Sex differences in weight gain, blood pressure control, and responses to melanocortin-4 receptor antagonism in offspring from lean and obese parents.

We examined potential sex differences in appetite and blood pressure (BP) responses to melanocortin-4 receptor (MC4R) blockade in offspring from lean and obese parents. Offspring from normal (N) diet-fed parents were fed N (NN) or high-fat (H) diets (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were also fed N (HN) or H diets (HH). Adult male and female offspring were implanted with BP telemetry probes and intracerebroventricular cannulas to infuse MC4R antagonist or vehicle. Infusion of the MC4R antagonist SHU-9119 (1 nmol/h) for 7 days caused larger increases in calorie intake and body weight in obese compared with lean offspring. In male offspring, HH and HN groups exhibited higher baseline BP compared with NN and NH, and HH showed a greater reduction in BP during SHU-9119 infusion. In female offspring, HH also showed higher baseline BP and greater reduction in BP during MC4R blockade. SHU-9119 reduced heart rate in all groups, but reductions were more pronounced in offspring from lean parents. Combined α and ß-adrenergic blockade reduced BP more in male HH offspring compared with NN controls. Losartan reduced BP more in male NH, HN, and HH offspring compared with NN controls. Losartan and α- and ß-adrenergic blockade reduced BP similarly in all female groups. These results suggest that endogenous MC4R activity contributes to elevated BP in obese offspring from obese parents. Our findings also indicate important sex differences in the mechanisms of BP control in male and female offspring of obese parents.

The Oligostilbene Gnetin H Is a Novel Glycolysis Inhibitor That Regulates Thioredoxin Interacting Protein Expression and Synergizes with OXPHOS Inhibitor in Cancer Cells.

Since aerobic glycolysis was first observed in tumors almost a century ago by Otto Warburg, the field of cancer cell metabolism has sparked the interest of scientists around the world as it might offer new avenues of treatment for malignant cells. Our current study claims the discovery of gnetin H (GH) as a novel glycolysis inhibitor that can decrease metabolic activity and lactic acid synthesis and displays a strong cytostatic effect in melanoma and glioblastoma cells. Compared to most of the other glycolysis inhibitors used in combination with the complex-1 mitochondrial inhibitor phenformin (Phen), GH more potently inhibited cell growth. RNA-Seq with the T98G glioblastoma cell line treated with GH showed more than an 80-fold reduction in thioredoxin interacting protein (TXNIP) expression, indicating that GH has a direct effect on regulating a key gene involved in the homeostasis of cellular glucose. GH in combination with phenformin also substantially enhances the levels of p-AMPK, a marker of metabolic catastrophe. These findings suggest that the concurrent use of the glycolytic inhibitor GH with a complex-1 mitochondrial inhibitor could be used as a powerful tool for inducing metabolic catastrophe in cancer cells and reducing their growth.

Parental obesity alters offspring blood pressure regulation and cardiovascular responses to stress: role of P2X7R and sex differences.

We examined the impact of parental obesity on offspring blood pressure (BP) regulation and cardiovascular responses to stress. Offspring from normal (N) diet-fed C57BL/6J parents were fed either N (NN) or a high-fat (H) diet (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were also fed N (HN) or H diet (HH). Body weight, calorie intake, and fat mass were measured at 22 wk of age when cardiovascular phenotyping was performed. Male and female HH offspring were 15% heavier than NH and 70% heavier than NN offspring. Male HH and HN offspring had elevated BP (121 ± 2 and 115 ± 1 mmHg, by telemetry) compared with male NH and NN offspring (108 ± 6 and 107 ± 3 mmHg, respectively) and augmented BP responses to angiotensin II, losartan, and hexamethonium. Male HH and HN offspring also showed increased BP responses to air-jet stress (37 ± 2 and 38 ± 2 mmHg) compared with only 24 ± 3 and 25 ± 3 mmHg in NH and NN offspring. Baseline heart rate (HR) and HR responses to air-jet stress were similar among groups. In females, BP and cardiovascular responses to stress were similar among all offspring. Male H diet-fed offspring from obese H diet-fed purinoreceptor 7-deficient (HH-P2X7R-KO) parents had normal BP that was similar to control NN-P2X7R-KO offspring from lean parents. These results indicate that parental obesity leads to increased BP and augmented BP responses to stress in their offspring in a sex-dependent manner, and the impact of parental obesity on male offspring BP regulation is markedly attenuated in P2X7R-KO mice.

