Substance P is a sensory nerve neuropeptide located near coronary vessels in the heart. Therefore, substance P may be one of the first mediators released in the heart in response to hypertension, and can contribute to adverse myocardial remodeling via interactions with the neurokinin-1 receptor. We asked: 1) whether substance P promoted cardiac hypertrophy, including the expression of fetal genes known to be re-expressed during pathological hypertrophy; and 2) the extent to which substance P regulated collagen production and fibrosis.
The sensory neuropeptide, ?-calcitonin gene-related peptide (?-CGRP) is protective against hypertension-induced heart damage and cardiac ischemia/reperfusion injury. To determine whether this neuropeptide is also cardioprotective in heart failure, this study examined whether the absence of ?-CGRP exacerbated the adverse cardiac remodeling, dysfunction and mortality in pressure overload heart failure induced by transverse aortic constriction (TAC). Male ?-CGRP knockout (KO) and wild type (WT) mice had TAC or sham surgery at day 0 and were studied on days 3, 14, 21, and 28. The survival rate of TAC ?-CGRP KO mice was lower than the TAC WT mice over the duration of the protocol. Left ventricular ?-CGRP content in TAC WT mice was higher at days 3, 14, and 21 than sham WT mice. Echocardiography demonstrated greater adverse cardiac remodeling and dysfunction in the TAC ?-CGRP KO compared to the TAC WT mice. The lung/body weight ratios and left ventricular masses were higher in TAC ?-CGRP KO compared to the TAC WT mice. While there was increased cardiac fibrosis in the TAC WT mice compared to shams, the TAC ?-CGRP KO mice had markedly increased fibrosis above that of the TAC WT mice. TAC WT mice had greater cardiac inflammation, cell death, and adaptive angiogenesis compared to sham mice. Importantly, the TAC ?-CGRP KO mice had greater inflammation, cell death, and attenuation of angiogenesis compared to TAC WT hearts. Thus, ?-CGRP plays a significant protective role in TAC-induced heart failure which may be mediated by decreased inflammation, cell death, and fibrosis.
The tachykinin, substance P, is found primarily in sensory nerves. In the heart, substance P-containing nerve fibers are often found surrounding coronary vessels, making them ideally situated to sense changes in the myocardial environment. Recent studies in rodents have identified substance P as having dual roles in the heart, depending on disease etiology and/or timing. Thus far, these studies indicate that substance P may be protective acutely following ischemia-reperfusion, but damaging long-term in non-ischemic induced remodeling and heart failure. Sensory nerves may be at the apex of the cascade of events leading to heart failure, therefore, they make a promising potential therapeutic target that warrants increased investigation.
This review is focused on gender differences in cardiac remodeling secondary to sustained increases in cardiac volume (VO) and generated pressure (PO). Estrogen has been shown to favorably alter the course of VO-induced remodeling. That is, the VO-induced increased extracellular matrix proteolytic activity and mast cell degranulation responsible for the adverse cardiac remodeling in males and ovariectomized rodents do not occur in intact premenopausal females. While less is known regarding the mechanisms responsible for female cardioprotection in PO-induced stress, gender differences in remodeling have been reported indicating the ability of premenopausal females to adequately compensate. In view of the fact that, in male mice with PO, mast cells have been shown to play a role in the adverse remodeling suggests favorable estrogen modification of mast cell phenotype may also be responsible for cardioprotection in females with PO. Thus, while evidence is accumulating regarding premenopausal females being cardioprotected, there remains the need for in-depth studies to identify critical downstream molecular targets that are under the regulation of estrogen and relevant to cardiac remodeling. Such studies would result in the development of therapy which provides cardioprotection while avoiding the adverse effects of systemic estrogen delivery.
Inflammatory cells play a major role in the pathology of heart failure by stimulating cardiac fibroblasts to regulate the extracellular matrix in an adverse way. In view of the fact that inflammatory cells have estrogen receptors, we hypothesized that estrogen provides cardioprotection by decreasing the ability of cardiac inflammatory cells to influence fibroblast function.
Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases. These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase. These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts function. However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation. Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease. This method requires a familiarity with the anatomical location of the rats xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.
