Dr. Ian M.C. Dixon
Molecular Cardiology, Institute of Cardiovascular Sciences
Department of Physiology, University of Manitoba
The work carried out by Dr. Dixon and the members of our lab deals with investigation of the signaling pathways responsible for cardiac fibrosis in developing overt congestive heart failure. In many types of heart disease, cardiac fibrosis “sneaks up” on the myocardium and is a primary contributor of dysfunction of cardiac muscle relaxation, leading to problems in heart ventricle filling following contraction. Reduced filling equates to heart failure with preserved ejection fraction (HFpEF) in fibrosed hearts, and may slip through normal methods of detection (M-mode echocardiography). In this context, we study the cell biology of cardiac myofibroblasts. Myofibroblasts are activated fibroblasts, and play a large role in contributing to cardiac fibrosis. Myofibroblasts are not normally found in healthy heart muscle, but are very common in diseased and failing hearts of various etiologies. We are interested in investigating cellular control of myofibroblast phenotype and function (migration, activation, contraction) as these functions all contribute to wound healing and remodeling 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, and even in athletes subjected to intensive training regimens. 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. Heart fibroblasts are unique – in some ways they are not comparable to lung or skin or vascular fibroblasts. Their unique biology supports their own investigation.
As cardiac myofibroblasts numbers are elevated in heart disease, we want to determine cellular control points for cell death and viability. In this context, we are investigating mechanisms that trigger autophagy and apoptosis in these cells, which ultimately may control their numbers in the myocardium, and possibly regulate the rate of cardiac fibrosis. Autophagy may be involved in non-apoptotic roles in these cells. We are also trying to determine whether autophagy is linked to activation of cardiac myofibroblasts, or whether it is linked to their death via apoptosis, or both. The mechanisms involved in stimulation of cardiac fibrosis are not fully understood. Usually the marginal attenuation of cardiac fibrosis because of a 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. Our efforts are directed on those signaling proteins that either stimulate or repress fibroblast and myofibroblast activity in the heart. We are well known for our work on profibrotic Smad proteins in heart and for our studies to investigate endogenous inhibitors Smad-based mechanisms 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 group 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 Ski and 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 development of a new drug and applying it to failing hearts may allow us to alter the course of the progression of heart disease. If we can slow down the progression of fibrosis present in the vast majority of cardiac disease, we can then preserve cardiac function after the disease has affected the function of heart.
What techniques and equipment are used in this laboratory?
The majority of our work makes use of primary myofibroblast culture, and we are expert at mechanisms in the activation of cardiac fibroblasts to myofibroblasts in culture. We use viral constructs 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. Pathways of interest to us include Smads and their inhibitors, and Hippo pathway effectors such as YAP1 and WWTR1. In collaboration with members of the Institute we employ extensive microscopy in our research using highly refined IF techniques to study the cell biology of myofibroblasts. We are expert in creating models of chronic heart failure after heart attack (25 years of experimental investigation with surgical ligation of the LAD in rat model). Recently we have developed inducible knockout mouse lines using mice floxed for Ski and other target genes, to allow us to expand our isolated cell culture findings to in vivo models to validate our discoveries into mechanisms of cardiac fibrosis.
We combine techniques in molecular biology with those of cell biology, microscopy and whole animal physiology to accomplish our research goals. We are also well versed in whole animal physiology, and can carry out combined in vivo and in vitro work using models of myocardial infarction. With the latter model, we have almost two decades of research experience.
We have an interest in studying autophagy and apoptosis in cardiac myofibroblasts, and we have developed several techniques to assess activation of these pathways in these cells. Further, we are investigating the effects of trans fatty acids on cellular viability with an emphasis on how different trans fatty acids affect these cells. Vaccennic trans fatty acid is found in dairy products, and we have noted that it is less cytotoxic than other transfats associated with processed foods eg, elaidic acid.
Besides collaborations with investigators in the CIHR group, Dr. Dixon has developed several 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 has also collaborated with Drs. Wayne Giles (University of Calgary) wherein we have focused on the electrical excitability of myofibroblasts in culture conditions, and how intracellular Ca movements are affected. Assistance from Drs. Boris Hinz (U of Toronto), M. Fujii (NIH, Bethesda, MD), Wayne R. Giles (Calgary), Francesco Ramirez (Mount Sinai, New York), Miyazono (Tokyo), Dietz (Baltimore), Molkentin (Cincinnati) and Paul Benya (UCLA) and within the U of Manitoba, Jeff Wigle, Mike Czubryt, Andrew Halayko, Tom Klonisch and Grant N. Pierce have allowed us to focus on the projects mentioned above.