Obesity, Hypertension, and Cardiac Dysfunction: Novel Roles of Immunometabolism in Macrophage Activation and Inflammation.

Obesity and hypertension, which often coexist, are major risk factors for heart failure and are characterized by chronic, low-grade inflammation, which promotes adverse cardiac remodeling. While macrophages play a key role in cardiac remodeling, dysregulation of macrophage polarization between the proinflammatory M1 and anti-inflammatory M2 phenotypes promotes excessive inflammation and cardiac injury. Metabolic shifting between glycolysis and mitochondrial oxidative phosphorylation has been implicated in macrophage polarization. M1 macrophages primarily rely on glycolysis, whereas M2 macrophages rely on the tricarboxylic acid cycle and oxidative phosphorylation; thus, factors that affect macrophage metabolism may disrupt M1/M2 homeostasis and exacerbate inflammation. The mechanisms by which obesity and hypertension may synergistically induce macrophage metabolic dysfunction, particularly during cardiac remodeling, are not fully understood. We propose that obesity and hypertension induce M1 macrophage polarization via mechanisms that directly target macrophage metabolism, including changes in circulating glucose and fatty acid substrates, lipotoxicity, and tissue hypoxia. We discuss canonical and novel proinflammatory roles of macrophages during obesity-hypertension-induced cardiac injury, including diastolic dysfunction and impaired calcium handling. Finally, we discuss the current status of potential therapies to target macrophage metabolism during heart failure, including antidiabetic therapies, anti-inflammatory therapies, and novel immunometabolic agents.

Dimethyl fumarate preserves left ventricular infarct integrity following myocardial infarction via modulation of cardiac macrophage and fibroblast oxidative metabolism.

Myocardial infarction (MI) is one of the leading causes of mortality and cardiovascular disease worldwide. MI is characterized by a substantial inflammatory response in the infarcted left ventricle (LV), followed by transition of quiescent fibroblasts to active myofibroblasts, which deposit collagen to form the reparative scar. Metabolic shifting between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is an important mechanism by which these cell types transition towards reparative phenotypes. Thus, we hypothesized that dimethyl fumarate (DMF), a clinically approved anti-inflammatory agent with metabolic actions, would improve post-MI remodeling via modulation of macrophage and fibroblast metabolism. Adult male C57BL/6J mice were treated with DMF (10 mg/kg) for 3-7 days after MI. DMF attenuated LV infarct and non-infarct wall thinning at 3 and 7 days post-MI, and decreased LV dilation and pulmonary congestion at day 7. DMF improved LV infarct collagen deposition, myofibroblast activation, and angiogenesis at day 7. DMF also decreased pro-inflammatory cytokine expression (Tnf) 3 days after MI, and decreased inflammatory markers in macrophages isolated from the infarcted heart (Hif1a, Il1b). In fibroblasts extracted from the infarcted heart at day 3, RNA-Seq analysis demonstrated that DMF promoted an anti-inflammatory/pro-reparative phenotype. By Seahorse analysis, DMF did not affect glycolysis in either macrophages or fibroblasts at day 3, but enhanced macrophage OXPHOS while impairing fibroblast OXPHOS. Our results indicate that DMF differentially affects macrophage and fibroblast metabolism, and promotes anti-inflammatory/pro-reparative actions. In conclusion, targeting cellular metabolism in the infarcted heart may be a promising therapeutic strategy.

Sex differences in the impact of parental obesity on offspring cardiac SIRT3 expression, mitochondrial efficiency, and diastolic function early in life.

