Proteogenomics Reveals Orthologous Alternatively Spliced Proteoforms in the Same Human and Mouse Brain Regions with Differential Abundance in an Alzheimer's Disease Mouse Model.

Alternative splicing (AS) may increase the number of proteoforms produced by a gene. Alzheimer's disease (AD) is a neurodegenerative disease with well-characterized AS proteoforms. In this study, we used a proteogenomics strategy to build a customized protein sequence database and identify orthologous AS proteoforms between humans and mice on publicly available shotgun proteomics (MS/MS) data of the corpus callosum (CC) and olfactory bulb (OB). Identical proteotypic peptides of six orthologous AS proteoforms were found in both species: PKM1 (gene PKM/Pkm), STXBP1a (gene STXBP1/Stxbp1), Isoform 3 (gene HNRNPK/Hnrnpk), LCRMP-1 (gene CRMP1/Crmp1), SP3 (gene CADM1/Cadm1), and PKCßII (gene PRKCB/Prkcb). These AS variants were also detected at the transcript level by publicly available RNA-Seq data and experimentally validated by RT-qPCR. Additionally, PKM1 and STXBP1a were detected at higher abundances in a publicly available MS/MS dataset of the AD mouse model APP/PS1 than its wild type. These data corroborate other reports, which suggest that PKM1 and STXBP1a AS proteoforms might play a role in amyloid-like aggregate formation. To the best of our knowledge, this report is the first to describe PKM1 and STXBP1a overexpression in the OB of an AD mouse model. We hope that our strategy may be of use in future human neurodegenerative studies using mouse models.

Changes in cardiovascular responses to chemoreflex activation of rats recovered from protein restriction are not related to AT1 receptors.

NEW FINDINGS: What is the central question of this study? In this study, we sought to investigate whether cardiovascular responses to peripheral chemoreflex activation of rats recovered from protein restriction are related to activation of AT1 receptors. What is the main finding and its importance? This study highlights the fact that angiotensinergic mechanisms activated by AT1 receptors do not support increased responses to peripheral chemoreflex activation by KCN in rats recovered from protein restriction. Also, we found that protein restriction led to increased resting ventilation in adult rats, even after recovery. The effects of a low-protein diet followed by recovery on cardiorespiratory responses to peripheral chemoreflex activation were tested before and after systemic angiotensin II type 1 (AT1 ) receptor antagonism. Male Fischer rats were divided into control and recovered (R-PR) groups after weaning. The R-PR rats were fed a low-protein (8%) diet for 35 days and recovered with a normal protein (20%) diet for 70 days. Control rats received a normal protein diet for 105 days (CG105 ). After cannulation surgery, mean arterial pressure, heart rate, respiratory frequency, tidal volume and minute ventilation were acquired using a digital recording system in freely moving rats. The role of angintensin II was evaluated by systemic antagonism of AT1 receptors with losartan (20 mg kg-1 i.v.). The peripheral chemoreflex was elicited by increasing doses of KCN (20-160 µg kg min-1 , i.v.). At baseline, R-PR rats presented increased heart rate and minute ventilation (372 ± 34 beats min-1 and 1.274 ± 377 ml kg-1  min-1 ) compared with CG105 animals (332 ± 22 beats min-1 and 856 ± 112 ml kg-1  min-1 ). Mean arterial pressure was not different between the groups. Pressor and bradycardic responses evoked by KCN (60 µg kg-1 ) were increased in R-PR (+45 ± 13 mmHg and -77 ± 47 beats min-1 ) compared with CG105 rats (+25 ± 17 mmHg and -27 ± 28 beats min-1 ), but no difference was found in the tachypnoeic response. These differences were preserved after losartan. The data suggest that angiotensin II acting on AT1 receptors may not be associated with the increased heart rate, increased minute ventilation and acute cardiovascular responses to peripheral chemoreflex activation in rats that underwent postweaning protein restriction followed by recovery.

Exercise training starting at weaning age preserves cardiac pacemaker function in adulthood of diet-induced obese rats.

