Survival following myocardial infarction (MI) has improved substantially over the last 40 years; however, the incidence of subsequent congestive heart failure has dramatically increased as a consequence. Discovering plasma markers that signify adverse cardiac remodeling may allow high-risk patients to be recognized earlier and may provide an improved way to assess treatment efficacy. Alterations in extracellular matrix (ECM) regulate cardiac remodeling following MI and potentially provide a large array of candidate indicators.
The field of cardiac proteomics has progressed rapidly over the past 20 years, since publication of the first two-dimensional electrophoretic gels of left ventricle proteins. Proteomic approaches are now routinely utilized to better understand how the left ventricle responds to injury.
In this review, we will discuss how methods have developed to allow comprehensive evaluation of the ECM proteome. We will explain how ECM proteomic data can be used to predict adverse remodeling for an individual patient and highlight future directions. Although this review will focus on the use of ECM proteomics to better understand post-MI remodeling responses, these approaches have applicability to a wide-range of cardiac pathologies, including pressure overload hypertrophy, viral myocarditis, and non-ischemic heart failure.
proteomics; remodeling; myocardial infarction; biomarker; MMP
The cardiac extracellular matrix (ECM) provides a platform for cells to maintain structure and function, which in turn maintains tissue function. In response to injury, the ECM undergoes remodeling that involves synthesis, incorporation, and degradation of matrix proteins, with the net outcome determined by the balance of these processes. The major goals of this review are a) to serve as an initial resource for students and investigators new to the cardiac ECM remodeling field, and b) to highlight a few of the key exciting avenues and methodologies that have recently been explored. While we focus on cardiac injury and responses of the left ventricle (LV), the mechanisms reviewed here have pathways in common with other wound healing models.
extracellular matrix; left ventricular remodeling; matrix metalloproteinases; inflammation; fibrosis; review
MMP-9 deletion has been shown to improve remodeling of the left ventricle (LV) post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48±3 in wt and 45±3% in null), indicating that both groups received an equal injury stimulus. LV infarct tissue was homogenized and analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix (ECM) proteins were identified in these spots by mass spectrometry, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.
cardiac remodeling; MMP-9; extracellular matrix; mice; proteomics; myocardial infarction
The concept that extracellular matrix (ECM) turnover occurs during cardiac remodeling is a well-accepted paradigm. To date, a multitude of studies document that remodeling is accompanied by increases in the synthesis and deposition of ECM components as well as increases in extracellular proteases, especially matrix metalloproteinases (MMPs), which break down ECM components. Further, soluble ECM fragments generated from enzymatic action serve to stimulate cell behavior and have been proposed as candidate plasma biomarkers of cardiac remodeling. This review briefly summarizes our current knowledge base on cardiac ECM turnover following myocardial infarction (MI), but more importantly extends discussion by defining avenues that remain to be explored to drive the ECM remodeling field forward. Specifically, this review will discuss cause and effect roles for the ECM changes observed following MI and the potential role of the ECM changes that may serve as trigger points to regulate remodeling. While the pattern of remodeling following MI is qualititatively similar but quantitively different from various types of injury, the basic theme in remodeling is repeated. Therefore, while we use the MI model as the prototype injury model, the themes discussed here are also relevant to cardiac remodeling due to other types of injury.
LIM and cysteine-rich domains 1; cardiac hypertrophy; editorial
editorial; diastolic compliance; PKC
hypertrophy; tumor necrosis factor α converting enzyme; matrix metalloproteinase; editorial
Following myocardial infarction (MI), circulating blood monocytes respond to chemotactic factors, migrate into the infarcted myocardium, and differentiate into macrophages. At the injury site, macrophages remove necrotic cardiac myocytes and apoptotic neutrophils; secrete cytokines, chemokines, and growth factors; and modulate phases of the angiogenic response. As such, the macrophage is a primary responder cell type that is involved in the regulation of post-MI wound healing at multiple levels. This review summarizes what is currently known about macrophage functions post-MI and borrows literature from other injury and inflammatory models to speculate on additional roles. Basic science and clinical avenues that remain to be explored are also discussed.
macrophage; myocardial infarction; matrix metalloproteinases; left ventricular remodeling; angiogenesis; fibrosis
hypertrophy; congestive heart failure; 5′-AMP activated protein kinase; editorial
angiotensin converting enzyme inhibitors; matrix metalloproteinase-9; editorial
Polymorphonuclear granulocytes (PMNs; neutrophils) serve as key effector cells in the innate immune system and provide the first line of defense against invading microorganisms. In addition to producing inflammatory cytokines and chemokines and undergoing a respiratory burst that stimulates the release of reactive oxygen species, PMNs also degranulate to release components that kill pathogens. Recently, neutrophil extracellular traps have been shown to be an alternative way to trap microorganisms and contain infection. PMN-derived granule components are also involved in multiple non-infectious inflammatory processes, including the response to myocardial infarction (MI). In this review, we will discuss the biological characteristics, recruitment, activation, and removal of PMNs, as well as the roles of PMN-derived granule proteins in inflammation and innate immunity, focusing on the MI setting when applicable. We also discuss future perspectives that will direct research in PMN biology.
