Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Attenuates the Development of Angiotensin II-Mediated Abdominal Aortic Aneurysm in Obese ob/ob Mice.

The infiltration and activation of macrophages as well as lymphocytes within the aorta contribute to the pathogenesis of abdominal aortic aneurysm (AAA). Invariant natural killer T (iNKT) cells are unique subset of T lymphocytes and have a crucial role in atherogenesis. However, it remains unclear whether iNKT cells also impact on the development of AAA. Ob/ob mice were administered angiotensin II (AngII, 1,000 ng/kg/min) or phosphate-buffered saline (PBS) by osmotic minipumps for 4 weeks and further divided into 2 groups; α-galactosylceramide (αGC; PBS-αGC; n = 5 and AngII-αGC; n = 12), which specifically activates iNKT cells, and PBS (PBS-PBS; n = 10, and AngII-PBS; n = 6). Maximal abdominal aortic diameter was comparable between PBS-PBS and PBS-αGC, and was significantly greater in AngII-PBS than in PBS-PBS. This increase was significantly attenuated in AngII-αGC without affecting blood pressure. αGC significantly enhanced iNKT cell infiltration compared to PBS-PBS. The ratio of F4/80-positive macrophages or CD3-positive T lymphocytes area to the lesion area was significantly higher in AngII-PBS than in PBS-PBS, and was significantly decreased in AngII-αGC. Gene expression of M2-macrophage specific markers, arginase-1 and resistin-like molecule alpha, was significantly greater in aortic tissues from AngII-αGC compared to AngII-PBS 1 week after AngII administration, and this increase was diminished at 4 weeks. Activation of iNKT cells by αGC can attenuate AngII-mediated AAA in ob/ob mice via inducing anti-inflammatory M2 polarized state. Activation of iNKT cells by the bioactive lipid αGC may be a novel therapeutic target against the development of AAA.

Positioning an ECG electrode to the dorsal side can record higher amplitude of CMAPs during cryoballoon ablation.

PURPOSE: Phrenic nerve injury (PNI) is one of the important complications during cryoballoon (CB) ablation. Recording diaphragmatic compound motor action potentials (CMAPs) during CB ablation can predict PNI. CMAP monitoring may be inaccurate when CMAP amplitudes are low. We examined the effect of positioning an electrocardiography (ECG) electrode at the dorsal side. METHODS: We retrospectively analyzed the cases of 197 consecutive patients who underwent CB ablation for pulmonary vein isolation (PVI) (April 2016 to December 2018) at our institution. CMAP amplitudes were monitored using two recording methods just before cryoapplication. (a) Conventional method: right-arm ECG electrode positioned 5 cm above the xiphoid on the ventral side; left-arm ECG electrode positioned along the costal margin. (b) Our original method: right-arm electrode positioned 5 cm above the xiphoid on the dorsal side; left-arm electrode positioned along the costal margin. RESULTS: The CMAP amplitude during right phrenic nerve pacing was significantly higher at the dorsal side than the ventral side (0.80 ± 0.31 mV vs 0.66 ± 0.29 mV, P < .01). Similarly, the CMAP amplitude during left phrenic nerve pacing was significantly higher at the dorsal side than the ventral side (0.92 ± 0.39 mV, 0.73 ± 0.37 mV, P < .01). PNI occurred in six patients (3.0%); three patients experienced transient PNI, another three patients experienced persistent PNI, and none developed permanent PNI. CONCLUSIONS: CMAP amplitudes were significantly high at the dorsal side compared to the ventral side. Monitoring phrenic nerve function using an ECG electrode at the dorsal side is a simple and easy procedure.

Dipeptidyl peptidase-4 inhibitor improved exercise capacity and mitochondrial biogenesis in mice with heart failure via activation of glucagon-like peptide-1 receptor signalling.

