Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

TNFR1 Contributes to Activation-Induced Cell Death of Pathological CD4+ T Lymphocytes During Ischemic Heart Failure.

CD4+ T cells turn pathological during heart failure (HF). We show that the expression of tumor necrosis factor (TNF)-α and tumor necrosis factor receptor (TNFR1) increases in HF-activated CD4+ T cells. However, the role of the TNF-α/TNFR1 axis in T-cell activation/proliferation is unknown. We show that TNFR1 neutralization during T-cell activation (ex vivo) or the loss of TNFR1 in adoptively transferred HF-activated CD4+ T cells (in vivo) augments their prosurvival and proliferative signaling. Importantly, TNFR1 neutralization does not affect CD69 expression or the pathological activity of HF-activated TNFR1-/- CD4+ T cells. These results show that during HF TNFR1 plays an important role in quelling prosurvival and proliferative signals in CD4+ T cells without altering their pathological activity.

CLIC4 localizes to mitochondrial-associated membranes and mediates cardioprotection.

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

CD4+ T-lymphocytes exhibit biphasic kinetics post-myocardial infarction.

CD4+ T-cells facilitate wound healing post-myocardial infarction (MI) but promote left-ventricular (LV) remodeling during ischemic heart failure (HF; 8 weeks post-MI). Therefore, it is critical to understand if sustained CD4+ T-cell activation leads to this pathological response, or if phenotypically different T-cells are activated during MI vs. HF. Using flow cytometry, we found that cardiac CD4+ T-cells exhibit two distinct patterns of transmigration. First pattern consisted of a rapid CD4+ T-cell response with maximal levels seen at 3 days post-MI which return to baseline by 14 days. However, during HF we observed a 2nd phase of activation and CD4+ T-cells were ∼20-fold higher in HF as compared to sham-operated mice. Importantly, these biphasic kinetics were observed with all major T-cell subsets such as Th1, Th2, Th17, and regulatory T-cells suggesting a global change. To determine the role of this 2nd peak of T-cell activation, CD4-iDTR mice were generated and treated with DT every 10 from 28 days post-MI to deplete CD4+ T-cells during chronic HF. While littermate control mice showed increased end-systolic and end-diastolic volumes (ESV and EDV) and decreased ejection fraction (EF) from 4 to 8 weeks post-MI, depletion of CD4+ T-cells in Cre + mice significantly blunted LV remodeling and inhibited progressive increases in the EDV and ESV, and reduction in EF. This suggests that CD4+ T-cell responses occurring during HF are different than those occurring during MI and promote LV remodeling and progressive cardiac dysfunction. Temporal immunomodulation of CD4+ T-cells could be a translatable modality for ischemic HF.

Mineralocorticoid receptor antagonists and glucocorticoids differentially affect skeletal muscle inflammation and pathology in muscular dystrophy.

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.

Estrogen Receptor-ß Agonists Modulate T-Lymphocyte Activation and Ameliorate Left Ventricular Remodeling During Chronic Heart Failure.

BACKGROUND: CD4+ T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. METHODS: RNA-sequencing of cardiac CD4+ T cells and flow cytometric analysis of immune cells was conducted. RESULTS: RNA-sequencing of CD4+ T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4+ T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERß agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4+ T cells without affecting other myeloid and lymphoid cells in the HF mice. CONCLUSIONS: Our studies indicate that ERß agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4+ T cells during chronic HF.

BEX1 is a critical determinant of viral myocarditis.

Viral infection of the heart is a common but underappreciated cause of heart failure. Viruses can cause direct cardiac damage by lysing infected cardiomyocytes. Inflammatory immune responses that limit viral replication can also indirectly cause damage during infection, making regulatory factors that fine-tune these responses particularly important. Identifying and understanding these factors that regulate cardiac immune responses during infection will be essential for developing targeted treatments for virus-associated heart failure. Our laboratory has discovered Brain Expressed X-linked protein 1 (BEX1) as a novel stress-regulated pro-inflammatory factor in the heart. Here we report that BEX1 plays a cardioprotective role in the heart during viral infection. Specifically, we adopted genetic gain- and loss-of-function strategies to modulate BEX1 expression in the heart in the context of coxsackievirus B3 (CVB3)-induced cardiomyopathy and found that BEX1 limits viral replication in cardiomyocytes. Interestingly, despite the greater viral load observed in mice lacking BEX1, inflammatory immune cell recruitment in the mouse heart was profoundly impaired in the absence of BEX1. Overall, the absence of BEX1 accelerated CVB3-driven heart failure and pathologic heart remodeling. This result suggests that limiting inflammatory cell recruitment has detrimental consequences for the heart during viral infections. Conversely, transgenic mice overexpressing BEX1 in cardiomyocytes revealed the efficacy of BEX1 for counteracting viral replication in the heart in vivo. We also found that BEX1 retains its antiviral role in isolated cells. Indeed, BEX1 was necessary and sufficient to counteract viral replication in both isolated primary cardiomyocytes and mouse embryonic fibroblasts suggesting a broader applicability of BEX1 as antiviral agent that extended to viruses other than CVB3, including Influenza A and Sendai virus. Mechanistically, BEX1 regulated interferon beta (IFN-ß) expression in infected cells. Overall, our study suggests a multifaceted role of BEX1 in the cardiac antiviral immune response.