Previously, we have reported sex differences in the cardiac remodeling response to ventricular volume overload whereby male and ovariectomized (OVX) female rats develop eccentric hypertrophy, and intact (Int) female rats develop concentric hypertrophy. In males, this adverse remodeling has been attributed to an initial cascade of events involving myocardial mast cell and matrix metalloproteinase activation and extracellular collagen matrix degradation. The objective of this study was to determine the effect of female hormones on this initial cascade. Accordingly, an aortocaval fistula (Fist) was created in 7-wk-old Int and OVX rats, which, together with sham-operated (sham) controls, were studied at 1, 3, and 5 days postsurgery. In Int-Fist rats, myocardial mast cell density, collagen volume fraction, endothelin (ET)-1, stem cell factor (SCF), and TNF-? remained at control levels or were minimally elevated throughout the study period. This was not the case in the OVX-Fist group, where the initial response included significant increases in mast cell density, collagen degradation, ET-1, SCF, and TNF-?. These events in the OVX-Fist group were abolished by prefistula treatment with a mast cell stabilizer nedocromil. Of note was the observation that ET-1, TNF-?, SCF, and collagen volume fraction values for the OVX-sham group were greater than those of the Int-sham group, suggesting that the reduction of female hormones alone results in major myocardial changes. We concluded that female hormone-related cardioprotection to the volume stressed myocardium is the result of an altered mast cell phenotype and/or the prevention of mast cell activation.
Substance P and neurokinin A (NKA) are sensory nerve neuropeptides encoded by the TAC1 gene. Substance P is a mast cell secretagogue and mast cells are known to play a role in adverse myocardial remodelling. Therefore, we wondered whether substance P and/or NKA modulates myocardial remodelling via a mast cell-mediated mechanism.
The mast cell product, tryptase, has recently been implicated to mediate fibrosis in the hypertensive heart. Tryptase has been shown to mediate noncardiac fibroblast function via activation of protease-activated receptor 2 and subsequent activation of the mitogen-activated protein kinase pathway, including extracellular signal-regulated kinase 1/2. Therefore, we hypothesized that this pathway may be a mechanism leading to fibrosis in the hypertensive heart. Isolated adult cardiac fibroblasts were treated with tryptase, which induced activation of extracellular signal-regulated kinase 1/2 via protease-activated receptor 2. Blockade of protease activated receptor 2 with FSLLRY (10 ?mol/L) and inhibition of the extracellular signal-regulated kinase pathway with PD98059 (10 ?mol/L) prevented collagen synthesis in isolated cardiac fibroblasts stimulated with tryptase. In contrast, p38 mitogen-activated protein kinase and stress-activated protein/c-Jun N-terminal kinase were not activated by tryptase. Cardiac fibroblasts isolated from spontaneously hypertensive rats showed this same pattern of activation. Treatment of spontaneously hypertensive rats with FSLLRY prevented fibrosis in these animals, indicating the in vivo applicability of the cultured fibroblast findings. Also, tryptase induced a myofibroblastic phenotype indicated by elevations in ?-smooth muscle actin and extra type III domain A (ED-A) fibronectin. Thus, the results from this study demonstrate the importance of tryptase for inducing a cardiac myofibroblastic phenotype, ultimately leading to the development of cardiac fibrosis. Specifically, tryptase causes cardiac fibroblasts to increase collagen synthesis via a mechanism involving activation of protease-activated receptor 2 and subsequent induction of extracellular signal-regulated kinase signaling.
Cardiovascular remodeling leading to heart failure is common in the elderly. Testing effective pharmacological treatment of human heart failure requires a suitable animal model that adequately mimics the human disease state.
Increased numbers of mast cells have been reported in explanted human hearts with dilated cardiomyopathy and in animal models of experimentally induced hypertension, myocardial infarction, and chronic volume overload secondary to aortocaval fistula and mitral regurgitation. Accordingly, mast cells have been implicated to have a major role in the pathophysiology of these cardiovascular disorders. In vitro studies have verified that mast cell proteases are capable of activating collagenase, gelatinases and stromelysin. Recent results have shown that with chronic ventricular volume overload, there is an elevation in mast cell density, which is associated with a concomitant increase in matrix metalloproteinase (MMP) activity and extracellular matrix degradation. However, the role of the cardiac mast cell is not one dimensional, with evidence from hypertension and cardiac transplantation studies suggesting that they can also assume a pro-fibrotic phenotype in the heart. These adverse events do not occur in mast cell deficient rodents or when cardiac mast cells are pharmacologically prevented from degranulating. This review is focused on the regulation and dual roles of cardiac mast cells in: (i) activating MMPs and causing myocardial fibrillar collagen degradation and (ii) causing fibrosis in the stressed, injured or diseased heart. Moreover, there is strong evidence that premenopausal female cardioprotection may at least partly be due to gender differences in cardiac mast cells. This too will be addressed.
