Effect of Low-Level Tragus Stimulation on Cardiac Metabolism in Heart Failure with Preserved Ejection Fraction: A Transcriptomics-Based Analysis.

Abnormal cardiac metabolism precedes and contributes to structural changes in heart failure. Low-level tragus stimulation (LLTS) can attenuate structural remodeling in heart failure with preserved ejection fraction (HFpEF). The role of LLTS on cardiac metabolism is not known. Dahl salt-sensitive rats of 7 weeks of age were randomized into three groups: low salt (0.3% NaCl) diet (control group; n = 6), high salt diet (8% NaCl) with either LLTS (active group; n = 8), or sham stimulation (sham group; n = 5). Both active and sham groups received the high salt diet for 10 weeks with active LLTS or sham stimulation (20 Hz, 2 mA, 0.2 ms) for 30 min daily for the last 4 weeks. At the endpoint, left ventricular tissue was used for RNA sequencing and transcriptomic analysis. The Ingenuity Pathway Analysis tool (IPA) was used to identify canonical metabolic pathways and upstream regulators. Principal component analysis demonstrated overlapping expression of important metabolic genes between the LLTS, and control groups compared to the sham group. Canonical metabolic pathway analysis showed downregulation of the oxidative phosphorylation (Z-score: -4.707, control vs. sham) in HFpEF and LLTS improved the oxidative phosphorylation (Z-score = -2.309, active vs. sham). HFpEF was associated with the abnormalities of metabolic upstream regulators, including PPARGC1α, insulin receptor signaling, PPARα, PPARδ, PPARGC1ß, the fatty acid transporter SLC27A2, and lysine-specific demethylase 5A (KDM5A). LLTS attenuated abnormal insulin receptor and KDM5A signaling. HFpEF is associated with abnormal cardiac metabolism. LLTS, by modulating the functioning of crucial upstream regulators, improves cardiac metabolism and mitochondrial oxidative phosphorylation.

Sex differences in the incidence and mode of death in rats with heart failure with preserved ejection fraction.

NEW FINDINGS: What is the central question of this study? Prior studies failed to address the role of sex in modifying the pathophysiology and response to therapy in heart failure with preserved ejection fraction (HFpEF), potentially introducing bias into translational findings. We aimed to explore sex differences in outcomes and sought to identify the underlying mechanisms in a well-established rat model of HFpEF. What is the main finding and its importance? Male rats with HFpEF exhibited worse survival compared with females and were at a higher risk for sudden death, attributable in part to QT prolongation, autonomic dysregulation and enhanced inflammation. These data might provide the basis for the development of sex-specific interventions in HFpEF targeting these abnormalities. ABSTRACT: Heart failure with preserved ejection fraction (HFpEF) accounts for 50% of heart failure, and sudden death is the leading cause of mortality. We aimed to explore sex differences in outcomes in rats with HFpEF and sought to identify the underlying mechanisms. Dahl salt-sensitive rats of either sex were randomized into high-salt diet (HS diet; 8% NaCl, n = 46, 50% female) or low-salt diet (LS diet; 0.3% NaCl; n = 24, 50% female) at 7 weeks of age. After 6 and 10 weeks of LS or HS diets, the ECG, heart rate variability, cytokines and echocardiographic parameters were measured. The animals were monitored daily for development of HFpEF and survival. Over 6 weeks of HS diet, rats developed significant hypertension and signs of HFpEF. Compared with female HS diet-fed rats, males exhibited more left ventricular dilatation, a longer QT interval, and worse autonomic tone, as assessed by heart rate variability and elevated inflammatory cytokines. Ten of 23 (46%) male rats died during follow-up, compared with two of 23 (9%) female rats (P = 0.01). There were four sudden deaths in males (with ventricular tachycardia documented in one rat), whereas the females died of heart failure. In conclusion, male rats with HFpEF exhibit worse survival compared with females and are at a higher risk for sudden death, attributable in part to QT prolongation, autonomic dysregulation and enhanced inflammation. These data might provide the basis for the development of sex-specific interventions in HFpEF targeting these abnormalities.

Inhibition of Dephosphorylation of Dolichyl Diphosphate Alters the Synthesis of Dolichol and Hinders Protein N-Glycosylation and Morphological Transitions in Candida albicans.

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.

The role of Alg13 N-acetylglucosaminyl transferase in the expression of pathogenic features of Candida albicans.