Previous studies suggest that parental obesity may adversely impact long-term metabolic health of the offspring. We tested the hypothesis that parental (paternal + maternal) obesity impairs cardiac function in the offspring early in life. Within 1-3 days after weaning, offspring from obese rats fed a high-fat diet (HFD-Offs) and age-matched offspring from lean rats (ND-Offs) were submitted to echocardiography and cardiac catheterization for assessment of pressure-volume relationships. Then, hearts were digested and isolated cardiomyocytes were used to determine contractile function, calcium transients, proteins related to calcium signaling, and mitochondrial bioenergetics. Female and male HFD-Offs were heavier (72 ± 2 and 61 ± 4 g vs. 57 ± 2 and 49 ± 1 g), hyperglycemic (112 ± 8 and 115 ± 12 mg/dL vs. 92 ± 10 and 96 ± 8 mg/dL) with higher plasma insulin and leptin concentrations compared with female and male ND-Offs. When compared with male controls, male HFD-Offs exhibited similar systolic function but impaired diastolic function as indicated by increased IVRT (22 ± 1 vs. 17 ± 1 ms), E/E' ratio (29 ± 2 vs. 23 ± 1), and tau (5.7 ± 0.2 vs. 4.8 ± 0.2). The impaired diastolic function was associated with reduced resting free Ca2+ levels and phospholamban protein expression, increased activated matrix metalloproteinase 2, and reduced SIRT3 protein expression, mitochondrial ATP reserve, and ATP-linked respiration. These results indicate that male and female Offs from obese parents have multiple metabolic abnormalities early in life (1-3 days after weaning) and that male, but not female, Offs have impaired diastolic function as well as reductions in cardiac SIRT3, resting free Ca2+ levels, and mitochondrial biogenesis.NEW & NOTEWORTHY Parental obesity contributes to diastolic dysfunction in young offspring (1-3 days after weaning) in a sex-dependent manner, as well as reduced cardiac SIRT3 expression and altered mitochondrial bioenergetics, resting Ca2+ levels, and reduced phospholamban protein levels.

Novel roles of immunometabolism and nonmyocyte metabolism in cardiac remodeling and injury.

Changes in cardiomyocyte metabolism have been heavily implicated in cardiac injury and heart failure (HF). However, there is emerging evidence that metabolism in nonmyocyte populations, including cardiac fibroblasts, immune cells, and endothelial cells, plays an important role in cardiac remodeling and adaptation to injury. Here, we discuss recent advances and insights into nonmyocyte metabolism in the healthy and injured heart. Metabolic switching from mitochondrial oxidative phosphorylation to glycolysis is critical for immune cell (macrophage and T lymphocyte) and fibroblast phenotypic switching in the inflamed and fibrotic heart. On the other hand, cardiac endothelial cells are heavily reliant on glycolytic metabolism, and thus impairments in glycolytic metabolism underlie endothelial cell dysfunction. Finally, we review current and ongoing metabolic therapies for HF and the potential implications for nonmyocyte metabolism.

Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to angiogenesis.

Cardiac fibroblasts are the major producers of extracellular matrix (ECM) to form infarct scar. We hypothesized that fibroblasts undergo a spectrum of phenotype states over the course of myocardial infarction (MI) from early onset to scar formation. Fibroblasts were isolated from the infarct region of C57BL/6J male mice (3-6 months old, n = 60) at days 0 (no MI control) and 1, 3, or 7 after MI. Whole transcriptome analysis was performed by RNA-sequencing. Of the genes sequenced, 3371 were differentially expressed after MI. Enrichment analysis revealed that MI day 1 fibroblasts displayed pro-inflammatory, leukocyte-recruiting, pro-survival, and anti-migratory phenotype through Tnfrsf9 and CD137 signaling. MI day 3 fibroblasts had a proliferative, pro-fibrotic, and pro-angiogenic profile with elevated Il4ra signaling. MI day 7 fibroblasts showed an anti-angiogenic homeostatic-like myofibroblast profile and with a step-wise increase in Acta2 expression. MI day 7 fibroblasts relied on Pik3r3 signaling to mediate Tgfb1 effects and Fgfr2 to regulate PI3K signaling. In vitro, the day 3 MI fibroblast secretome stimulated angiogenesis, while day 7 MI fibroblast secretome repressed angiogenesis through Thbs1 signaling. Our results reveal novel mechanisms for fibroblasts in expressing pro-inflammatory molecules and regulating angiogenesis following MI.

