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.

Guidelines on the use of sex and gender in cardiovascular research.

In cardiovascular research, sex and gender have not typically been considered in research design and reporting until recently. This has resulted in clinical research findings from which not only all women, but also gender-diverse individuals have been excluded. The resulting dearth of data has led to a lack of sex- and gender-specific clinical guidelines and raises serious questions about evidence-based care. Basic research has also excluded considerations of sex. Including sex and/or gender as research variables not only has the potential to improve the health of society overall now, but it also provides a foundation of knowledge on which to build future advances. The goal of this guidelines article is to provide advice on best practices to include sex and gender considerations in study design, as well as data collection, analysis, and interpretation to optimally establish rigor and reproducibility needed to inform clinical decision-making and improve outcomes. In cardiovascular physiology, incorporating sex and gender is a necessary component when optimally designing and executing research plans. The guidelines serve as the first guidance on how to include sex and gender in cardiovascular research. We provide here a beginning path toward achieving this goal and improve the ability of the research community to interpret results through a sex and gender lens to enable comparison across studies and laboratories, resulting in better health for all.

Sex as a biological variable for cardiovascular physiology.

There have been ongoing efforts by federal agencies and scientific communities since the early 1990s to incorporate sex and/or gender in all aspects of cardiovascular research. Scientific journals provide a critical function as change agents to influence transformation by encouraging submissions for topic areas, and by setting standards and expectations for articles submitted to the journal. As part of ongoing efforts to advance sex and gender in cardiovascular physiology research, the American Journal of Physiology-Heart and Circulatory Physiology recently launched a call for papers on Considering Sex as a Biological Variable. This call was an overwhelming success, resulting in 78 articles published in this collection. This review summarizes the major themes of the collection, including Sex as a Biological Variable Within: Endothelial Cell and Vascular Physiology, Cardiovascular Immunity and Inflammation, Metabolism and Mitochondrial Energy, Extracellular Matrix Turnover and Fibrosis, Neurohormonal Signaling, and Cardiovascular Clinical and Epidemiology Assessments. Several articles also focused on establishing rigor and reproducibility of key physiological measurements involved in cardiovascular health and disease, as well as recommendations and considerations for study design. Combined, these articles summarize our current understanding of sex and gender influences on cardiovascular physiology and pathophysiology and provide insight into future directions needed to further expand our knowledge.

Regulation of cardiac fibroblasts by lymphocytes after a myocardial infarction: playing in the major league.

Cardiac fibrosis is a pathological condition characterized by excessive accumulation of extracellular matrix components within the myocardium, which can lead to impaired cardiac function and heart failure. Studies have shown that lymphocytes including B and T cells play important roles in the development and progression of cardiac fibrosis after a myocardial infarction. In this review, we focus on the regulation of cardiac fibrosis by lymphocyte subsets, with a particular emphasis on CD4+ and CD8+ T cells and their effects on fibroblasts and cardiac remodeling. We also highlight areas for further exploration of the interactions between T cells and fibroblasts necessary for understanding and treating cardiac fibrosis and heart failure.

Endothelial mechanical stretch regulates the immunological synapse interface of renal endothelial cells in a sex-dependent manner.

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.

Cardiac and vascular complications in lupus: Is there a role for sex?

Systemic lupus erythematosus (SLE) is a common systemic autoimmune disorder and is characterized by autoantibody formation and subsequent immune complex deposition into target organs. SLE affects nearly nine women to every one man worldwide. Patients with SLE are at an enhanced risk for cardiovascular disease (CVD) morbidity and mortality. CVD is the leading cause of death worldwide and includes heart and blood vessel disorders, cerebrovascular disease, and rheumatic heart disease. Specific mechanisms by which cardiac and vascular pathophysiology develops in patients with SLE are still not fully known. Not only do we not understand this correlation between SLE and CVD, but there is also a critical gap in scientific knowledge on the contribution of sex. In this review, we will discuss the cardiac and vascular pathological disease states that are present in some patients with SLE. More importantly, we will discuss the potential mechanisms for the role of sex and sex hormones in the development of CVD with SLE.

Harnessing the Plasma Proteome to Mirror Current and Predict Future Cardiac Remodeling After Myocardial Infarction.