Myeloid mineralocorticoid receptors contribute to skeletal muscle repair in muscular dystrophy and acute muscle injury.

Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.

Estrogenic bias in T-Lymphocyte biology: Implications for cardiovascular disease.

Gender bias in cardiovascular disease has been extensively documented in epidemiological and clinical studies. Despite this, the precise molecular mechanisms underlying these disparities between men and women are poorly understood. It is clear that physiological concentrations of estradiol, such as those present in pre-menopausal women, exert cardioprotective effects that are absent in men or in post-menopausal women. These cardioprotective effects, in part, are due to the estrogen receptor-mediated modulation of the immune system including T-cells. Estrogen receptors (ERs) are widely expressed in different T-cell subsets which are known to play an indispensable role in the progression of cardiovascular disease. Because T-cells can be polarized into several distinct subsets depending on the activation milieu, they can have many different, potentially opposing functions, and it is unclear what roles estrogen receptor signaling may play in mediating these functions. This is further complicated by the discrete and often antagonistic actions of different ERs on T-cell biology which dictate the balance between numerous ER-dependent signaling pathways. While myriad effects of estrogen in T-cells are relevant for many cardiovascular diseases, their widespread effects on several other (patho)physiological systems introduce several obstacles to understanding ER signaling and its precise effects on the immune system. This review aims to provide a more comprehensive summary of the mechanisms of estrogen receptor-mediated modulation of T-cell function, polarization, and cytokine production in the context of cardiovascular disease.

Early Inflammation in Muscular Dystrophy Differs between Limb and Respiratory Muscles and Increases with Dystrophic Severity.

Duchenne muscular dystrophy (DMD) is a genetic, degenerative, striated muscle disease exacerbated by chronic inflammation. Mdx mice in the genotypic DMD model poorly represent immune-mediated pathology observed in patients. Improved understanding of innate immunity in dystrophic muscles is required to develop specific anti-inflammatory treatments. Here, inflammation in mdx mice and the more fibrotic utrn+/-;mdx Het model was comprehensively investigated. Unbiased analysis showed that mdx and Het mice contain increased levels of numerous chemokines and cytokines, with further increased in Het mice. Chemokine and chemokine receptor gene expression levels were dramatically increased in 4-week-old dystrophic quadriceps muscles, and to a lesser extent in diaphragm during the early injury phase, and had a small but consistent increase at 8 and 20 weeks. An optimized direct immune cell isolation method prevented loss of up to 90% of macrophages with density-dependent centrifugation previously used for mdx flow cytometry. Het quadriceps contain higher proportions of neutrophils and infiltrating monocytes than mdx, and higher percentages of F4/80Hi, but lower percentages of F4/80Lo cells and patrolling monocytes compared with Het diaphragms. These differences may restrict regenerative potential of dystrophic diaphragms, increasing pathologic severity. Fibrotic and inflammatory gene expression levels are higher in myeloid cells isolated from Het compared with mdx quadriceps, supporting Het mice may represent an improved model for testing therapeutic manipulation of inflammation in DMD.

Immune cell Dilemma in Ischemic Cardiomyopathy: To Heal or Not to Heal.

Inflammation is a double-edged sword for sterile tissue injury such as in myocardial infarction (MI). After ischemic injury, inflammatory immune responses activate repair processes, clear tissue-debris, form a stable scar and initiate angiogenesis in the myocardium for efficient wound-healing. However, incomplete immune resolution or sustained low-grade inflammation lead to ischemic cardiomyopathy (IC) characterized by maladaptive tissue remodeling and left-ventricular dilatation. It is clear that a delicate balance of cytokines, chemokines, prostaglandins, resolvins, and the innate and adaptive immune systems is critical for adequate healing as both insufficient- or overt-activation of inflammatory responses can either enhance rupture incidence or exacerbate cardiac dysfunction in the long-term. Among all the players, immune cells are the most critical as they are not only a source for all of the inflammatory protein mediators, but are also a target. However, phenotypic complexities associated with different immune subtypes, their interdependence, phasic-activations and varied functionalities often make it difficult to segregate the effects of one immune cell from another. In this review, we briefly summarize the role of several innate and adaptive immune cells to acquaint readers with complex immune-networks that dictate the extent of wound-healing post-MI and maladaptive remodeling during IC.

