Phenylephrine Treatment Research Articles

In Vivo Stimulation of α- and β-Adrenoceptors in Mice Differentially Alters Small RNA Content of Circulating Extracellular Vesicles.

When myocardial function is compromised as in heart failure (HF), there is activation of the sympathetic nervous system with elevated circulating catecholamine levels. These catecholamines activate cardiac and extra-cardiac adrenergic receptors (ARs). Interest in secreted extracellular vesicles (EVs) from the heart is growing and in HF, it is not known whether excessive activation of α- or β-adrenergic receptors (ARs) could induce specific changes in EV content. In this study, we have evaluated, by next generation sequencing, the small RNA content, including micro-RNAs (miRs), of circulating EVs of mice exposed to chronic selective α- or β- AR stimulation. EVs from mouse blood were purified by differential ultracentrifugation resulting in EVs with an average size of 116.6 ± 4.8 nm that by immunoblotting included protein markers of EVs. We identified the presence of miRs in blood EVs using miR-21-5p and -16-5p real-time PCR as known constituents of blood exosomes that make up a portion of EVs. We next performed next generation sequencing (NGS) of small non-coding RNAs found in blood EVs from mice following 7 days of chronic treatment with isoproterenol (ISO) or phenylephrine (PE) to stimulate α- or β-ARs, respectively. PE increased the percent of genomic repeat region reads and decreased the percent of miR reads. In miR expression analysis, PE and ISO displayed specific patterns of miR expression that suggests differential pathway regulation. The top 20 KEGG pathways predicted by differential expressed miRs show that PE and ISO share 11 of 20 pathways analyzed and reveal also key differences including three synapse relative pathways induced by ISO relative to PE treatment. Both α-and β-AR agonists can alter small RNA content of circulating blood EVs/exosomes including differential expression and loading of miRs that indicate regulation of distinct pathways. This study provides novel insight into chronic sympathetic nervous system activation in HF where excessive catecholamines may not only participate in pathological remodeling of the heart but alter other organs due to secretion of EVs with altered miR content.

Metabolic Interventions to Prevent Hypertrophy-Induced Alterations in Contractile Properties In Vitro

(1) Background: The exact mechanism(s) underlying pathological changes in a heart in transition to hypertrophy and failure are not yet fully understood. However, alterations in cardiac energy metabolism seem to be an important contributor. We characterized an in vitro model of adrenergic stimulation-induced cardiac hypertrophy for studying metabolic, structural, and functional changes over time. Accordingly, we investigated whether metabolic interventions prevent cardiac structural and functional changes; (2) Methods: Primary rat cardiomyocytes were treated with phenylephrine (PE) for 16 h, 24 h, or 48 h, whereafter hypertrophic marker expression, protein synthesis rate, glucose uptake, and contractile function were assessed; (3) Results: 24 h PE treatment increased expression of hypertrophic markers, phosphorylation of hypertrophy-related signaling kinases, protein synthesis, and glucose uptake. Importantly, the increased glucose uptake preceded structural and functional changes, suggesting a causal role for metabolism in the onset of PE-induced hypertrophy. Indeed, PE treatment in the presence of a PAN-Akt inhibitor or of a GLUT4 inhibitor dipyridamole prevented PE-induced increases in cellular glucose uptake and ameliorated PE-induced contractile alterations; (4) Conclusions: Pharmacological interventions, forcing substrate metabolism away from glucose utilization, improved contractile properties in PE-treated cardiomyocytes, suggesting that targeting glucose uptake, independent from protein synthesis, forms a promising strategy to prevent hypertrophy and hypertrophy-induced cardiac dysfunction.

TMEM173 protects against pressure overload-induced cardiac hypertrophy by modulating autophagy.

TMEM173 has been reported to participate in endoplasmic reticulum stress, inflammation and immunology, all of which closely involved with cardiac hypertrophy. But its role in autophagy is not fully figured out. In our research, Tmem173 global knockout (KO) mice manifested more deteriorated hypertrophy, fibrosis, inflammatory infiltration and cardiac malfunction compared with wild type C57BL/6 mice after 6 weeks of transverse aortic constriction. And KO mice showed inhibited autophagosome degradation in myocardium observed under transmission electron microscope and in protein level. In in vitro experiments conducted in neonatal rat cardiomyocytes under phenylephrine treatment, the abundance of Tmem173 gene was negatively related to the abundance of LC3-Ⅱ and the number of red and yellow fluorescent dots, of which reflected the capacity of autophagosome degradation. These results indicated that TMEM173 might be a promoter of autophagic flux and protected against pressure overload-induced cardiac hypertrophy. It may serve as a potential therapeutic target for cardiac hypertrophy in the future.

