Many etiologies of heart disease are characterized by expansion and remodeling of the cardiac extracellular matrix (ECM or matrix) which results in cardiac fibrosis. Cardiac fibrosis is mediated in cardiac fibroblasts by TGF-β1/R-Smad2/3 signaling. Matrix component proteins are synthesized by activated resident cardiac fibroblasts known as myofibroblasts (MFB). These events are causal to heart failure with diastolic dysfunction and reduced cardiac filling. We have shown that exogenous Ski, a TGF-β1/Smad repressor, localizes in the cellular nucleus and deactivates cardiac myofibroblasts. This deactivation is associated with reduction of myofibroblast marker protein expression in vitro, including alpha smooth muscle actin (α-SMA) and extracellular domain-A (ED-A) fibronectin. We hypothesize that Ski also acutely regulates MMP expression in cardiac MFB. While acute Ski overexpression in cardiac MFB in vitro was not associated with any change in intracellular MMP-9 protein expression versus LacZ-treated controls,exogenous Ski caused elevated MMP-9 mRNA expression and increased MMP-9 protein secretion versus controls. Zymographic analysis revealed increased MMP-9-specific gelatinase activity in myofibroblasts overexpressing Ski versus controls. Moreover, Ski expression was attended by reduced paxillin and focal adhesion kinase phosphorylation (FAK - Tyr 397) versus controls. As myofibroblasts are hypersecretory and less motile relative to fibroblasts, Ski's reduction of paxillin and FAK expression may reflect the relative deactivation of myofibroblasts. Thus, in addition to its known antifibrotic effects, Ski overexpression elevates expression and extracellular secretion/release of MMP-9 and thus may facilitate internal cytoskeletal remodeling as well as extracellular ECM components. Further, as acute TGF-β1 treatment of primary cardiac MFB is known to cause rapid translocation of Ski to the nucleus, our data support an autoregulatory role for Ski in mediating cardiac ECM accumulation.
PGC-1α plays a key role in cardiac metabolism by regulating downstream target genes involved in fatty acid oxidation and mitochondrial biogenesis. Our objective was to examine PGC-1α expression during cardiomyocyte hypoxia. Hypoxia (12h) decreased PGC-1α expression and promoter histone acetylation. Hypoxia-induced PGC-1α down-regulation was attenuated by a histone deacetylase inhibitor, and was accelerated by an ERRα inverse agonist. We found that ERRα regulates PGC-1α gene expression via a conserved binding site in the PGC-1α promoter. Over-expression of ERRα in isolated cardiomyocytes was sufficient to induce PGC-1α expression and rescued the loss of PGC-1α expression during hypoxia. Extending hypoxia to 24h resulted in recovery of PGC-1α expression, an effect attenuated by an AMP kinase inhibitor or exaggerated by removal of glucose. Hypoxia thus exerts a biphasic effect on PGC-1α expression: initial down-regulation due to histone deacetylation, followed by restoration via activation of AMP kinase. While ERRα was not involved in hypoxia-mediated PGC-1α down-regulation, ERRα over-expression rescued this loss, and ERRα expression was required for basal PGC-1α expression. Our results describe a novel regulatory mechanism for PGC-1α expression which may contribute to metabolic derangement during ischemia and heart failure. Supported by the Canadian Institutes of Health Research.
Arterial stiffness (AS) is an impairment in arterial relaxation and function in blood pressure regulation and is associated with the development of multiple cardiovascular diseases including hypertension, atherosclerosis, and adverse outcomes such as stroke, heart failure, and death. AS comprises an increase in vascular wall components (extracellular matrix proteins or vascular smooth muscle cell (VSMC) composition) or cellular malfunction, or a combination of these factors. Preliminary data from our lab showed that using the Transverse Aortic Constriction (TAC) model, the transcription factor scleraxis is upregulated in the aortic wall on the pre-TAC/high pressure side versus post-TAC/low pressure side. Scleraxis also induces cellular phenotype activation of fibroblasts to myofibroblasts in cardiac fibrosis and mediates epithelial to mesenchymal transition in development. Angiotensin II (AngII) induces hypertension and increases stiffness in arteries, and we found that it also up-regulates scleraxis expression, suggesting the possibility that scleraxis may alter arterial wall composition. In this study, we investigated the role of scleraxis expression in VSMC, and its effect when over-expressed along with AngII treatment in small resistance arteries and aorta. In vivo, we upregulated scleraxis in VSMC using a Cre/LoxP approach and the tamoxifen-inducible Myh11cre mouse, with or without AngII delivery by micro-osmotic pump, then isolated 3rd order mesenteric arteries and assessed arterial function and stiffness by pressure myography. We harvested aortas for immunohistology staining and gene expression analysis. In vitro, we upregulated scleraxis in VSMC with or without AngII treatment and measured their proliferation by flow cytometry. Our findings show that scleraxis induces stiffness in mesenteric arteries, but not aorta by induction of VSMC proliferation. Scleraxis upregulation with AngII infusion exacerbates vascular stiffness in mesenteric arteries due to significant changes in arterial wall components, reducing internal lumen diameter and smooth muscle relaxation. At the molecular level, extracellular matrix protein expression is reduced in aortas. In in vitro studies, scleraxis-AngII treated VSMC exhibit lower proliferative capacity, distinct morphological changes and higher contractile gene expression, indicative of a hypertrophic contractile phenotype altering overall arterial stiffness. Our study shows that scleraxis alone can induce AS by regulating VSMC phenotype toward proliferation. In contrast, arterial stiffness is exacerbated in scleraxis overexpression and AngII-infused vessels by inducing VSMC towards a hypertrophic contractile phenotype. Therefore, scleraxis alters vascular smooth muscle phenotypic switching and arterial stiffness through different mechanisms, depending on the presence of AngII, which may have different pathological impacts depending on the specific underlying conditions. This work is supported by funding from Heart & Stroke Foundation and BMO Studentship. