Jin Endo

Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction

Acute myocardial infarction (MI) causes sterile inflammation, which is characterized by recruitment and activation of innate and adaptive immune system cells. Here we delineate the temporal dynamics of immune cell accumulation following MI by flow cytometry. Neutrophils increased immediately to a peak at 3 days post-MI. Macrophages were numerically the predominant cells infiltrating the infarcted myocardium, increasing in number over the first week post-MI. Macrophages are functionally heterogeneous, whereby the first responders exhibit high expression levels of proinflammatory mediators, while the late responders express high levels of the anti-inflammatory cytokine IL-10; these macrophages can be classified into M1 and M2 macrophages, respectively, based on surface-marker expression. M1 macrophages dominated at 1-3 days post-MI, whereas M2 macrophages represented the predominant macrophage subset after 5 days. The M2 macrophages expressed high levels of reparative genes in addition to proinflammatory genes to the same levels as in M1 macrophages. The predominant subset of dendritic cells (DCs) was myeloid DC, which peaked in number on day 7. Th1 and regulatory T cells were the predominant subsets of CD4(+) T cells, whereas Th2 and Th17 cells were minor populations. CD8(+) T cells, γδT cells, B cells, natural killer (NK) cells and NKT cells peaked on day 7 post-MI. Timely reperfusion reduced the total number of leukocytes accumulated in the post-MI period, shifting the peak of innate immune response towards earlier and blunting the wave of adaptive immune response. In conclusion, these results provide important knowledge necessary for developing successful immunomodulatory therapies.

Metabolic Remodeling Induced by Mitochondrial Aldehyde Stress Stimulates Tolerance to Oxidative Stress in the Heart

Rationale: Aldehyde accumulation is regarded as a pathognomonic feature of oxidative stress–associated cardiovascular disease. Objective: We investigated how the heart compensates for the accelerated accumulation of aldehydes. Methods and Results: Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2&agr; phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress–resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.) Conclusions: The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione–redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.

Cardioprotective mechanism of omega-3 polyunsaturated fatty acids

Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid and docosahexaenoic acid, are widely regarded as cardioprotective. Several large-scale, randomized clinical trials have shown that dietary intake of omega-3 PUFAs improves the prognosis of patients with symptomatic heart failure or recent myocardial infarction. Therefore, dietary consumption of omega-3 PUFA is recommended in international guidelines for the general population to prevent the occurrence of cardiovascular diseases (CVDs). However, the precise mechanisms underlying the cardioprotective effects of omega-3 PUFAs are not fully understood. Omega-3 PUFAs can be incorporated into the phospholipid bilayer of cell membranes and can affect membrane fluidity, lipid microdomain formation, and signaling across membranes. Omega-3 PUFAs also modulate the function of membrane ion channels, such as Na and L-type Ca channels, to prevent lethal arrhythmias. Moreover, omega-3 PUFAs also prevent the conversion of arachidonic acid into pro-inflammatory eicosanoids by serving as an alternative substrate for cyclooxygenase or lipoxygenase, resulting in the production of less potent products. In addition, a number of enzymatically oxygenated metabolites derived from omega-3 PUFAs were recently identified as anti-inflammatory mediators. These omega-3 metabolites may contribute to the beneficial effects against CVDs that are attributed to omega-3 PUFAs.

Administration of granulocyte colony-stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair.

The administration of granulocyte colony‐stimulating factor (G‐CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)‐derived c‐kit+ cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM‐derived hematopoietic cells mobilized by G‐CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)‐transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G‐CSF for 10 days. In the acute phase, a number of GFP+ cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and α‐smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G‐CSF administration, and the enhanced myofibroblast‐rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G‐CSF‐mobilized monocytes were harvested from the peripheral blood of GFP‐transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP+ myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G‐CSF promotes this recruitment and enhances cardiac protection. Disclosure of potential conflicts of interest is found at the end of this article.

4-hydroxy-2-nonenal protects against cardiac ischemia-reperfusion injury via the Nrf2-dependent pathway.