PMNs; Myocardial infarction; Inflammation; Innate immunity; Degranulation; Matrix metalloproteinases
The naked mole rat is an extremely long-lived (>31 years) small (35 g) rodent. Moreover, it maintains good health for most of its long life. We hypothesized that naked mole rats also show attenuated cardiac aging. With age, cardiac muscle can become less compliant, causing a decline in early diastolic filling (E) and a compensatory increase in atrial contraction-induced late filling (A). This results in decreased left ventricular E/A ratio. Doppler imaging showed no significant differences in E/A ratios (p = .48) among old (18–20 years) breeders and nonbreeders despite differences in estrogen levels. A cross-sectional study of 1- to 20-year-old naked mole rats (n = 76) revealed that E/A ratios declined with age in females (n = 40; p = .002) but not in males (n = 36; p = 0.45). Despite this, neither gender shows increased morbidity or mortality with age. These findings suggest that, notwithstanding the previously observed high lipid peroxidation in heart tissue, NMRs must possess mechanisms to stave off progression to fatal cardiac disease.
Left ventricular function; Diastolic dysfunction; Naked mole rat; Hypogonadic; Cardiac imaging
Editorial; Extracellular Matrix Proteomics; Extracellular Matrix; Ischemia-Reperfusion; Proteomics
Following a myocardial infarction (MI), the homeostatic balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) is disrupted as part of the left ventricle (LV) response to injury. The full complement of responses to MI has been termed LV remodeling and includes changes in LV size, shape and function. The following events encompass the LV response to MI: 1) inflammation and LV wall thinning and dilation, 2) infarct expansion and necrotic myocyte resorption, 3) accumulation of fibroblasts and scar formation, and 4) endothelial cell activation and neovascularization.1, 2 In this review, we will summarize MMP and TIMP roles during these events, focusing on the spatiotemporal localization and MMP and TIMP effects on cellular and tissue-level responses. We will review MMP and TIMP structure and function, and discuss specific MMP roles during both the acute and chronic phases post-MI, which may provide insight into novel therapeutic targets to limit adverse remodeling in the MI setting.
myocardial infarction; left ventricular remodeling; matrix metalloproteinases; tissue inhibitors of metalloproteinases; review
Cardiac aging is characterized by diastolic dysfunction of the left ventricle (LV), which is due in part to increased LV wall stiffness. In the diastolic phase, myocytes are relaxed and extracellular matrix (ECM) is a critical determinant to the changes of LV wall stiffness. To evaluate the effects of ECM composition on cardiac aging, we developed a mathematical model to predict LV dimension and wall stiffness changes in aging mice by integrating mechanical laws and our experimental results. We measured LV dimension, wall thickness, LV mass, and collagen content for wild type (WT) C57/BL6J mice of ages ranging from 7.3 months to those of 34.0 months. The model was established using the thick wall theory and stretch-induced tissue growth to an isotropic and homogeneous elastic composite with mixed constituents. The initial conditions of the simulation were set based on the data from the young mice. Matlab simulations of this mathematical model demonstrated that the model captured the major features of LV remodeling with age and closely approximated experimental results. Specifically, the temporal progression of the LV interior and exterior dimensions demonstrated the same trend and order-of-magnitude change as our experimental results. In conclusion, we present here a validated mathematical model of cardiac aging that applies the thick-wall theory and stretch-induced tissue growth to LV remodeling with age.
About 6 million Americans suffer from heart failure and 70% of heart failure cases are caused by myocardial infarction (MI). Following myocardial infarction, increased cytokines induce two major types of macrophages: classically activated macrophages which contribute to extracellular matrix destruction and alternatively activated macrophages which contribute to extracellular matrix construction. Though experimental results have shown the transitions between these two types of macrophages, little is known about the dynamic progression of macrophages activation. Therefore, the objective of this study is to analyze macrophage activation patterns post-MI.
We have collected experimental data from adult C57 mice and built a framework to represent the regulatory relationships among cytokines and macrophages. A set of differential equations were established to characterize the regulatory relationships for macrophage activation in the left ventricle post-MI based on the physical chemistry laws. We further validated the mathematical model by comparing our computational results with experimental results reported in the literature. By applying Lyaponuv stability analysis, the established mathematical model demonstrated global stability in homeostasis situation and bounded response to myocardial infarction.