Effect of Adrenergic Agonists on High-Fat Diet-Induced Hepatic Steatosis in Mice.

The autonomic nervous system, consisting of sympathetic and parasympathetic branches, plays an important role in regulating metabolic homeostasis. The sympathetic nervous system (SNS) regulates hepatic lipid metabolism by regulating adrenergic receptor activation, resulting in the stimulation of hepatic very-low-density lipoprotein-triglyceride (TG) production in vivo. However, only a few studies on the relationship between SNS and hepatic steatosis have been reported. Here, we investigate the effect of adrenergic receptor agonists on hepatic steatosis in mice fed a high-fat diet (HFD). The α-adrenergic receptor agonist phenylephrine (10 mg/kg/d) or the β-adrenergic receptor agonist isoproterenol (30 mg/kg/d) was coadministered with HFD to male mice. After five weeks, hepatic steatosis, TG levels, and hepatic fat metabolism-related biomarkers were examined. HFD treatment induced hepatic steatosis, and cotreatment with phenylephrine, but not isoproterenol, attenuated this effect. Phenylephrine administration upregulated the mRNA levels of hepatic peroxisome proliferator-activated receptor alpha and its target genes (such as carnitine palmitoyltransferase 1) and increased hepatic β-hydroxybutyrate levels. Additionally, phenylephrine treatment increased the expression of the autophagosomal marker LC3-II but decreased that of p62, which is selectively degraded during autophagy. These results indicate that phenylephrine inhibits hepatic steatosis through stimulation of β-oxidation and autophagy in the liver.

Injections of the α-2 adrenoceptor agonist clonidine into the dorsal raphe nucleus increases food intake in satiated rats

The present study aimed to evaluate the effects of pharmacological manipulation of α-adrenergic agonists in the dorsal raphe nucleus (DR) on food intake in satiated rats. Adult male Wistar rats with chronically implanted cannula in the DR were injected with adrenaline (AD) or noradrenaline (NA) (both at doses of 6, 20 and 60 nmol), or α-1 adrenergic agonist phenylephrine (PHE) or α-2 adrenergic agonist clonidine (CLO) (both at doses of 6 and 20 nmol). The injections were followed by the evaluation of ingestive behaviors. Food and water intake were evaluated for 60 min. Administration of AD and NA at 60 nmol and CLO at 20 nmol increased food intake and decreased latency to start consumption in satiated rats. The ingestive behavior was not significantly affected by PHE treatment in the DR. CLO treatment increased Fos expression in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) in rats that were allowed to eat during the experimental recording (AF group). However, when food was not offered during the experiment (WAF group), PVN neurons were not activated, whereas, neuronal activity remained high in the ARC when compared to control group. Noteworthy, ARC POMC neurons expressed Fos in the AF group. However, double-labeled POMC/Fos cells were absent in the ARC of the WAF group, although an increase in Fos expression was observed in non-POMC cells after CLO injections in the WAF group. In conclusion, the data from the present study highlight that the pharmacological activation of DR α-adrenoceptors affects food intake in satiated rats. The feeding response evoked by CLO injections into DR was similar to that induced by NA or AD injections, suggesting that the hyperphagia after NA or AD treatment depends on α-2 adrenoceptors activation. Finally, we have demonstrated that CLO injections into DR impact neuronal activity in the ARC, possibly evoking a homeostatic response toward food intake.

Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice.

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. We employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5–7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, our data suggest that Dysbindin’s role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted.