Peripheral sympathetic overdrive in young obese subjects contributes to further aggravation of insulin resistance, diabetes, and hypertension, thus inducing worsening clinical conditions in adulthood. Exercise training has been considered a strategy to repair obesity autonomic dysfunction, thereby reducing the cardiometabolic risk. Therefore, the aim of this study was to assess the effect of early exercise training, starting immediately after weaning, on cardiac autonomic control in diet-induced obese rats. Male Wistar rats (weaning) were divided into four groups: (i) a control group (n = 6); (ii) an exercise-trained control group (n = 6); (iii) a diet-induced obesity group (n = 6); and (iv) an exercise-trained diet-induced obesity group (n = 6). The development of obesity was induced by 9 weeks of palatable diet intake, and the training program was implemented in a motor-driven treadmill (5 times per week) during the same period. After this period, animals were submitted to vein and artery catheter implantation to assess cardiac autonomic balance by methylatropine (3 mg/kg) and propranolol (4 mg/kg) administration. Exercise training increased running performance in both groups (p < 0.05). Exercise training also prevented the increased resting heart rate in obese rats, which seemed to be related to cardiac pacemaker activity preservation (p < 0.05). Additionally, the training program preserved the pressure and bradycardia responses to autonomic blockade in obese rats (p < 0.05). An exercise program beginning at weaning age prevents cardiovascular dysfunction in obese rats, indicating that exercise training may be used as a nonpharmacological therapeutic strategy for the treatment of cardiometabolic diseases.

Excitatory amino acid receptors in the dorsomedial hypothalamus are involved in the cardiovascular and behavioural chemoreflex responses.

The present study investigated the role of the dorsomedial hypothalamus (DMH) on cardiovascular and behavioural responses of chemoreflex activation in conscious rats. The arterial chemoreflex was activated by potassium cyanide (KCN, 40 µg, i.v.) before and after bilateral microinjection of lidocaine (2%) or kynurenic acid (2.7 nmol) into the DMH. Locomotor activity was measured to assess the chemoreflex behavioural response. Bilateral microinjection of lidocaine into the DMH produced a significant reduction in the pressor response induced by chemoreflex activation (+51 ± 4 versus +34 ± 5 mmHg, n = 5, P < 0.05). A similar reduction in the pressor chemoreflex response was also observed after microinjection of kynurenic acid into the DMH (+50 ± 3 versus +22 ± 5 mmHg, n = 6, P < 0.05). Strikingly, the behaviour/locomotor activity induced by chemoreflex activation was virtually abolished after blockade of excitatory amino acid receptors in the DMH with kynurenic acid (44 ± 6 versus 5 ± 4 cm, n = 6, P < 0.05). There was no correlation between the reduction in pressor and behavioural chemoreflex responses (r = -0.186, P > 0.05). The bradycardic response of the chemoreflex was not altered by lidocaine or kynurenic acid microinjected into the DMH. These results strongly suggest that the excitatory amino acid receptors in the DMH are essential for full expression of the behavioural response of the chemoreflex and participate, at least in part, in the integration of the pressor response of the chemoreflex in conscious rats.

Effect of L-NAME microinjected into the nucleus tractus solitarius on jejunal glucose and electrolyte absorption in anesthetized rats.

The aim of the present study was to investigate the effects of N(G)-nitro-L-arginine-methyl-ester (L-NAME) microinjected into the rostral nucleus tractus solitarius (NTS) on jejunal glucose, sodium and potassium absorption. Male Wistar rats (210-250 g, n=6-12) were anesthetized and submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and perform a subdiaphragmatic truncal vagotomy or sympathectomy. Either 0.9% NaCl or L-NAME (10 nmol 100 nl⁻¹) was microinjected into the rostral NTS using a stereotaxic instrument. Tyrode solution (pH 8) containing twice the usual concentrations of glucose, sodium and potassium was infused (0.5 ml min⁻¹) into the jejunal loop and samples were taken at 10-min intervals during the 40-min experiment. Results were expressed by the difference between influx and efflux. L-NAME into the NTS increased glucose absorption and decreased potassium absorption when compared to the saline group (38.8 ± 3.8 vs. 50.3 ± 3.3 mg/dl and 0.6±0.01 vs. 0.4 ± 0.03 mM, respectively; p<0.05). Sympathectomy inhibited the glucose absorption caused by L-NAME alone (50.3 ± 3.3 vs. 30.7 ± 4.6 mg/dl; p<0.05), whereas vagotomy inhibited the L-NAME effect on potassium absorption (0.40 ± 0.02 vs. 0.70 ± 0.05; p<0.05). Moreover, increased sodium absorption was observed only in the group that received 30 nmol of L-NAME into NTS (33.0 ± 4.2 vs. 48.4 ± 3.9). In conclusion, the results suggest the participation of endogenous nitric oxide (NO) in the NTS in modulating intestinal glucose and potassium absorption mediated by the autonomic nervous system.

Altered cardiovascular reflexes responses in conscious Angiotensin-(1-7) receptor Mas-knockout mice.

