Neutrophils infiltrate early in myocardial infarction (MI) to initiate necrotic debris removal by launching a proinflammatory response. The extent of cardiomyocyte necrosis within the first 24 h of MI establishes the degree of wall thinning that will occur in the infarct zone. As neutrophils coordinate early tissue remodeling by degranulating proteases to oversee tissue breakdown, we hypothesized neutrophil protein expression might mirror the extent of infarct wall thinning at MI day 1. We examined a dataset collected following permanent coronary artery ligation in 3-6 months old C57BL/6J male mice. The dataset included echocardiography assessment of wall thickness and aptamer-based proteomic evaluation of neutrophils isolated from the left ventricle of control day 0 (n=10) or MI day 1 (n=10) mice. The wall thinning index was calculated as 1/wall thickness; all analyses were performed as comparisons to infarct wall thickness at systole. By regression analysis of 123 neutrophil proteins to wall thinning index at both day 0 and MI day 1, there were 4 proteins positively associated, with all 4 proteins increasing with greater wall thinning. These were histone H1.2 (r=0.62, p=0.004), S100A9 (r=0.60, p=0.005), histone H3.1 (r=0.55, p=0.01), and fibrinogen (r=0.47, p=0.04). Our results reveal protein candidates that may be therapeutic targets to limit MI wall thinning.
Neutrophils are key effector cells of the innate immune system, serving as a first line of defense in the response to injury and playing essential roles in the wound healing process. Following myocardial infarction (MI), neutrophils infiltrate into the infarct region to propagate inflammation and begin the initial phase of cardiac wound repair. Pro-inflammatory neutrophils release proteases to degrade extracellular matrix (ECM), a necessary step for the removal of necrotic myocytes as a prelude for scar formation. Neutrophils transition their phenotype over time to regulate MI inflammation resolution and stabilize scar formation. Neutrophils contribute to the evolution from inflammation to resolution and scar formation by serving anti-inflammatory and repair functions. As anti-inflammatory cells, neutrophils contribute ECM proteins during scar formation, in particular fibronectin, galectin-3, and vimentin. The diverse and polarizing functions that contribute to MI wound repair make this innate immune cell a viable target to improve MI outcomes. Thus, understanding the signaling involved in neutrophil physiology in the context of MI may help to identify novel therapeutic targets.
Myocardial Infarction (MI) initiates a series of wound healing events that begins with up-regulation of an inflammatory response and culminates in scar formation. The extracellular matrix (ECM) is intricately involved in all stages from initial break down of existing ECM to synthesis of new ECM to form the scar. This review will summarize our current knowledge on the processes involved in ECM remodeling after MI and identify the gaps that still need to be filled.
Cardiac remodeling following myocardial infarction (MI) leads to structural and vascular changes in the myocardium. To better understand the mechanisms involved, we used photoacoustic ECG‐gated Kilohertz Visualization imaging (PA EKV) to measure oxygen saturation in the myocardium. Photoacoustic imaging is a noninvasive imaging technique that uses near‐infrared laser light to optically excite molecules in vivo . Oxygenated hemoglobin absorbs laser light at a different wavelength (850nm) from non‐oxygenated hemoglobin (750nm) which allows for the detection of the relative oxygen saturation (sO 2 ) of a tissue and the amount of hemoglobin present (HbT). In this study, photoacoustic imaging was combined with EKV imaging for continuous measurement of sO 2 and HbT throughout the contraction cycle. Imaging was done on C57Bl/6J mice (F, 4–5 months of age) to measure baseline sO 2 levels in the anterior and posterior myocardium. An MI was induced by ligation of the left anterior descending coronary artery. Mice were imaged at days 1, 7, and 14 after MI to measure changes in myocardial sO 2 . All data presented here are from diastole of the long axis orientation and similar results were obtained from short axis measurements. Oxygen saturation on the anterior myocardial wall decreased from 74.7% pre‐MI to 19.9% 1 day after MI (p<0.0001). Oxygen saturation showed recovery at 7 (54.9%, p=0.023) and 14 (49.9%, p=0.007) days post infarct, yet remained significantly reduced from pre‐MI levels. Oxygen saturation was lower on the posterior myocardium (57.2%) compared to the anterior myocardium but did not change following MI. Our results demonstrate oxygen saturation begins to recover 7 days after MI as the heart adapts to ligation and neoangiogenesis is stimulated. This study validates the use of PA EKV for the measurement of oxygen saturation in the infarcted myocardium. Support or Funding Information We acknowledge funding from National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505. The UNMC Ultrasound Core is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institution of Health under grant P30 GM127200.
Neutrophils infiltrate into the left ventricle (LV) early after myocardial infarction (MI) and launch a proinflammatory response. Along with neutrophil infiltration, LV wall thinning due to cardiomyocyte necrosis also peaks at
Myocardial infarction (MI) initiates an intense inflammatory response that induces neutrophil infiltration into the infarct region. Neutrophils commence the pro-inflammatory response that includes upregulation of cytokines and chemokines (e.g., interleukin-1 beta) and degranulation of pre-formed proteases (e.g., matrix metalloproteinases -8 and -9) that degrade existing extracellular matrix to clear necrotic tissue. An increase or complete depletion of neutrophils both paradoxically impair MI resolution, indicating a complex role of neutrophils in cardiac wound healing. Following pro-inflammation, the neutrophil shifts to a reparative phenotype that promotes inflammation resolution and aids in scar formation. Across the shifts in phenotype, the neutrophil communicates with other cells to coordinate repair and scar formation. This review summarizes our current understanding of neutrophil crosstalk with cardiomyocytes and macrophages during MI wound healing.