To identify plasma proteins that mirror current and predict future remodeling after myocardial infarction (MI), we retrospectively interrogated plasma proteomes of day (D)0 control (n = 16) and D3 MI (n = 15) from C57BL/6 J mice (20 ± 1 months). A total of 165 unique proteins were correlated with cardiac physiology variables. We prospectively tested the hypothesis that candidates identified retrospectively would predict cardiac physiology at an extended timepoint (D7 MI) in a second cohort of mice (n = 4 ± 1 months). We also examined human plasma from healthy controls (n = 18) and patients 48 h after presentation for MI (n = 41). Retrospectively, we identified 5 strong reflectors of remodeling (all r ≥ 0.60 and p < 0.05). Prospectively, ApoA1, IgA, IL-17E, and TIMP-1 mirrored current and predicted future remodeling. In humans, cytokine-cytokine receptor signaling was the top enriched KEGG pathway for all candidates. In summary, we identified plasma proteins that serve as useful prognostic indicators of adverse remodeling and progression to heart failure.

Riding the wave: a quantitative report of electrocardiogram utilization for myocardial infarction confirmation.

The purpose of this study was to generate a quantitative profile of electrocardiograms (ECGs) for confirming surgical success of permanent coronary artery ligation. An ECG was recorded at baseline, and 0, 1, and 5 min after ligation and analyzed using iWorkx LabScribe software. Cohort 1 (C57Bl6/J, n = 8/sex) was enrolled to determine ECG characteristics that were confirmed in cohort 2 (C57Bl6/J, n = 6/sex; CD8-/-n = 6 males/4 females). Of the 16 mice in cohort 1, 12 (6/sex) had an infarct ≥35% and four mice (2/sex) had <35% based on 2,3,5-triphenyltetrazolium chloride staining. After ligation, the QRS complex and R-S amplitude were significantly different compared with baseline. No differences were observed in the R-S amplitude between mice with infarcts ≥35% versus <35% at any time point, whereas the QRS complex was significant 1 min after ligation. Receiver operating characteristic (ROC) curve linked changes in the QRS complex but not the R-S amplitude at 1 and 5 min with surgical success. Data were normalized to baseline values to calculate fold change. ROC analysis of the normalized QRS data indicated strong sensitivity and specificity for infarcts ≥35%; normalized R-S amplitude remained nonsignificant. With a cutoff generated by ROC analysis of cohort 1 (>80% sensitivity; >90% specificity), the non-normalized QRS complex of cohort 2 had an 86% success rate (2 false positives; 1 false negative). The normalized data had a 77% success rate (2 false positives; 3 false negatives). Neither sex nor genotype was associated with false predictions (P = 0.18). Our data indicate that the area under the QRS complex 1 min after ligation can improve reproducibility in MI surgeries.NEW & NOTEWORTHY Our study describes a quantitative method for using an electrocardiogram (ECG) to determine which animals have infarcts that reflect coronary artery ligation. Using a quantitative ECG, investigators will have the benefit of having real-time feedback during the procedure, which will ultimately decrease the amount of time investigators spend performing experiments. This overall increase in efficiency will help investigators decrease animal numbers used due to better surgical outcomes.

Chronic Porphyromonas gingivalis lipopolysaccharide induces adverse myocardial infarction wound healing through activation of CD8+ T cells.

Oral and gum health have long been associated with incidence and outcomes of cardiovascular disease. Periodontal disease increases myocardial infarction (MI) mortality by sevenfold through mechanisms that are not fully understood. The goal of this study was to evaluate whether lipopolysaccharide (LPS) from a periodontal pathogen accelerates inflammation after MI through memory T-cell activation. We compared four groups [no MI, chronic LPS, day 1 after MI, and day 1 after MI with chronic LPS (LPS + MI); n = 68 mice] using the mouse heart attack research tool 1.0 database and tissue bank coupled with new analyses and experiments. LPS + MI increased total CD8+ T cells in the left ventricle versus the other groups (P < 0.05 vs. all). Memory CD8+ T cells (CD44 + CD27+) were 10-fold greater in LPS + MI than in MI alone (P = 0.02). Interleukin (IL)-4 stimulated splenic CD8+ T cells away from an effector phenotype and toward a memory phenotype, inducing secretion of factors associated with the Wnt/ß-catenin signaling that promoted monocyte migration and decreased viability. To dissect the effect of CD8+ T cells after MI, we administered a major histocompatibility complex-I-blocking antibody starting 7 days before MI, which prevented effector CD8+ T-cell activation without affecting the memory response. The reduction in effector cells diminished infarct wall thinning but had no effect on macrophage numbers or MertK expression. LPS + MI + IgG attenuated macrophages within the infarct without effecting CD8+ T cells, suggesting these two processes were independent. Overall, our data indicate that effector and memory CD8+ T cells at post-MI day 1 are amplified by chronic LPS to potentially promote infarct wall thinning.NEW & NOTEWORTHY Although there is a well-documented link between periodontal disease and heart health, the mechanisms are unclear. Our study indicates that in response to circulating periodontal endotoxins, memory CD8+ T cells are activated, resulting in an acceleration of macrophage-mediated inflammation after MI. Blocking activation of effector CD8+ T cells had no effect on the macrophage numbers or wall thinning at post-MI day 1, indicating that this response was likely due in part to memory CD8+ T cells.

