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This study analyzed how glyphosate exposure in the gestational period affects vascular function in their female offspring and whether oxidative stress is involved in this effect. To this, pregnant Wistar rats were exposed through drinking water to 0.2% of a glyphosate commercial formulation, and we analyzed the response to acetylcholine and phenylephrine in the aorta from offspring of Glyphosate-based herbicide (O-GBH) and controls (O-CON) rats at six months of age. Relaxation to acetylcholine was reduced in O-GBH than in O-CON. Acute Indomethacin and Apocynin increased relaxation to acetylcholine in O-GBH. The aorta from O-GBH was hyperactive to phenylephrine; the preincubation with N-nitro-L-arginine methyl ester (L-NAME) increased contraction to phenylephrine more in O-CON than O-GBH. TEMPOL similarly reduced phenylephrine response, and L-NAME prevented this effect. The TBARS and GSH levels were increased in O-GBH than in O-CON. Results reinforce the concept that oxidative stress during the perinatal period contributes to the development of vascular changes in adulthood. Results also reveal that oxidative stress parameters altered, and the current levels considered safe for exposure to Glyphosate deserve further investigation, especially in the female gender., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study analyzed how glyphosate exposure in the gestational period affects vascular function in their offspring, focusing on the influence of age and whether oxidative stress is involved in this effect. To this, pregnant Wistar rats were exposed through drinking water to 0.2% of a glyphosate commercial formulation, and we analyzed the response to acetylcholine and phenylephrine in the aorta from offspring of glyphosate herbicide-based (O-GHB) and controls (O-CON) rats at 3, 6, and 12 months of age. O-GHB groups showed no changes in arterial blood pressure or aorta histological analysis. Relaxation to acetylcholine was reduced in O-GHB than O-CON. Acute TEMPOL increased relaxation to acetylcholine in O-GHB at 6 and 12 months of age. The aorta from O-GHB was hyperactive to phenylephrine only at 6 months of age. Preincubation with N-nitro-L-arginine methyl ester (L-NAME) increased contraction to phenylephrine more in O-CON than O-GHB. TEMPOL similarly reduced phenylephrine response. This effect was prevented by L-NAME. Results reinforce the concept that oxidative stress during the perinatal period contributes to the development of vascular changes in adulthood. Results also reveal that although no changes in cardiac or histological parameters have been demonstrated, the current levels considered safe for exposure to glyphosate deserve further investigation, especially during pregnancy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The heart is a high energy demand organ and enhancing mitochondrial function is proposed as the next-generation therapeutics for heart failure. Our previous study found that anthelmintic drug niclosamide enhanced mitochondrial respiration and increased adenosine triphosphate (ATP) production in cardiomyocytes, therefore, this study aimed to determine the effect of niclosamide on heart failure in mice and the potential molecular mechanisms. The heart failure model was induced by transverse aortic constriction (TAC) in mice. Oral administration of niclosamide improved TAC-induced cardiac hypertrophy, cardiac fibrosis, and cardiac dysfunction in mice. Oral administration of niclosamide reduced TAC-induced increase of serum IL-6 in heart failure mice. In vitro, niclosamide within 0.1 μM increased mitochondrial respiration and ATP production in mice heart tissues. At the concentrations more than 0.1 μM, niclosamide reduced the increased interleukin- 6 (IL-6) mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 and THP-1 derived macrophages. In cultured primary cardiomyocytes and cardiac fibroblasts, niclosamide (more than 0.1 μM) suppressed IL-6- and phenylephrine-induced cardiomyocyte hypertrophy, and inhibited collagen secretion from cardiac fibroblasts. In conclusion, niclosamide attenuates heart failure in mice and the underlying mechanisms include enhancing mitochondrial respiration of cardiomyocytes, inhibiting collagen secretion from cardiac fibroblasts, and reducing the elevated serum inflammatory mediator IL-6. The present study suggests that niclosamide might be therapeutic for heart failure., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Peroxisomal biogenesis factor 5 (PEX5) is a member of peroxisome biogenesis protein family which serves as a shuttle receptor for the import of peroxisome matrix protein. The function of PEX5 on cardiomyocyte hypertrophy remained to be elucidated. Our study demonstrated that the protein expression level of PEX5 was declined in primary neonatal rat cardiomyocytes treated with phenylephrine (PE) and hearts from cardiac hypertrophic rats induced by abdominal aortic constriction (AAC). Overexpression of PEX5 alleviated cardiomyocyte hypertrophy induced by PE, while silencing of PEX5 exacerbated cardiomyocyte hypertrophy. PEX5 improved redox imbalance by decreasing cellular reactive oxygen species level and preserving peroxisomal catalase. Moreover, PEX5 knockdown aggravated PE-induced activation of redox-sensitive signaling pathways, including mitogen-activated protein kinase (MAPK) pathway and signal transducer and activator of transcription 3 (STAT3); whereas PEX5 overexpression suppressed activation of MAPK and STAT3. But PEX5 did not affect PE-induced phosphorylation of mammalian target of rapamycin (mTOR). In conclusion, the present study suggests that PEX5 protects cardiomyocyte against hypertrophy via regulating redox homeostasis and inhibiting redox-sensitive signaling pathways MAPK and STAT3., (Copyright © 2021. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Pinoresinol diglucoside (PDG), the active compound extracted from Eucommia ulmoides, Styrax sp. and Forsythia suspensa, plays the roles in regulating hypertension, inflammation and oxidative stress., Aims: Considering that hypertension and inflammation has been proved to contribute to cardiac remodeling, we tested the effects of PDG on cardiac hypertrophy (CM)., Methods: Male Sprague Dawley (SD) rats were used to construct hypertrophic rats by partial abdominal aortic constriction (AAC)-surgery. PDG solution (2 mg/ml) was used to treat AAC-induced rats by intraperitoneal injection at low dose (L-PDG, 2.5 mg/kg per day), medium dose (M-PDG, 5 mg/kg per day), and high dose (H-PDG, 7.5 mg/kg per day) for 3 weeks post AAC-surgery. CM was evaluated by the ratio of left ventricular weight to body weight ratio (LVW/BW), left ventricular wall thickness by H&E staining, and collagen content deposit by Masson's staining. Further, isoproterenol (ISO) and phenylephrine (PE) were used to produce cellular models of CM in neonatal rat ventricular cardiomyocytes (NRVMs). PDG pre-treated NRVMs 2 h at low dose (L-PDG, 2.5 μg/ml), medium dose (M-PDG, 5 μg/ml), and high dose (H-PDG, 7.5 μg/ml) for 24 h with or without PE- and ISO-stimulation. CM was evaluated by the expressions of hypertrophic biomarkers. Next, the hypertrophic biomarkers and pro-inflammatory cytokines were measured using quantitative real-time PCR (qRT-PCR), the expressions of protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/transcription factor nuclear factor-kappa B (NF-kB) signaling pathway were determined by Western blotting., Results: PDG treatment prevented cardiac histomorphology damages, decreased upregulations of hypertrophic biomarkers, and prevented fibrosis and inflammation after pressure overload resulting from AAC-surgery. Consistently, PDG remarkably inhibited the changes of cardiomyocyte hypertrophic biomarkers and inflammatory responses in cellular models of CM. Interestingly, PDG administration inhibited the activation of AKT/mTOR/NF-kB signaling pathway both in vivo and in vitro., Conclusions: PDG prevents AAC-induced CM in vivo, PE- and ISO-induced CM in vitro. The AKT/mTOR/NF-kB signaling pathway could be the potential therapeutic target involved in the protection of PDG. These findings provide novel evidence that PDG might be a promising therapeutic strategy for CM., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Abstract: Nuclear factor of activated T cells, cytoplasmic 4 (NFATc4), a nuclear transcription factor, has been implicated in cardiac hypertrophy through the enhancement of hypertrophic gene expression. However, the role of NFATc4 in mitochondrial modulation is mostly unknown. The current study aimed to investigate the role of NFATc4 in regulating mitochondrial function during phenylephrine (PE)-induced cardiac hypertrophy. Our results showed that overexpression of NFATc4 aggravated the PE-induced decrease in mitochondrial genesis, membrane potential, and mitochondrial gene expression as well as impaired mitochondrial respiration. However, knockdown of NFATc4 relieved PE-induced perturbations in mitochondria and cardiomyocyte hypertrophy. Mechanistically, by activating phosphoinositide-dependent kinase 1 and promoting a combination of AKT and phosphoinositide-dependent kinase 1, phosphorylation and sequential acetylation of PGC-1α were aggravated by NFATc4 and suppressed the activity of PGC-1α. In conclusion, NFATc4-regulated factors were shown to be associated with mitochondrial function and exacerbated PE-induced mitochondrial dysfunction. These findings revealed new roles of NFATc4 in cardiac hypertrophy., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Prenatal glucocorticoid overexposure has been shown to programme adult cardiovascular function in a range of species, but much less is known about the long-term effects of neonatal glucocorticoid overexposure. In horses, prenatal maturation of the hypothalamus-pituitary-adrenal axis and the normal prepartum surge in fetal cortisol occur late in gestation compared to other precocious species. Cortisol levels continue to rise in the hours after birth of full-term foals and increase further in the subsequent days in premature, dysmature and maladapted foals. Thus, this study examined the adult cardiovascular consequences of neonatal cortisol overexposure induced by adrenocorticotropic hormone administration to full-term male and female pony foals. After catheterisation at 2-3 years of age, basal arterial blood pressures (BP) and heart rate were measured together with the responses to phenylephrine (PE) and sodium nitroprusside (SNP). These data were used to assess cardiac baroreflex sensitivity. Neonatal cortisol overexposure reduced both the pressor and bradycardic responses to PE in the young adult males, but not females. It also enhanced the initial hypotensive response to SNP, slowed recovery of BP after infusion and reduced the gain of the cardiac baroreflex in the females, but not males. Basal diastolic pressure and cardiac baroreflex sensitivity also differed with sex, irrespective of neonatal treatment. The results show that there is a window of susceptibility for glucocorticoid programming during the immediate neonatal period that alters cardiovascular function in young adult horses in a sex-linked manner.