LncRNA ZEB2‑AS1 stimulates cardiac hypertrophy by downregulating PTEN

Cardiac hypertrophy (CH) is closely related to a range of cardiovascular diseases, including heart failure and sudden cardiac death. The present study aimed to elucidate the role of long non-coding RNA (lncRNA) ZEB2 antisense RNA 1 (ZEB2-AS1) in regulating the hypertrophic process of cardiomyocytes and the potential underlying mechanism. An in vivo CH mouse model was established by performing transverse aortic constriction procedures. An in vitro CH model was established in primary cardiomyocytes isolated from mice by phenylephrine (PE) treatment. The relative protein levels of BNP, ANP and PTEN in cells with different groups (CH group and control group) were determined by western blotting. Relative expression levels of ZEB2-AS1, natriuretic peptide A (ANP) and brain natriuretic peptide (BNP) were determined in both in vivo and in vitro CH models. The regulatory effects of ZEB2-AS1/phosphatase and tensin homolog (PTEN) on cell surface area, and the relative expression levels of ANP and BNP were explored. ZEB2-AS1, ANP and BNP expression levels were increased in both in vivo and in vitro CH models compared with the sham and negative control groups, respectively. ZEB2-AS1 knockdown decreased cell surface area, and downregulated ANP and BNP expression levels in PE-treated primary cardiomyocytes. Similarly, PTEN overexpression reduced cell surface area, and downregulated ANP and BNP expression levels in PE-treated primary cardiomyocytes. Moreover, PTEN reversed the regulatory effects of ZEB2-AS1 on hypertrophic cardiomyocytes. Therefore, the present study suggested that lncRNA ZEB2-AS1 may influence the progression of CH by downregulating PTEN.

The effect of spinal hypotension and anesthesia-to-delivery time interval on neonatal outcomes in planned cesarean delivery

Maternal hypotension after spinal anesthesia, and time from anesthesia to delivery, are potentially modifiable risk factors for neonatal acidosis. This study aimed to examine the relationship between the time from spinal anesthesia to delivery and spinal hypotension in planned cesarean deliveries and their effect on neonatal outcome, primarily neonatal acidosis. We performed a retrospective analysis of women with singleton pregnancy undergoing spinal anesthesia for planned cesarean delivery between 37 0/7 and 41 6/7 weeks' gestation using electronic medical records. The occurrence of spinal hypotension and anesthesia-to-incision and incision-to-delivery intervals (minutes) were the primarily studied variables. In addition, spinal hypotension index was developed to account for the duration and magnitude of maternal hypotension. The 90th percentile for the spinal hypotension index defined the sustained spinal hypotension group. The primary outcome was neonatal acidosis (pH of ≤7.1 or base deficit of ≥12.0). The odds ratios were calculated using univariate and multivariate logistic regression models. The multivariate analysis included sporadic spinal hypotension or sustained spinal hypotension, use of vasopressor treatment, and anesthesia-to-incision and incision-to-delivery intervals. We included 3150 women in the study. Notably, 43.4% experienced at least 1 event of spinal hypotension (sporadic) and 14.8% experienced sustained spinal hypotension. Neonatal acidosis occurred in 3.4% cases of sporadic spinal hypotension (odds ratio, 1.83; 95% confidence interval, 2.27-2.87) and in 5.8% cases of sustained hypotension (odds ratio, 3.00; 95% confidence interval, 1.87-4.80). Both anesthesia-to-incision and incision-to-delivery intervals were significantly associated with neonatal acidosis as follows: at 90th percentile cutoff, the odds ratios for neonatal acidosis were 3.82 (95% confidence interval, 2.03-7.19) and 2.94 (95% confidence interval, 1.70-5.10), respectively. The use of ephedrine (odds ratio, 2.42; 95% confidence interval, 1.35-4.32) but not phenylephrine (odds ratio, 0.76; 95% confidence interval, 0.34-1.72) treatment was also associated with more cases of neonatal acidosis. The woman's age, gestational age, neonatal birthweight, fetal presentation, and the number of previous cesarean deliveries were not associated with neonatal acidosis. In multivariate analysis, anesthesia-to-incision and incision-to-delivery intervals, use of vasopressor treatment, and sustained spinal hypotension were independently associated with neonatal acidosis. After adjustment, the risk for neonatal acidosis did not increase in women who experienced sporadic spinal hypotension only. Neither neonatal acidosis nor the primary research variables were associated with neonatal complications such as transient tachypnea of the newborn, respiratory distress, or admission to the neonatal unit. Neonatal acidosis in planned cesarean delivery was common. However, serious perinatal consequences were rare. The adverse effects of sustained spinal hypotension and prolonged anesthesia-to-incision and incision-to-delivery intervals on neonatal acid-base balance were additive. This supports the adoption of prevention strategies for spinal hypotension, which is widely evidenced based on the obstetrical anesthesia literature, but still not universally used. Whether the reduction in intraoperative time intervals would benefit the neonate should be determined by future prospective studies.