BACKGROUND: The pathogenic potential of Candida albicans depends on adhesion to the host cells mediated by highly glycosylated adhesins, hyphae formation and growth of biofilm. These factors require effective N-glycosylation of proteins. Here, we present consequences of up- and down-regulation of the newly identified ALG13 gene encoding N-acetylglucosaminyl transferase, a potential member of the Alg7p/Alg13p/Alg14p complex catalyzing the first two initial reactions in the N-glycosylation process. METHODS: We constructed C. albicans strain alg13∆::hisG/TRp-ALG13 with one allele of ALG13 disrupted and the other under the control of a regulatable promoter, TRp. Gene expression and enzyme activity were measured using RT-qPCR and radioactive substrate. Cell wall composition was estimated by HPLC DIONEX. Protein glycosylation status was analyzed by electrophoresis of HexNAcase, a model N-glycosylated protein in C. albicans. RESULTS: Both decreased and elevated expression of ALG13 changed expression of all members of the complex and resulted in a decreased activity of Alg7p and Alg13p and under-glycosylation of HexNAcase. The alg13 strain was also defective in hyphae formation and growth of biofilm. These defects could result from altered expression of genes encoding adhesins and from changes in the carbohydrate content of the cell wall of the mutant. GENERAL SIGNIFICANCE: This work confirms the important role of protein N-glycosylation in the pathogenic potential of C. albicans.

Transcutaneous Vagus Nerve Stimulation Ameliorates the Phenotype of Heart Failure With Preserved Ejection Fraction Through Its Anti-Inflammatory Effects.

BACKGROUND: A systemic proinflammatory state plays a central role in the development of heart failure with preserved ejection fraction (HFpEF). Low-level transcutaneous vagus nerve stimulation (LLTS) suppresses inflammation in animals and humans, mediated by an α7nAchR (alpha7 nicotinic acetylcholine receptor)-dependent pathway. We examined the effects of LLTS on cardiac function, inflammation, and fibrosis in the presence of α7nAchR pharmacological blockade in a rat model of HFpEF. METHODS: Dahl salt-sensitive rats at 7 weeks of age were treated with high-salt diet for 6 weeks to induce HFpEF, followed by 4 weeks of (1) LLTS, (2) LLTS with the α7nAchR blocker methyllycaconitine, (3) sham, and (4) olmesartan. Blood pressure, cardiac function by echocardiography, heart rate variability, and serum cytokines were measured at 13 and 17 weeks of age. Cardiac fibrosis, inflammatory cell infiltration, and gene expression were determined at 17 weeks. RESULTS: LLTS attenuated the increase in blood pressure; improved cardiac function; decreased inflammatory cytokines, macrophage infiltration, and fibrosis; and improved survival compared with other groups. Methyllycaconitine attenuated these effects, whereas olmesartan did not improve cardiac function or fibrosis despite maintaining similar blood pressure as LLTS. Heart rate variability was similarly improved in the LLTS and LLTS plus methyllycaconitine groups but remained low in the other groups. LLTS reversed the dysregulated inflammatory signaling pathways in HFpEF hearts. CONCLUSIONS: Neuromodulation with LLTS improved cardiac function in a rat model of HFpEF through its anti-inflammatory and antifibrotic effects. These results provide the basis for further clinical trials in humans.

Neuromodulation of Inflammation to Treat Heart Failure With Preserved Ejection Fraction: A Pilot Randomized Clinical Trial.

Background A systemic proinflammatory state plays a central role in the development of heart failure with preserved ejection fraction. Low-level transcutaneous vagus nerve stimulation suppresses inflammation in humans. We conducted a sham-controlled, double-blind, randomized clinical trial to examine the effect of chronic low-level transcutaneous vagus nerve stimulation on cardiac function, exercise capacity, and inflammation in patients with heart failure with preserved ejection fraction. Methods and Results Patients with heart failure with preserved ejection fraction and at least 2 additional comorbidities (obesity, diabetes, hypertension, or age ≥65 years) were randomized to either active (tragus) or sham (earlobe) low-level transcutaneous vagus nerve stimulation (20 Hz, 1 mA below discomfort threshold), for 1 hour daily for 3 months. Echocardiography, 6-minute walk test, quality of life, and serum cytokines were assessed at baseline and 3 months. Fifty-two patients (mean age 70.4±9.2 years; 70% female) were included (active, n=26; sham, n=26). Baseline characteristics were balanced between the 2 arms. Adherence to the protocol of daily stimulation was >90% in both arms (P>0.05). While the early mitral inflow Doppler velocity to the early diastolic mitral annulus velocity ratio did not differ between groups, global longitudinal strain and tumor necrosis factor-α levels at 3 months were significantly improved in the active compared with the sham arm (-18.6%±2.5% versus -16.0%±2.4%, P=0.002; 8.9±2.8 pg/mL versus 11.3±2.9 pg/mL, P=0.007, respectively). The reduction in tumor necrosis factor-α levels correlated with global longitudinal strain improvement (r=-0.73, P=0.001). Quality of life was better in the active arm. No device-related side effects were observed. Conclusions Neuromodulation with low-level transcutaneous vagus nerve stimulation over 3 months resulted in a significant improvement in global longitudinal strain, inflammatory cytokines, and quality of life in patients with heart failure with preserved ejection fraction. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03327649.