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In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%-1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Rationale: The constrained mitochondria in cardiomyocytes communicate with each other, through mitochondrial kissing or nanotunneling, forming a dynamically continuous network to share content and transfer signals. However, the molecular mechanism of cardiac inter-mitochondrial communication is unclear. Objective: To determine the molecular mechanism underlying the robust inter-mitochondrial communication and its pathophysiological relevance in the heart. Methods and Results: By mitochondria-targeted expressing the photoactivatable green fluorescent protein, we revealed that most mitochondrial nanotubes bridge communicating mitochondrial pairs were associated with microtubules. Miro2 (mitochondrial Rho GTPase), the outer mitochondrial membrane protein which usually mediates mitochondrial transport within cells, accompanied with mitochondrial nanotubes along microtubules in adult cardiomyocytes. Adenovirus mediated expression of Miro2 in cardiomyocytes accelerated inter-mitochondrial communication through increasing mitochondrial nanotunneling and mitochondrial kissing between adjacent mitochondrial pairs. In transverse aortic constriction-induced hypertrophic mouse hearts Miro2 protein was declined, accompanied with decreased inter-mitochondrial communication. Miro2 transgenic mice showed ameliorated cardiac function, increased mitochondrial nanotube formation and inter-mitochondrial communication, and improved mitochondrial function after transverse aortic constriction. E3 ubiquitin ligase Parkin was increased in transverse aortic constriction mouse hearts and phenylephrine stimulation-induced hypertrophic cardiomyocytes. Inhibition of proteasome blocked phenylephrine-induced decrease of Miro2, and Parkin overexpression led to the decrease of Miro2. Conclusions: Mitochondrial Miro2 expression levels regulate inter-mitochondrial communication along microtubules in adult cardiomyocytes, and degradation of Miro2 through Parkin-mediated ubiquitination contributes to impaired inter-mitochondrial communication and cardiac dysfunction during hypertrophic heart diseases.Visual Overview: An online visual overview is available for this article.

Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional secreted protein that can be induced by fasting, hypoxia and glucocorticoids. ANGPTL4 has been associated with a variety of diseases; however, the role of ANGPTL4 in cardiac hypertrophy remains poorly understood. In our study, we aimed to explore the effect of ANGPTL4 on phenylephrine-induced cardiomyocyte hypertrophy. Our results showed that knockdown of ANGPTL4 expression significantly exacerbated cardiomyocyte hypertrophy, as demonstrated by increased hypertrophic marker expression, including ANP and cell surface area. Moreover, significantly reduced fatty acid oxidation, as featured by decreased CPT-1 levels, was observed in hypertrophic cardiomyocytes following ANGPTL4 down-regulation. Furthermore, knockdown of ANGPLT4 led to down-regulated expression of peroxisome proliferator-activated receptor α (PPARα), which is the key regulator of cardiac fatty acid oxidation. In addition, ANGPTL4 silencing promoted the activation of JNK1/2, and JNK1/2 signaling blockade could restore the level of PPARα and significantly ameliorate the ANGPTL4 knockdown-induced cardiomyocyte hypertrophy. Therefore, our study demonstrated that ANGPTL4 regulates PPARα through JNK1/2 signaling and is required for the inhibition of cardiomyocyte hypertrophy., (© 2019 The Author(s).)

Host-guest interaction of two significant drugs, phenylephrine hydrochloride and synephrine with α and β-cyclodextrins were studied systematically. Initially two simple but reliable physicochemical techniques namely conductance and surface tension were employed to find out saturation concentration for the inclusion and its stoichiometry. The obtained 1:1 stoichiometry was further confirmed by two spectrometric methods, UV-Vis study and spectrofluorimetry. Significant shifts in IR stretching frequency also support the inclusion process. Relative stabilities of the inclusion complexes were established by the association constants obtained from UV-Vis spectroscopic measurements, program based mathematical calculation of conductivity data. Calculations of the thermodynamic parameters dictates thermodynamic feasibility of the inclusion process. Spectrofluorometric measurement scaffolds the UV-Vis spectroscopic measurement validating stability of the ICs once again. Mass spectroscopic measurement gives the molecular ion peaks corresponding to the inclusion complex of 1:1 molar ratio of host and guest molecules. The mechanism of inclusion was drawn by 1 H-NMR and 2D ROESY spectroscopic analysis. Surface texture of the inclusion complexes was studied by SEM. Finally, the cytotoxic activities of the inclusion complexes were analyzed and found, Cell viability also balances for non-toxic behavior of the ICs. Moreover, all the studies reveal the formation of inclusion complexes of two ephedra free, alternatively emerging drugs (after their banned product having ephedra) SNP, PEH with α and β-CD which enriches the drug delivery system with their regulatory release without any chemical modification.

Cytochrome c oxidase (CCO) is a copper-dependent enzyme of mitochondrial respiratory chain. In pressure overload-induced cardiac hypertrophy, copper level and CCO activity are both depressed, along with disturbance in mitochondrial fusion and fission dynamics. Copper repletion leads to recovery of CCO activity and normalized mitochondrial dynamics. The present study was undertaken to define the link between CCO activity and mitochondrial dynamic changes. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine to induce cell hypertrophy. Hypertrophic cardiomyocytes were then treated with copper to reverse hypertrophy. In the hypertrophic cardiomyocytes, CCO activity was depressed and mitochondrial fusion was suppressed. Upon copper repletion, CCO activity was recovered and mitochondrial fusion was reestablished. Depression of CCO activity by siRNA targeting CCO assembly homolog 17 (COX17), a copper chaperone for CCO, led to fragmentation of mitochondria, which was not recoverable by copper supplementation. This study thus demonstrates that copper-dependent CCO is critical for mitochondrial fusion in the regression of cardiomyocyte hypertrophy.

