Jason S. Huber

Adipose Tissue Insulin Action and IL-6 Signaling after Exercise in Obese Mice

INTRODUCTION Adipose tissue insulin action is impaired in obesity and is associated with inflammation, macrophage infiltration, and polarization toward a proinflammatory phenotype. Acute exercise can reduce markers of adipose inflammation, including interleukin (IL) 6, in parallel with improvements in insulin action; however, others have provided evidence that IL-6 has anti-inflammatory properties. PURPOSE This study aimed to examine the relation between IL-6 signaling, macrophage infiltration, and polarization and insulin action in inguinal fat after acute exercise in obese, insulin-resistant mice. METHODS Male C57BL/6 mice were fed a low-fat diet (10% kcal lard) or a high-fat diet (HFD, 60% kcal lard) for 7 wk and then underwent an acute bout of exercise (2-h treadmill running: 15 m·min, 5% incline). RESULTS The HFD resulted in increased body mass, glucose intolerance, and attenuated insulin-induced AKT Thr308 phosphorylation in inguinal fat. This was accompanied by increases in indices of macrophage infiltration (F4/80, CD68, and monocyte chemoattractant protein-1 expression) and polarization toward an M1 phenotype (increased expression of CD11c, CD11c/galactose-type C-type lectin 1, and inducible nitric oxide synthase). Immunofluorescence imaging demonstrated increased F4/80- and CD11c-positive cells with the HFD. Two hours after exercise, the insulin-induced activation of AKT Th308 phosphorylation was recovered in HFD mice. This was accompanied by an upregulation of IL-6 and IL-10 signaling, as demonstrated by increased expression of IL-6, IL-10, and SOCS3 as well as STAT3 phosphorylation. Furthermore, acute exercise resulted in a shift toward reduction in M1 polarization indicated by a decrease in the ratio of CD11c to galactose-type C-type lectin 1 mRNA as well as a decline in F4/80- and CD11c-positive cells. CONCLUSIONS The results suggest a link between exercise-induced increases in IL-6, reductions in indices of M1 macrophages, and increased IL-10, a reputed anti-inflammatory cytokine with insulin-sensitizing properties.

Exercise mediated IL-6 signaling occurs independent of inflammation and is amplified by training in mouse adipose tissue

The purpose of this investigation was to determine whether exercise-induced increases in adipose tissue interleukin 6 (IL-6) signaling occurred as part of a larger proinflammatory response to exercise and whether the induction of IL-6 signaling with acute exercise was altered in trained mice in parallel with changes in the IL-6 receptor complex. Sedentary and trained C57BL/6J mice were challenged with an acute bout of exercise. Adipose tissue and plasma were collected immediately and 4 h afterward and analyzed for changes in indices of IL-6 signaling, circulating IL-6, markers of adipose tissue inflammation, and expression/content of IL-6 receptor and glycoprotein 130 (gp130). In untrained mice, IL-6 mRNA increased immediately after exercise, and increases in indices of IL-6 signaling were increased 4 h after exercise in epididymal, but not inguinal adipose tissue. This occurred independent of increases in plasma IL-6 and alterations in markers of inflammation. When compared with untrained mice, in trained mice, acute exercise induced the expression of gp130 and IL-6 receptor alpha (IL-6Rα), and training increased the protein content of these. Acute exercise induced the expression, and training increased the protein content, of glycoprotein 130 and IL-6Rα and was associated with a more rapid increase in markers of IL-6 signaling in epididymal adipose tissue from trained compared with untrained mice. The ability of exogenous IL-6 to increase phosphorylation of STAT3 was similar between groups. Our findings demonstrate that acute exercise increases IL-6 signaling in a depot-dependent manner, likely through an autocrine/paracrine mechanism. This response is initiated more rapidly after exercise in trained mice, potentially as a result of increases in IL-6Rα and gp130.

Central-acting therapeutics alleviate respiratory weakness caused by heart failure–induced ventilatory overdrive