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 88 full-length research papers and 18 book chapters with 124 abstracts. Collaborating with Dr. Jeffrey Wigle, Dixon recently co-edited a book entitled “Cardiac fibrosis and heart failure: Cause or effect?”. Dr. Dixon is a past MRC (CIHR) scholar, MHRC scholar, and has held studentships and post-doctoral fellowships from the Heart and Stroke Foundation and the Medical Research Council of Canada. He is the past winner of the Heart and Stroke Foundation of Manitoba Robert E. Beamish Memorial Award for excellence in cardiovascular research (2002 and 2007), and a recipient of the Myles Robinson Heart Health Scholarship. He has served for many national and international review committees in basic cardiovascular sciences including Chairing for reviews within the Heart and Stroke Foundation (Canada), and as committee member for CIHR and current membership with the U.S. National Institute of Health (NIH). Dixon has reviewed for Wellcome Trust, Alberta Heritage Foundation, and the Michael Smith Foundation.
Dr. Dixon has supervised the research activities of 26 graduate students (graduated) and is supervising four graduate 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 – Camille (22) and Keith (19).
For more information, please contact:
Ian M.C. Dixon
Tel. (204) 235.3419
Fax. (204) 233.6723
Epigenetics, heart disease and cardiac fibrosis
Epigenetics refers to changes in gene expression that does not involve changes to the underlying DNA sequence. Our lab is particularly interested in how cells in the heart that synthesize extracellular matrix (matrix) undergo phenoconversion without changes in genotype. Disease states such as heart failure after myocardial infarction may influence epigenetic modifications in cells, and these may lead to phenoconversion of normally quiescent cells such as cardiac fibroblasts to cardiac myofibroblasts, which are hyper synthetic and physically contractile. Too much matrix is necessarily a “bad thing” and is synonymous with cardiac fibrosis and heart failure. Members of our lab have discovered that Ski (a phylogenetically ancient protein that inhibits TGF-b and Smad signaling) may reverse fibroblast phenoconversion when overexpressed in cells. We would like to investigate the epigenetic mechanisms that control this class of proteins, and are specifically interested in processes that govern SUMOylation, phosphorylation, and ubiquitination of Ski and Sno proteins.
Thus we would design experiments to help us track:
- The addition of a Small Ubiquitin-like Modifier (SUMO) protein to Ski as the target. As this modification is involved in trafficking proteins to the nucleus, we would like to know whether this plays a role in compartmentalizing Ski in the cytosol in cells active in chronic wound healing.
- Whether ubiquitination is important for regulating Ski in myofibroblasts in the failing heart. The addition of one or a chain of ubiquitin proteins to a protein target. It often targets the protein for degradation but can also alter its localization, conformation, or binding.
- Whether phosphorylation of serine or threonine residues of Ski is relevant to its cellular function, by altering protein conformation.
- Deletion or induction of Ski or Sno using CRISPR technology. We would use this genome editing system to target Ski or Sno DNA regions of interest by a specific guide RNA linked to Cas9 to induce DNA double strand breaks. It may be particularly useful in avoiding off-target effects, and for simplicity of implementation.
Our goal is to gain a better understanding of the cellular control of Ski and Sno, and thus understand this mode of fibroblast phenoconversion, and subsequent induction of cardiac fibrosis in heart failure.
What is Cardiac Fibrosis?
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.
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.
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.
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.
Myocytes = muscle cells – cardiomyocytes are cardiac muscle cells.
The Extracellular Matrix or Matrix = the proteins that lie between the muscle cells – the matrix provides tissue rigidity and holds the heart (and all other organs) together. This is where fibroblasts are found.
Fibroblasts = cells which are responsible for healing wounds and contributing to the cardiac matrix (they become hypersynthetic myofibroblasts when activated). When activated (by calcium) they become stimulated to release collagen to create scar tissue. Fibroblasts are found throughout the body, but the heart is unique in the way these proteins continue to work even after their job is done. In other parts of the body they die after a scar has formed, in the heart, they continue to release collagen, eventually leading to a condition called Fibrosis.
Collagen = a major component of the matrix, like a molecular cement, holds muscle cells together. Release is controlled by Fibroblasts. Too much, and the heart can’t function properly.
Fibrosis = A condition which occurs following heart trauma (heart attack) or in the progression of heart disease. Trauma or disease activates Fibroblasts which pump out excessive amounts of collagen creating excess cardiac scarring. Eventually, there is too much scar tissue and the heart can no longer relax or contract properly, causing death. This is why some people may survive a heart attack and then suffer from compensated heart failure later in life.