Guidelines for in vivo mouse models of myocardial infarction.

Despite significant improvements in reperfusion strategies, acute coronary syndromes all too often culminate in a myocardial infarction (MI). The consequent MI can, in turn, lead to remodeling of the left ventricle (LV), the development of LV dysfunction, and ultimately progression to heart failure (HF). Accordingly, an improved understanding of the underlying mechanisms of MI remodeling and progression to HF is necessary. One common approach to examine MI pathology is with murine models that recapitulate components of the clinical context of acute coronary syndrome and subsequent MI. We evaluated the different approaches used to produce MI in mouse models and identified opportunities to consolidate methods, recognizing that reperfused and nonreperfused MI yield different responses. The overall goal in compiling this consensus statement is to unify best practices regarding mouse MI models to improve interpretation and allow comparative examination across studies and laboratories. These guidelines will help to establish rigor and reproducibility and provide increased potential for clinical translation.

Multicellular Human Cardiac Organoids Transcriptomically Model Distinct Tissue-Level Features of Adult Myocardium.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.

Reperfused vs. nonreperfused myocardial infarction: when to use which model.

There is a lack of understanding in the cardiac remodeling field regarding the use of nonreperfused myocardial infarction (MI) and reperfused MI in animal models of MI. This Perspectives summarizes the consensus of the authors regarding how to select the optimum model for your experiments and is a part of ongoing efforts to establish rigor and reproducibility in cardiac physiology research.

Organized Chaos: Deciphering Immune Cell Heterogeneity's Role in Inflammation in the Heart.

During homeostasis, immune cells perform daily housekeeping functions to maintain heart health by acting as sentinels for tissue damage and foreign particles. Resident immune cells compose 5% of the cellular population in healthy human ventricular tissue. In response to injury, there is an increase in inflammation within the heart due to the influx of immune cells. Some of the most common immune cells recruited to the heart are macrophages, dendritic cells, neutrophils, and T-cells. In this review, we will discuss what is known about cardiac immune cell heterogeneity during homeostasis, how these cell populations change in response to a pathology such as myocardial infarction or pressure overload, and what stimuli are regulating these processes. In addition, we will summarize technologies used to evaluate cell heterogeneity in models of cardiovascular disease.

Immune regulation of cardiac fibrosis post myocardial infarction.

Pathological changes resulting from myocardial infarction (MI) include extracellular matrix alterations of the left ventricle, which can lead to cardiac stiffness and impair systolic and diastolic function. The signals released from necrotic tissue initiate the immune cascade, triggering an extensive inflammatory response followed by reparative fibrosis of the infarct area. Immune cells such as neutrophils, monocytes, macrophages, mast cells, T-cells, and dendritic cells play distinct roles in orchestrating this complex pathological condition, and regulate the balance between pro-fibrotic and anti-fibrotic responses. This review discusses how molecular signals between fibroblasts and immune cells mutually regulate fibrosis post-MI, and outlines the emerging pharmacological targets and therapies for modulating inflammation and cardiac fibrosis associated with MI.

Fibroblasts: The arbiters of extracellular matrix remodeling.