The Apolipoprotein A-I Mimetic L-4F Attenuates Monocyte Activation and Adverse Cardiac Remodeling after Myocardial Infarction.

Excessive inflammation after myocardial infarction (MI) can promote infarct expansion and adverse left ventricular (LV) remodeling. L-4F, a mimetic peptide of apolipoprotein A-I (apoA-I), exhibits anti-inflammatory and anti-atherogenic properties; however, whether L-4F imparts beneficial effects after myocardial infarction (MI) is unknown. Here we demonstrate that L-4F suppresses the expansion of blood, splenic, and myocardial pro-inflammatory monocytes and macrophages in a mouse model of reperfused MI. Changes in immune cell profiles were accompanied by alleviation of post-MI LV remodeling and dysfunction. In vitro, L-4F also inhibited pro-inflammatory and glycolytic gene expression in macrophages. In summary, L-4F treatment prevents prolonged and excessive inflammation after MI, in part through modulation of pro-inflammatory monocytes and macrophages, and improves post-MI LV remodeling. These data suggest that L-4F could be a used as a therapeutic adjunct in humans with MI to limit inflammation and alleviate the progression to heart failure.

Fibroblast growth factor-inducible 14 mediates macrophage infiltration in heart to promote pressure overload-induced cardiac dysfunction.

AIMS: Heart failure (HF) is characterized by compromised cardiac structure and function. Previous work has identified a link between upregulation of pro-inflammatory cytokines and HF. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory cytokine, which binds to fibroblast growth factor inducible 14 (Fn14), a ubiquitously expressed cell-surface receptor. The objective of this study was to investigate the role of TWEAK/Fn14 pathway in promoting cardiac inflammation under non ischemic stress conditions. MAIN METHODS: Wild type (WT) and Fn14 knock out (Fn14-/-) mice were subjected to pressure overload [transaortic constriction (TAC)] for 1 or 6 weeks. A subset of WT TAC animals were treated with the Fn14 antagonist L524-0366. Cardiac function was measured by echocardiography. Cardiac fibrosis and macrophage infiltration were quantified using immunohistochemistry and flow cytometry, respectively. Cardiac fibroblasts were isolated for quantifying TWEAK-induced chemokine release. KEY FINDINGS: Fn14-/- mice displayed improved cardiac function, reduced fibrosis and lower macrophage infiltration in heart compared to WT following TAC. L524-0366 mitigated maladaptive remodeling with TAC. TWEAK induced secretion of the pro-inflammatory chemokine, monocyte chemoattractant protein 1 from WT but not Fn14-/- fibroblasts in vitro, in part through activation of non-canonical NF-κB signaling. Finally, Fn14 expression was increased in mouse following TAC and in human failing hearts. SIGNIFICANCE: Our findings support an important role for the TWEAK/Fn14 promoting macrophage infiltration and fibrosis in heart under non-ischemic stress, with potential for therapeutic intervention to improve cardiac function in the setting of HF.

Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts.

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints.NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-µm filter can replace density-mediated mononuclear cell separation which usually results in 50-70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

Cardiovascular inflammation: RNA takes the lead.

Inflammation has recently gained tremendous attention as a key contributor in several chronic diseases. While physiological inflammation is essential to counter a wide variety of damaging stimuli and to improve wound healing, dysregulated inflammation such as in the myocardium and vasculature can promote cardiovascular diseases. Given the high severity, prevalence, and economic burden of these diseases, understanding the factors involved in the regulation of physiological inflammation is essential. Like other complex biological phenomena, RNA-based processes are emerging as major regulators of inflammatory responses. Among such processes are cis-regulatory elements in the mRNA of inflammatory genes, noncoding RNAs directing the production or localization of inflammatory cytokines/chemokines, or pathogenic RNA driving inflammatory responses. In this review, we describe several specific RNA-based molecular mechanisms by which physiological inflammation pertaining to cardiovascular diseases is regulated. These include the role of AU-rich element-containing mRNAs, long non-coding RNAs, microRNAs, and viral RNAs.

Dysfunctional and Proinflammatory Regulatory T-Lymphocytes Are Essential for Adverse Cardiac Remodeling in Ischemic Cardiomyopathy.