Reactive oxygen species (ROS) attack polyunsaturated fatty acids of the membrane and trigger lipid peroxidation, which results in the generation of alpha,beta-unsaturated aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). There is compelling evidence that high concentrations of aldehydes are responsible for much of the damage elicited by cardiac ischemia-reperfusion injury, while sublethal concentrations of aldehydes stimulate stress resistance pathways, to achieve cardioprotection. We investigated the mechanism of cardioprotection mediated by 4-HNE. For cultured cardiomyocytes, 4-HNE was cytotoxic at higher concentrations (>or=20 microM) but had no appreciable cytotoxicity at lower concentrations. Notably, a sublethal concentration (5muM) of 4-HNE primed cardiomyocytes to become resistant to cytotoxic concentrations of 4-HNE. 4-HNE induced nuclear translocation of transcription factor NF-E2-related factor 2 (Nrf2), and enhanced the expression of gamma-glutamylcysteine ligase (GCL) and the core subunit of the Xc(-) high-affinity cystine transporter (xCT), thereby increasing 1.45-fold the intracellular GSH levels. Cardiomyocytes treated with either Nrf2-specific siRNA or the GCL inhibitor l-buthionine sulfoximine (BSO) were less tolerant to 4-HNE. Moreover, the cardioprotective effect of 4-HNE pretreatment against subsequent glucose-free anoxia followed by reoxygenation was completely abolished in these cells. Intravenous administration of 4-HNE (4 mg/kg) activated Nrf2 in the heart and increased the intramyocardial GSH content, and consequently improved the functional recovery of the left ventricle following ischemia-reperfusion in Langendorff-perfused hearts. This cardioprotective effect of 4-HNE was not observed for Nrf2-knockout mice. In summary, 4-HNE activates Nrf2-mediated gene expression and stimulates GSH biosynthesis, thereby conferring on cardiomyocytes protection against ischemia-reperfusion injury.

Heart failure causes cholinergic transdifferentiation of cardiac sympathetic nerves via gp130-signaling cytokines in rodents

Although several cytokines and neurotrophic factors induce sympathetic neurons to transdifferentiate into cholinergic neurons in vitro, the physiological and pathophysiological roles of this remain unknown. During congestive heart failure (CHF), sympathetic neural tone is upregulated, but there is a paradoxical reduction in norepinephrine synthesis and reuptake in the cardiac sympathetic nervous system (SNS). Here we examined whether cholinergic transdifferentiation can occur in the cardiac SNS in rodent models of CHF and investigated the underlying molecular mechanism(s) using genetically modified mice. We used Dahl salt-sensitive rats to model CHF and found that, upon CHF induction, the cardiac SNS clearly acquired cholinergic characteristics. Of the various cholinergic differentiation factors, leukemia inhibitory factor (LIF) and cardiotrophin-1 were strongly upregulated in the ventricles of rats with CHF. Further, LIF and cardiotrophin-1 secreted from cultured failing rat cardiomyocytes induced cholinergic transdifferentiation in cultured sympathetic neurons, and this process was reversed by siRNAs targeting Lif and cardiotrophin-1. Consistent with the data in rats, heart-specific overexpression of LIF in mice caused cholinergic transdifferentiation in the cardiac SNS. Further, SNS-specific targeting of the gene encoding the gp130 subunit of the receptor for LIF and cardiotrophin-1 in mice prevented CHF-induced cholinergic transdifferentiation. Cholinergic transdifferentiation was also observed in the cardiac SNS of autopsied patients with CHF. Thus, CHF causes target-dependent cholinergic transdifferentiation of the cardiac SNS via gp130-signaling cytokines secreted from the failing myocardium.

Glucocorticoid protects rodent hearts from ischemia/reperfusion injury by activating lipocalin-type prostaglandin D synthase–derived PGD2 biosynthesis

