Consequences of Decreased Brain Serotonin in the Pyridoxine Deficient Young Rat

The concentrations of serotonin in various brain areas were significantly decreased in the pyridoxine-deficient young rat. 2. There was no change in the concentration of dopamine. 3. Both Bmax and Kid of [3H]serotonin binding to membrane preparations from cerebral cortex were increased in deficiency and were restored to normal upon pyridoxine supplementation. 4. There was no change in [3H]spiroperidol binding to corpus striatal membrane preparations in pyridoxine-deficient rats.

Sympathetic Stimulation and Hypertension in the Pyridoxine-Deficient Adult Rat

Pyridoxal phosphate is the coenzyme of various decarboxylases involved in the formation of monoamine neurotransmitters such as y-aminobutyric acid , serotonin , dopamine, and norepinephrine . Adult male Sprague-Dawley rats placed on a pyridoxine -deficient diet for 8 weeks showed significant hypertension compared with pyridoxine -supplemented controls . Hypothalamic contents of pyridoxal phosphate , y-aminobutyric acid, and serotonin in the pyridoxine - deficient rats were significantly lower than those in pyridoxine -supplemented controls . Hypertension was associated with sympathetic stimulation . Treatment of pyridoxine-deficient rats with a single dose of pyridoxine (10 mg/kg body weight) reversed the blood pressure to normal levels within 24 hours, with concomitant restorations of hypothalamic serotonin and y-aminobutyric acid as well as the return of plasma norepinephrine and epinephrine to normal levels . Also, pyridoxine treatment reversed the hypothalamic hypothyroidism observed in pyridoxine -deficient rats . These results indicate an association between pyridoxine deficiency and sympathetic stimulation leading to hypertension.

Enhancement Of High Affinity Y-Aminobutyric Acid Receptor Binding In Cerebellum Of Pyridoxine-Deficient Rat

The high-affinity of [3H]y-aminobutyric acid (GABA) to GABAA receptors and [3H]baclofen to GABAB receptors were studied in the cerebellum of pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding ( Bmax) of both GABAA and GABAB receptors with no significant difference in their binding affinities (Kd). The changes observed suggest a supersensitivity of GABAA and GABAB receptors which seems to correlate negatively with the concentration of GABA in the cerebellum of pyridoxine-deficient rats.

Age-related and sex-related alterations in f3-adrenergic receptors in different regions of rat brain

The binding of (-)[ 3H ]dihydroalprenolol , an antagonist of norepinephrine , to $-adrenergic receptors in different regions of the brain of male and female rats of various ages was measured . The binding to the synaptosomal fraction of corpus striatum , hypothalamus, cerebral cortex, cerebellum and the brainstems shows a significant decrease in the binding in old rats of both sexes . Only in the female corpus striatal region, the binding in the adult and the old is the same . In the case of females, the highest binding is seen in the young. In the male, an increase in binding occurs up to adulthood , after which it declines, suggesting a definite sex-related difference in the Q-adrenergic receptor.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

Probiotic administration attenuates myocardial hypertrophy and heart failure following myocardial infarction in the rat

Background—Probiotics are extensively used to promote gastrointestinal health and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut. Here, we determined whether oral probiotic administration can alter the progression of post-infarction heart failure. Methods and Results—Rats were subjected to six weeks of sustained coronary artery occlusion and administered the probiotic Lactobacillus rhamnosus GR-1 or placebo in the drinking water ad libitum. Culture and 16s rRNA sequencing showed no evidence of GR-1 colonization or a significant shift in the composition of the cecal microbiome. However, animals administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy based on tissue weight assessment as well as gene expression of atrial natriuretic peptide. Moreover, these animals demonstrated improved hemodynamic parameters reflecting both improved systolic and diastolic left ventricular function. Serial echocardiography revealed significantly improved left ventricular parameters throughout the six week follow-up period including a marked preservation of left ventricular ejection fraction as well as fractional shortening. Beneficial effects of GR-1 were still evident in those animals in which GR-1 was withdrawn at four weeks suggesting persistence of the GR-1 effects following cessation of therapy. Investigation of mechanisms showed a significant increase in the leptin to adiponectin plasma concentration ratio in rats subjected to coronary ligation which was abrogated by GR-1. Metabonomic analysis showed differences between sham control and coronary artery ligated hearts particularly with respect to preservation of myocardial taurine levels. Conclusions—The study suggests that probiotics offer promise as a potential therapy for the attenuation of heart failure.