Are microRNAs responsible for cardiac hypertrophy in fish and mammals? What we can learn in the activation process in a zebrafish ex vivo model

Recent studies have correlated dysregulated miRNA expression with diseased hearts. With the aim of developing an easily manipulated experimental model, phenylephrine (PE) was added to cultured zebrafish hearts to study the expression of miR1 and miR133a by qRT-PCR. Both miRs were downregulated, with greater downregulation leading to higher hypertrophy. The involvement of this miRs was confirmed by the in-vivo inoculation of complementary sequences (AmiR1 and AmiR133a). HSP70 (involved in transporting proteins and in anti-apoptosis processes) was increased in both treatments. Hyperplasia was observed in the epicardium based on WT1 expression (embryonic epicardial cell marker) in both the PE treatment and AmiR133a treatment. The treatment with AmiR1 showed only cardiomyocyte hypertrophy. This ex-vivo model revealed that miR1 and miR133a play a key role in activating early processes leading to myocardium hypertrophy and epicardium hyperplasia and confirmed the expected similarities with hypertrophic disease that occurs in humans.

Small Nppa and Myl2 Promoters Are Sufficient to Maintain Chamber‐specific Expression on an AAV9 Platform

ObjectiveTo generate an AAV9 vector with small promoters that selectively express transgenes in a chamber‐specific manner.IntroductionThe adeno‐associated virus serotype 9 (AAV9) vector exhibits high transduction efficiency and cardiotropism which has allowed for the elucidation of tissue‐specific functions of genes implicated in cardiac diseases. However, cardiac diseases such as atrial fibrillation, require a more selective strategy to allow for the study of gene function in a chamber‐specific manner. AAV9 vectors carrying chamber‐specific promoters, such as Nppa and SLN for atrial‐specific expression, have been published; however, these vectors contain 632 and 1000 base pairs (bp), respectively, limiting the carrying capacity for transgenes.HypothesisLess than 500 bp of the Nppa and Myl2 promoters is sufficient to confer potent AAV9‐mediated atrial and ventricular cardiomyocyte specific expression, respectively.MethodsFragments of the Nppa promoter, consisting of about 600 and 400 bp, and of the Myl2 promoter, consisting of about 700 and 300 bp, were inserted into a Green Fluorescent Protein (GFP) expressing vector. These vectors were electroporated into primary neonatal rat atrial and ventricular cardiomyocytes (NRAMS and NRVMs). Because pathological hypertrophy is known to upregulate expression of Nppa in ventricular cardiomyocytes, NRAMs and NRVMs were treated with 50 uM of phenylephrine (PE), a chemical inducer of hypertrophy, for 48 hours, to assess the faithfulness of our promoters to maintain chamber‐specific GFP expression. Promoter activity was analyzed by immunoblot (IB) for GFP and fluorescent microscopy. The promoters which expressed GFP robustly and retained chamber‐specificity with PE treatment were selected for testing in mice in an AAV9 vector. The mouse model used was a two‐color fluorescent Cre Recombinase (Cre) reporter, consisting of membrane targeted Tomato (mT) and GFP (mG). In this model, mT fluorescence is exclusively expressed; upon introduction of Cre, mT is excised, allowing mG to express. We generated AAV9‐Cre driven by the Nppa or Myl2 promoters, injected mice via tail‐vein with 1×1011 viral particles, performed TAC for 4 weeks, and determined chamber‐specific expression of Cre by IB detection of GFP from atrial and ventricular tissue.ResultsIn NRAMs and NRVMs, IB and fluorescent microscopy confirmed that promoters as small as 400 bp and 300 bp for Nppa and Myl2, respectively, elicited robust atrial and ventricular specific GFP expression, respectively. Interestingly, 600 bp of the Nppa promoter expressed GFP in NRVMs with PE treatment, while 400 bp did not. In an AAV9‐Cre vector, 400 and 300 bp of the Nppa and Myl2 promoter, respectively, were sufficient to mediate chamber‐specific excision of mT, as determined by IB for GFP.ConclusionSmall promoter regions of Nppa and Myl2, consisting of about 400 and 300 bp, respectively, can be utilized in an AAV9 vector to selectively express transgenes in atrial and ventricular cardiomyocytes, respectively. Importantly, the smaller size of the Nppa and Myl2 promoters leaves room for larger transgenes, alleviating the limitations imposed upon the small carrying capacity of AAV9 vectors.Support or Funding InformationAmerican Heart AssociationNational Institute of Health