Myocardial infarction remodeling that progresses to heart failure: a signaling misunderstanding.

After myocardial infarction, remodeling of the left ventricle involves a wound-healing orchestra involving a variety of cell types. In order for wound healing to be optimal, appropriate communication must occur; these cells all need to come in at the right time, be activated at the right time in the right amount, and know when to exit at the right time. When this occurs, a new homeostasis is obtained within the infarct, such that infarct scar size and quality are sufficient to maintain left ventricular size and shape. The ideal scenario does not always occur in reality. Often, miscommunication can occur between infarct and remote spaces, across the temporal wound-healing spectrum, and across organs. When miscommunication occurs, adverse remodeling can progress to heart failure. This review discusses current knowledge gaps and recent development of the roles of inflammation and the extracellular matrix in myocardial infarction remodeling. In particular, the macrophage is one cell type that provides direct and indirect regulation of both the inflammatory and scar-forming responses. We summarize current research efforts focused on identifying biomarker indicators that reflect the status of each component of the wound-healing process to better predict outcomes.

LXR/RXR signaling and neutrophil phenotype following myocardial infarction classify sex differences in remodeling.

Sex differences in heart failure development following myocardial infarction (MI) are not fully understood. We hypothesized that differential MI signaling could explain variations in outcomes. Analysis of the mouse heart attack research tool 1.0 (422 mice; young = 5.4 ± 0.1; old = 23.3 ± 0.1 months of age) was used to dissect MI signaling pathways, which was validated in a new cohort of mice (4.8 ± 0.2 months of age); and substantiated in humans. Plasma collected at visit 2 from the MI subset of the Jackson Heart Study (JHS; a community-based study consisting of middle aged and older adults of African ancestry) underwent glycoproteomics grouped by outcome: (1) heart failure hospitalization after visit 2 (n = 3 men/12 women) and (2) without hospitalization through 2012 (n = 24 men/21 women). Compared to young male mice, the infarct region of young females had fewer, but more efficient tissue clearing neutrophils with reduced pro-inflammatory gene expression. Apolipoprotein (Apo) F, which acts upstream of the liver X receptors/retinoid X receptor (LXR/RXR) pathway, was elevated in the day 7 infarcts of old mice compared to young controls and was increased in both men and women with heart failure. In vitro, Apo F stimulated CD36 and peroxisome proliferator-activated receptor (PPAR)γ activation in male neutrophils to turn off NF-κB activation and stimulate LXR/RXR signaling to initiate resolution. Female neutrophils were desensitized to Apo F and instead relied on thrombospondin-1 stimulation of CD36 to upregulate AMP-activated protein kinase, resulting in an overall better wound healing strategy. With age, female mice were desensitized to LXR/RXR signaling, resulting in enhanced interleukin-6 activation, a finding replicated in the JHS community cohort. This is the first report to uncover sex differences in post-MI neutrophil signaling that yielded better outcomes in young females and worse outcomes with age.

Mapping macrophage polarization over the myocardial infarction time continuum.

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.

Matrix metalloproteinase-12 as an endogenous resolution promoting factor following myocardial infarction.

Following myocardial infarction (MI), timely resolution of inflammation promotes wound healing and scar formation while limiting excessive tissue damage. Resolution promoting factors (RPFs) are agents that blunt leukocyte trafficking and inflammation, promote necrotic and apoptotic cell clearance, and stimulate scar formation. Previously identified RPFs include mediators derived from lipids (resolvins, lipoxins, protectins, and maresins), proteins (glucocorticoids, annexin A1, galectin 1, and melanocortins), or gases (CO, H2S, and NO). Matrix metalloproteinase-12 (MMP-12; macrophage elastase) has shown promising RPF qualities in a variety of disease states. We review here the evidence that MMP-12 may serve as a novel RPF with potential therapeutic efficacy in the setting of MI.