Lipocalin-type prostaglandin D synthase (L-PGDS), which was originally identified as an enzyme responsible for PGD2 biosynthesis in the brain, is highly expressed in the myocardium, including in cardiomyocytes. However, the factors that control expression of the gene encoding L-PGDS and the pathophysiologic role of L-PGDS in cardiomyocytes are poorly understood. In the present study, we demonstrate that glucocorticoids, which act as repressors of prostaglandin biosynthesis in most cell types, upregulated the expression of L-PGDS together with cytosolic calcium-dependent phospholipase A2 and COX2 via the glucocorticoid receptor (GR) in rat cardiomyocytes. Accordingly, PGD2 was the most prominently induced prostaglandin in vivo in mouse hearts and in vitro in cultured rat cardiomyocytes after exposure to GR-selective agonists. In isolated Langendorff-perfused mouse hearts, dexamethasone alleviated ischemia/reperfusion injury. This cardioprotective effect was completely abrogated by either pharmacologic inhibition of COX2 or disruption of the gene encoding L-PGDS. In in vivo ischemia/reperfusion experiments, dexamethasone reduced infarct size in wild-type mice. This cardioprotective effect of dexamethasone was markedly reduced in L-PGDS-deficient mice. In cultured rat cardiomyocytes, PGD2 protected against cell death induced by anoxia/reoxygenation via the D-type prostanoid receptor and the ERK1/2-mediated pathway. Taken together, these results suggest what we believe to be a novel interaction between glucocorticoid-GR signaling and the cardiomyocyte survival pathway mediated by the arachidonic acid cascade.

Intramolecular control of protein stability, subnuclear compartmentalization, and coactivator function of peroxisome proliferator-activated receptor γ coactivator 1α

Peroxisome proliferator-activated receptor γ coactivator (PGC)-1 is a critical transcriptional regulator of energy metabolism. Here we found that PGC-1α is a short lived and aggregation-prone protein. PGC-1α localized throughout the nucleoplasm and was rapidly destroyed via the ubiquitin-proteasome pathway. Upon proteasome inhibition, PGC-1α formed insoluble polyubiquitinated aggregates. Ubiquitination of PGC-1α depended on the integrity of the C terminus-containing arginine-serine-rich domains and an RNA recognition motif. Interestingly, ectopically expressed C-terminal fragment of PGC-1α was autonomously ubiquitinated and aggregated with promyelocytic leukemia protein. Cooperation of the N-terminal region containing two PEST-like motifs was required for prevention of aggregation and targeting of the polyubiquitinated PGC-1α for degradation. This region thereby negatively controlled the aggregation properties of the C-terminal region to regulate protein turnover and intranuclear compartmentalization of PGC-1α. Exogenous expression of the PGC-1α C-terminal fragment interfered with degradation of full-length PGC-1α and enhanced its coactivation properties. We concluded that PGC-1α function is critically regulated at multiple steps via intramolecular cooperation among several distinct structural domains of the protein.

18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload–induced maladaptive cardiac remodeling

Macrophage-derived 18-HEPE protects mice from cardiac remodeling by preventing proinflammatory activation of cardiac fibroblasts and subsequent fibrosis.

Bone Marrow–Derived Cells Are Involved in the Pathogenesis of Cardiac Hypertrophy in Response to Pressure Overload

Background— Bone marrow (BM) cells possess broad differentiation potential and can form various cell lineages in response to pathophysiological cues. The present study investigated whether BM-derived cells contribute to the pathogenesis of cardiac hypertrophy, as well as the possible cellular mechanisms involved in such a role. Methods and Results— Lethally irradiated wild-type mice were transplanted with BM cells from enhanced green fluorescent protein–transgenic mice. The chimeric mice were subjected to either prolonged hypoxia or transverse aortic constriction. BM-derived enhanced green fluorescent protein–expressing cardiomyocytes increased in number over time, emerging predominantly in the pressure-overloaded ventricular myocardium, although they constituted <0.01% of recipient cardiomyocytes. To determine whether BM-derived cardiomyocytes were derived from cell fusion or transdifferentiation at the single-cell level, lethally irradiated Cre mice were transplanted with BM cells from the double-conditional Cre reporter mouse line Z/EG. BM-derived cardiomyocytes were shown to arise from both cell fusion and transdifferentiation. Interestingly, BM-derived myofibroblasts expressing both vimentin and &agr;-smooth muscle actin were concentrated in the perivascular fibrotic area. These cells initially expressed MAC-1/CD14 but lost expression of these markers during the chronic phase, which suggests that they were derived from monocytes. A similar phenomenon occurred in cultured human monocytes, most of which ultimately expressed vimentin and &agr;-smooth muscle actin. Conclusions— We found that BM-derived cells were involved in the pathogenesis of cardiac hypertrophy via the dual mechanisms of cell fusion and transdifferentiation. Moreover, the present results suggest that BM-derived monocytes accumulating in the perivascular space might play an important role in the formation of perivascular fibrosis via direct differentiation into myofibroblasts.