Although there is a correlation between hypertension and levels of interleukin (IL) 6, the exact role this cytokine plays in myocardial remodeling is unknown. This is complicated by the variable tissue and circulating levels of IL-6 reported in numerous experimental models of hypertension. Accordingly, we explored the hypothesis that elevated levels of IL-6 mediate adverse myocardial remodeling. To this end, adult male Sprague-Dawley rats were infused with IL-6 (2.5 microg . kg(-1) . h(-1), IP) for 7 days via osmotic minipump and compared with vehicle-infused, aged-matched controls. Left ventricular function was evaluated using a blood-perfused isolated heart preparation. Myocardial interstitial collagen volume fraction and isolated cardiomyocyte size were also assessed. Isolated adult cardiac fibroblast experiments were performed to determine the importance of the soluble IL-6 receptor in mediating cardiac fibrosis. IL-6 infusions in vivo resulted in concentric left ventricular hypertrophy, increased ventricular stiffness, a marked increase in collagen volume fraction (6.2% versus 1.7%; P<0.001), and proportional increases in cardiomyocyte width and length, all independent of blood pressure. The soluble IL-6 receptor in combination with IL-6 was found to be essential to producing increased collagen concentration by isolated cardiac fibroblasts and also played a role in mediating a phenotypic conversion to myofibroblasts. These novel observations demonstrate that IL-6 induces a myocardial phenotype almost identical to that of the hypertensive heart, identifying IL-6 as potentially important in this remodeling process.
TNF-alpha is known to cause adverse myocardial remodeling. While we have previously shown a role for cardiac mast cells in mediating increases in myocardial TNF-alpha, however, matrix metalloproteinase (MMP) activation of TNF-alpha may also be contributory. We sought to determine the relative roles of MMPs and cardiac mast cells in the activation of TNF-alpha in the hearts of rats subjected to chronic volume overload. Interventions with the broad spectrum MMP inhibitor, GM6001, or the mast cell stabilizer, nedocromil, were performed in the rat aortocaval fistula (ACF) model of volume overload. Myocardial TNF-alpha levels were significantly increased in the ACF. This increase was prevented by MMP inhibition with GM6001 (p< or =0.001 vs. ACF). Conversely, myocardial TNF-alpha levels were increased in the ACF+nedocromil treated fistula groups (p< or =0.001 vs. sham). The degradation of interstitial collagen volume fraction seen in the untreated ACF group was prevented in both the GM6001 and nedocromil treated hearts. Significant increases in LV myocardial ET-1 levels also occurred in the ACF group at 3days post-fistula. Whereas administration of GM6001 significantly attenuated this increase, mast cell stabilization with nedocromil markedly exacerbated the increase, producing ET-1 levels 6.5 fold and 2 fold greater than that in the sham-operated control and ACF group, respectively. The efficacy of the MMP inhibitor, GM6001, to prevent increased levels of myocardial TNF-alpha is indicative of MMP-mediated cleavage of latent extracellular membrane-bound TNF-alpha protein as the primary source of bioactive TNF-alpha in the myocardium of the volume overload heart.
Chronic activation of the sympathetic nervous system is a key component of cardiac hypertrophy and fibrosis. However, previous studies have provided evidence that also implicate inflammatory cells, including mast cells (MCs), in the development of cardiac fibrosis. The current study investigated the potential interaction of cardiac MCs with the sympathetic nervous system. Eight-week-old male spontaneously hypertensive rats were sympathectomized to establish the effect of the sympathetic nervous system on cardiac MC density, myocardial remodeling, and cytokine production in the hypertensive heart. Age-matched Wistar Kyoto rats served as controls. Cardiac fibrosis and hypertension were significantly attenuated and left ventricular mass normalized, whereas cardiac MC density was markedly increased in sympathectomized spontaneously hypertensive rats. Sympathectomy normalized myocardial levels of interferon-gamma, interleukin 6, and interleukin 10, but had no effect on interleukin 4. The effects of norepinephrine and substance P on isolated cardiac MC activation were investigated as potential mechanisms of interaction between the two. Only substance P elicited MC degranulation. Substance P was also shown to induce the production of angiotensin II by a mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes, and macrophages. These results demonstrate the ability of neuropeptides to regulate inflammatory cell function, providing a potential mechanism by which the sympathetic nervous system and afferent nerves may interact with inflammatory cells in the hypertensive heart.
Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine that has been implicated in the pathogenesis of heart failure. In contrast, we have recently shown that myocardial levels of TNF-alpha are acutely elevated in the aortocaval (AV) fistula model of heart failure. Based on these observations, we hypothesized that progression of adverse myocardial remodeling secondary to volume overload would be prevented by inhibition of TNF-alpha with etanercept. Furthermore, a principal objective of this study was to elucidate the effect of TNF-alpha inhibition during different phases of the myocardial remodeling process. Eight-week-old male Sprague-Dawley rats were randomly divided into the following three groups: sham-operated controls, untreated AV fistulas, and etanercept-treated AV fistulas. Each group was further subdivided to study three different time points consisting of 3 days, 3 wk, and 8 wk postfistula. Etanercept was administered subcutaneously at 1 mg/kg body wt. Etanercept prevented collagen degradation at 3 days and significantly attenuated the decrease in collagen at 8 wk postfistula. Although TNF-alpha antagonism did not prevent the initial ventricular dilatation at 3 wk postfistula, etanercept was effective at significantly attenuating the subsequent ventricular hypertrophy, dilatation, and increased compliance at 8 wk postfistula. These positive adaptations achieved with etanercept administration translated into significant functional improvements. At a cellular level, etanercept also markedly attenuated increases in cardiomyocyte length, width, and area at 8 wk postfistula. These observations demonstrate that TNF-alpha has a pivotal role in adverse myocardial remodeling and that treatment with etanercept can attenuate the progression to heart failure.
Correlative data suggest that cardiac mast cells are a component of the inflammatory response that is important to hypertension-induced adverse myocardial remodeling. However, a causal relationship has not been established. We hypothesized that adverse myocardial remodeling would be inhibited by preventing the release of mast cell products that may interact with fibroblasts and other inflammatory cells. Eight-week-old male spontaneously hypertensive rats were treated for 12 weeks with the mast cell stabilizing compound nedocromil (30 mg/kg per day). Age-matched Wistar-Kyoto rats served as controls. Nedocromil prevented left ventricular fibrosis in the spontaneously hypertensive rat independent of hypertrophy and blood pressure, despite cardiac mast cell density being elevated. The mast cell protease tryptase was elevated in the spontaneously hypertensive rat myocardium and was normalized by nedocromil. Treatment of isolated adult spontaneously hypertensive rat cardiac fibroblasts with tryptase induced collagen synthesis and proliferation, suggesting this as a possible mechanism of mast cell-mediated fibrosis. In addition, nedocromil prevented macrophage infiltration into the ventricle. The inflammatory cytokines interferon-gamma and interleukin (IL)-4 were increased in the spontaneously hypertensive rat and normalized by nedocromil, whereas IL-6 and IL-10 were decreased in the spontaneously hypertensive rat, with nedocromil treatment normalizing IL-6 and increasing IL-10 above the control. These results demonstrate for the first time a causal relationship between mast cell activation and fibrosis in the hypertensive heart. Furthermore, these results identify several mechanisms, including tryptase, inflammatory cell recruitment, and cytokine regulation, by which mast cells may mediate hypertension-induced left ventricular fibrosis.
In the abdominal aortocaval (AV) fistula model of heart failure, we have shown that the acute doubling of cardiac mature mast cell (MC) density involved the maturation, but not proliferation, of a resident population of immature cardiac MCs. An increase in stem cell factor (SCF) may be responsible for this MC maturation process. Thus, the purpose of this study was to determine if: 1) myocardial SCF levels are increased following the initiation of cardiac volume overload; 2) the incubation of left ventricular (LV) tissue slices with SCF results in an increase in mature MC density; and 3) chemical degranulation of mature cardiac MCs in LV tissue slices results in an increase in SCF and mature MC density via MC chymase. Male rats with either sham or AV fistula surgery were studied at 6h and 1 and 3 days post-surgery. LV slices from normal male rat hearts were incubated for 16h with media alone or media containing one of the following: 1) recombinant rat SCF (20 ng/ml) to determine the effects of SCF on MC maturation; 2) the MC secretagogue compound 48/80 (20 ?g/ml) to determine the effects of MC degranulation on SCF levels and mature MC density; 3) media containing compound 48/80 and anti-SCF (5 ?g/ml) to block the effects of SCF; 4) chymase (100 nM) to determine the effects of chymase on SCF; and 5) compound 48/80 and chymostatin (chymase inhibitor, 10 ?M) to block the effects of MC chymase. In AV fistula animals, myocardial SCF was significantly elevated above that in the sham group at 6h and 1 day post fistula by 2 and 1.8 fold, respectively, and then returned to normal by 3 days; this increase slightly preceded significant increases in MC density. Incubation of LV slices with SCF resulted in a doubling of mature MC density and this was concomitant with a significant decrease in the number of immature mast cells. Incubation of LV slices with compound 48/80 increased media SCF levels and mature MC density and with anti-SCF and chymostatin prevented these compound 48/80-induced increases. Incubation with chymase increased media SCF levels and mature MC density. These findings indicate that activated mature cardiac mast cells are responsible, in a paracrine fashion, for the increase in mature MC density post AV fistula by rapidly increasing SCF levels via the release of chymase.