Dissecting genetic factors affecting phenylephrine infusion rates during anesthesia: a genome-wide association study employing EHR data

BackgroundThe alpha-adrenergic agonist phenylephrine is often used to treat hypotension during anesthesia. In clinical situations, low blood pressure may require prompt intervention by intravenous bolus or infusion. Differences in responsiveness to phenylephrine treatment are commonly observed in clinical practice. Candidate gene studies indicate genetic variants may contribute to this variable response.MethodsPharmacological and physiological data were retrospectively extracted from routine clinical anesthetic records. Response to phenylephrine boluses could not be reliably assessed, so infusion rates were used for analysis. Unsupervised k-means clustering was conducted on clean data containing 4130 patients based on phenylephrine infusion rate and blood pressure parameters, to identify potential phenotypic subtypes. Genome-wide association studies (GWAS) were performed against average infusion rates in two cohorts: phase I (n = 1205) and phase II (n = 329). Top genetic variants identified from the meta-analysis were further examined to see if they could differentiate subgroups identified by k-means clustering.ResultsThree subgroups of patients with different response to phenylephrine were clustered and characterized: resistant (high infusion rate yet low mean systolic blood pressure (SBP)), intermediate (low infusion rate and low SBP), and sensitive (low infusion rate with high SBP). Differences among clusters were tabulated to assess for possible confounding influences. Comorbidity hierarchical clustering showed the resistant group had a higher prevalence of confounding factors than the intermediate and sensitive groups although overall prevalence is below 6%. Three loci with P < 1 × 10−6 were associated with phenylephrine infusion rate. Only rs11572377 with P = 6.09 × 10−7, a 3′UTR variant of EDN2, encoding a secretory vasoconstricting peptide, could significantly differentiate resistant from sensitive groups (P = 0.015 and 0.018 for phase I and phase II) or resistant from pooled sensitive and intermediate groups (P = 0.047 and 0.018).ConclusionsRetrospective analysis of electronic anesthetic records data coupled with the genetic data identified genetic variants contributing to variable sensitivity to phenylephrine infusion during anesthesia. Although the identified top gene, EDN2, has robust biological relevance to vasoconstriction by binding to endothelin type A (ETA) receptors on arterial smooth muscle cells, further functional as well as replication studies are necessary to confirm this association.

LncRNA PEG10 aggravates cardiac hypertrophy through regulating HOXA9.

To uncover the role of long non-coding RNA (lncRNA) PEG10 in the progression of cardiac hypertrophy by regulating HOXA9. In vivo cardiac hypertrophy model was established by performing transverse aortic constriction model (TAC) procedures in mice. Relative levels of PEG10, ANP and BNP in mice undergoing TAC procedures or sham operations were determined. In vitro cardiac hypertrophy model was established by phenylephrine (PE) treatment in primary cardiomyocytes. Relative levels of PEG10, ANP and BNP in cardiomyocytes were determined as well. Regulatory effects of HOXA9 on surface area of cardiomyocytes and relative levels of ANP and BNP were assessed. Finally, potential influences of PEG10/HOXA9 regulatory loop on cell surface area and relative levels of ANP and BNP were explored. Compared with mice in sham group, those in TAC group presented higher levels of PEG10, ANP and BNP. PE treatment markedly upregulated PEG10, ANP and BNP in primary cardiomyocytes, which were downregulated by transfection of si-PEG10. Besides, surface area of cardiomyocytes was enlarged by PE treatment, which was reduced after silence of PEG10. Silence of HOXA9 presented a similar effect as that of PEG10 in cardiomyocytes. Transfection of si-HOXA9 reversed the expanded cell surface area, and upregulated ANP and BNP in cardiomyocytes overexpressing PEG10. PEG10 is upregulated in hypertrophic cardiomyocytes. PEG10 aggravates cardiac hypertrophy by positively regulating HOXA9.