AIMS: Exercise capacity is reduced in heart failure (HF) patients, due mostly to skeletal muscle abnormalities including impaired energy metabolism, mitochondrial dysfunction, fibre type transition, and atrophy. Glucagon-like peptide-1 (GLP-1) has been shown to improve exercise capacity in HF patients. We investigated the effects of the administration of a dipeptidyl peptidase (DPP)-4 inhibitor on the exercise capacity and skeletal muscle abnormalities in an HF mouse model after myocardial infarction (MI). METHODS AND RESULTS: MI was created in male C57BL/6J mice by ligating the left coronary artery, and a sham operation was performed in other mice. The mice were then divided into two groups according to the treatment with or without a DPP-4 inhibitor, MK-0626 [1 mg/kg body weight (BW)/day] provided in the diet. Four weeks later, the exercise capacity evaluated by treadmill test was revealed to be limited in the MI mice, and it was ameliorated in the MI + MK-0626 group without affecting the infarct size or cardiac function. The citrate synthase activity, mitochondrial oxidative phosphorylation capacity, supercomplex formation, and their quantity were reduced in the skeletal muscle from the MI mice, and these decreases were normalized in the MI + MK-0626 group, in association with the improvement of mitochondrial biogenesis. Immunohistochemical staining also revealed that a shift toward the fast-twitch fibre type in the MI mice was also reversed by MK-0626. Favourable effects of MK-0626 were significantly inhibited by treatment of GLP-1 antagonist, Exendin-(9-39) (150 pmol/kg BW/min, subcutaneous osmotic pumps) in MI + MK-0626 mice. Similarly, exercise capacity and mitochondrial function were significantly improved by treatment of GLP-1 agonist, Exendin-4 (1 nmol/kg/BW/h, subcutaneous osmotic pumps). CONCLUSIONS: A DPP-4 inhibitor may be a novel therapeutic agent against the exercise intolerance seen in HF patients by improving the mitochondrial biogenesis in their skeletal muscle.

Serum myostatin levels are independently associated with skeletal muscle wasting in patients with heart failure.

BACKGROUND: It has been reported that skeletal muscle mass and strength are decreased in patients with heart failure (HF), and HF is associated with both reduced exercise capacity and adverse clinical outcomes. Myostatin has been known as a negative regulator of muscle growth, follistatin as the myostatin antagonist, maintaining tissue homeostasis. We thus determined serum myostatin levels in HF patients and whether they are associated with skeletal muscle wasting. METHODS AND RESULTS: Forty one consecutive HF patients (58±15years old, New York Heart Association class I-III) and 30 age-matched healthy subjects as controls (53±8years old) were studied. Serum myostatin levels were significantly lower in HF patients than controls (18.7±7.4 vs. 23.6±5.2ng/mL, P<0.001). Circumference of the thickest part of the right thigh was significantly small (468±72 vs. 559±37mm, P=0.001) and lower extremity muscular strength was lower in patients with HF (129±55 vs. 219±52N×m, P<0.001). Fourteen HF patients (34%) had muscle wasting. By univariate analysis, higher age, higher serum follistatin, and lower serum myostatin were significantly associated with the presence of muscle wasting. By multivariate analysis, serum myostatin levels were independently associated with muscle wasting (OR=0.77, 95% CI [0.58, 0.93], P=0.02). CONCLUSION: Serum myostatin levels were significantly decreased in HF patients and associated with lower extremity muscle wasting, suggesting that myostatin may be an important factor for maintaining skeletal muscle mass and strength in HF.

The experimental model of transition from compensated cardiac hypertrophy to failure created by transverse aortic constriction in mice.