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have recently demonstrated that the amino-terminal domain of G protein coupled receptor kinase (GRK) type 5, (GRK5-NT) inhibits NFκB activity in cardiac cells leading to a significant amelioration of LVH. Since GRK5-NT is known to bind calmodulin, this study aimed to evaluate the functional role of GRK5-NT in the regulation of calcium-calmodulin-dependent transcription factors. We found that the overexpression of GRK5-NT in cardiomyoblasts significantly reduced the activation and the nuclear translocation of NFAT and its cofactor GATA-4 in response to phenylephrine (PE). These results were confirmed in vivo in spontaneously hypertensive rats (SHR), in which intramyocardial adenovirus-mediated gene transfer of GRK5-NT reduced both wall thickness and ventricular mass by modulating NFAT and GATA-4 activity. To further verify in vitro the contribution of calmodulin in linking GRK5-NT to the NFAT/GATA-4 pathway, we examined the effects of a mutant of GRK5 (GRK5-NTPB), which is not able to bind calmodulin. When compared to GRK5-NT, GRK5-NTPB did not modify PE-induced NFAT and GATA-4 activation. In conclusion, this study identifies a double effect of GRK5-NT in the inhibition of LVH that is based on the regulation of multiple transcription factors through means of different mechanisms and proposes the amino-terminal sequence of GRK5 as a useful prototype for therapeutic purposes., Competing Interests: The Authors declare no conflicts of interest.

Cardiac hypertrophy is one of the major risk factors of cardiovascular morbidity and mortality. Autophagy is acknowledged to be an important mechanism regulating cardiac hypertrophy. Sestrin 1, a downstream target gene of p53, has been proven to regulate autophagy. However, the role of Sestrin 1 in cardiac hypertrophy remains unknown. Our study showed that Sestrin 1 mRNA and protein expression declined in pressure overload cardiac hypertrophy and phenylephrine (PE)-induced cardiac hypertrophy. Knockdown of Sestrin 1 by RNAi deteriorated PE-induced cardiac hypertrophy, whereas the overexpression of Sestrin 1 by adenovirus transfection blunted hypertrophy. We discovered that knockdown of Sestrin 1 resulted in impaired autophagy while overexpression of Sestrin 1 resulted in increased autophagy without affecting lysosomal function. In addition, the antihypertrophic effect of Sestrin 1 overexpression was eliminated by autophagy blockade. Importantly, Sestrin 1 targets at the AMPK/mTORC1/autophagy pathway to inhibit cardiac hypertrophy by interaction with AMPK which is responsible for autophagy regulation. Taken together, our data indicate that Sestrin 1 regulates AMPK/mTORC1/autophagy axis to attenuate cardiac hypertrophy., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Evidence suggests that store-operated Ca2+ entry (SOCE) is involved in the hypertrophy of cardiomyocytes. The signaling mechanisms of SOCE contributing to cardiac hypertrophy following phenylephrine (PE) stimulation are not fully understood. Ca 2+ /calmodulin-dependent protein kinase II δ (CaMKIIδ) plays an important role in regulating intracellular Ca 2+ hemostasis and function in the cardimyocytes. This study is aimed to determine the role of CaMKIIδ in regulating the PE-induced myocardial hypertrophy and the associated molecular signaling mechanisms. We used primary cultures of neonatal cardimyocytes isolated from the left ventricle of Sprague Dawley rats to investigate the effects of CaMKIIδ on myocardial hypertrophy and intracellular Ca 2+ mobilization. We found that the expression of CaMKIIδ was enhanced in PE-induced hypertrophic cardiomyocytes. CaMKIIδ siRNA, CaMKII inhibitor KN93, and SOCE blocker BTP2 attenuated the increase in the expression of CaMKIIδ and normalized the hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide, and size of cardiomyocytes induced by PE stimulation. The protein level of stromal interaction molecule 1 and Orai1, the essential components of the SOCE, is also enhanced in hypertrophic cardiomyocytes, which were normalized by CaMKIIδ siRNA and KN93 treatment. Hypertrophic cardiomyocytes showed an increase in the peak of Ca 2+ transient following store depletion, which was inhibited by SOCE blocker BTP2, CaMKIIδ siRNA, and KN93. The Ca 2+ currents through Ca 2+ release-activated Ca 2+ channels were increased in PE-treated cardiomyocytes and were attenuated by CaMKIIδ siRNA and KN93. These data indicate that PE-induced myocardial hypertrophy requires a complex signaling pathway that involves activation of both CaMKIIδ and SOCE. In conclusion, these studies reveal that up-regulation of CaMKIIδ may contribute to the PE-induced myocardial hypertrophy through the activation of SOCE expressed in the cardiomyocytes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Previous animal studies have shown that the administration of probiotic Lactobacillus rhamnosus can provide a protective effect against ischemia/reperfusion and necrotic injury to the intestine, liver, and heart, as well as a therapeutic effect to the outcome of ischemic injury to the heart, including cardiac hypertrophy and heart failure. We hypothesized that L. rhamnosus GR-1 major secreted protein 1 (MSP-1), also known as p75, plays a major role in this phenomenon. Experiments using neonatal rat ventricular cardiomyocytes showed that live and dead GR-1 bacteria, probiotic-conditioned media, and other probiotic species and strains inhibited the α1-adrenergic receptor agonist phenylephrine-induced hypertrophy as assessed by markers atrial natriuretic peptide and α-skeletal actin. However, using a mutant strain, we showed that this MSP-1 was not required for the inhibition. The ability of factors produced by lactobacilli to improve cardiac function warrants further study for the management of cardiac hypertrophy and heart failure., Competing Interests: The authors have declared that no competing interests exist.

Phenylephrine (PHE), a selective α1-adrenergic receptor agonist, is often used as a decongestant for mydriasis prior to cataract surgery, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of PHE to the cornea and its cellular and molecular mechanisms remain unknown. To provide references for secure medication and prospective therapeutic interventions of PHE, we investigated the cytotoxicity of PHE to corneal stroma and its possible mechanisms using an in vitro model of human corneal stromal (HCS) cells and an in vivo model of cat keratocytes. We found that PHE, above the concentration of 0.0781125% (1/128 of its clinical therapeutic dosage), had a dose- and time-dependent cytotoxicity to HCS cells by inducing morphological abnormality and viability decline, as well as S phase arrest. Moreover, PHE induced apoptosis of HCS cells by inducing plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation. Furthermore, PHE could induce activations of caspase-3 and -9, disruption of mitochondrial transmembrane potential, downregulation of anti-apoptotic Bcl-xL, upregulation of pro-apoptotic Bax, along with upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. The cytotoxic and pro-apoptotic effects of PHE were also proven by the induced apoptotic-like ultrastructural alterations of keratocytes in vivo. Taken together, our results suggest that PHE has a significant cytotoxicity to corneal stroma cells both in vitro and in vivo by inducing cell apoptosis, and the pro-apoptotic effect of PHE is achieved via a Bcl-2 family proteins-mediated mitochondrion-dependent pathway., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Objective: In Thailand, hypotension after spinal anesthesia for cesarean section is routinely treated by ephedrine. As incidence of fetal acidosis reportedly increases resulting from placental transfer of ephedrine, phenylephrine, an alpha-1 agonist with less lipid solubility, becomes an alternative. However, the potential development of serious bradycardia after phenylephrine is a concern. The objectives of this study were to investigate the incidence of serious bradycardia and identify risk factors associated with phenylephrine-induced serious bradycardia and other side effects of phenylephrine., Material and Method: This descriptive cross-sectional study was conducted between July 1, 2014 and March 15, 2015 on 509 parturients undergoing cesarean section under spinal anesthesia. Predelivery hypotension was treated by intravenous phenylephrine 100 mcg and pretherapeutic heart rate (pHR) was recorded. If serious bradycardia (HR < 60 bpm and hypotension or HR <45 bpm) developed, atropine 0.6 mg was administered intravenously. Data were analyzed using multivariable logistic regression and AuROC., Results: Incidence of serious bradycardia was 11% (95% CI: 8.0-14.0). A one bpm increment increase in pHR reduced this incidence by 4% (adjusted OR: 0.96; 95% CI: 0.94-0.98, p < 0.001; AuROC: 0.76). As compared to apHR greater than 80 bpm, apHR of 61 to 80 bpm and a pHR of 60 bpm or lower increased the risk of serious bradycardia by 3.55 times and 12.81 times, respectively. Other risk factors were height (adjusted OR: 0.94; 95% CI: 0.89-0.98, p = 0.015), baseline DBP (adjusted OR: 0.97; 95% CI: 0.94-0.99,p = 0.03), and anesthetic level at first minute (adjusted OR: 1.13; 95% CI: 1.02-1.23, p = 0.02). Benign and temporary abnormal ECG readings were noted., Conclusion: Phenylephrine for antihypotensive treatment in spinal anesthesia induces bradycardia. Findings indicate an association between slower HR at time phenylephrine is administered and serious bradycardia. Close ECG monitoring and prompt treatment are required.