Spinal-Induced Hypotension in Preeclamptic and Healthy Parturients Undergoing Cesarean Section.

BACKGROUND:There is a widespread belief that spinal anaesthesia in patients with preeclampsia might cause severe hypotension and decreased uteroplacental perfusion. This study aimed to evaluate the incidence and severity of spinal induced-hypotension in preeclamptics and healthy parturients.METHODS:Total of 78 patients (40 healthy and 38 preeclamptic) undergoing a C-Section with spinal anaesthesia were included. Spinal anaesthesia was performed with a mixture of 8-9 mg isobaric 0.5% bupivacaine, 20 mcg fentanyl and 100 mcg morphine (total volume 2.2-2.4 ml). Blood pressures (BP)-SBP, DBP, MAP were recorded non-invasively before performing spinal anaesthesia and at 2.5 minutes after a spinal puncture.RESULTS:The BP falls (%) from baseline were significantly greater in the healthy parturients compared to those with preeclampsia (25.8% ± 10.1 vs 18.8% ± 17.0 for SBP, 28.5% ± 8.8 vs 22.5% ± 10.4 for DBP, and 31.2% ± 14.2 vs 18.2% ± 12.6% for MAP, p < 0.05). The incidence rate of hypotension in the preeclamptics was 25% compared to 53% in healthy parturients (p < 0.001). Higher doses of vasopressors both ephedrine (16.5 ± 8.6 vs 6.0 ± 2.0 mg) and phenylephrine (105 ± 25 mg) in the healthy women were required. There was no need for phenylephrine treatment in the preeclamptic group.CONCLUSION:This study showed that the incidence and severity of spinal-induced hypotension in preeclamptic patients are less than in healthy women. The use of low dose spinal anaesthesia also contributed to this statement.

A Novel Method of Isolating Myofibrils From Primary Cardiomyocyte Culture Suitable for Myofibril Mechanical Study.

Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca2+-activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm2 (resting tension), 146.8 ± 13.8 mN/mm2 (maximal active tension, P0), 5.4 ± 0.22 s−1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s−1 (linear relaxation rate), and 10.8 ± 1.3 s−1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca2+-sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.

In vitro evidence that endothelium-dependent vasodilatation induced by clozapine is mediated by an ATP-sensitive potassium channel.

There is a definite association between antipsychotic drugs and arterial hypertension. However, endothelium functions are scarcely considered. This investigation was carried out to study the mechanisms involved in clozapine endothelium-dependent vascular reactivity. The experimental animals were male Wistar rats with a mean age of 70-90 days (250-300 g). The endothelium-dependent vascular reactivity was studied by measuring the isometric force and then constructing clozapine concentration-response curves. The force registrations were obtained in the aorta rings with and without the endothelium precontracted with phenylephrine (PE10-6M) treatment; this followed incubation for 30 min in "organ chambers" with different inhibitors: l- NAME (nitric oxide/cGMP); indomethacin (PGI2/cAMP); tetraethylammonium (TEA), and specific hyperpolarization blockers (paxillin, apamin, glibenclamide). The data were presented as the mean ± standard error of the mean (SEM) and were compared by one-way ANOVA or two-way ANOVA followed by the Bonferroni post-test. The primary outcomes were: 1) Clozapine-induced endothelium-dependent relaxation was not inhibited by indomethacin, l-NAME, ODQ, and methylene blue (MB); 2) The combination of l-NAME + indomethacin partially prevented the relaxation; 3) Clozapine did not induce relaxation in vessels contracted with KCl; 4) TEA did not block the clozapine-induced relaxation in vessels precontracted with PE (10-6 M); 5) The potassium channel blockers paxillin and apamin did not prevent relaxation but glibenclamide did. Concerning the mechanisms involved in clozapine endothelium-dependent vascular reactivity, the present study suggests that there is synergistic participation that probably occurs through a crosstalk mechanism of the cAMP, cGMPpathways and hyperpolarization.