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly upregulated or downregulated (>2-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly-investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were upregulated in AVMs vs NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were upregulated (e.g. NRK, JAK2, STK38L) or downregulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. Conclusions. This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.

Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 mu M epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.

Intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy

Important sex differences in cardiovascular disease outcomes exist, including conditions of hypertrophic cardiomyopathy and cardiac ischemia. Studies of sex differences in the extent to which load-independent (primary) hypertrophy modulates the response to ischemia-reperfusion (I/R) damage have not been characterized. We have previously described a model of primary genetic cardiac hypertrophy, the hypertrophic heart rat (HHR). In this study the sex differences in HHR cardiac function and responses to I/R [compared to control normal heart rat (NHR)] were investigated ex vivo. The ventricular weight index was markedly increased in HHR female (7.82 ± 0.49 vs. 4.80 ± 0.10 mg/g; P < 0.05) and male (5.76 ± 0.22 vs. 4.62 ± 0.07 mg/g; P < 0.05) hearts. Female hearts of both strains exhibited a reduced basal contractility compared with strain-matched males [maximum first derivative of pressure (dP/dtmax): NHR, 4,036 ± 171 vs. 4,258 ± 152 mmHg/s; and HHR, 3,974 ± 160 vs. 4,540 ± 259 mmHg/s; P < 0.05]. HHR hearts were more susceptible to I/R (I = 25 min, and R = 30 min) injury than NHR hearts (decreased functional recovery, and increased lactate dehydrogenase efflux). Female NHR hearts exhibited a significantly greater recovery (dP/dtmax) post-I/R relative to male NHR (95.0 ± 12.2% vs. 60.5 ± 9.4%), a resistance to postischemic dysfunction not evident in female HHR (29.0 ± 5.6% vs. 25.9 ± 6.3%). Ventricular fibrillation was suppressed, and expression levels of Akt and ERK1/2 were selectively elevated in female NHR hearts. Thus the occurrence of load-independent primary cardiac hypertrophy undermines the intrinsic resistance of female hearts to I/R insult, with the observed abrogation of endogenous cardioprotective signaling pathways consistent with a potential mechanistic role in this loss of protection.

Auranofin increases apoptosis and ischaemia-reperfusion injury in the rat isolated heart

1. Auranofin, an antirheumatic gold compound, is an inhibitor of selenocysteine enzymes, such as thioredoxin reductase and glutathione peroxidase. These enzymes play an important role in protecting cardiac tissue from oxidative stress generated during ischaemia–reperfusion.

2. Auranofin (100 mg/kg) was administered to rats and their hearts were subjected to an in vitro model of ischaemia–reperfusion. The activity of thioredoxin reductase and glutathione peroxidase was determined in liver and heart tissues in an attempt to correlate enzymatic activity with heart recovery after ischaemia–reperfusion.

3. There was significantly less thioredoxin reductase activity in rat liver extracts, whereas the level of glutathione activity remained unchanged, demonstrating that the dose of auranofin used was able to selectively inhibit one of these enzyme systems. Rats administered auranofin displayed significantly impaired recovery from ischaemic insult. The end diastolic pressure was increased, whereas the rate pressure product was significantly decreased.

4. The level of postischaemic apoptosis was also assessed by examining caspase-3 activity in tissue homogenates. Auranofin significantly increased the degree of postischaemic apoptosis, leading to poor postischaemic recovery.