This study evaluated the physiological importance of Angiotensin-(1-7) receptor Mas on reflex control of circulation. Experiments were performed in male Mas-knockout (Mas-KO) and Wild Type (WT) conscious mice (12-20 wk of age). Baroreceptor reflex was evaluated by the bradycardic response induced by phenylephrine (0.25 µg/5 µl, i.v.). Bezold-Jarisch reflex was evaluated by phenylbiguanide (0.5 µg/5 µl, i.v.) and chemoreflex by potassium cyanide (2.5 µg/5 µl, i.v.). Baseline mean arterial pressure was higher in Mas-KO (n=14) as compared with WT mice (n=18) (118±1 mmHg vs. 109±2 mmHg); however, heart rate was similar in both strains (615±30 bpm vs. 648±13 bpm). Baroreflex bradycardia was lower (0.78±0.44 ms/mmHg vs. 1.30±0.14 ms/mmHg) in Mas-KO compared with WT mice. The depressor (-17±5 mmHg vs. -45±6 mmHg) and bradycardic (-212±36 bpm vs. -391±29 bpm) components of the Bezold-Jarisch reflex were also lower in Mas-KO mice. In addition, chemoreflex pressor response (+20±3 mmHg vs. +12±0.8 mmHg) and bradycardic response (-250±74 bpm vs. -52±26 bpm) were significantly higher in Mas-KO. These results further advances previous studies by showing that the lack of Mas receptor induced important imbalance in the neural control of blood pressure, altering not only the baroreflex but also the chemo- and Bezold-Jarisch reflexes.

The enhanced hyperglycemic response to hemorrhage hypotension in obese rats is related to an impaired baroreflex.

The aim of the present study was to assess the metabolic adjustments in adult rats with autonomic imbalance induced by hypercaloric diet. Male Wistar rats (4 weeks of age) were fed a chow diet (CD, n = 12) or hypercaloric diet (HD, n = 13) for 19 weeks. Body weight and dietary intake were measured every week and the basal metabolic rate was assessed. After 19 weeks of diet, six animals from each group were anesthetized with a lethal dose of barbital sodium (100 mg/Kg body weight, intraperitoneal; i.p.). Lee index was evaluated and adipose pads weighted. The remaining animals had a silastic cannula placed into the jugular vein for drug administration, blood collecting, and hemorrhage (1.2 mL/100 g bw/2 min). A polyethylene catheter (PE50) was inserted into the abdominal artery through the femoral artery for cardiovascular monitoring. The assessment of autonomic balance was done by evaluation of baroreflex sensitivity (intravenous (IV) injection of phenylephrine and sodium nitroprusside) and hemorrhage (1.2 mL/100 g bw/2 min). As expected, the HD induced obesity; increased weight gain (28%), adipose pads weight, and baroreflex dysfunction. The plasma level of free fatty acids and triacylglycerols were increased in HD rats by about 124% and 424%, respectively, as well as the basal metabolic rate measured at 19th weeks of diet (p < 0.01). We observed that baroreflex sensitivity to phenylephrine was reduced by about 50%, and the hyperglycemic response to hemorrhage hypotension was increased by 128% in HD rats. We found also a negative correlation between the alteration in baroreflex sensitivity and the increase in hyperglycemic response to hemorrhage in the obese rats (r = 0.72, p < 0.01) and a strong positive correlation between the increased Lee index and the hemorrhagic hyperglycemia (r = 0.93, p < 0.01). Our data demonstrate that obesity induced by hypercaloric diet in Wistar rats promotes an autonomic imbalance, which interferes with metabolic responses dependent on baroreflex sensitivity. In addition, we showed the existence of close correlation between the loss of baroreflex sensitivity and the degree of obesity.

Malnutrition enhances cardiovascular responses to chemoreflex activation in awake rats.

Several studies in the literature suggest that low-protein intake is associated with increases in sympathetic efferent activity and cardiovascular disease. Among the possible mechanisms, changes in the neurotransmission of cardiovascular reflexes have been implicated. Therefore, the present study comprised the evaluation of chemoreflex responsiveness in rats subjected to a low-protein diet during the 35 days after weaning. As a result, we observed that malnourished rats presented higher levels of baseline mean arterial pressure and heart rate and exhibited a mild increase in the pressor response to chemoreflex activation. They also exhibited a massive bradycardic response to chemoreflex activation. Interestingly, bilateral ligature of the carotid body arteries further increased baseline mean arterial pressure and heart rate in malnourished animals. The data suggest severe autonomic imbalance and/or change in the central interplay between neural and cardiovascular mechanisms.

Hyperglycemic response to hemorrhage is modulated by baroreceptors unloading but not by peripheral chemoreceptors activation.