Chronic Ethanol Administration Prevents Compensatory Cardiac Hypertrophy in Pressure Overload.

BACKGROUND: Alcohol is among the most commonly abused drugs worldwide and affects many organ systems, including the heart. Alcoholic cardiomyopathy is characterized by a dilated cardiac phenotype with extensive hypertrophy and extracellular matrix (ECM) remodeling. We have previously shown that chronic ethanol (EtOH) administration accelerates the progression to heart failure in a rat model of volume overload. However, the mechanism by which this decompensation occurs is unknown. For this study, we hypothesized that chronic EtOH administration would prevent compensatory hypertrophy and cardiac remodeling in a rodent model of pressure overload (PO). METHODS: Abdominal aortic constriction was used to create PO in 8-week-old male Wistar rats. Alcohol administration was performed via chronic intermittent EtOH vapor inhalation for 2 weeks prior to surgery and for the duration of the 8-week study. Echocardiography measurements were taken to assess ventricular functional and structural changes. RESULTS: PO increased posterior wall thickness and the hypertrophic markers, atrial and B-type natriuretic peptides (ANP and BNP). With the added stressor of EtOH, wall thickness, ANP, and BNP decreased in PO animals. The combination of PO and EtOH resulted in increased wall stress compared to PO alone. PO also caused increased expression of collagen I and III, whereas EtOH alone only increased collagen III. The combined stresses of PO and EtOH led to an increase in collagen I expression, but collagen III did not change, resulting in an increased collagen I/III ratio in the PO rats treated with EtOH. Lastly, Notch1 expression was significantly increased only in the PO rats treated with EtOH. CONCLUSIONS: Our data indicate that chronic EtOH may limit the cardiac hypertrophy induced by PO which may be associated with a Notch1 mechanism, resulting in increased wall stress and altered ECM profile.

Exposure to chronic alcohol accelerates development of wall stress and eccentric remodeling in rats with volume overload.

Chronic alcohol abuse is one of the leading causes of dilated cardiomyopathy (DCM) in the United States. Volume overload (VO) also produces DCM characterized by left ventricular (LV) dilatation and reduced systolic and diastolic function, eventually progressing to congestive heart failure. For this study, we hypothesized that chronic alcohol exposure would exacerbate cardiac dysfunction and remodeling due to VO. Aortocaval fistula surgery was used to induce VO, and compensatory cardiac remodeling was allowed to progress for either 3days (acute) or 8weeks (chronic). Alcohol was administered via chronic intermittent ethanol vapor (EtOH) for 2weeks before the acute study and for the duration of the 8week chronic study. Temporal alterations in LV function were assessed by echocardiography. At the 8week end point, pressure-volume loop analysis was performed by LV catheterization and cardiac tissue collected. EtOH did not exacerbate LV dilatation (end-systolic and diastolic diameter) or systolic dysfunction (fractional shortening, ejection fraction) due to VO. The combined stress of EtOH and VO decreased the eccentric index (posterior wall thickness to end-diastolic diameter ratio), increased end-diastolic pressure (EDP), and elevated diastolic wall stress. VO also led to increases in posterior wall thickness, which was not observed in the VO+EtOH group, and wall thickness significantly correlated with LV BNP expression. VO alone led to increases in interstitial collagen staining (picrosirius red), which while not statistically significant, tended to be decreased by EtOH. VO increased LV collagen I protein expression, whereas in rats with VO+EtOH, LV collagen I was not elevated relative to Sham. The combination of VO and EtOH also led to increases in LV collagen III expression relative to Sham. Rats with VO+EtOH had significantly lower collagen I/III ratio than rats with VO alone. During the acute remodeling phase of VO (3days), VO significantly increased collagen III expression, whereas this effect was not observed in rats with VO+EtOH. In conclusion, chronic EtOH accelerates the development of elevated wall stress and promotes early eccentric remodeling in rats with VO. Our data indicate that these effects may be due to disruptions in compensatory hypertrophy and extracellular matrix remodeling in response to volume overload.

Alcohol Vapor Inhalation as a Model of Alcohol-Induced Organ Disease.