Background/aims: Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA., Methods: The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE, 50 µmol/L, 48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 µg/ml, 48 h). The cell surface area was determined by immunofluorescent staining for α-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio. The miR-199a-5p expression level was quantified in PE-treated cardiomyocytes and heart samples from acute myocardial infarction (AMI) mouse model. MiR-199a-5p overexpression was used to determine its role in the anti-hypertrophic effect of QL on cardiomyocytes., Results: PE induced obvious enlargement of cell surface in cardiomyocytes, paralleling with increased ANP, BNP, and MYH7 mRNA levels and elevated protein/DNA ratio. All these changes were reversed by the treatment with QL. Meanwhile, miR-199a-5p was increased in AMI mouse heart tissues. Of note, the increase of miR-199a-5p in PE-treated cardiomyocytes was reversed by the treatment with QL. Moreover, overexpression of miR-199a-5p abolished the anti-hypertrophic effect of QL on cardiomyocytes., Conclusion: QL prevents PE-induced cardiac hypertrophy. MiR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

A hollow microneedle (HM) was prepared to deliver a phenylephrine (PE) solution into the anal sphincter muscle as a method for treating fecal incontinence. The goal of this study was the local targeted delivery of PE into the sphincter muscle through the perianal skin with minimal pain using hollow microneedles, resulting in the increase of resting anal sphincter pressure. PE was administered on the left and the right sides of the anus of a rat through the perianal skin using 1.5mm long HM. An in vivo imaging system study was conducted after injection of Rhodamine B, and a histological study was performed after injection of gentian violet. The resting anal sphincter pressure in response to various drug doses was measured by using an air-charged catheter. Anal pressure change produced by HM administration was compared with change produced by intravenous injection (IV), subcutaneous (SC) injection and intramuscular (IM) injection. The change in mean blood pressure produced by HM administration as a function of PE dose was compared with change produced by PBS injection. A pharmacokinetic study of the new HM administration method was performed. A model drug solution was localized in the muscle layer under the perianal skin at the injection site and then diffused out over time. HM administration of PE induced significant contraction of internal anal sphincter pressure over 12h after injection, and the maximum anal pressure was obtained between 5 and 6h. Compared to IV, SC and IM treatments, HM treatment produced greater anal pressure. There was no increase in blood pressure after HM administration of PE within the range of predetermined concentration. Administration of 800μg/kg of PE using HM produced 0.81±0.38h of tmax. Our study suggests that HM administration enables local delivery of a therapeutic dose of PE to the anal sphincter muscle layer with less pain. This new treatment has great potential as a clinical application because of the ease of the procedure, minimal pain, and dose-dependent response., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aims: Accumulating evidence suggest that sarcomere signalling complexes play a pivotal role in cardiomyocyte hypertrophy by communicating stress signals to the nucleus to induce gene expression. Ankyrin repeat domain 1 (ANKRD1) is a transcriptional regulatory protein that also associates with sarcomeric titin; however, the exact role of ANKRD1 in the heart remains to be elucidated. We therefore aimed to examine the role of ANKRD1 in cardiomyocyte hypertrophic signalling., Methods and Results: In neonatal rat ventricular myocytes, we found that ANKRD1 is part of a sarcomeric signalling complex that includes ERK1/2 and cardiac transcription factor GATA4. Treatment with hypertrophic agonist phenylephrine (PE) resulted in phosphorylation of ERK1/2 and GATA4 followed by nuclear translocation of the ANKRD1/ERK/GATA4 complex. Knockdown of Ankrd1 attenuated PE-induced phosphorylation of ERK1/2 and GATA4, inhibited nuclear translocation of the ANKRD1 complex, and prevented cardiomyocyte growth. Mice lacking Ankrd1 are viable with normal cardiac function. Chronic PE infusion in wild-type mice induced significant cardiac hypertrophy with reactivation of the cardiac fetal gene program which was completely abrogated in Ankrd1 null mice. In contrast, ANKRD1 does not play a role in haemodynamic overload as Ankrd1 null mice subjected to transverse aortic constriction developed cardiac hypertrophy comparable to wild-type mice., Conclusion: Our study reveals a novel role for ANKRD1 as a selective regulator of PE-induced signalling whereby ANKRD1 recruits and localizes GATA4 and ERK1/2 in a sarcomeric macro-molecular complex to enhance GATA4 phosphorylation with subsequent nuclear translocation of the ANKRD1 complex to induce hypertrophic gene expression., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Background: Transient preceding brief ischemia provides potent cardioprotection against subsequent long ischemia, termed ischemic preconditioning. Here, we hypothesized that transient short-term hypertrophic stimulation would induce the expression of hypertrophy regression genes and render the heart resistant to subsequent hypertrophic stress, and slow the progression to heart failure, as well., Methods and Results: Cardiomyocyte hypertrophy was induced in mice by either transverse aortic constriction or an infusion of phenylephrine, and in neonatal rat ventricular cardiomyocytes by norepinephrine exposures. In the preconditioning groups, hypertrophic stimulation was provided for 1 to 7 days and then withdrawn for several days by either aortic debanding or discontinuing phenylephrine or norepinephrine treatment, followed by subsequent reexposure to the hypertrophic stimulus for the same period as in the control group. One or 6 weeks after transverse aortic constriction, the heart weight/body weight ratio was lower in the preconditioning group than in the control group, whereas the lung weight/body weight ratio was significantly decreased 6 weeks after transverse aortic constriction. Similar results were obtained in mice receiving phenylephrine infusion and neonatal rat ventricular cardiomyocytes stimulated with norepinephrine. Both mRNA and protein expression of S100A8 and S100A9 showed significant upregulation after the removal of hypertrophic stimulation and persisted for 6 weeks in response to reimposition of transverse aortic constriction. The treatment with recombinant S100A8/A9 inhibited norepinephrine-induced myocyte hypertrophy and reduced the expression of calcineurin and NFATc3, but the silencing of S100A8/A9 prevented such changes., Conclusions: Preconditioning with prohypertrophic factors exerts an antihypertrophic effect and slows the progression of heart failure, indicating the existence of the phenomenon for hypertrophic preconditioning., (© 2015 The Authors.)