Drugs that penetrate the blood-brain barrier normalize ventilatory function and prevent diaphragm atrophy in heart failure. A brainy treatment for heart failure Respiratory difficulty and diaphragm weakness are known symptoms of heart failure, but they are usually attributed to pulmonary edema damaging the diaphragm through physical stress. Now, Foster et al. have determined that this is not the only contributing factor, using mouse models to demonstrate that diaphragm weakness develops even in heart failure without pulmonary edema. The authors linked this observation to changes in angiotensin II and β-adrenergic signaling, which result in centrally controlled ventilatory overdrive. As a result, the researchers found that drugs targeting β-adrenergic signaling were effective in preventing ventilatory overdrive and subsequent diaphragmatic injury but only if they penetrated the blood-brain barrier. Diaphragmatic weakness is a feature of heart failure (HF) associated with dyspnea and exertional fatigue. Most studies have focused on advanced stages of HF, leaving the cause unresolved. The long-standing theory is that pulmonary edema imposes a mechanical stress, resulting in diaphragmatic remodeling, but stable HF patients rarely exhibit pulmonary edema. We investigated how diaphragmatic weakness develops in two mouse models of pressure overload–induced HF. As in HF patients, both models had increased eupneic respiratory pressures and ventilatory drive. Despite the absence of pulmonary edema, diaphragmatic strength progressively declined during pressure overload; this decline correlated with a reduction in diaphragm cross-sectional area and preceded evidence of muscle weakness. We uncovered a functional codependence between angiotensin II and β-adrenergic (β-ADR) signaling, which increased ventilatory drive. Chronic overdrive was associated with increased PERK (double-stranded RNA–activated protein kinase R–like ER kinase) expression and phosphorylation of EIF2α (eukaryotic translation initiation factor 2α), which inhibits protein synthesis. Inhibition of β-ADR signaling after application of pressure overload normalized diaphragm strength, Perk expression, EIF2α phosphorylation, and diaphragmatic cross-sectional area. Only drugs that were able to penetrate the blood-brain barrier were effective in treating ventilatory overdrive and preventing diaphragmatic atrophy. These data provide insight into why similar drugs have different benefits on mortality and symptomatology, despite comparable cardiovascular effects.

Dissecting the role of the myofilament in diaphragm dysfunction during the development of heart failure in mice

Dyspnea and reduced exercise capacity, caused, in part, by respiratory muscle dysfunction, are common symptoms in patients with heart failure (HF). However, the etiology of diaphragmatic dysfunction has not been identified. To investigate the effects of HF on diaphragmatic function, models of HF were surgically induced in CD-1 mice by transverse aortic constriction (TAC) and acute myocardial infarction (AMI), respectively. Assessment of myocardial function, isolated diaphragmatic strip function, myofilament force-pCa relationship, and phosphorylation status of myofilament proteins was performed at either 2 or 18 wk postsurgery. Echocardiography and invasive hemodynamics revealed development of HF by 18 wk postsurgery in both models. In vitro diaphragmatic force production was preserved in all groups while morphometric analysis revealed diaphragmatic atrophy and fibrosis in 18 wk TAC and AMI groups. Isometric force-pCa measurements of myofilament preparations revealed reduced Ca(2+) sensitivity of force generation and force generation at half-maximum and maximum Ca(2+) activation in 18 wk TAC. The rate of force redevelopment (ktr) was reduced in all HF groups at high levels of Ca(2+) activation. Finally, there were significant changes in the myofilament phosphorylation status of the 18 wk TAC group. This includes a decrease in the phosphorylation of troponin T, desmin, myosin light chain (MLC) 1, and MLC 2 as well as a shift in myosin isoforms. These results indicate that there are multiple changes in diaphragmatic myofilament function, which are specific to the type and stage of HF and occur before overt impairment of in vitro force production.

Pulmonary Flow as an Improved Method for Determining Cardiac Output in Mice after Myocardial Infarction

Background: Echocardiography is a valuable noninvasive technique to estimate cardiac output (CO) from the left ventricle (LV) not only in clinical practice but also in small‐animal experiments. CO is used to grade cardiac function and is especially important when investigating cardiac injury (e.g., myocardial infarction [MI]). Critically, MI deforms the LV, invalidating the assumptions fundamental to calculating of cardiac volumes directly from the LV. Thus, the purpose of this study was to determine if Doppler‐derived blood flow through the pulmonary trunk (pulmonary flow [PF]) was an improved method over conventional LV–dependent echocardiography to accurately determine CO after MI. Methods: Variations in CO were induced either by transverse aortic constriction or MI. Echocardiography was performed in healthy (n = 27), transverse aortic constriction (n = 25), and MI (n = 41) mice. CO calculated from PF (pulsed‐wave Doppler) was internally compared with CO calculated from left ventricular images using M‐mode (Teichholz formula) and the single‐plane ellipsoid two‐dimensional (2D) formula and externally compared with the gold standard, flow probe CO. Results: In healthy mice, all three echocardiographic methods (M‐mode, 2D, and PF) correlated well with flow probe–derived CO. In MI mice, only PF CO values correlated well with flow probe values. Bland‐Altman analysis confirmed that PF was improved over M‐mode and 2D echocardiography. Inter‐ and intrauser variability of PF CO was reduced, and both inter‐ and intraclass correlation coefficients were improved compared with either M‐mode or 2D CO calculations. Conclusions: PF CO calculated from pulsed‐wave Doppler through the pulmonary trunk was an improved method of estimating CO over LV–dependent formulas after MI. HighlightsLeft ventricular infarction drastically alters chamber shape and wall kinetics.This invalidates assumptions for calculating cardiac output (CO).Pulmonary flow (PF) precisely and accurately determines CO.PF is easily determined in experimental models of heart failure.PF reduces user bias and variability in determining CO.