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Myofibroblasts are the primary mediators of cardiac remodeling, and their persistence in the myocardium is implicated in the development of fibrosis. Thus, inhibiting myofibroblast differentiation represents a novel approach for treating cardiac fibrosis. Scleraxis is a transcription factor required for development of collagen rich tissues. Interestingly, the same stimuli that induce differentiation also cause increased scleraxis levels in vitro and in vivo. Thus, we hypothesized that scleraxis promotes the myofibroblast phenotype. To test this, we over‐expressed scleraxis via adenovirus in primary cardiac fibroblasts (CFs) and measured changes in key myofibroblast markers. We observed that scleraxis over‐expression in CFs caused increased mRNA and protein levels of these markers. Since myofibroblasts are also distinguished from fibroblasts by their contractility, we used a contraction assay to determine the functional implications of increased marker levels. We found that over‐expressing scleraxis in CFs caused a significant increase in contraction. However, over‐expression of a dominant negative DNA binding‐deficient scleraxis mutant did not induce contraction, indicating a requirement for intact scleraxis. These results demonstrate that scleraxis promotes the cardiac myofibroblast phenotype, implicating scleraxis as a potential target for the intervention of cardiac fibrosis. Grant Funding Source : Supported by Canadian Institute of Healthy Research, St. Boniface Hospital Foundation
Therapeutic approaches to managing cardiac fibrosis are lacking. Our objective is to examine the role of the transcription factor scleraxis (Scx) in cardiac type I collagen production, and to evaluate its potential for anti-fibrotic therapy development. We noted Scx expression in various collagen-producing cell types, including cardiac fibroblasts. Scx expression increases concomitant with collagen production, e.g. in response to TGF-β1 in vitro and in cardiac infarct scar or in pressure-overloaded myocardium in vivo. We have identified sites in the human collagen Iα2 (COLIα2) gene promoter mediating transactivation by scleraxis, and have shown that Scx is sufficient to induce ColIα2 expression in cardiac fibroblasts. Scx expression is regulated by the pro-fibrotic Smad signalling pathway downstream of TGF-β1, which regulates COLIα2 expression additively with Scx. A dominant-negative Scx mutant abrogated TGF-β1-mediated ColIα2 gene expression, suggesting that Scx activity is required for collagen expression in agreement with our finding of reduced cardiac collagen gene expression in Scx null mice. Scx thus plays a central and critical role in collagen expression in the heart, and targeting Scx activity or expression may provide a novel approach in the development of new anti-fibrotic therapies. Supported by the Canadian Institutes of Health Research & the St. Boniface Hospital Research Foundation.
Vascular dysfunction underlies numerous significant diseases including diabetes, atherosclerosis and hypertension. Vascular dysfunction can be a result of altered smooth muscle contraction/relaxation, and impaired endothelial cell function within the vessel wall. Vascular fibrosis involves an increase in the thickness of vessel wall. This contributes to either an increase in extracellular matrix (ECM) synthesis or induced vascular smooth muscle proliferation within the vessel wall, or both, causing stiffer vessels with impaired tone and reduced lumen diameter.Our lab identified the transcription factor scleraxis as a novel master regulator of fibrotic signaling in the myocardium, showing scleraxis is sufficient to induce fibroblast to myofibroblast phenotype conversion, a critical step in the development of fibrosis, and directly up-regulates ECM genes in cardiac fibroblasts. Angiotensin II (AngII) was reported to induce vascular fibrosis via activation of the transcription factor Smad3 in aortic vascular smooth muscle cells. Our lab has shown that Smad3 physically interacts with scleraxis, and critically requires scleraxis to drive TGFβ/Smad fibrotic signaling in cardiac fibroblasts.Our preliminary data has revealed that scleraxis is detectable in the arterial wall, and scleraxis expression is elevated in high pressure versus low pressure regions of vessels. Loss of scleraxis in the aortas of scleraxis knock-out mice reveals a discontinuation and disarrangement in the structure of vascular wall. We thus hypothesize that scleraxis is sufficient and necessary to induce vascular fibrosis.Pressure myography data reveals an increase in vascular stiffness and thickness of 3rd order mesenteric arteries of smooth muscle-specific scleraxis overexpression mice. Also, our data shows that vascular stiffness is significantly increased in AngII-induced scleraxis overexpression mice with a relative increase in telemetry blood pressure measurements and pulse wave velocity. Histological sections suggest a reduction of the translamellar ECM accumulations in AngII-induced scleraxis overexpression aortas. To summarize, scleraxis may contribute to vascular stiffness by inducing vascular smooth muscle proliferation.
Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.