Purpose: The aim of this study was to investigate the effect of intracameral injection of bisulfite-containing phenylephrine on rabbit corneal endothelium cells in rabbits., Methods: Fifteen eyes of 15 New Zealand white rabbits were divided into 3 groups. Five eyes from each group were injected with 0.5% (0.5 mL, 0.05% bisulfite, group 1), 1.0% bisulfite-containing phenylephrine (0.5 mL, 0.1% bisulfite, group 2), or an equal volume of balanced salt solution as a control (group 3). Specular microscopy, corneal pachymetry, and a slit-lamp examination were used to evaluate corneal endothelial cell loss and other complications before injection, and at 1 hour and 1 week after injection. For morphological studies, corneal buttons were excised at 1 week and stained with alizarin red and trypan blue. Optical microscopy was used to examine 1 cornea from each of the 3 groups., Results: Within each group, there were no changes in specular microscopy and corneal pachymetry findings over the 1-week duration of the study. Although all groups showed the presence of several giant cells in the endothelium, the hexagonal shape of the corneal endothelium was morphologically preserved, and no abnormal endothelial cells were observed., Conclusions: Intracameral injection of bisulfite-containing phenylephrine (up to 1.0%) does not affect endothelial cell viability or morphology of the rabbit cornea.

Recent evidence suggests that polydatin (PD), a resveratrol glucoside, may have beneficial actions on the cardiac hypertrophy. Therefore, the current study focused on the underlying mechanism of the PD anti-hypertrophic effect in cultured cardiomyocytes and in progression from cardiac hypertrophy to heart failure in vivo. Experiments were performed on cultured neonatal rat, ventricular myocytes as well as adult mice subjected to transverse aortic constriction (TAC). Treatment of cardiomyocytes with phenylephrine for three days produced a marked hypertrophic effect as evidenced by significantly increased cell surface area and atrial natriuretic peptide (ANP) protein expression. These effects were attenuated by PD in a concentration-dependent manner with a complete inhibition of hypertrophy at the concentration of 50 µM. Phenylephrine increased ROCK activity, as well as intracellular reactive oxygen species production and lipid peroxidation. The oxidizing agent DTDP similarly increased Rho kinase (ROCK) activity and induced hypertrophic remodeling. PD treatment inhibited phenylephrine-induced oxidative stress and consequently suppressed ROCK activation in cardiomyocytes. Hypertrophic remodeling and heart failure were demonstrated in mice subjected to 13 weeks of TAC. Upregulation of ROCK signaling pathway was also evident in TAC mice. PD treatment significantly attenuated the increased ROCK activity, associated with a markedly reduced hypertrophic response and improved cardiac function. Our results demonstrated a robust anti-hypertrophic remodeling effect of polydatin, which is mediated by inhibition of reactive oxygen species dependent ROCK activation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cardiac hypertrophy is a major risk factor of cardiovascular morbidity and mortality. Autophagy is established to be involved in regulating cardiac hypertrophy. REDD1, a stress-responsive protein, is proved to contribute in autophagy induction. However, the role of REDD1 in cardiac hypertrophy remains unknown. Our study demonstrated that REDD1 knockdown by RNAi exacerbated phenylephrine (PE)-induced cardiac hypertrophy, manifested by increased hypertrophic markers such as ANP and cell surface area. In addition, we discovered that ERK1/2 signaling pathway was involved in the effect of REDD1 on hypertrophy. Moreover, our study showed that REDD1 knockdown impaired autophagy in hypertrophied cardiomyocytes. mTOR, a signaling molecule governing autophagy induction, was activated by the knockdown of REDD1 under PE stress. Importantly, the pro-hypertrophic effect of REDD1 knockdown was significantly reversed by the autophagy enhancer rapamycin. Taken together, we firstly prove that REDD1 is essential for inhibiting cardiac hypertrophy by enhancing autophagy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cluster of differentiation 73 (CD73) is an ecto-5' nucleotidase which catalyzes the conversion of AMP to adenosine. One of the many functions of adenosine is to suppress the activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme important in regulating intracellular calcification. Since myocardial calcification is associated with various cardiac disease states, we studied the individual roles and crosstalk between CD73 and TNAP in regulating myocyte responses to the α1 adrenoceptor agonist phenylephrine in terms of calcification and hypertrophy. Cultured neonatal rat cardiomyocytes were treated with 10 µM phenylephrine for 24 h in the absence or presence of the stable adenosine analog 2-chloro-adenosine, the TNAP inhibitor tetramisole or the CD73 inhibitor α,β-methylene ADP. Phenylephrine produced marked hypertrophy as evidenced by significant increases in myocyte surface area and ANP gene expression, as well as calcification determined by Alizarin Red S staining. These responses were associated with reduced CD73 gene and protein expression and CD73 activity. Conversely, TNAP expression and activity were significantly increased although both were suppressed by 2-chloro-adenosine. CD73 inhibition alone significantly reduced myocyte-derived adenosine levels by >50 %, and directly induced hypertrophy and calcification in the absence of phenylephrine. These responses and those to phenylephrine were abrogated by TNAP inhibition. We conclude that TNAP contributes to the hypertrophic effect of phenylephrine, as well as its ability to produce cardiomyocyte calcification. These responses are minimized by CD73-dependent endogenously produced adenosine.

The aim of the study was to examine the reorganization of left ventricular myocardium (LVM) in ovariectomized rats with experimental heart failure (EHF) as affected by melatonin administration. Rat LVM was studied using the methods of light microscopy, immunocytochemistry and morphometry. At day 14 of EHF, marked heteromorphism of contractile cardiomyocytes (CMC), signs of dystrophy, overcontraction and damage of muscle fibers, karyopyknosis, paranuclear edema, local sarcoplasm destruction were detected together with CMC showing signs of apoptosis. At day 28 after EHF modeling, a further increase in the number of CMC with significant changes in the morphological and tinctorial properties was found as well as the cells with signs of apoptosis. In rats given melatonin for 14 days, there was a regression of pathological changes, with the decrease in the hypertrophy of CMC and their nuclei, reduction of the number of CMC with the signs of apoptosis. Cardioprotective effect of melatonin on LVM of ovariectomized rats with EHF is discussed.

Arterial hypertension is a major risk factor for ischemic stroke. However, the management of preexisting hypertension is still controversial in the treatment of acute stroke in hypertensive patients. The present study evaluates the influence of preserving hypertension during focal cerebral ischemia on stroke outcome in a rat model of chronic hypertension, the spontaneously hypertensive rats (SHR). Focal cerebral ischemia was induced by transient (1h) occlusion of the middle cerebral artery, during which mean arterial blood pressure was maintained at normotension (110-120mm Hg, group 1, n=6) or hypertension (160-170mm Hg, group 2, n=6) using phenylephrine. T2-, diffusion- and perfusion-weighted MRI were performed serially at five different time points: before and during ischemia, and at 1, 4 and 7 days after ischemia. Lesion volume and brain edema were estimated from apparent diffusion coefficient maps and T2-weighted images. Regional cerebral blood flow (rCBF) was measured within and outside the perfusion deficient lesion and in the corresponding regions of the contralesional hemisphere. Neurological deficits were evaluated after reperfusion. Infarct volume, edema, and neurological deficits were significantly reduced in group 2 vs. group 1. In addition, higher values and rapid restoration of rCBF were observed in group 2, while rCBF in both hemispheres was significantly decreased in group 1. Maintaining preexisting hypertension alleviates ischemic brain injury in SHR by increasing collateral circulation to the ischemic region and allowing rapid restoration of rCBF. The data suggest that maintaining preexisting hypertension is a valuable approach to managing hypertensive patients suffering from acute ischemic stroke., (Published by Elsevier B.V.)