BACKGROUND: Transverse aortic constriction (TAC) operation is used as an experimental model of left ventricular (LV) hypertrophy and LV failure in mice. The severity of LV remodeling or failure may depend on the degree of TAC, but is variable among operated animals. Therefore, we tried to identify the optimal diameter of TAC to create this model with ease and high reproducibility. METHODS AND RESULTS: To produce TAC in C57BL/6J mice (7-9 weeks, body weight 19-26 g, n = 109), a 7-0 nylon suture ligature was tightly tied around the transverse aorta against needles with 3 different diameters (mm); 0.40, 0.385 and 0.375. LV wall thickness, end-diastolic dimension, fractional shortening were measured by echocardiography. At 4 weeks after TAC, no mouse with the 0.400 mm gauge progressed in LV failure. The 0.385 mm pin gauge mouse kept a more survival rate compared with the 0.375 mm (59% vs 48%), representing same efficient in LV failure. With the 0.385 mm pin gauge, hearts of mice remained LV hypertrophy at 1 week after TAC, followed by LV failure at 4 weeks. CONCLUSION: TAC with the diameter of 0.385 mm can effectively induce the transition from LV hypertrophy to failure in mice with relatively preserved survival.

Serum brain-derived neurotropic factor level predicts adverse clinical outcomes in patients with heart failure.

BACKGROUND: Brain-derived neurotropic factor (BDNF) is involved in cardiovascular diseases as well as skeletal muscle energy metabolism and depression. We investigated whether serum BDNF level was associated with prognosis in patients with heart failure (HF). METHODS AND RESULTS: We measured the serum BDNF level in 58 patients with HF (59.2 ± 13.7 years old, New York Heart Association functional class I-III) at baseline, and adverse events, including all cardiac deaths and HF rehospitalizations, were recorded during the median follow-up of 20.3 months. In a univariate analysis, serum BDNF levels were significantly associated with peak oxygen capacity (ß = 0.547; P = .003), anaerobic threshold (ß = 0.929; P = .004), and log minute ventilation/carbon dioxide production slope (ß = -10.15; P = .005), but not Patient Health Questionnaire scores (ß = -0.099; P = .586). A multivariate analysis demonstrated that serum BDNF level was an independent prognostic factor of adverse events (hazard ratio 0.41, 95% confidence interval 0.20-0.84; P = .003). The receiver operating characteristic curve demonstrated that low levels of BDNF (<17.4 ng/mL) were associated with higher rates of adverse events compared with high levels of BDNF (≥17.4 ng/mL; log rank test: P < .001). CONCLUSIONS: Decreased serum BDNF levels were significantly associated with adverse outcomes in HF patients, suggesting that these levels can be a useful prognostic biomarker.

Angiotensin II can directly induce mitochondrial dysfunction, decrease oxidative fibre number and induce atrophy in mouse hindlimb skeletal muscle.

NEW FINDINGS: What is the central question of this study? Does angiotensin II directly induce skeletal muscle abnormalities? What is the main finding and its importance? Angiotensin II induces skeletal muscle abnormalities and reduced exercise capacity. Mitochondrial dysfunction and a decreased number of oxidative fibres are manifest early, while muscle atrophy is seen later. Thus, angiotensin II may play an important role in the skeletal muscle abnormalities observed in a wide variety of diseases. Skeletal muscle abnormalities, such as mitochondrial dysfunction, a decreased percentage of oxidative fibres and atrophy, are the main cause of reduced exercise capacity observed in ageing and various diseases, including heart failure. The renin-angiotensin system, particularly angiotensin II (Ang II), is activated in the skeletal muscle in these conditions. Here, we examined whether Ang II could directly induce these skeletal muscle abnormalities and investigated their time course. Angiotensin II (1000 ng kg(-1)  min(-1) ) or vehicle was administered to male C57BL/6J mice (10-12 weeks of age) via subcutaneously implanted osmotic minipumps for 1 or 4 weeks. Angiotensin II significantly decreased body and hindlimb skeletal muscle weights compared with vehicle at 4 weeks. In parallel, muscle cross-sectional area was also decreased in the skeletal muscle at 4 weeks. Muscle RING finger-1 and atrogin-1 were significantly increased in the skeletal muscle from mice treated with Ang II. In addition, cleaved caspase-3 and terminal deoxynucleotidyl trasferase-mediated dUTP nick-positive nuclei were significantly increased in mice treated with Ang II at 1 and 4 weeks, respectively. Mitochondrial oxidative enzymes, such as citrate synthase, complex I and complex III activities were significantly decreased in the skeletal muscle from mice treated Ang II at 1 and 4 weeks. NAD(P)H oxidase-derived superoxide production was increased. NADH staining revealed that type I fibres were decreased and type IIb fibres increased in mice treated with Ang II at 1 week. The work and running distance evaluated by a treadmill test were significantly decreased in mice treated with Ang II at 4 weeks. Thus, Ang II could directly induce the abnormalities in skeletal muscle function and structure.