BACKGROUND: Heart failure (HF) is a state of inappropriately sustained inflammation, suggesting the loss of normal immunosuppressive mechanisms. Regulatory T-lymphocytes (Tregs) are considered key suppressors of immune responses; however, their role in HF is unknown. We hypothesized that Tregs are dysfunctional in ischemic cardiomyopathy and HF, and they promote immune activation and left ventricular (LV) remodeling. METHODS: Adult male wild-type C57BL/6 mice, Foxp3-diphtheria toxin receptor transgenic mice, and tumor necrosis factor (TNF) α receptor-1 (TNFR1)-/- mice underwent nonreperfused myocardial infarction to induce HF or sham operation. LV remodeling was assessed by echocardiography as well as histological and molecular phenotyping. Alterations in Treg profile and function were examined by flow cytometry, immunostaining, and in vitro cell assays. RESULTS: Compared with wild-type sham mice, CD4+Foxp3+ Tregs in wild-type HF mice robustly expanded in the heart, circulation, spleen, and lymph nodes in a phasic manner after myocardial infarction, beyond the early phase of wound healing, and exhibited proinflammatory T helper 1-type features with interferon-γ, TNFα, and TNFR1 expression, loss of immunomodulatory capacity, heightened proliferation, and potentiated antiangiogenic and profibrotic properties. Selective Treg ablation in Foxp3-diphtheria toxin receptor mice with ischemic cardiomyopathy reversed LV remodeling and dysfunction, alleviating hypertrophy and fibrosis, while suppressing circulating CD4+ T cells and systemic inflammation and enhancing tissue neovascularization. Tregs reconstituted after ablation exhibited restoration of immunosuppressive capacity and normalized TNFR1 expression. Treg dysfunction was also tightly coupled to Treg-endothelial cell contact- and TNFR1-dependent inhibition of angiogenesis and the mobilization and tissue infiltration of CD34+Flk1+ circulating angiogenic cells in a C-C chemokine ligand 5/C-C chemokine receptor 5-dependent manner. Anti-CD25-mediated Treg depletion in wild-type mice imparted similar benefits on LV remodeling, circulating angiogenic cells, and tissue neovascularization. CONCLUSIONS: Proinflammatory and antiangiogenic Tregs play an essential pathogenetic role in chronic ischemic HF to promote immune activation and pathological LV remodeling. The restoration of normal Treg function may be a viable approach to therapeutic immunomodulation in this disease.

CCR2+ Monocyte-Derived Infiltrating Macrophages Are Required for Adverse Cardiac Remodeling During Pressure Overload.

Although chronic inflammation is a central feature of heart failure (HF), the immune cell profiles differ with different underlying causes. This suggests that for immunomodulatory therapy in HF to be successful, it needs to be tailored to the specific etiology. Here, the authors demonstrate that monocyte-derived C-C chemokine receptor 2 (CCR2)+ macrophages infiltrate the heart early during pressure overload in mice, and that blocking this response either pharmacologically or with antibody-mediated CCR2+ monocyte depletion alleviates late pathological left ventricular remodeling and dysfunction, T-cell expansion, and cardiac fibrosis. Hence, suppression of CCR2+ monocytes/macrophages may be an important immunomodulatory therapeutic target to ameliorate pressure-overload HF.

Activated T Lymphocytes are Essential Drivers of Pathological Remodeling in Ischemic Heart Failure.

BACKGROUND: Inappropriately sustained inflammation is a hallmark of chronic ischemic heart failure (HF); however, the pathophysiological role of T lymphocytes is unclear. METHODS AND RESULTS: Permanent coronary ligation was performed in adult C57BL/6 mice. When compared with sham-operated mice, mice with HF (8 weeks after ligation) exhibited the following features: (1) significant (P<0.05) expansion of circulating CD3+CD8+ cytotoxic and CD3+CD4+ helper (Th) T lymphocytes, together with increased Th1, Th2, Th17, and regulatory T-cell (Treg) CD4+ subsets; (2) significant expansion of CD8+ and CD4+ T cells in failing myocardium, with increased Th1, Th2, Th17, and Treg CD4+ subsets, marked reduction of the Th1/Th2 ratio, augmentation of the Th17/Treg ratio, and upregulation of Th2 cytokines; and (3) significantly increased Th1, Th2, Th17 cells, and Tregs, in the spleen and mediastinal lymph nodes, with expansion of splenic antigen-experienced effector and memory CD4+ T cells. Antibody-mediated CD4+ T-cell depletion in HF mice (starting 4 weeks after ligation) reduced cardiac infiltration of CD4+ T cells and prevented progressive left ventricular dilatation and hypertrophy, whereas adoptive transfer of splenic CD4+ T cells (and, to a lesser extent, cardiac CD3+ T cells) from donor mice with HF induced long-term left ventricular dysfunction, fibrosis, and hypertrophy in naive recipient mice. CONCLUSIONS: CD4+ T lymphocytes are globally expanded and activated in chronic ischemic HF, with Th2 (versus Th1) and Th17 (versus Treg) predominance in failing hearts, and with expansion of memory T cells in the spleen. Cardiac and splenic T cells in HF are primed to induce cardiac injury and remodeling, and retain this memory on adoptive transfer.