Objective: Prolonged action potential and decreased outward K(+) currents are consistent findings in hypertrophic myocardium. The relation between action potential prolongation and myocyte hypertrophy has remained unclear. The present study investigated the temporal relation between action potential prolongation and myocyte hypertrophy, and the effect of enhancing repolarization on myocyte hypertrophy induced by phenylephrine., Methods: Neonatal rabbit ventricular myocytes were cultured and treated with 10 μmol/l phenylephrine. At 6 and 48 h after phenylephrine stimulation, myocyte hypertrophic parameters (including myocyte volume, total protein content, and membrane capacitance), action potential duration (APD), and calcineurin activity were measured; meanwhile, the effect of human-ether-a-go-go-related gene (HERG; encoding the αsubunit of rapidly activating delayed rectifier potassium channel) transfection on the above parameters at 48 h of phenylephrine stimulation was also measured., Results: At 6 h after phenylephrine treatment, APD at 90% repolarization of neonatal rabbit ventricular myocytes was prolonged by 14.3% (P<.05), but myocyte hypertrophy was not found. At 48 h after phenylephrine stimulation, APD at 90% repolarization of neonatal rabbit ventricular myocytes was furthermore prolonged by 18.8% (P<.05); at the same time, myocyte volume, total protein content, membrane capacitance, and calcineurin activity were increased by 40.0%, 41.8%, 36.4%, and 124.1%, respectively (P<.01). Neonatal rabbit ventricular myocytes transfected by pcDNA3-HERG overexpressed I(HERG,tail) current, which was about fourfold higher than I(Kr) (rapidly activating delayed rectifier K(+) current) of neonatal rabbit ventricular myocytes without transfection of HERG. HERG overexpression could accelerate repolarization and shorten APD at 90% repolarization prolonged by phenylephrine and partially inhibit myocyte hypertrophy and calcineurin activation., Conclusions: During the myocyte hypertrophy induced by phenylephrine, prolongation of APD at 90% repolarization is not secondary to but precedes myocyte hypertrophy. HERG overexpression could enhance the repolarization and inhibit the calcineurin activation and myocyte hypertrophy induced by phenylephrine., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Traumatic brain injury (TBI) contributes to morbidity in children, and boys are disproportionately represented. Endothelin-1 (ET-1) contributes to impaired autoregulation via oxygen (O₂⁻) after TBI in piglets, but its relative role in males compared with females has not been previously investigated. Increased cerebral perfusion pressure (CPP) via phenylephrine (Phe) sex dependently improves impairment of autoregulation after TBI through modulation of extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK) upregulation, aggravated in males, but blocked in females. Activation of adenosine-5'-triphosphate (ATP) and Ca sensitive K channels produce vasodilation, contributing to autoregulation. We hypothesized that ET-1 upregulation is greater in males after TBI and that disturbed autoregulation will be prevented by Phe in a sex-dependent manner through modulation of ET-1, O₂⁻, and ERK. Results show that ET-1 release was greater in males after fluid percussion injury (FPI), blunted by Phe in females, but aggravated in males. K channel vasodilation was impaired more in males than in females after TBI. Phe prevented reductions in K channel vasodilation in females, but further reduced dilation in males after TBI. Co-administration of BQ-123, U0126, or PEG-SOD (ET-1, ERK antagonist, and O₂⁻ scavenger) with Phe restored dilation to K agonists and hypotension in males after TBI. ERK upregulation was blocked by BQ-123 and PEG-SOD. These data indicate that TBI upregulates ET-1 more in males than in females. Elevation of CPP with Phe sex dependently prevents impairment of cerebral autoregulation after TBI through modulation of ET-1, O₂⁻, and ERK mediated impairment of K channel vasodilation. These observations advocate for the consideration of development of sex-based therapies for the treatment of hemodynamic sequelae of pediatric TBI.

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 μg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 μg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 μM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 μM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and β) effects.

Angiopoietins were thought to be endothelial cell-specific via the tie2 receptor. We showed that angiopoietin-1 (ang1) also interacts with integrins on cardiac myocytes (CMs) to increase survival. Because ang1 monomers bind and activate integrins (not tie2), we determined their function in vivo. We examined monomer and multimer expressions during physiological and pathological cardiac remodeling and overexpressed ang1 monomers in phenylephrine-induced cardiac hypertrophy. Cardiac ang1 levels (mRNA, protein) increased during postnatal development and decreased with phenylephrine-induced cardiac hypertrophy, whereas tie2 phosphorylations were unchanged. We found that most or all of the changes during cardiac remodeling were in monomers, offering an explanation for unchanged tie2 activity. Heart tissue contains abundant ang1 monomers and few multimers (Western blotting). We generated plasmids that produce ang1 monomers (ang1-256), injected them into mice, and confirmed cardiac expression (immunohistochemistry, RT-PCR). Ang1 monomers localize to CMs, smooth muscle cells, and endothelial cells. In phenylephrine-induced cardiac hypertrophy, ang1-256 reduced left ventricle (LV)/tibia ratios, fetal gene expressions (atrial and brain natriuretic peptides, skeletal actin, beta-myosin heavy chain), and fibrosis (collagen III), and increased LV prosurvival signaling (akt, MAPK(p42/44)), and AMPK(T172). However, tie2 phosphorylations were unchanged. Ang1-256 increased integrin-linked kinase, a key regulator of integrin signaling and cardiac health. Collectively, these results suggest a role for ang1 monomers in cardiac remodeling.

Background and Purpose: Bradycardia is a risk factor for the development of torsade de pointes (TdP). The aim of this work was to compare the importance of changes in heart rate and arterial blood pressure in the development of drug-induced TdP and to investigate the role of vagal influences., Experimental Approach: Experiments were performed in open-chest, pentobarbital-anaesthetized, male rabbits which were given clofilium (20, 60 and 200 nmol kg(-1) min(-1)) with rising doses of either phenylephrine (75, 150, 225 and 300 nmol kg(-1) min(-1)), angiotensin II (0.25, 0.5, 0.75 and 1 nmol kg(-1) min(-1)) or saline. A fourth group received phenylephrine and cloflium after bilateral vagotomy. ECGs, haemodynamics and epicardial monophasic action potentials were recorded., Key Results: TdP occurred in 57% of rabbits given phenylephrine and clofilium. Replacement of phenylephrine with saline or angiotensin II reduced the incidence of TdP to 0 and 17%, respectively. Vagotomy prevented TdP in rabbits given phenylephrine and clofilium. Increases in blood pressure induced by phenylephrine and angiotensin II were similar. Bradycardia only occurred with phenylephrine and was reduced but not abolished by vagotomy. Neither short-term variability of repolarization nor action potential triangulation could predict TdP., Conclusions and Implications: These results indicate that reflex activation of vagal nerve activity is essential for the induction of drug-induced TdP in alpha1-adrenoceptor-stimulated anaesthetized rabbits. This implies that alterations in vagal activity may also precipitate episodes of drug-induced TdP in man and that this should be considered in selecting models used in drug development.

A zinc finger protein, GATA4, is one of the hypertrophy-responsive transcription factors and increases its DNA binding and transcriptional activities in response to hypertrophic stimuli in cardiac myocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. However, p300-targeted acetylated sites of GATA4 during myocardial cell hypertrophy have not been identified. By mutational analysis, we showed that 4 lysine residues located between amino acids 311 and 322 are required for synergistic activation of atrial natriuretic factor and endothelin-1 promoters by GATA4 and p300. A tetra-mutant GATA4, in which these 4 lysine residues were simultaneously mutated, retained the ability to localize in nuclei and to interact with cofactors including FOG-2, GATA6, and p300 but lacked p300-induced acetylation, DNA binding, and transcriptional activities. Furthermore, coexpression of the tetra-mutant GATA4 with wild-type GATA4 impaired the p300-induced acetylation, DNA binding, and transcriptional activities of the wild type. When we expressed the tetra-mutant GATA4 in neonatal rat cardiac myocytes using a lentivirus vector, this mutant suppressed phenylephrine-induced increases in cell size, protein synthesis, and expression of hypertrophy-responsive genes. However, its expression did not affect the basal state. Thus, we have identified the most critical lysine residues acting as p300-mediated acetylation targets in GATA4 during hypertrophic responses in cardiac myocytes. The results also demonstrate that GATA4 with simultaneous mutation of these sites specifically suppresses hypertrophic responses as a dominant-negative form, providing further evidence for the acetylation of GATA4 as one of critical nuclear events in myocardial cell hypertrophy.