(Pro)renin receptor in skeletal muscle is involved in the development of insulin resistance associated with postinfarct heart failure in mice.

We previously reported that insulin resistance was induced by the impairment of insulin signaling in the skeletal muscle from heart failure (HF) via NAD(P)H oxidase-dependent oxidative stress. (Pro)renin receptor [(P)RR] is involved in the activation of local renin-angiotensin system and subsequent oxidative stress. We thus examined whether (P)RR inhibitor, handle region peptide (HRP), could ameliorate insulin resistance in HF after myocardial infarction (MI) by improving oxidative stress and insulin signaling in the skeletal muscle. C57BL6J mice were divided into four groups: sham operated (Sham, n = 10), Sham treated with HRP (Sham+HRP, 0.1 mg·kg(-1)·day(-1), n = 10), MI operated (MI, n = 10), and MI treated with HRP (MI+HRP, 0.1 mg/kg/day, n = 10). After 4 wk, MI mice showed left ventricular dysfunction, which was not affected by HRP. (P)RR was upregulated in the skeletal muscle after MI (149% of sham, P < 0.05). The decrease in plasma glucose after insulin load was smaller in MI than in Sham (21 ± 2 vs. 44 ± 3%, P < 0.05), and was greater in MI+HRP (38 ± 2%, P < 0.05) than in MI. Insulin-stimulated serine phosphorylation of Akt and glucose transporter 4 translocation were decreased in the skeletal muscle from MI by 48 and 49% of Sham, both of which were ameliorated in MI+HRP. Superoxide production and NAD(P)H oxidase activities were increased in MI, which was inhibited in MI+HRP. HRP ameliorated insulin resistance associated with HF by improving insulin signaling via the inhibition of NAD(P)H oxidase-induced superoxide production in the skeletal muscle. The (P)RR pathway is involved in the development of insulin resistance, at least in part, via the impairment of insulin signaling in the skeletal muscle from HF.

Pioglitazone ameliorates the lowered exercise capacity and impaired mitochondrial function of the skeletal muscle in type 2 diabetic mice.

We have reported that exercise capacity is reduced in high fat diet (HFD)-induced diabetic mice, and that this reduction is associated with impaired mitochondrial function in skeletal muscle (SKM). However, it remains to be clarified whether the treatment of diabetes ameliorates the reduced exercise capacity. Therefore, we examined whether an insulin-sensitizing drug, pioglitazone, could improve exercise capacity in HFD mice. C57BL/6J mice were fed a normal diet (ND) or HFD, then treated with or without pioglitazone (3 mg/kg/day) to yield the following 4 groups: ND+vehicle, ND+pioglitazone, HFD+vehicle, and HFD+pioglitazone (n=10 each). After 8 weeks, body weight, plasma glucose, and insulin in the HFD+vehicle were significantly increased compared to the ND+vehicle group. Pioglitazone normalized the insulin levels in HFD-fed mice, but did not affect the body weight or plasma glucose. Exercise capacity determined by treadmill tests was significantly reduced in the HFD+vehicle, and this reduction was almost completely ameliorated in HFD+pioglitazone mice. ADP-dependent mitochondrial respiration, complex I and III activities, and citrate synthase activity were significantly decreased in the SKM of the HFD+vehicle animals, and these decreases were also attenuated by pioglitazone. NAD(P)H oxidase activity was significantly increased in the HFD+vehicle compared with the ND+vehicle, and this increase was ameliorated in HFD+pioglitazone mice. Pioglitazone improved the exercise capacity in diabetic mice, which was due to the improvement in mitochondrial function and attenuation of oxidative stress in the SKM. Our data suggest that pioglitazone may be useful as an agent for the treatment of diabetes mellitus.

