Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Cortical bone stem cells modify cardiac inflammation after myocardial infarction by inducing a novel macrophage phenotype.

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-ß, but not IL-4. CBSC therapy increased the number of galectin-3+ macrophages, CD4+ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-ß, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.

Cortical bone-derived stem cell therapy reduces apoptosis after myocardial infarction.

Ischemic heart diseases such as myocardial infarction (MI) are the largest contributors to cardiovascular disease worldwide. The resulting cardiac cell death impairs function of the heart and can lead to heart failure and death. Reperfusion of the ischemic tissue is necessary but causes damage to the surrounding tissue by reperfusion injury. Cortical bone stem cells (CBSCs) have been shown to increase pump function and decrease scar size in a large animal swine model of MI. To investigate the potential mechanism for these changes, we hypothesized that CBSCs were altering cardiac cell death after reperfusion. To test this, we performed TUNEL staining for apoptosis and antibody-based immunohistochemistry on tissue from Göttingen miniswine that underwent 90 min of lateral anterior descending coronary artery ischemia followed by 3 or 7 days of reperfusion to assess changes in cardiomyocyte and noncardiomyocyte cell death. Our findings indicate that although myocyte apoptosis is present 3 days after ischemia and is lower in CBSC-treated animals, myocyte apoptosis accounts for <2% of all apoptosis in the reperfused heart. In addition, nonmyocyte apoptosis trends toward decreased in CBSC-treated hearts, and although CBSCs increase macrophage and T-cell populations in the infarct region, the occurrence of apoptosis in CD45+ cells in the myocardium is not different between groups. From these data, we conclude that CBSCs may be influencing cardiomyocyte and noncardiomyocyte cell death and immune cell recruitment dynamics in the heart after MI, and these changes may account for some of the beneficial effects conferred by CBSC treatment.NEW & NOTEWORTHY The following research explores aspects of cell death and inflammation that have not been previously studied in a large animal model. In addition, apoptosis and cell death have not been studied in the context of cell therapy and myocardial infarction. In this article, we describe interactions between cell therapy and inflammation and the potential implications for cardiac wound healing.

Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial Infarction.

RATIONALE: Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients. OBJECTIVE: To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model. METHODS AND RESULTS: Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia-reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5-ethynyl-2'deoxyuridine (EdU)-a thymidine analog-containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals. CONCLUSIONS: CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.

Protein Kinase C Inhibition With Ruboxistaurin Increases Contractility and Reduces Heart Size in a Swine Model of Heart Failure With Reduced Ejection Fraction.

Inotropic support is often required to stabilize the hemodynamics of patients with acute decompensated heart failure; while efficacious, it has a history of leading to lethal arrhythmias and/or exacerbating contractile and energetic insufficiencies. Novel therapeutics that can improve contractility independent of beta-adrenergic and protein kinase A-regulated signaling, should be therapeutically beneficial. This study demonstrates that acute protein kinase C-α/ß inhibition, with ruboxistaurin at 3 months' post-myocardial infarction, significantly increases contractility and reduces the end-diastolic/end-systolic volumes, documenting beneficial remodeling. These data suggest that ruboxistaurin represents a potential novel therapeutic for heart failure patients, as a moderate inotrope or therapeutic, which leads to beneficial ventricular remodeling.

Ethanol consumption in the Sprague-Dawley rat increases sensitivity of the dorsal raphe nucleus to 5,7-dihydroxytryptamine.

Alcoholism afflicts 1 in 13 US adults, and comorbidity with depression is common. Levels of serotonin (5-HT) metabolites in alcoholic or depressed humans and rat strains are lower compared to healthy counterparts. Rats bred for ethanol (EtOH) preference are common in EtOH studies, however out-bred strains better model the range of EtOH consumption in humans. We examined voluntary EtOH consumption in out-bred Sprague-Dawley (SD) rats placed in the 20% EtOH intermittent access drinking paradigm (IA). Acquisition of 20% EtOH consumption (g EtOH/kg/24h) was assessed during the first 6-8 weeks of IA. Rats naturally separated into two groups (Drinkers or Non-drinkers) based on EtOH intake above or below 0.5 g/kg/24h prior to treatment intervention. We examined the effect of central 5-HT depletion on EtOH consumption by infusing 5,7-dihyroxytryptamine (5,7-DHT; i.c.v., 200-300 µg) or vehicle and measured EtOH consumption for 4 weeks post-operatively in IA. Compared to baseline, there was no effect of vehicle or 5,7-DHT on EtOH consumption during the post-operative period. Quantification of 5-HT depletion in the dorsal raphe nucleus (DRN) using tryptophan hydroxylase-2 (TPH2) immunohistochemistry resulted in a 76% decrease in staining with 5,7-DHT treatment. Interestingly, preservation of the ventromedial (VM) sub-regions was evident in all animals treated with 5,7-DHT, regardless of drinking behavior. In addition, Drinkers treated with 5,7-DHT had significantly more TPH2 depletion in the DRN compared to Non-drinkers. Our findings indicate that out-bred SD rats exhibit a natural EtOH consumption behavior (Drinker or Non-drinker) that is stable across time and independent of 5-HT depletion in the CNS. In addition, rats that regularly consumed >0.5 g EtOH/kg had greater sensitivity to 5,7-DHT in the DRN, indicating an interaction between EtOH and sensitivity of DRN 5-HT cells to neurotoxic substances. This may contribute to the dysfunctionality of the 5-HT system in alcoholic humans and lead to a better understanding of current pharmacological treatments for this addiction.