction event eg, not all portions of the cell contract similar distances, nor does the cell contract in a well-coordinated manner. Myofibroblasts express - smooth muscle actin and SMemb (embryonic smooth muscle myosin) among other contractile proteins, and their uncoordinated contraction may be due to the heterogenous distribution of these proteins within the cell. Thus, stress fibres in these cells (not visible in this movie) may exist in a number of orientations in each cell.

How is myofibroblast contraction useful in wound healing of the infarct scar in post-myocardial infarction (heart attack)? Contraction of these cells resists opposing or retractile forces in the beating heart, and assists in the shrinkage of the wound. Contraction of these cells also deformed the surrounding extracellular matrix and may liberate latent cytokines stored in the matrix, including the activation of latent TGF-1, which may then stimulate even more wound healing in damaged hearts. Thus a positive feedback cycle ensues. If this goes unchecked, then the heart may undergo inappropriate deposition of matrix associated with cardiac fibrosis and eventual heart failure.

Human tissue studies - Primary human ventricular fibroblasts sourced from anterior apical infarct. The Dixon lab is now embarking upon a series of studies to characterize TGF-b signaling in these cells. Many thanks to Dr. Darren Freed for providing these cells to our laboratory.

Detection of myofibroblasts in left ventricular infarct scar 4 weeks after infarction: Panel A,A1, A2: Triple-fluorescence staining of a cardiac infarcted area, 4 weeks postmyocardial infarction, for, A, anti-α-smooth muscle actin (green), A1, anti-DDR2 (red), A2, Hoechst 33342 (blue) as well as a anti-α-smooth muscle actin and anti-DDR2. Areas of overlap between green and red stained items indicate presence of cells with myofibroblast characteristics. The white sizing bar in A2 = 350 μM. Panel A3 shows a close-up image of the white boxed area within Panel A2; several cells can be discerned that express both markers (see arrows in A3). Panel B: Triple staining of cardiac infarcted area for antivimentin (green), anti-DDR2 (red) and Hoechst 33342. The image is consistent with coexpression of vimentin and DDR2 (markers of fibroblastic cells) by these cells. Panel C: Section of the infarct region in the test heart treated with secondary antibodies (fluorescein-conjugated anti-mouse, Texas-Red conjugated anti-rabbit immunoglobulin antibodies, as well as Hoechst 33342) in the absence of primary antibodies. The section indicates minimal non-specific staining. The white sizing bars in A3, B and C are 50 μM. Panels D and D1: Images from an infarcted heart section stained, respectively, for desmin (green; cardiac and smooth muscle marker), or desmin (green) and nuclei (blue). Panels Panels E and E1: Images from an infarcted heart section stained, respectively, for SMemb (green; myofibroblast marker), or SMemb (green) and nuclei (blue). Panels F and F1: Images from an infarcted heart section stained, respectively, for ED-A fibronectin (myofibroblast marker), or ED-A fibronectin (green) and nuclei (blue). Pink arrows point to infarct areas which stain negative for desmin, but positive for SMemb or ED-A fibronectin. The sizing bar in D-F1 = 100 μM. All panels are sections of infarcted left ventricle taken from 4 week MI rat models.