Cardiac hypertrophy is regulated by a complex interplay of pro- and anti-hypertrophic factors. Here, we report a novel anti-hypertrophic pathway composed of catalase, protein kinase CK2 (CK2), and apoptosis repressor with caspase recruitment domain (ARC). Our results showed that ARC phosphorylation levels, CK2 activity, and catalase expression levels were decreased in the hearts of the angiotensinogen transgenic mice and in cardiomyocytes treated with the hypertrophic stimuli, including phenylephrine, tumor necrosis factor-alpha, and angiotensin II. To understand the role of ARC in hypertrophy, we observed that enforced expression of ARC could inhibit hypertrophy. Knockdown of endogenous ARC or inhibition of its phosphorylation could sensitize cardiomyocytes to undergoing hypertrophy. The phosphorylatable, but not the nonphosphorylatable, ARC could inhibit hypertrophy. Thus, ARC is able to inhibit hypertrophy in a phosphorylation-dependent manner. In exploring the molecular mechanism by which CK2 activity is reduced, we found that CK2 was carbonylated in angiotensinogen transgenic mice and in cardiomyocytes treated with the hypertrophic stimuli. The decrease in catalase expression led to an elevated level of reactive oxygen species. The latter oxidatively modified CK2, resulting in its carbonylation. CK2 lost its catalytic activity upon carbonylation. ARC is phosphorylated by CK2, and ARC phosphorylation levels were reduced as a consequence of the decrease of CK2 activity. To understand the molecular mechanism by which ARC inhibits hypertrophy, we observed that ARC could inhibit the activation of mitochondrial permeability transition. These results suggest that catalase, CK2, and ARC constitute an anti-hypertrophic pathway in the heart.

Rhinitis of allergic and viral origin is often self-treated by patients with locally applied vasoconstrictor decongestant drugs. In turn, prolonged use of these agents produce an inflammatory condition termed rhinitis medicamentosa. Cationic drugs are sequestered into cells via various mechanisms, including mitochondrial concentration and V-ATPase-driven trapping in vacuoles that swell by an osmotic mechanism. We hypothesized that receptor-independent endomembrane sequestration of topically applied concentrated alpha-adrenoceptor agonists (decongestants, mydriatics) could contribute to their toxicity and prolonged duration of action. The morphological and functional effects of phenylephrine and xylometazoline on rabbit aortic smooth muscle cells were examined and their possible sequestration evaluated using the contractility of rabbit aorta rings. Synthetic agonists produced V-ATPase-dependent cell vacuolization (prevented by bafilomycin A1; xylometazoline 250 microM, phenylephrine 2.5 mM). V-ATPase-mediated cytotoxicity was slow (24 h; phenylephrine only, 5-10 mM); a rapid xylometazoline-induced cytotoxicity (> or =500 microM, 4 h) correlated to mitochondrial functional alterations. Xylometazoline had slower contraction and relaxation kinetics than the other alpha-adrenoceptor agonists in the aorta; bafilomycin pre-treatment influenced its kinetics (accelerated contraction and relaxation) and concentration-effect relationship (potentiation). V-ATPase-driven sequestration contributed to a component of the tissue reservoir of both phenylephrine and xylometazoline as assessed by aortic rings contracted with the concentrated agonists and subsequently washed. Phenylephrine and xylometazoline caused the V-ATPase-dependent cytopathology at a fraction of the usual topical concentrations; this form of sequestration influenced the toxicity and pharmacology of individual agents.

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca(2+) transport, and cytoplasmic Ca(2+) signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [(14)C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca(2+)-ATPase protein levels and Ca(2+) transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca(2+) signaling kinetics, measured with a Ca(2+)-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca(2+) signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca(2+) signaling is partially compensated by alternate Ca(2+) transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

The present study was designed to test the hypothesis that arterial baroreflex dysfunction promotes the development of atherosclerosis. Experiment 1: the baroreflex sensitivity (BRS) was measured in 30 Sprague-Dawley (SD) rats in conscious state with a computerized blood pressure monitoring system. Four weeks later, the rats were administered with Vitamin D3, and fed with the high-cholesterol diet for 8 weeks to induce atherosclerosis. The hearts and aortae were removed for pathological examination. A negative correlation was found between BRS and the scores of coronary (r=-0.464, P<0.01) or aortic atherosclerosis (r=-0.524, P<0.01) in SD rats. Experiment 2: sinoaortic denervation (SAD) was performed in SD rats. Then atherosclerosis was also induced. The atherosclerosis scores in SAD rats were significantly higher than those in sham-operated rats (aortic score: 1.50+/-0.41 versus 1.10+/-0.39, P<0.05; coronary score: 1.70+/-0.35 versus 1.25+/-0.54, P<0.05). Using immunohistochemistry and Western blotting methods, it was found that the expressions of C-reactive protein, intercellular adhesion molecule-1 and vascular-cell adhesion molecule-1 in coronary artery and aorta were increased in SAD rats compared with sham-operated rats. These results indicate that arterial baroreflex dysfunction promotes the development of atherosclerosis in rats, and that inflammation may be involved in this process.

Conventional therapy to treat hypertension often involves arterial vasodilation. Decrease of blood pressure by vasodilators is normally associated with adverse effects because of their low vascular selectivity. This is of interest to develop new molecules with potential for clinical use and fewer side effects. Recently, a new bioactive compound of the N-acylhydrazone class, LASSBio-294, was shown to produce a cardioinotropic effect and vasodilation. In this report, new derivatives of LASSBio-294 were designed and tested on the contractile response of vascular smooth muscle from Wistar rats. Phenylephrine-induced contracture in the aorta was inhibited by the derivatives LASSBio-785 and LASSBio-788. The concentrations necessary to cause 50% reduction of the maximal vascular response (IC50) were 10.2 +/- 0.5 and 67.9 +/- 6.5 microM. Vasodilation induced by both derivatives is likely to be mediated by a direct effect on smooth muscle because it was not dependent on the integrity of vascular endothelium. LASSBio-785 was seven times more potent than the reference compound LASSBio-294 (IC50 = 74 microM) in producing an endothelium-independent vasodilator effect.

Background: Transgenic mouse models expressing a missense mutation (R92Q) or a splice donor site mutation (trunc) in the cardiac troponin T (cTnT) model familial hypertrophic cardiomyopathy (FHC) in humans. Although males from these strains share the unusual property of having significantly smaller ventricles and cardiac myocytes, they differ with regard to systolic function, fibrosis, and gene expression. Little is known about how these phenotypes affect the responses to additional pathological stimuli., Methods and Results: We tested the ability of hearts of both sexes of wild-type and mutant mice to respond to defined pathological, pharmacological, hypertrophic stimuli in vivo. Hearts of mutant cTnT models of both sexes were able to undergo hypertrophy in response to at least one stimulus, but the extent differed between the 2 mutants and was sex specific. Interestingly, the trunc-mutant mouse heart was resistant to the development of fibrosis in response to pharmacological stimuli. Stimulation with 2 adrenergic agonists led to sudden cardiac death of all male but not female mutant animals, which suggests altered adrenergic responsiveness in these 2 models of FHC., Conclusions: Hypertrophic signaling is differentially affected by distinct mutations in cTnT and is sex modified. Hearts can respond with either an augmented hypertrophic and fibrotic response or a diminished hypertrophy and resistance to fibrosis. Sudden cardiac death is related to adrenergic stress and is independent of the development of fibrosis but occurred only in male mice. These results suggest that patients with certain TnT mutations may respond to certain pathological situations with a worsened phenotype.