We have established and validated a mathematical model for macrophage activation post-MI. The stability analysis provided a possible strategy to intervene the balance of classically and alternatively activated macrophages in this study. The results will lay a strong foundation to understand the mechanisms of left ventricular remodelling post-MI.
We have previously shown that cardiac sarcopenia occurs with age in C57/BL6J mice. However, underlying mechanisms and plasma biomarkers of cardiac aging have not been identified. Accordingly, the objective of this study was to identify and evaluate plasma biomarkers that reflect cardiac aging phenotypes.
Methods and Results
Plasma from adult (7.5±0.5 months old, n=27) and senescent (31.7±0.5 months old, n=25) C57/BL6J mice was collected and levels of 69 markers were measured by multi-analyte profiling. Of these, 26 analytes were significantly increased and 3 were significantly decreased in the senescent group compared to the adult group. The majority of analytes that increased in the senescent group were inflammatory markers associated with macrophage functions, including matrix metalloproteinase-9 (MMP-9) and monocyte chemotactic protein-1 (MCP-1/CCL-2). Immunoblotting (n=12/ group) showed higher MMP-9 and MCP-1 levels in the left ventricle (LV) of senescent mice (p<0.05), and their expression levels in the LV correlated with plasma levels (rho=0.50 for MMP-9 and rho=0.62 for MCP1, p<0.05). Further, increased plasma MCP-1 and MMP-9 levels correlated with the increase in end diastolic dimensions that occurs with senescence. Immunohistochemistry (n=3/ group) for Mac-3, a macrophage marker, showed increased macrophage densities in the senescent LV; and dual labeling immunohistochemistry of Mac-3 and MMP-9 revealed robust co-localization of MMP-9 to the macrophages in the senescent LV sections, indicating that the macrophage is a major contributor of MMP-9 in the senescent LV.
Our results suggest that MCP-1 and MMP-9 are potential plasma markers for cardiac aging and that augmented MCP-1 and MMP-9 levels and macrophage content in the LV could provide an underlying inflammatory mechanism of cardiac aging.
aging; biomarker; macrophage; metalloproteinase; myocardium
Following myocardial infarction (MI), matrix metalloproteinase-9 (MMP-9) levels increase, and MMP-9 deletion improves post-MI remodeling of the left ventricle (LV). We provide here a technical report on plasma-analysis from wild type (WT) and MMP-9 null mice using fractionation and mass-spectrometry-based proteomics. MI was induced by coronary artery ligation in male WT and MMP-9 null mice (4–8 months old; n = 3/genotype). Plasma was collected on days 0 (pre-) and 1 post-MI. Plasma proteins were fractionated and proteins in the lowest (fraction 1) and highest (fraction 12) molecular weight fractions were separated by 1-D SDS-PAGE, digested in-gel with trypsin and analyzed by HPLC-ESI-MS/MS on an Orbitrap Velos. We tried five different fractionation protocols, before reaching an optimized protocol that allowed us to identify over 100 proteins. Serum amyloid A substantially increased post-MI in both genotypes, while alpha-2 macroglobulin increased only in the null samples. In fraction 12, extracellular matrix proteins were observed only post-MI. Interestingly, fibronectin-1, a substrate of MMP-9, was identified at both day 0 and day 1 post-MI in the MMP-9 null mice but was only identified post-MI in the WT mice. In conclusion, plasma fractionation offers an improved depletion-free method to evaluate plasma changes following MI.
Clinical studies suggest that rosiglitazone (RSG) treatment may increase the incidence of heart failure in diabetic patients. In this study, we examined whether a high corn oil diet with RSG treatment in insulin resistant aging mice exerted metabolic and pro-inflammatory effects that stimulate cardiac dysfunction. We also evaluated whether fish oil attenuated these effects. Female C57BL/6J mice (13 months old) were divided into 5 groups: (1) lean control (LC), (2) corn oil, (3) fish oil, (4) corn oil + RSG and (5) fish oil + RSG. Mice fed a corn oil enriched diet and RSG developed hypertrophy of the left ventricle (LV) and decreased fractional shortening, despite a significant increase in total body lean mass. In contrast, LV hypertrophy was prevented in RSG treated mice fed a fish oil enriched diet. Importantly, hyperglycemia was controlled in both RSG groups. Further, fish oil + RSG decreased LV expression of atrial and brain natriuretic peptides, fibronectin and the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α, concomitant with increased interleukin-10 and adiponectin levels compared to the corn oil + RSG group. Fish oil + RSG treatment suppressed inflammation, increased serum adiponectin, and improved fractional shortening, attenuating the cardiac remodeling seen in the corn oil + RSG diet fed C57BL/6J insulin resistant aging mice. Our results suggest that RSG treatment has context-dependent effects on cardiac remodeling and serves a negative cardiac role when given with a corn oil enriched diet.