Non-invasive vagus nerve stimulation attenuates proinflammatory cytokines and augments antioxidant levels in the brainstem and forebrain regions of Dahl salt sensitive rats.

The anti-inflammatory effects of vagus nerve stimulation are well known. It has recently been shown that low-level, transcutaneous stimulation of vagus nerve at the tragus (LLTS) reduces cardiac inflammation in a rat model of heart failure with preserved ejection fraction (HFpEF). The mechanisms by which LLTS affect the central neural circuits within the brain regions that are important for the regulation of cardiac vagal tone are not clear. Female Dahl salt-sensitive rats were initially fed with either low salt (LS) or high salt (HS) diet for a period of 6 weeks, followed by sham or active stimulation (LLTS) for 30 min daily for 4 weeks. To study the central effects of LLTS, four brainstem (SP5, NAb, NTS, and RVLM) and two forebrain sites (PVN and SFO) were examined. HS diet significantly increased the gene expression of proinflammatory cytokines in the SP5 and SFO. LLTS reversed HS diet-induced changes at both these sites. Furthermore, LLTS augmented the levels of antioxidant Nrf2 in the SP5 and SFO. Taken together, these findings suggest that LLTS has central anti-inflammatory and antioxidant properties that could mediate the neuromodulation of cardiac vagal tone in the rat model of HFpEF.

Spatial memory formation differentially affects c-Fos expression in retrosplenial areas during place avoidance training in rats.

The retrosplenial cortex is involved in spatial memory function, but the contribution of its individual areas is not well known. To elucidate the involvement of retrosplenial cortical areas 29c and 30 in spatial memory, we analyzed the expression of c-Fos in these areas in the experimental group of rats that were trained in a spatial place avoidance task, i.e. to avoid shocks presented in an unmarked sector of a stable arena under light conditions. Control rats were trained in the same context as the experimental rats either without (Control-noUS) or with shocks (Control-US) that were delivered in a random, noncontingent manner for three days. On the first day of place avoidance learning, the experimental group exhibited c-Fos induction in area 29c, similar to both control groups. In area 30, similarly high levels of c-Fos expression were observed in the experimental and Control-US groups. On the third day of training, when the experimental group efficiently avoided c-Fos expression in areas 29c and 30 was lower compared with the first day of training. In area 29c c-Fos level was also lower in the experimental than in comparison to the Control-US group. In area 30, c-Fos expression in the experimental group was lower than in both control groups. In conclusion, areas 29c and 30 appear to be activated during spatial memory acquisition on the first day of training, whereas area 30 seems suppressed during long-term memory functioning on the third day of training when rats effectively avoid.

[Agility in treatment of children with type 1 diabetes--pilot study]. / Wykorzystanie agility jako formy rekreacji w leczeniu dzieci z cukrzyca typu 1 - doniesienie wstepne.

INTRODUCTION: Physical activity is a very important element in treatment of children with type 1 diabetes. However, it is difficult to find suitable exercises for the children due to their specific needs and psychophysiological condition. The aim of this study was to examine the effects of agility as a physical activity used to improve parameters of metabolic control in children with type 1 diabetes. Additionally, we hope that this form of recreation could induce a more health-oriented behavior in children. MATERIAL AND METHODS: The experimental group consisted of seven girls aged 8-10 years, the patients of the Clinic of Pediatrics, Endocrinology, Diabetology, Metabolic Disorders and Cardiology of the Developmental Age in Szczecin. The children were qualified for the study after the prior assessment of their metabolic control under the conditions of the one-day hospital stay program. The physical condition of the patients was controlled with a 6-minute walk test and the test of perceived exertion (Borg scale). All the patients were treated using a personal insulin pump and the basal-bolus therapy. The applied research method used the scheme of physical exercise performed 3 times a week, 45 min each, reaching the intensity of 75% of the maximum heart rate under effort performed by a given patient. Before the exercises and directly after their completion, sugar level was measured in the blood of the patients (Accu-chek Active). During the exercises, the heart rate was measured with a pulsometer Bauer PM 80. The exercises included outdoor games and agility sessions. Especially the latter received a positive response and high involvement. This form includes a dog going through an obstacle course, with a child as a guide. RESULTS AND CONCLUSIONS: After three months of the exercise scheme, the analysis of the collected results showed a statistically significant (p <0.05) decrease in the insulin doses (bolus) during morning hours (7-8 am), and in the evening at 9 pm and 10.30 pm, with an unchanged basal. No such decrease was observed in the control group. This study showed that an interesting physical activity resulted in a more eager and systematic effort among examined diabetic children. Its proper organization in time may help in the metabolic control in children with type 1 diabetes.