Possible Impact of Spinal Anesthesia and Phenylephrine on Sublingual Microcirculation of Cesarean Delivery Patients.

BackgroundThis study was a proof of concept of a novel means to evaluate microcirculatory changes during spinal anesthesia for cesarean delivery. It sought to examine the distributive circulatory effects of spinal anesthesia and evaluate the impact of phenylephrine administration on the microcirculation of these women.MethodsAfter Research Ethics Board approval, healthy, non-laboring pregnant women with singleton, term pregnancies scheduled for elective cesarean delivery were recruited. Participants were randomly assigned to receive either phenylephrine infusion or phenylephrine bolus. Spinal anesthesia was standardized. A sidestream dark-field (SDF) MicroScan® video microscope was applied to the sublingual mucosa to obtain microcirculation videos in five different visual fields. Videos were made before and after spinal anesthesia. The resultant videos were analyzed randomly and blindly. The mean microvascular flow index (MFI) values were compared before and after spinal anesthesia. The difference in MFI following spinal anesthesia was compared between phenylephrine infusion and bolus groups.ResultsThirty-two patients were recruited for the study; 22 patients had complete video sets for analysis. Baseline characteristics were similar between the two groups, including preoperative hemodynamics. There were no significant differences between pre- and post-spinal MFI. The post-spinal MFI within the infusion group (mean ± standard deviation: 2.74 ± 0.21) was not significantly different from the bolus group (2.56 ± 0.42, P = 0.22).ConclusionDespite theoretical physiological implications of spinal anesthesia and phenylephrine on the microcirculation, significant alteration of the MFI was not observed between pre- and post-spinal anesthesia (within group). Additionally, despite an eight-fold larger phenylephrine dose for continuous infusion prophylaxis used in this group of women, this did not result in a significant alteration of the microcirculation compared to those who received phenylephrine treatment for hypotension (between groups).

Association between intraoperative phenylephrine administration and umbilical artery pH in women with hypertensive disorders of pregnancy: a retrospective cohort study

In healthy pregnant women undergoing Cesarean section, treatment of post-spinal hypotension (PSH) with phenylephrine is reportedly more efficacious than ephedrine in preventing a decline in umbilical artery pH (UApH), which is an indicator of fetal acidosis. However, phenylephrine has the potential to reduce placental blood flow and consequently decrease UApH in women who develop hypertensive disorders of pregnancy (HDP), due to decreased cardiac function and placental vascular abnormalities. We aimed to verify the association between decreased UApH and phenylephrine administered before delivery to treat PSH in women with HDP. This single-center retrospective cohort study was conducted between April 2008 and October 2016 by assessing the records of pregnant women with confirmed or suspected HDP who underwent Cesarean section under spinal anesthesia. The association between phenylephrine and the primary endpoint of decreased UApH was investigated using logistic regression analysis and propensity score matching. In the original cohort of 107 pregnant women with HDP, neither univariate nor multivariate analysis indicated a significant association between phenylephrine treatment and decreased UApH [crude odds ratio (OR) = 2.26; 95% confidence interval (CI) 0.75-6.77; P = 0.14], (adjusted OR = 1.80; 95% CI 0.55-5.93; P = 0.33). Thirty patients each who were not treated with phenylephrine (control group) and were treated with phenylephrine (phenylephrine group) were matched by propensity score analysis. UApH [median (interquartile range)] was not significantly different between control and phenylephrine groups [7.30 (7.25-7.35) vs. 7.27 (7.25-7.30); P = 0.14]. Intraoperative phenylephrine administration to treat PSH in women with HDP was not associated with decreased UApH.