Combination of exercise training and diet restriction normalizes limited exercise capacity and impaired skeletal muscle function in diet-induced diabetic mice.

Exercise training (EX) and diet restriction (DR) are essential for effective management of obesity and insulin resistance in diabetes mellitus. However, whether these interventions ameliorate the limited exercise capacity and impaired skeletal muscle function in diabetes patients remains unexplored. Therefore, we investigated the effects of EX and/or DR on exercise capacity and skeletal muscle function in diet-induced diabetic mice. Male C57BL/6J mice that were fed a high-fat diet (HFD) for 8 weeks were randomly assigned for an additional 4 weeks to 4 groups: control, EX, DR, and EX+DR. A lean group fed with a normal diet was also studied. Obesity and insulin resistance induced by a HFD were significantly but partially improved by EX or DR and completely reversed by EX+DR. Although exercise capacity decreased significantly with HFD compared with normal diet, it partially improved with EX and DR and completely reversed with EX+DR. In parallel, the impaired mitochondrial function and enhanced oxidative stress in the skeletal muscle caused by the HFD were normalized only by EX+DR. Although obesity and insulin resistance were completely reversed by DR with an insulin-sensitizing drug or a long-term intervention, the exercise capacity and skeletal muscle function could not be normalized. Therefore, improvement in impaired skeletal muscle function, rather than obesity and insulin resistance, may be an important therapeutic target for normalization of the limited exercise capacity in diabetes. In conclusion, a comprehensive lifestyle therapy of exercise and diet normalizes the limited exercise capacity and impaired muscle function in diabetes mellitus.

Activation of invariant natural killer T cells by α-galactosylceramide ameliorates myocardial ischemia/reperfusion injury in mice.

Invariant natural killer T (iNKT) cells orchestrate tissue inflammation via regulating various cytokine productions. However the role of iNKT cells has not been determined in myocardial ischemia/reperfusion (I/R) injury. The purpose of this study was to examine whether the activation of iNKT cells by α-galactosylceramide (α-GC), which specifically activates iNKT cells, could affect myocardial I/R injury. I/R or sham operation was performed in male C57BL/6J mice. I/R mice received the injection of either αGC (I/R+αGC, n=48) or vehicle (I/R+vehicle, n=49) 30 min before reperfusion. After 24h, infarct size/area at risk was smaller in I/R+αGC than in I/R+vehicle (37.8 ± 2.7% vs. 47.1 ± 2.5%, P<0.05), with no significant changes in area at risk. The numbers of infiltrating myeloperoxidase- and CD3-positive cells were lower in I/R+αGC. Apoptosis evaluated by TUNEL staining and caspase-3 protein was also attenuated in I/R+αGC. Myocardial gene expression of tumor necrosis factor-α and interleukin (IL)-1ß in I/R+αGC was lower to 46% and 80% of that in I/R+vehicle, respectively, whereas IL-10, IL-4, and interferon (IFN)-γ were higher in I/R+αGC than I/R+vehicle by 2.0, 4.1, and 9.6 folds, respectively. The administration of anti-IL-10 receptor antibody into I/R+αGC abolished the protective effects of αGC on I/R injury (infarct size/area at risk: 53.1 ± 5.2% vs. 37.4 ± 3.5%, P<0.05). In contrast, anti-IL-4 and anti-IFN-γ antibodies did not exert such effects. In conclusion, activated iNKT cells by αGC play a protective role against myocardial I/R injury through the enhanced expression of IL-10. Therapies designed to activate iNKT cells might be beneficial to protect the heart from I/R injury.