Pathophysiological Mapping of Experimental Heart Failure: Left and Right Ventricular Remodeling in Transverse Aortic Constriction Is Temporally, Kinetically and Structurally Distinct

A growing proportion of heart failure (HF) patients present with impairments in both ventricles. Experimental pressure-overload (i.e., transverse aortic constriction, TAC) induces left ventricle (LV) hypertrophy and failure, as well as right ventricle (RV) dysfunction. However, little is known about the coordinated progression of biventricular dysfunction that occurs in TAC. Here we investigated the time course of systolic and diastolic function in both the LV and RV concurrently to improve our understanding of the chronology of events in TAC. Hemodynamic, histological, and morphometric assessments were obtained from the LV and RV at 2, 4, 9, and 18 weeks post-surgery. Results: Systolic pressures peaked in both ventricles at 4 weeks, thereafter steadily declining in the LV, while remaining elevated in the RV. The LV and RV followed different structural and functional timelines, suggesting the patterns in one ventricle are independent from the opposing ventricle. RV hypertrophy/fibrosis and pulmonary arterial remodeling confirmed a progressive right-sided pathology. We further identified both compensation and decompensation in the LV with persistent concentric hypertrophy in both phases. Finally, diastolic impairments in both ventricles manifested as an intricate progression of multiple parameters that were not in agreement until overt systolic failure was evident. Conclusion: We establish pulmonary hypertension was secondary to LV dysfunction, confirming TAC is a model of type II pulmonary hypertension. This study also challenges some common assumptions in experimental HF (e.g., the relationship between fibrosis and filling pressure) while addressing a knowledge gap with respect to temporality of RV remodeling in pressure-overload.

Moderate and severe hypoxia elicit divergent effects on cardiovascular function and physiological rhythms

In the present study, we provide evidence for divergent physiological responses to moderate compared to severe hypoxia, addressing an important knowledge gap related to severity, duration and after‐effects of hypoxia encountered in cardiopulmonary situations. The physiological responses to moderate and severe hypoxia were not proportional, linear or concurrent with the time‐of‐day. Hypoxia elicited severity‐dependent physiological responses that either persisted or fluctuated throughout normoxic recovery. The physiological basis for these distinct cardiovascular responses implicates a shift in the sympathovagal set point and probably not molecular changes at the artery resulting from hypoxic stress.

Sodium nitrate supplementation alters mitochondrial H2O2 emission but does not improve mitochondrial oxidative metabolism in the heart of healthy rats

Supplementation with dietary inorganic nitrate ([Formula: see text]) is increasingly recognized to confer cardioprotective effects in both healthy and clinical populations. While the mechanism(s) remains ambiguous, in skeletal muscle oral consumption of NaNO3 has been shown to improve mitochondrial efficiency. Whether NaNO3 has similar effects on mitochondria within the heart is unknown. Therefore, we comprehensively investigated the effect of NaNO3 supplementation on in vivo left ventricular (LV) function and mitochondrial bioenergetics. Healthy male Sprague-Dawley rats were supplemented with NaNO3 (1 g/l) in their drinking water for 7 days. Echocardiography and invasive hemodynamics were used to assess LV morphology and function. Blood pressure (BP) was measured by tail-cuff and invasive hemodynamics. Mitochondrial bioenergetics were measured in LV isolated mitochondria and permeabilized muscle fibers by high-resolution respirometry and fluorometry. Nitrate decreased ( P < 0.05) BP, LV end-diastolic pressure, and maximal LV pressure. Rates of LV relaxation (when normalized to mean arterial pressure) tended ( P = 0.13) to be higher with nitrate supplementation. However, nitrate did not alter LV mitochondrial respiration, coupling efficiency, or oxygen affinity in isolated mitochondria or permeabilized muscle fibers. In contrast, nitrate increased ( P < 0.05) the propensity for mitochondrial H2O2 emission in the absence of changes in cellular redox state and decreased the sensitivity of mitochondria to ADP (apparent Km). These results add to the therapeutic potential of nitrate supplementation in cardiovascular diseases and suggest that nitrate may confer these beneficial effects via mitochondrial redox signaling.