Approximately 50% of Americans have hypertension, which significantly increases the risk of heart failure. In response to increased peripheral resistance in hypertension, intensified mechanical stretch in the myocardium induces cardiomyocyte hypertrophy and fibroblast activation to withstand increased pressure overload. This changes the structure and function of the heart, leading to pathological cardiac remodeling and eventual progression to heart failure. In the presence of hypertensive stimuli, cardiac fibroblasts activate and differentiate to myofibroblast phenotype capable of enhanced extracellular matrix secretion in coordination with other cell types, mainly cardiomyocytes. Both systemic and local renin-angiotensin-aldosterone system activation lead to increased angiotensin II stimulation of fibroblasts. Angiotensin II directly activates fibrotic signaling such as transforming growth factor β/SMAD and mitogen-activated protein kinase (MAPK) signaling to produce extracellular matrix comprised of collagens and matricellular proteins. With the advent of single-cell RNA sequencing techniques, heterogeneity in fibroblast populations has been identified in the left ventricle in models of hypertension and pressure overload. The various clusters of fibroblasts reveal a range of phenotypes and activation states. Select antihypertensive therapies have been shown to be effective in limiting fibrosis, with some having direct actions on cardiac fibroblasts. The present review focuses on the fibroblast-specific changes that occur in response to hypertension and pressure overload, the knowledge gained from single-cell analyses, and the effect of antihypertensive therapies. Understanding the dynamics of hypertensive fibroblast populations and their similarities and differences by sex is crucial for the advent of new targets and personalized medicine.
Inflammation presides early after myocardial infarction (MI) as a key event in cardiac wound healing. Ischemic cardiomyocytes secrete inflammatory cues to stimulate infiltration of leukocytes, predominantly macrophages and neutrophils. Infiltrating neutrophils degranulate to release a series of proteases including matrix metalloproteinase (MMP)-9 to break down extracellular matrix and remove necrotic myocytes to create space for the infarct scar to form. While neutrophil to macrophage communication has been explored, the reverse has been understudied. We used a proteomics approach to catalogue the macrophage secretome at MI day 1. Murinoglobulin-1 (MUG1) was the highest-ranked secreted protein (4.1-fold upregulated at MI day 1 vs. day 0 pre-MI cardiac macrophages, p = 0.004). By transcriptomics evaluation, galectin-3 (Lgals3) was 2.2-fold upregulated (p = 0.008) in MI day 1 macrophages. We explored the direct roles of MUG1 and Lgals3 on neutrophil degranulation. MUG1 blunted while Lgals3 amplified neutrophil degranulation in response to phorbol 12-myristate 13-acetate or interleukin-1β, as measured by MMP-9 secretion. Lgals3 itself also stimulated MMP-9 secretion. To determine if MUG1 regulated Lgals3, we co-stimulated neutrophils with MUG1 and Lgals3. MUG1 limited degranulation stimulated by Lgals3 by 64% (p < 0.001). In vivo, MUG1 was elevated in the infarct region at MI days 1 and 3, while Lgals3 increased at MI day 7. The ratio of MUG1 to Lgals3 positively correlated with infarct wall thickness, revealing that MUG1 attenuated infarct wall thinning. In conclusion, macrophages at MI day 1 secrete MUG1 to limit and Lgals3 to accentuate neutrophil degranulation to regulate infarct wall thinning.
Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While IL‐4 is an in vitro stimulus for anti‐inflammation, the MI myocardium does not express IL‐4. We hypothesized that continuous exogenous IL‐4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. C57BL/6J male mice (3–6 months of age) were subcutaneously infused with either saline (n=17) or IL‐4 (20 ng/g/day; n=17) and evaluated at MI day 3. Macrophages and neutrophils were isolated ex vivo from the infarct region and examined for pro‐inflammatory genes (Ccl3, Ccl5, Il1b, Il12a, and Tnfa) and anti‐inflammatory genes (Arg1, Ym1, Mrc1, Il10, and Tgfb, Il6). Exogenous IL‐4 decreased pro‐inflammatory Ccl3, IL‐12a, Tnfa, and Tgfb1 in neutrophils while increasing anti‐inflammatory Arg1 and Ym1 in macrophages (all p<0.05); none of the other markers were different. Tissue clearance by IL‐4 treated neutrophils was not different, while phagocytosis of neutrophils, but not myocytes, doubled in IL‐4 treated macrophages (p<0.05). Because macrophage cell physiology was influenced by IL‐4, whole transcriptome analysis of macrophages isolated from the infarct regions of IL‐4 (n=10) or saline (n=11) treated mice was performed by RNA‐sequencing. Of the 24,341 genes sequenced, 2,042 genes were differentially expressed with IL‐4 stimulation (all p<0.05). Pdgfc gene expression was ranked first by p value, increasing from 1.8±0.3 in saline treated macrophages to 5.4±0.2 FPKM units in macrophages stimulated with IL‐4 (p=1×10 −9 ). Importantly, changes in macrophage physiology and transcriptome occurred before differences in cardiac physiology evaluated by echocardiography, which allowed the isolation of effects in cell phenotype not induced by global myocardial effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF‐CC protein (10 μg/ml) or PDGF‐CC blocking antibody (10 μg/ml) revealed no effect on Arg1 or Ym1 expression, indicating the in vivo effect of IL‐4 to stimulate macrophage gene expression was independent of PDGF‐CC. Altogether, our results indicate that exogenous IL‐4 promotes inflammation resolution by turning off pro‐inflammation in neutrophils while stimulating anti‐inflammation in macrophages to induce neutrophil phagocytosis. Support or Funding Information National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505