The aim of this study was to assess the relative participation of carotid baro- and chemoreceptors on plasma glucose and lactate level in response to hemorrhagic hypotension. We also evaluated the effects of selective activation of carotid chemoreceptors. One week before the experiments, male Wistar rats (250-300 g) were submitted to bilateral total carotid denervation (BCD-group), or to bilateral ligature of the carotid body artery (ChD-group). During the same surgical procedure, a chronic jugular catheter for blood sampling and hemorrhage (1.2 mL/100 g/2 min) and polyethylene cannula was inserted into the left femoral artery for cardiovascular monitoring. One group submitted to fictitious surgery was used as a surgical control (Sham-group). Carotid chemoreceptors were selectively activated by sodium cyanide (NaCN, 40 microg/0.1 mL i.v.) in the Sham and ChD group. The results showed that hyperglycemic response to hemorrhage in the BCD-group was reduced whereas in the ChD-group there was no significant change in this parameter compared to the Sham group (8.6 +/- 0.5 mM, Sham-hemorrhaged, n = 8; 7.2 +/- 0.3 mM, BCD-hemorrhaged, n = 8 and 9.4 +/- 0.6 mM, ChD, n = 8, p < 0.05). Increased plasma lactate levels following hemorrhage were observed in all the three experimental groups throughout the experimental period and there were no differences between the groups. Chemoreceptor stimulation by NaCN also produced hyperglycemia, as well as an increase in blood pressure and bradycardia but did not affect plasma lactate concentration. Ligature of the carotid body artery annulled the cardiovascular responses induced by NaCN, but did not change the hyperglycemic response to hypoxia. In conclusion, our data indicate that carotid chemoreceptors do not play any major role in overall metabolic response to hypoxia or hemorrhagic hypotension. Furthermore, the results suggest that carotid baroreceptors unloading play a predominant role as main source of afferent impulses leading to the hyperglycemic response to hemorrhage. In addition our data shows that the metabolic response and cardiovascular adjustment to hypoxia can be dissociated by ligature of the carotid body artery.

The hypothalamic paraventricular nucleus and carotid receptors modulate hyperglycemia induced by hemorrhage.

The aim of this study was to assess the role of cholinergic transmission in the paraventricular nucleus of the hypothalamus (PVN) and carotid body receptors in mediating a rise in plasma glucose levels in response to hemorrhagic hypotension in rats. Methylatropine (1x10(-9) mol) or 0.15 M NaCl (0.2 microl) was injected into the PVN of Wistar rats weighing 250-300 g bearing a chronic jugular catheter for blood sampling and hemorrhage (1.2 ml/100 g/2 min). Polyethylene cannulae (PE-10) were inserted into the left femoral artery for cardiovascular monitoring. In the other experimental protocol, hemorrhage was performed on rats submitted to bilateral carotid receptor denervation (H-CD). The results show that the hyperglycemic response to hemorrhage was decreased by either methylatropine (H-MA) treatment or bilateral carotid receptor denervation (10.3+/-0.4 mM, control, n=15 vs. 7.7+/-0.2 mM, H-MA, n=12, and 7.6+/-0.3 mM, H-CD, n=5, p<0.01). Furthermore, methylatropine did not affect the recovery of blood pressure after hemorrhage-induced hypotension, suggesting that the metabolic and pressor adjustments have different efferent pathways. Our data demonstrate that cholinergic input from the PVN and carotid receptors (chemo- and/or baroreceptors) might participate in the same neural pathway activated by hemorrhage-induced hypotension that produces hyperglycemia.

Reflexos cardiovasculares e hipertensäo arterial / Cardiovascular reflexes and arterial hypertension

Os reflexos originados nos pressorreceptores arteriais e nos receptores de estiramento da regiäo cardiopulmonar säo os principais mecanismos de controle efetivo da pressäo arterial a curto prazo. O reflexo pressorreceptor é considerado um sistema de controle de alto ganho que mantém a pressäo arterial dentro de limites normais em períodos de segundos a minutos. Assim como o diagnóstico e tratamento da hipertensäo arterial focado no nível basal da pressäo sangüínea determina grande reduçäo da morbi-mortalidade da populaçäo, a variabilidade momento a momento da pressäo arterial por si, cujo controle é funçäo do barorreflexo, é também de importante significado clínico. Estudos clínicos têm mostrado, por exemplo, que uma reduzida sensibilidade do barorreflexo está associada com a morte súbita que se segue ao infarto agudo do miocárdio. Uma série de outros reflexos participam na regulaçäo da pressäo sangüínea em animais e indivíduos normotensos, como por exemplo, os reflexos originados em metaborreceptores musculares, reflexos originados na área trigeminal e quimiorreflexo. Alteraçöes no funcionamento desses reflexos na hipertensäo näo tem sido exploradas de forma sistemática, com exceçäo ao quimiorreflexo, originado por receptores arteriais ativados por hipóxia, hipercapnia, acidose e isquemia. No presente artigo pretendemos discutir as alteraçöes do reflexos cardiovasculares na hipertensäo arterial, enfocando os distúrbios no funcionamento do barorreflexo e quimiorreflexo.