BACKGROUND: Chronic intermittent ethanol vapor (CIEV) exposure has been used extensively to produce rodent models of alcohol dependence, but unlike other models of alcohol abuse, CIEV has not been assessed as a model of end-organ damage. The purpose of this study was to characterize the effects of CIEV on peripheral organ systems affected by alcohol abuse, including the liver, lungs, and cardiovascular system. METHODS: Adult male Sprague-Dawley rats were exposed to daily CIEV for a period of 8 weeks (14HR ON/10HR OFF), producing blood alcohol levels of ~200 mg/dl. Controls were exposed to room air. After 8 weeks, echocardiography was performed to assess cardiac function. Indices of liver injury (alanine and aspartate aminotransferases [ALT and AST]; cytochrome p450 2E1 [CYP2E1]; alcohol dehydrogenase [ADH]; Oil Red O and triglyceride content; lipid peroxidation; inflammatory cytokine expression; and macrophage infiltration), and lung inflammatory cell count, proinflammatory cytokine expression, and lipid peroxidation were measured. RESULTS: Left ventricular posterior wall thickness was significantly decreased, and systolic blood pressure was significantly elevated by CIEV compared with air controls. CIEV led to a significant increase in plasma ALT and triglycerides compared with room air controls. CIEV did not affect liver triglyceride content, lipid staining or peroxidation, but increased CYP2E1 and chemokine (C-C motif) ligand 2 (CCL2) protein expression, while decreasing ADH expression. CIEV significantly increased numbers of both polymorphonuclear neutrophils and lymphocytes in the bronchoalveolar lavage fluid, indicative of pulmonary inflammation. However, CIEV did not produce significant changes in lung mass, pulmonary lipid peroxidation, inflammatory cytokine expression, or edema. CONCLUSIONS: These results show that CIEV produces hepatic, pulmonary, and cardiovascular effects in rats similar to those found in other models of chronic alcohol administration. Alcohol vapor administration is a novel method of alcohol-induced tissue injury with high potential for widespread use in alcohol toxicology research.

Alcohol modulation of cardiac matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs favors collagen accumulation.

BACKGROUND: Chronic alcohol consumption has been shown in human and animal studies to result in collagen accumulation, myocardial fibrosis, and heart failure. Cardiac fibroblasts produce collagen and regulate extracellular matrix (ECM) homeostasis through the synthesis and activity of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), with the balance of MMPs/TIMPs determining the rate of collagen turnover. Dynamic changes of MMP and TIMP expression were reported in alcohol-induced hepatic fibrosis; however, the effect of alcohol on MMP/TIMP balance in the heart and cardiac fibroblasts is unknown. We hypothesized that alcohol exposure alters cardiac fibroblast MMP and TIMP expression to promote collagen accumulation in the heart. METHODS: Cardiac fibroblasts isolated from adult rats were cultured in the presence of alcohol (12.5 to 200 mM) for 48 hours. MMP, TIMP, and collagen type I and III expression were assayed by Western blot analysis. Hydroxyproline (HPro) was used as a marker of collagen production. The in vivo cardiac effects of ethanol (EtOH) were determined using rats exposed to EtOH vapor for 2 weeks, resulting in blood alcohol levels of 150 to 200 mg/dl. Cardiac collagen volume fraction (CVF), as well as MMP, TIMP, and collagen expression, was assessed. RESULTS: EtOH-exposed rats exhibited up-regulation of TIMP-1, TIMP-3 and TIMP-4 in the heart, with no significant increases in MMPs. Cardiac fibroblasts exhibited transformation to a profibrotic phenotype following exposure to alcohol. These changes were reflected by increased α-smooth muscle actin and collagen I and III expression, as well as increased collagen secretion. In vivo EtOH exposure also produced fibrosis, indicated by increased CVF and expression of collagens. CONCLUSIONS: Alcohol exposure modulates cardiac fibroblast MMP/TIMP expression favoring a profile associated with collagen accumulation. Our data suggest that this disrupted MMP/TIMP profile may contribute to the development of myocardial fibrosis and cardiac dysfunction resulting from chronic alcohol abuse.