We have examined the effect of an intradermal injection of phenylephrine (1mg/0.1 ml), an alpha-1-adrenoceptor agonist in normal subjects, and patients with sympathetically-independent (SIP) and sympathetically-maintained pain (SMP). Normal subjects and SIP patients experienced only brief stinging pain, while subsets of both sympathectomized and non-sympathectomized SMP patients (6/9 and 4/8, respectively) experienced an additional abnormal pain response accompanied by mechano-allodynia around the injection site. Both the normal and abnormal pain response after intradermal phenylephrine are similar to those observed with intradermal norepinephrine. In contrast to previous reports in the literature, we found that three sympathectomized SMP patients (who, however, had failed to experience pain relief after surgical sympathectomy despite very good relief after sympathetic blocks) also experienced abnormal pain and mechano-allodynia when phenylephrine was injected to a limb contralateral to the symptomatic sympathectomized extremity. Abnormal pain response evoked by norepinephrine or phenylephrine injection in the ipsilateral symptomatic limb of SMP patients may be due to injury-evoked nociceptor responsiveness to catecholamines. However, such a response in contralateral asymptomatic limbs suggests an additional factor that more likely than not is of central origin and may or may not be related to sympathectomy and its success or failure to treat pain.

Background: Atypical prostate hyperplasia, resembling PIN lesions in men, together with chronic inflammatory exudates, have been reported after chronic administration of phenylephrine (PE). The present study aims to characterize the evolution of the expression of typical leukocyte markers immunohistochemically as correlated to the promotion of PIN lesions., Methods: Adolescent rats received injections of PE (subcutaneously) (10 mg/kg BW/per day) and were euthanized 3, 8 hr, 1, 3, 7, 14 days thereafter. The dissected prostates were fixed in formalin, and paraffin embedded sections were cut and immunoreacted., Results: PE exerted a time related bi-phasic effect on the rat prostate. A first inflammatory reaction phase took place at 3-8 hr post injection characterized by vascular dilatation, congestion, edema, and massive leukocytic infiltrate, mainly of ED1+ cells. At 24 hr, the amount of ED1+ cells decreased approaching the equivalent values of ED2+, CD8+ cells, and mastocytes. Their values remained relatively high for the rest of the experimental period. A second phase of proliferative changes, consisting of healing fibrosis as well as dysplastic epithelial lesions, similar to human PIN lesions, appeared on the 7th day., Conclusions: Neuro-immune interactions promote prostatic fibrosis and dysplastic changes; these being preceded by an acute and transient inflammatory reaction., (Copyright 2003 Wiley-Liss, Inc.)

Ethylene dibromide (EDB) has been used as a model compound for eliciting hepato- and nephrotoxicity. Conjugation with glutathione (GSH) has been shown to play a role in the bioactivation of EDB. The aim of this study was to determine whether activation of alpha(1)-adrenergic receptors, which causes a decrease in cellular GSH levels, could modulate the nephrotoxicity of EDB. For this purpose, male ICR mice were treated with EDB and/or the alpha-adrenergic agonist, phenylephrine (Pe), or the alpha-adrenergic antagonist, phentolamine (Phe). Animals treated with EDB (40 mg/kg, i.p.) had a 9.3-fold increase in urinary gamma-glutamyltranspeptidase (GGTP: EC 2.3.2.2) activity and a 38% decrease in renal non-protein bound sulfhydryl (NPSH) levels; however, animals co-treated with EDB and Pe (50 mg/kg, i.p.) exhibited a 27.8-fold increase in urinary GGTP activity and a 60% decrease in NPSH levels. The enhanced presence of urinary GGTP and decrease in cellular levels of NPSH was nearly blocked by treating animals concomitantly with EDB and Phe (10 mg/kg, i.p.) or EDB, Pe, and Phe. Histopathological examination revealed the enhanced degree of tissue damage and necrosis following treatment with EDB and Pe, and the protective effect of Phe at ameliorating EDB toxicity. These results indicate that factors that can influence alpha-adrenergic receptors may be critical in assessing dose-response data used in the risk assessment process.

Cardiac hypertrophy is a major predictor of future morbidity and mortality. Recent investigation has centered around identifying the molecular signaling pathways that regulate cardiac myocyte reactivity with the goal of modulating pathologic hypertrophic programs. One potential regulator of cardiomyocyte hypertrophy is the calcium-sensitive phosphatase calcineurin. We show here that calcineurin enzymatic activity, mRNA, and protein levels are increased in cultured neonatal rat cardiomyocytes by hypertrophic agonists such as angiotensin II, phenylephrine, and 1% fetal bovine serum. This induction of calcineurin activity was associated with an increase in calcineurin Abeta (CnAbeta) mRNA and protein, but not in CnAalpha or CnAgamma. Agonist-dependent increases in calcineurin enzymatic activity were specifically inhibited with an adenovirus expressing a noncompetitive peptide inhibitor of calcineurin known as cain [Lai, M. M., Burnett, P. E., Wolosker, H., Blackshaw, S. & Snyder, S. H. (1998) J. Biol. Chem. 273, 18325-18331]. Targeted inhibition of calcineurin with cain or an adenovirus expressing only the calcineurin inhibitory domain of AKAP79 attenuated cardiomyocyte hypertrophy and atrial natriuretic factor expression in response to angiotensin II, phenylephrine, and 1% fetal bovine serum. These data demonstrate that calcineurin is an important regulator of cardiomyocyte hypertrophy in response to certain agonists and suggest that cyclosporin A and FK506 function to attenuate cardiac hypertrophy by specifically inhibiting calcineurin.

Neuropeptide FF (NPFF), a morphine modulatory peptide, is localized within discrete autonomic regions including the brainstem nucleus tractus solitarius (NTS) and the parabrachial nucleus (PBN). We investigated the activation of NPFF neurons in the NTS of rats induced by cardiovascular challenge and centrally generated opiate withdrawal. For hypotensive stimulation, we used systemic infusions of sodium nitroprusside (NP) or hemorrhage (HEM), and hypertension was achieved by intravenous phenylephrine (PHENYL) or angiotensin II (AII). In rats that received continuous intracerebroventricular injections of morphine, intraperitoneal injections of naloxone precipitated behavioural signs of opioid withdrawal. Activated NTS neurons were identified by using a combined immunohistochemistry for Fos and NPFF, and neurons projecting to the PBN were determined with a retrograde tracer. HEM, administration of vasoactive drugs, and opiate withdrawal produced a very robust activation of NTS neurons. In NP and HEM groups, 25.6+/-3.2% and 7.6+/-1.3% of NPFF neurons were activated, respectively. Lesser numbers of NPFF neurons were activated in the PHENYL (4.6+/-1.6%) and AII (2.4+/-0.8%) groups. However, following opiate withdrawal, virtually no Fos expression was observed in NPFF neurons. NPFF neurons activated during NP infusion constituted the largest number of cells projecting to the PBN. This study shows that NPFF neurons in NTS that project to the PBN respond selectively to NP as opposed to other cardiovascular challenges or opiate withdrawal. These data support an emerging and important role for NPFF in the context of central cardiovascular regulation.

The pressor response induced by phenylephrine in the rat isolated mesenteric arterial bed was significantly increased following treatment with nocodazole, a drug that disassembles microtubules (10 microM, 90 min). This increase was even greater in the presence of nitric oxide (NO) synthase inhibition, and completely reversed by paclitaxel (20 microM), a stabilizer of microtubules. These results demonstrate that disassembly of microtubules enhances vasoconstriction to receptor activation, suggesting that the microtubules modulate the transduction of intracellular signals in endothelium and vascular smooth muscle cells.