Effect of sodium selenite-enriched reperfusion solutions on rat cardiac ischemia reperfusion injury

Cardiac surgery often generates oxidative stress leading to ischemia reperfusion injury (I-R). Antioxidants have been shown to prevent this injury and have been added to cardioplegic solutions to assist in recovery. In this study, we tested the effectiveness of sodium selenite in protecting against ischemia reperfusion injury and investigated the mechanisms behind this protection. Hearts from male Wistar rats were subjected to ischemia reperfusion using the Langendorf model. Krebs-Henseleit perfusion solutions were supplemented with 0,0.1, 0.5, 1.0, and 10μM sodium selenite. Hearts were perfused for 30 min and then subjected to 22.5 min of global ischemia followed by 45 min reperfusion. Heart rate, ischemic contracture, end diastolic pressure, and developed ventricular pressure were monitored. At the completion of the experiment, hearts were homogenized and tissue extracts were assayed for glutathione peroxidase (GSH-Px) and thioredoxin reductase (Thx-Red) activity. Sodium selenite, at a concentration of 0.5 μM, demonstrated a protective effect on the recovery of cardiac function following I-R, as evidenced by a lower end diastolic pressure and enhanced recovery of rate pressure product. There was no beneficial effect observed in hearts perfused with 0.1 μM sodium selenite-supplemented buffer, whereas poorer functional recovery was observed in hearts perfused with 10 μM sodium selenite-supplemented buffer. The beneficial effect of sodium selenite was not mediated through increased activity of GSH-Px or Thx-Red. This study demonstrates that the addition of sodium selenite to reperfusion solutions, at an optimal concentration of 0.5 μM, assists in cardiac recovery following ischemia reperfusion.

PHARMACOLOGICAL EVALUATION OF THE INHIBITORY EFFECT OF EXTRACTS FROM ANCHIETIA-SALUTARIS ON THE HISTAMINE-RELEASE INDUCED IN THE RAT AND THE GUINEA-PIG

The tea made with leaves and stems of plant Anchietia salutaris is traditionally used in Brazil to treat allergies. We examined the effects of a crude aqueous extract and of purified fractions of this plant on the histamine release induced in rat and guinea pig tissues. The crude extract (3-10 mu g/ml) inhibits the histamine release induced by compound 48/80 (0.5 mu g/ml) and antigen in rat peritoneal mast cells. The inhibition is significant after 10 s of preincubation and is completed after 3 min. The crude extract dissolved in the perfusion fluid (1-30 mu g/ml) also inhibits the histamine release induced in guinea pig heart by cardiac anaphylaxis and in hearts from pretreated animals (10-100 mg/kg i.p.). In pretreated animals, the effect manifests after 3 h, is maximum after 12 h and disappears after 48 h. The histamine release induced in isolated guinea pig heart by ionophore A23187 is inhibited by similar doses as in antigen-induced histamine release. Extraction with solvents concentrated the active principle (s) in the hexane fractions, as demonstrated by the inhibition of the histamine release induced by antigen in isolated cells from guinea pig heart dispersed with collagenase. In subfractions produced by the fractionation of the hexane fraction, the active principle(s) concentrated in the subfractions obtained by extraction with hexane and ethyl acetate, which shows the low polarity of the compound(s). The same subfractions that inhibit the histamine release induced by antigen in cells from guinea pig heart also inhibit pulmonary cells. Our result show that A. salutaris contains low-polarity compound(s) that inhibit the histamine release induced by three different mechanisms in mast cells from two animal species. These facts suggest that the active principle(s) of A, salutaris could be useful in the treatment of allergies and/or as a tool for the study of mast cell secretions.

Smoking is associated with remodeling of gap junction in the rat heart: Smoker's paradox explanation?

Background: In a previous study utilizing the rat model, exposure to tobacco smoke for 5 weeks increased survival after AMI, despite similar age and infarct size between the smokers and nonsmokers, and absence of reperfusion. Objective: Thus, this study aimed to analyze the effects of exposure to tobacco smoke on intensity, distribution or phosphorylation of connexin 43 in the rat heart. Methods: Wistar rats weighing 100 g were randomly allocated into 2 groups: 1) Control (n = 25); 2) Exposed to tobacco smoke (ETS), n = 23. After 5 weeks, left ventricular morphometric analysis, immunohisthochemistry and western blotting for connexin 43 (Cx43) were performed. Results: Collagen volume fraction, cross-sectional areas, and ventricular weight were not statistically different between control and ETS. ETS showed lower stain intensity of Cx43 at intercalated disks (Control: 2.32 ± 0.19; ETS: 1.73 ± 0.18; p = 0.04). The distribution of CX43 at intercalated disks did not differ between the groups (Control: 3.73 ± 0.12; ETS: 3.20 ± 0.17; p = 0.18). ETS rats showed higher levels of dephosphorylated form of Cx43 (Control: 0.45 ± 0.11; ETS: 0.90 ± 0.11; p = 0.03). On the other hand, total Cx43 did not differ between control and ETS groups (Control: 0.75 ± 0.19; ETS: 0.93 ± 0.27; p = 0.58). Conclusion: Exposure to tobacco smoke resulted in cardiac gap junction remodeling, characterized by alterations in the quantity and phosphorylation of the Cx43, in rats hearts. This finding could explain the smoker's paradox observed in some studies.