Calcium = (Ca) an element which provides a very important and common cellular signal. Sustained elevation of Ca inside the cell eg, [Ca 2+]i , via increased Ca influx is important in the regulation of a number of cellular functions, including that of gene expression (collagens), or differentiation of cells. Thus Ca entry to the cell may control cell contraction or secretion of matrix.
NXC1 = The sodium-calcium exchanger. NCX1 is a protein abundant in different cardiac cells (including both myocytes and fibroblasts) which contributes to the movement of calcium. Dr. Hryshko is investigating NCX1 in myocytes, and Dixon is doing so in cardiac fibroblasts. A relatively new area of heart research is the exploration of new drugs that block the function of NCX1. Two types of these drugs include SEA 0400 and KBR7943. Dr. Dixon is examining the use of these drugs to inhibit the NCX1 in fibroblasts, and thereby inhibit fibroblast-based release of collagen.
From the Heart and Stroke Foundation
- Over one third (36%) of all deaths in Canada (1999) are due to heart disease and stroke
- 80.2% of Canadians (20-59 yrs) have at least one of the following risk factors for heart disease or stroke: daily smoking, physical inactivity, being overweight, self-reported high blood pressure or self-reported diabetes
- 37% of all female deaths and 35% of all male deaths result from heart disease and stroke
- While more men than women die from coronary artery disease and heart attack, more women than men die from congestive heart failure
- Heart disease and stroke combined are the leading cause of hospitalization for women (excluding childbirth and pregnancy)
- By the age of 70, 1 in 5 women and 1 in 4 men reported having been told by a physician that they had heart problems
- 50% of Canadians with heart problems or stroke required help with daily activities, while only 11.5% of individuals without heart problems or stroke required such helpeart disease and stroke combined are the number one cause of hospitalization among men and women in Canada
- The total cost of heart disease and stroke to the Canadian economy is approximately $18.5 billion – more than any other disease
Ghavami S, et al. Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts. Cell Death Dis. 2012 Jun 21;3:e330. doi: 10.1038/cddis.2012.61.
Cunnington RH, et al. Antifibrotic properties of c-Ski and its regulation of cardiac myofibroblast phenotype and contractility. Am J Physiol Cell Physiol. 2011 Jan;300(1):C176-86. Epub 2010 Oct 13.
Freed DH, et al. Role of myosin light chain kinase in cardiotrophin-1-induced cardiac myofibroblast cell migration. Am J Physiol Heart Circ Physiol. 2011 Aug;301(2):H514-22. Epub 2011 May 13
Santiago JJ, et al. Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts. Dev Dyn.
Dixon IM. The soluble interleukin 6 receptor takes its place in the pantheon of interleukin 6 signaling proteins: phenoconversion of cardiac fibroblasts to myofibroblasts.
Hypertension. 2010 Aug;56(2):193-5. Epub 2010 Jul 6. No abstract available.
Award of Merit – Manitoba Medical Student’s Association – Teaching Award 2010, 2011
Dr. R.E. Beamish Memorial Award HSFM 2007
Students and Fellows
Ryan H. Cunnington
- Henry Friesen Young Scientist Award 2011
- CIHR PDF fellowship (nominee), Faculty of Medicine
- Distinguished Dissertation Award (nominee)
Title: Inhibition of the Smad signal in post-MI heart failure
($50,000 per annum) July 1, 2011 – June 30, 2013
2. Canadian Institutes for Health Research
Title: Regulation of the extracellular matrix by myofibroblasts in cardiac hypertrophy and failure.
($136,111 per annum) October 1, 2008 – September 30, 2013.
3. Canadian Institutes for Health Research
Title: Role of Ski in myofibroblast regulation in heart failure.
($121,047 per annum). Notification of the award in December of 2011. (Termed out from April 2012 – May 2017).
4. Canada/USA grant with Dr. Robert Simari (Mayo Clinic, Rochester, MN) St.Boniface/Mayo Clinic joint research project:
Title: Myofibroblast diversity in tissue engineering. $125,000/2 years.
(September 1, 2012 – August 31, 2014).
5. Heart and Stroke Foundation of Canada – Grantees are Ian M.C. Dixon, M.P. Czubryt, and Jeff Wigle.
Title: Ski and Scleraxis form a negative feedback loop in regulation of cardiac myofibroblast function
($101,000 per annum) July 1, 2013 – June 30, 2017