Fisetin inhibits cardiac hypertrophy by suppressing oxidative stress

Cardiac hypertrophy is a pathophysiological response to various pathological stresses and ultimately leads to heart failure. Oxidative stress is one of the critical processes involved in hypertrophy development. Fisetin, a small molecular flavonoid, has been shown to have anti-oxidative, anti-proliferative and anti-inflammatory properties. However, the effect of fisetin on cardiac hypertrophy remains unknown. In our present study, we showed that fisetin inhibited pressure overload-induced cardiac hypertrophy, improved cardiac function in vivo and suppressed phenylephrine (PE)-induced cardiomyocyte hypertrophy in vitro. Reactive oxygen species (ROS) levels were markedly decreased by fisetin treatment in both hypertrophic hearts and cardiomyocytes. Moreover, fisetin significantly up-regulated the expression of antioxidative genes, including catalase (CAT), superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HO-1). Furthermore, co-treatment with N-acetylcysteine (NAC; ROS scavenger) and fisetin did not have synergistic inhibitory effects on PE-induced cardiomyocyte hypertrophy, indicating that the anti-hypertrophic effects of fisetin are mainly associated with the blockade of oxidative stress. Finally, the pro-hypertrophic signaling pathways, mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) kinase, were found to be suppressed by fisetin after pressure overload and PE treatment. In conclusion, our study revealed that fisetin protects against cardiac hypertrophy and that oxidative stress inhibition may be one of the pivotal mechanisms involved.

PARP1 interacts with HMGB1 and promotes its nuclear export in pathological myocardial hypertrophy.

High-mobility group box 1 (HMGB1) exhibits various functions according to its subcellular location, which is finely conditioned by diverse post-translational modifications, such as acetylation. The nuclear HMGB1 may prevent from cardiac hypertrophy, whereas its exogenous protein is proven to induce hypertrophic response. This present study sought to investigate the regulatory relationships between poly(ADP-ribose) polymerase 1 (PARP1) and HMGB1 in the process of pathological myocardial hypertrophy. Primary-cultured neonatal rat cardiomyocytes (NRCMs) were respectively incubated with three cardiac hypertrophic stimulants, including angiotensin II (Ang II), phenylephrine (PE), and isoproterenol (ISO), and cell surface area and the mRNA expression of hypertrophic biomarkers were measured. the catalytic activity of PARP1 was remarkably enhanced, meanwhile HMGB1 excluded from the nucleus. PARP1 overexpression by infecting with adenovirus PARP1 (Ad-PARP1) promoted the nuclear export of HMGB1, facilitated its secretion outside the cell, aggravated cardiomyocyte hypertrophy, which could be alleviated by HMGB1 overexpression. PE treatment led to the similar results, while that effect was widely depressed by PARP1 silencing or its specific inhibitor AG14361. Moreover, SD rats were intraperitoneally injected with 3-aminobenzamide (3AB, 20 mg/kg every day, a well-established PARP1 inhibitor) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks, echocardiography and morphometry of the hearts were measured. Pre-treatment of 3AB relieved AAC-caused the translocation of nuclear HMGB1 protein, cardiac hypertrophy, and heart dysfunction. Our research offers a novel evidence that PARP1 combines with HMGB1 and accelerates its translocation from nucleus to cytoplasm, and the course finally causes cardiac hypertrophy.

Abstract 211: Myocardial Hypertrophy and Circulating RNAs

While increased left ventricular mass (LVM) is strongly associated with incident heart failure (HF), events during transition from increased LVM to HF remain unclear. Extracellular non-coding RNAs (ex-RNAs) have been implicated in cardiac hypertrophy, though whether these ex-RNAs reflect important pathways in HF in humans is underexplored. In &gt;2,000 individuals with concomitant M-mode echocardiography and ex-RNA measurements in the Framingham Heart Study, we found that lower circulating concentrations of three ex-RNAs—miR-20a-5p, miR-106b-5p, miR-17-5p—were associated with (1) greater LVM (+ one other pre-clinical phenotype, e.g., left atrial dimension or LVEDV) and (2) greater incident HF risk over a median follow-up 7.7 years ( Fig. A ). These 3 miRNAs were members of a tight cluster, regulating 883 mRNAs in common, associated with “hypertension” (OMIM) and biological process relevant to HF, including TGF-β signaling. We observed an increase in myocardial expression of these miRNAs during different phases of hypertrophy/HF development ( Fig. C, D ). Using gain and loss of function in vitro , our preliminary results suggest up-regulation of cardiomyocyte miR-106b expression abrogates expression of pathologic hypertrophy markers (ANP and BNP) during phenylephrine treatment, consistent with in silico results suggesting broad connections between miR-106b targets and natriuretic peptide signaling ( Fig. B, E-F ). These results provide translational evidence that circulating miRNAs associated with hypertrophy in patients may be protective in the transition from hypertrophy to HF at the molecular level.