Aging; Corn oil; Fish oil; Cardiac remodeling; Inflammation; Left ventricle hypertrophy; Rosiglitazone
Classic therapeutics for ischemic heart disease are less effective in individuals with the metabolic syndrome. As the prevalence of the metabolic syndrome is increasing, better understanding of cardiac metabolism is needed to identify potential new targets for therapeutic intervention. Thioredoxin-interacting protein (Txnip) is a regulator of metabolism and an inhibitor of the antioxidant thioredoxins, but little is known about its roles in the myocardium. We examined hearts from Txnip-KO mice by polony multiplex analysis of gene expression and an independent proteomic approach; both methods indicated suppression of genes and proteins participating in mitochondrial metabolism. Consistently, Txnip-KO mitochondria were functionally and structurally altered, showing reduced oxygen consumption and ultrastructural derangements. Given the central role that mitochondria play during hypoxia, we hypothesized that Txnip deletion would enhance ischemia-reperfusion damage. Surprisingly, Txnip-KO hearts had greater recovery of cardiac function after an ischemia-reperfusion insult. Similarly, cardiomyocyte-specific Txnip deletion reduced infarct size after reversible coronary ligation. Coordinated with reduced mitochondrial function, deletion of Txnip enhanced anaerobic glycolysis. Whereas mitochondrial ATP synthesis was minimally decreased by Txnip deletion, cellular ATP content and lactate formation were higher in Txnip-KO hearts after ischemia-reperfusion injury. Pharmacologic inhibition of glycolytic metabolism completely abolished the protection afforded the heart by Txnip deficiency under hypoxic conditions. Thus, although Txnip deletion suppresses mitochondrial function, protection from myocardial ischemia is enhanced as a result of a coordinated shift to enhanced anaerobic metabolism, which provides an energy source outside of mitochondria.
The availability of temporal measurements on biological experiments has significantly promoted research areas in systems biology. To gain insight into the interaction and regulation of biological systems, mathematical frameworks such as ordinary differential equations have been widely applied to model biological pathways and interpret the temporal data. Hill equations are the preferred formats to represent the reaction rate in differential equation frameworks, due to their simple structures and their capabilities for easy fitting to saturated experimental measurements. However, Hill equations are highly nonlinearly parameterized functions, and parameters in these functions cannot be measured easily. Additionally, because of its high nonlinearity, adaptive parameter estimation algorithms developed for linear parameterized differential equations cannot be applied. Therefore, parameter estimation in nonlinearly parameterized differential equation models for biological pathways is both challenging and rewarding. In this study, we propose a Bayesian parameter estimation algorithm to estimate parameters in nonlinear mathematical models for biological pathways using time series data.
We used the Runge-Kutta method to transform differential equations to difference equations assuming a known structure of the differential equations. This transformation allowed us to generate predictions dependent on previous states and to apply a Bayesian approach, namely, the Markov chain Monte Carlo (MCMC) method. We applied this approach to the biological pathways involved in the left ventricle (LV) response to myocardial infarction (MI) and verified our algorithm by estimating two parameters in a Hill equation embedded in the nonlinear model. We further evaluated our estimation performance with different parameter settings and signal to noise ratios. Our results demonstrated the effectiveness of the algorithm for both linearly and nonlinearly parameterized dynamic systems.
Our proposed Bayesian algorithm successfully estimated parameters in nonlinear mathematical models for biological pathways. This method can be further extended to high order systems and thus provides a useful tool to analyze biological dynamics and extract information using temporal data.
Exercise has been shown to improve function of the left ventricle (LV) following myocardial infarction (MI). The mechanisms to explain this benefit have not been fully delineated, but may involve improved mechanics resulting in unloading effects and increased endothelial nitric oxide synthase levels.[1, 2] Accordingly, the goal of this study was to determine how the LV infarct proteome is altered by a post-MI exercise regimen. Sprague-Dawley rats underwent ligation of the left descending coronary artery to induce MI. Exercise training was initiated four weeks post-MI and continued for 8 weeks in n=12 rats. Compared with the sedentary MI group (n=10), the infarct region of rats receiving exercise showed 20 protein spots with altered intensities in two-dimensional gels (15 increased and 5 decreased; p<0.05). Of 52 proteins identified in 20 spots, decreased levels of voltage-dependent anion-selective channel 2 and increased levels of glutathione perioxidase and manganese superoxide were confirmed by immunoblotting. Cardiac function was preserved in rats receiving exercise training, and the beneficial effect was linked with changes in these 3 proteins. In conclusion, our results suggest that post-MI exercise training increases anti-oxidant levels and decreases ion channel levels, which may explain, in part, the improved cardiac function seen with exercise.
myocardial infarction; exercise; proteomics; anti-oxidant; two-dimensional gel electrophoresis; left ventricular remodeling