Extracellular matrix (ECM) is the foundation on which all cells and organs converge to orchestrate normal physiological functions. In the setting of pathology, the ECM is modified to incorporate additional roles, with modifications including turnover of existing ECM and deposition of new ECM. The fibroblast is center stage in coordinating both normal tissue homeostasis and response to disease. Understanding how fibroblasts work under normal conditions and are activated in response to injury or stress will provide mechanistic insight that triggers discovery of new therapeutic treatments for a wide range of disease. We highlight here fibroblast roles in the cancer, lung, and heart as example systems where fibroblasts are major contributors to homeostasis and pathology.

Exogenous IL-4 shuts off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to induce neutrophil phagocytosis following myocardial infarction.

INTRODUCTION: Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While interleukin (IL)-4 is an in vitro anti-inflammatory stimulus, the MI myocardium does not express a considerable amount of IL-4 but does express IL4 receptors. 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. METHODS: 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) beginning 24 h after MI and evaluated at MI day 3. RESULTS: Macrophages and neutrophils were isolated ex vivo from the infarct region and examined. Exogenous IL-4 decreased pro-inflammatory Ccl3, Il12a, Tnfa, and Tgfb1 in neutrophils and increased anti-inflammatory Arg1 and Ym1 in macrophages (all p < .05). Tissue clearance by IL-4 treated neutrophils was not different, while selective phagocytosis of neutrophils doubled in IL-4 treated macrophages (p < .05). Of 24,339 genes examined by RNA-sequencing, 2042 genes were differentially expressed in macrophages from IL-4 stimulated infarct (all FDR p < .05). Pdgfc gene expression was ranked first, increasing 3-fold in macrophages stimulated with IL-4 (p = 1 × 10-9). Importantly, changes in macrophage physiology and transcriptome occurred in the absence of global LV effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF-CC protein (10 µg/ml) or PDGF-CC blocking antibody (200 ng/ml) did not change Arg1 or Ym1 expression, indicating the in vivo effect of IL-4 to stimulate macrophage anti-inflammatory gene expression was independent of PDGF-CC. CONCLUSIONS: Our results indicate that exogenous IL-4 promotes inflammation resolution by turning off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to mediate removal of apoptotic neutrophils.

Differential effects of low-dose sacubitril and/or valsartan on renal disease in salt-sensitive hypertension.

Diuretics and renin-angiotensin system blockers are often insufficient to control the blood pressure (BP) in salt-sensitive (SS) subjects. Abundant data support the proposal that the level of atrial natriuretic peptide may correlate with the pathogenesis of SS hypertension. We hypothesized here that increasing atrial natriuretic peptide levels with sacubitril, combined with renin-angiotensin system blockage by valsartan, can be beneficial for alleviation of renal damage in a model of SS hypertension, the Dahl SS rat. To induce a BP increase, rats were challenged with a high-salt 4% NaCl diet for 21 days, and chronic administration of vehicle or low-dose sacubitril and/or valsartan (75 µg/day each) was performed. Urine flow, Na+ excretion, and water consumption were increased on the high-salt diet compared with the starting point (0.4% NaCl) in all groups but remained similar among the groups at the end of the protocol. Upon salt challenge, we observed a mild decrease in systolic BP and urinary neutrophil gelatinase-associated lipocalin levels (indicative of alleviated tubular damage) in the valsartan-treated groups. Sacubitril, as well as sacubitril/valsartan, attenuated the glomerular filtration rate decline induced by salt. Alleviation of protein cast formation and lower renal medullary fibrosis were observed in the sacubitril/valsartan- and valsartan-treated groups, but not when sacubitril alone was administered. Interestingly, proteinuria was mildly mitigated only in rats that received sacubitril/valsartan. Further studies of the effects of sacubitril/valsartan in the setting of SS hypertension, perhaps involving a higher dose of the drug, are warranted to determine if it can interfere with the progression of the disease.

T-cell regulation of fibroblasts and cardiac fibrosis.

Inflammation contributes to the development of heart failure (HF) through multiple mechanisms including regulating extracellular matrix (ECM) degradation and deposition. Interactions between cells in the myocardium orchestrates the magnitude and duration of inflammatory cell recruitment and ECM remodeling events associated with HF. More recently, studies have shown T-cells have signficant roles in post-MI wound healing. T-cell biology in HF illustrates the complexity of cross-talk between inflammatory cell types and resident fibroblasts. This review will focus on T-cell recruitment to the myocardium and T-cell specific factors that might influence cardiac wound healing and fibrosis in the heart with consideration of age and sex as important factors in T-cell activity.