Myocardial Remodeling after Large Infarcts in Rat Converts Post Rest-Potentiation in Force Decay

Background: Post-rest contraction (PRC) of cardiac muscle provides indirect information about the intracellular calcium handling. Objective: Our aim was to study the behavior of PRC, and its underlying mechanisms, in rats with myocardial infarction. Methods: Six weeks after coronary occlusion, the contractility of papillary muscles (PM) obtained from sham-operated (C, n = 17), moderate infarcted (MMI, n = 10) and large infarcted (LMI, n = 14) rats was evaluated, following rest intervals of 10 to 60 seconds before and after incubation with lithium chloride (Li+) substituting sodium chloride or ryanodine (Ry). Protein expression of SR Ca(2+)-ATPase (SERCA2), Na+/Ca2+ exchanger (NCX), phospholamban (PLB) and phospho-Ser(16)-PLB were analyzed by Western blotting. Results: MMI exhibited reduced PRC potentiation when compared to C. Opposing the normal potentiation for C, post-rest decays of force were observed in LMI muscles. In addition, Ry blocked PRC decay or potentiation observed in LMI and C; Li+ inhibited NCX and converted PRC decay to potentiation in LMI. Although MMI and LMI presented decreased SERCA2 (72 +/- 7% and 47 +/- 9% of Control, respectively) and phospho-Ser(16)-PLB (75 +/- 5% and 46 +/- 11%, respectively) protein expression, overexpression of NCX (175 +/- 20%) was only observed in LMI muscles. Conclusion: Our results showed, for the first time ever, that myocardial remodeling after MI in rats may change the regular potentiation to post-rest decay by affecting myocyte Ca(2+) handling proteins. (Arq Bras Cardiol 2012;98(3):243-251)

Insulin alone or with captopril: effects on signaling pathways (AKT and AMPK) and oxidative balance after ischemia-reperfusion in isolated hearts

Insulin and the inhibition of the reninangiotensin system have independent benefits for ischemiareperfusion injury, but their combination has not been tested. Our aim was to evaluate the effects of insulin+captopril on insulin/angiotensin signaling pathways and cardiac function in the isolated heart subjected to ischemiareperfusion. Isolated hearts were perfused (Langendorff technique) with KrebsHenseleit (KH) buffer for 25 min. Global ischemia was induced (20 min), followed by reperfusion (30 min) with KH (group KH), KH+angiotensin-I (group A), KH+angiotensin-I+captopril (group AC), KH+insulin (group I), KH+insulin+angiotensin-I (group IA), or KH+insulin+angiotensin-I+captopril (group IAC). Group A had a 24% reduction in developed pressure and an increase in end-diastolic pressure vs. baseline, effects that were reverted in groups AC, IA, and IAC. The phosphorylation of protein kinase B (AKT) was higher in groups I and IA vs. groups KH and A. The phosphorylation of AMP-activated protein kinase (AMPK) was similar to 31% higher in groups I, IA, and IAC vs. groups KH, A, and AC. The tert-butyl hydroperoxide (tBOOH)-induced chemiluminescence was lower (similar to 2.2 times) in all groups vs. group KH and was similar to 35% lower in group IA vs. group A. Superoxide dismutase content was lower in groups A, AC, and IAC vs. group KH. Catalase activity was similar to 28% lower in all groups (except group IA) vs. group KH. During reperfusion of the ischemic heart, insulin activates the AKT and AMPK pathways and inhibits the deleterious effects of angiotensin-I perfusion on SOD expression and cardiac function. The addition of captopril does not potentiate these effects.