Male, wild-type, FVB strain mice were fed a nutritionally complete liquid diet supplemented with 4% ethanol v/v over a time course of 1, 2, 4, 8, 12, and 14 weeks. Controls were offered an isocaloric liquid equivalent and pair fed with their ethanol counterparts. Changes in cardiac physiology were assessed at respective time points via echocardiography. Additionally, the use of histological techniques, mRNA analysis, apoptosis determination, and immunohistochemistry were employed to determine the functional and structural changes on the heart. Echocardiograph analysis revealed a compensatory phase that occurred early in the time course (1-8 weeks) and decompensation reverting toward heart failure at weeks 12 and 14. Throughout the study, an increase in cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis, TGF-?, and the presence of ?-SMA-positive cells were determined. A compensatory period in mice treated with ethanol occurred early followed by a transition to a dilated phenotype over time. A number of factors may be involved in this process including the activation of myofibroblasts and their fibrotic activities that is correlated with the presence of transforming growth factor beta.
Our laboratory has previously reported significant increases of the proinflammatory cytokine TNF-? in male hearts secondary to sustained volume overload. These elevated levels of TNF-? are accompanied by left ventricular (LV) dilatation and cardiac dysfunction. In contrast, estrogen has been shown to protect against this adverse cardiac remodeling in both female and male rats. The purpose of this study was to determine whether estrogen has an effect on inflammation-related genes that contribute to this estrogen-mediated cardioprotection. Myocardial volume overload was induced by aortocaval fistula in 8 wk old male Sprague-Dawley rats (n = 30), and genes of interest were identified using an inflammatory PCR array in Sham, Fistula, and Fistula + Estrogen-treated (0.02 mg/kg per day beginning 2 wk prior to fistula) groups. A total of 55 inflammatory genes were modified (?2-fold change) at 3 days postfistula. The number of inflammatory gene was reduced to 21 genes by estrogen treatment, whereas 13 genes were comparably modulated in both fistula groups. The most notable were TNF-?, which was downregulated by estrogen, and the TNF-? receptors, which were differentially regulated by estrogen. Specific genes related to arachidonic acid metabolism were downregulated by estrogen, including cyclooxygenase-1 and -2. Finally, gene expression for the ?1-integrin cell adhesion subunit was significantly upregulated in the LV of estrogen-treated animals. Protein levels reflected the changes observed at the gene level. These data suggest that estrogen provides its cardioprotective effects, at least in part, via genomic modulation of numerous inflammation-related genes.
The cytokine interferon-gamma (IFN-?) is the only known member of the type II family of interferons, and as such, binds to its own distinct receptor. It is important in host defense against infection, as well as adaptive immune responses. While a wide array of cytokines are known to be involved in adverse remodeling of the heart and the progression to heart failure, the role of IFN-? is unclear. Recent evidence from clinical studies, animal models of myocarditis and hypertension, as well as isolated cell studies, provide conflicting data as to whether IFN-? is pathological or protective in the heart. Thus, it is important to highlight these discrepant findings so that areas of future investigation can be identified to more clearly determine the precise role of IFN-? in the heart. Accordingly, this review will (1) discuss the source of IFN-? in the diseased heart; (2) summarize the data from animal studies; (3) discuss the effects of IFN-? on isolated cardiac fibroblasts and cardiomyocytes; (4) identify signaling mechanisms that may be invoked by IFN-? in the heart; and (5) present the clinical evidence supporting a role for IFN-? in heart failure.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.