Angiotensin II receptor blocker improves the lowered exercise capacity and impaired mitochondrial function of the skeletal muscle in type 2 diabetic mice.

NAD(P)H oxidase-induced oxidative stress is at least in part involved with lowered exercise capacity and impaired mitochondrial function in high-fat diet (HFD)-induced diabetic mice. NAD(P)H oxidase can be activated by activation of the renin-angiotensin system. We investigated whether ANG II receptor blocker can improve exercise capacity in diabetic mice. C57BL/6J mice were fed a normal diet (ND) or HFD, and each group of mice was divided into two groups: treatment with or without olmesartan (OLM; 3 mg·kg(-1)·day(-1) in the drinking water). The following groups of mice were studied: ND, ND+OLM, HFD, and HFD+OLM (n = 10 for each group). After 8 wk, HFD significantly increased body weight, plasma glucose, and insulin compared with ND, and OLM did not affect these parameters in either group. Exercise capacity, as determined by treadmill tests, was significantly reduced in HFD, and this reduction was ameliorated in HFD+OLM. ADP-dependent mitochondrial respiration was significantly decreased, and NAD(P)H oxidase activity and superoxide production by lucigenin chemiluminescence were significantly increased in skeletal muscle from HFD, which were attenuated by OLM. There were no such effects by OLM in ND. We concluded that OLM ameliorated the decrease in exercise capacity in diabetic mice via improvement in mitochondrial function and attenuation of oxidative stress in skeletal muscle. These data may have a clinical impact on exercise capacity in the medical treatment of diabetes mellitus.

Activation of natural killer T cells ameliorates postinfarct cardiac remodeling and failure in mice.

RATIONALE: Chronic inflammation in the myocardium is involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Invariant natural killer T (iNKT) cells have been shown to produce inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have determined the pathophysiological role of iNKT cells in post-MI LV remodeling. OBJECTIVE: The purpose of this study was to examine whether the activation of iNKT cells might affect the development of LV remodeling and failure. METHODS AND RESULTS: After creation of MI, mice received the injection of either α-galactosylceramide (αGC; n=27), the activator of iNKT cells, or phosphate-buffered saline (n=31) 1 and 4 days after surgery, and were followed during 28 days. Survival rate was significantly higher in MI+αGC than MI+PBS (59% versus 32%, P<0.05). LV cavity dilatation and dysfunction were significantly attenuated in MI+αGC, despite comparable infarct size, accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. The infiltration of iNKT cells were increased during early phase in noninfarcted LV from MI and αGC further enhanced them. It also enhanced LV interleukin (IL)-10 gene expression at 7 days, which persisted until 28 days. AntienIL-10 receptor antibody abrogated these protective effects of αGC on MI remodeling. The administration of αGC into iNKT cell-deficient Jα18(-/-) mice had no such effects, suggesting that αGC was a specific activator of iNKT cells. CONCLUSIONS: iNKT cells play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as IL-10.

Successful termination of recurrent ventricular arrhythmias by adaptive servo-ventilation in a patient with heart failure.

A 60-year-old woman who underwent operation due to severe aortic stenosis with left ventricular dysfunction had frequent nonsustained ventricular tachycardia (NSVT) at night. She had an increased apnea-hypopnea index and a reduction in minimum O2 saturation during sleep, which was closely associated with the frequency of NSVT. Adaptive servo-ventilation (ASV) therapy improved sleep disorder breathing (SDB) and also reduced ventricular arrhythmias. These effects were associated with the attenuation of the sympathetic nerve activities by the analysis of heart rate variability. ASV is expected to be effective in the treatment of ventricular tachyarrhythmias in patients with heart failure and SDB.