Targeting immunometabolism during cardiorenal injury: roles of conventional and alternative macrophage metabolic fuels.

Macrophages play critical roles in mediating and resolving tissue injury as well as tissue remodeling during cardiorenal disease. Altered immunometabolism, particularly macrophage metabolism, is a critical underlying mechanism of immune dysfunction and inflammation, particularly in individuals with underlying metabolic abnormalities. In this review, we discuss the critical roles of macrophages in cardiac and renal injury and disease. We also highlight the roles of macrophage metabolism and discuss metabolic abnormalities, such as obesity and diabetes, which may impair normal macrophage metabolism and thus predispose individuals to cardiorenal inflammation and injury. As the roles of macrophage glucose and fatty acid metabolism have been extensively discussed elsewhere, we focus on the roles of alternative fuels, such as lactate and ketones, which play underappreciated roles during cardiac and renal injury and heavily influence macrophage phenotypes.

Immunometabolism, extracellular vesicles and cardiac injury.

Recent evidence from our lab and others suggests that metabolic reprogramming of immune cells drives changes in immune cell phenotypes along the inflammatory-to-reparative spectrum and plays a critical role in mediating the inflammatory responses to cardiac injury (e.g. hypertension, myocardial infarction). However, the factors that drive metabolic reprogramming in immune cells are not fully understood. Extracellular vesicles (EVs) are recognized for their ability to transfer cargo such as microRNAs from remote sites to influence cardiac remodeling. Furthermore, conditions such as obesity and metabolic syndrome, which are implicated in the majority of cardiovascular disease (CVD) cases, can skew production of EVs toward pro-inflammatory phenotypes. In this mini-review, we discuss the mechanisms by which EVs may influence immune cell metabolism during cardiac injury and factors associated with obesity and the metabolic syndrome that can disrupt normal EV function. We also discuss potential sources of cardio-protective and anti-inflammatory EVs, such as brown adipose tissue. Finally, we discuss implications for future therapeutics.

Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction.

Introduction: Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. Methods: MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO). Results: By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone. Discussion: Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

Direct Cardiac Actions of Sodium-Glucose Cotransporter 2 Inhibition Improve Mitochondrial Function and Attenuate Oxidative Stress in Pressure Overload-Induced Heart Failure.

Clinical trials showed that sodium-glucose cotransporter 2 (SGLT2) inhibitors, a class of drugs developed for treating diabetes mellitus, improve prognosis of patients with heart failure (HF). However, the mechanisms for cardioprotection by SGLT2 inhibitors are still unclear. Mitochondrial dysfunction and oxidative stress play important roles in progression of HF. This study tested the hypothesis that empagliflozin (EMPA), a highly selective SGLT2 inhibitor, improves mitochondrial function and reduces reactive oxygen species (ROS) while enhancing cardiac performance through direct effects on the heart in a non-diabetic mouse model of HF induced by transverse aortic constriction (TAC). EMPA or vehicle was administered orally for 4 weeks starting 2 weeks post-TAC. EMPA treatment did not alter blood glucose or body weight but significantly attenuated TAC-induced cardiac dysfunction and ventricular remodeling. Impaired mitochondrial oxidative phosphorylation (OXPHOS) in failing hearts was significantly improved by EMPA. EMPA treatment also enhanced mitochondrial biogenesis and restored normal mitochondria morphology. Although TAC increased mitochondrial ROS and decreased endogenous antioxidants, EMPA markedly inhibited cardiac ROS production and upregulated expression of endogenous antioxidants. In addition, EMPA enhanced autophagy and decreased cardiac apoptosis in TAC-induced HF. Importantly, mitochondrial respiration significantly increased in ex vivo cardiac fibers after direct treatment with EMPA. Our results indicate that EMPA has direct effects on the heart, independently of reductions in blood glucose, to enhance mitochondrial function by upregulating mitochondrial biogenesis, enhancing OXPHOS, reducing ROS production, attenuating apoptosis, and increasing autophagy to improve overall cardiac function in a non-diabetic model of pressure overload-induced HF.