Oestrogen inhibits BMP4‐induced BMP4 expression in cardiomyocytes: a potential mechanism of oestrogen‐mediated protection against cardiac hypertrophy

Background and Purpose

Oestrogen inhibits cardiac hypertrophy and bone morphogenetic protein‐4 (BMP4) induces cardiac hypertrophy. Here we have studied the inhibition by oestrogen of BMP4 expression in cardiomyocytes.

Experimental Approach

Cultures of neonatal rat cardiomyocytes were used in in vitro experiments. Bilatαl ovariectomy (OVX) was carried out in female Kunming mice and cardiac hypertrophy was induced by transverse aortic constriction (TAC).

Key Results

Oestrogen inhibited BMP4‐induced cardiomyocyte hypertrophy and BMP4 expression in vitro. The inhibition of BMP4‐induced BMP4 protein expression by oestrogen was prevented by the inhibitor of oestrogen receptor‐β, PHTPP, but not by the inhibitor of oestrogen receptor‐α MPP. BMP4 induced smad1/5/8 activation, which was not affected by oestrogen in cardiomyocytes. BMP4 induced JNK but not ERK1/2 and p38 activation, and activated JNK was inhibited by oestrogen. Treatment with the p38 inhibitor SB203580 or the JNK inhibitor SP600125 inhibited BMP4‐induced BMP4 expression in cardiomyocytes, but the ERK1/2 inhibitor U0126 increased BMP4‐induced BMP4 expression, indicating that JNK, ERK1/2 and p38 MAPKs were all involved, although only JNK activation contributed to the inhibition of BMP4‐induced BMP4 expression by oestrogen. TAC induced significant heart hypertrophy in OVX mice in vivo and oestrogen replacement inhibited TAC‐induced heart hypertrophy in OVX mice. In parallel with the data of heart hypertrophy, oestrogen replacement significantly reduced the increased BMP4 protein expression in TAC‐treated OVX mice.

Conclusions and Implications

Oestrogen treatment inhibited BMP4‐induced BMP4 expression in cardiomyocytes through stimulating oestrogen receptor‐β and inhibiting JNK activation. Our results provide a novel mechanism underlying oestrogen‐mediated protection against cardiac hypertrophy.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

BMP4

bone morphogenetic protein‐4

ER‐α

oestrogen receptor‐α

ER‐β

oestrogen receptor‐β

MPP

1,3‐bis(4‐hydroxyphenyl)‐4‐methyl‐5‐[4‐(2‐piperidinylethoxy)phenol]‐1H‐pyrazole dihydrochloride

OVX

ovariectomy

PHTPP

4‐(2‐phenyl‐5,7‐bis(trifluoromethyl)pyrazolo[1,5‐a]pyrimidin‐3‐yl)phenol

TAC

transverse aortic constriction

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Activation of M 3 cholinoceptors attenuates vascular injury after ischaemia/reperfusion by inhibiting the Ca 2+/calmodulin‐dependent protein kinase II pathway

Background and Purpose

The activation of M ₃ cholinoceptors (M ₃ receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)‐induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) regulation.

Experimental Approach

Rats were given choline (10 mg·kg⁻¹, i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M ₃ receptor antagonist, 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), was injected (0.12 μg·kg⁻¹, i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca²⁺‐cycling proteins were also assessed.

Key Results

Choline treatment attenuated I/R‐induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up‐regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca²⁺‐cycling proteins, including Na⁺/Ca²⁺ exchanger, inositol 1,4,5‐trisphosphate receptor, sarcoplasmic reticulum Ca²⁺‐ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4‐DAMP.

Conclusions and Implications

Our data suggest that inhibition of the ROS‐mediated CaMKII pathway and modulation of Ca²⁺‐cycling proteins may be novel mechanisms underlying choline‐induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M ₃ receptors in the vasculature, providing a new therapeutic strategy for I/R‐induced vascular injury.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

4‐DAMP

4‐diphenylacetoxy‐N‐methylpiperidine methiodide

CaMKII

Ca²⁺/calmodulin‐dependent protein kinase II

DHE

dihydroethidium

I/R

ischaemia/reperfusion

IP₃R

inositol 1,4,5‐trisphosphate receptor

NAC

N‐acetyl‐L‐cysteine

NCX

Na⁺/Ca²⁺ exchanger

PLB

phospholamban

ROS

reactive oxygen species

SERCA

sarcoplasmic reticulum Ca²⁺‐ATPase

SNP

sodium nitroprusside

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Emerging role of hydrogen sulfide in hypertension and related cardiovascular diseases

Hydrogen sulfide (H₂S) has traditionally been viewed as a highly toxic gas; however, recent studies have implicated H₂S as a third member of the gasotransmitter family, exhibiting properties similar to NO and carbon monoxide. Accumulating evidence has suggested that H₂S influences a wide range of physiological and pathological processes, among which blood vessel relaxation, cardioprotection and atherosclerosis have been particularly studied. In the cardiovascular system, H₂S production is predominantly catalyzed by cystathionine γ‐lyase (CSE). Decreased endogenous H₂S levels have been found in hypertensive patients and animals, and CSE ^−/− mice develop hypertension with age, suggesting that a deficiency in H₂S contributes importantly to BP regulation. H₂S supplementation attenuates hypertension in different hypertensive animal models. The mechanism by which H₂S was originally proposed to attenuate hypertension was by virtue of its action on vascular tone, which may be related to effects on different ion channels. Both H₂S and NO cause vasodilatation and there is cross‐talk between these two molecules to regulate BP. Suppression of oxidative stress may also contribute to antihypertensive effects of H₂S. This review also summarizes the state of research on H₂S and hypertension in China. A better understanding of the role of H₂S in hypertension and related cardiovascular diseases will allow novel strategies to be devised for their treatment.

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This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviation

2K1C

two‐kidney‐one‐clip

Ang II

angiotensin II

AOA

aminooxyacetic acid

AT₁ receptor

angiotensin II type 1 receptor

CBS

cystathionine‐β‐synthase

CO

carbon monoxide

CSE

cystathionine‐γ‐lyase

DBP

diastolic BP

eNOS

endothelial NOS

H₂S

hydrogen sulfide

I/R

ischaemia/reperfusion

L‐NAME

N^G‐nitro‐l‐arginine methyl ester

MAP

mean arterial pressure

MPST

3‐mercaptopyruvate sulfurtransferase

MWT

medial wall thickness

PAAT

pulmonary arterial acceleration time

PAG

DL‐propargylglycine

PHT

pulmonary hypertension

RVET

right ventricular ejection time

RVH

right ventricular hypertrophy

SBP

systolic BP

SHR

spontaneously hypertensive rat

SMCs

smooth muscle cells

VEGFR‐1

soluble fms‐like tyrosine kinase 1

VD

vas deferens

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Dietary salt regulates epithelial sodium channels in rat endothelial cells: adaptation of vasculature to salt

Background and Purpose

The epithelial sodium channel (ENaC) is expressed in vascular endothelial cells and is a negative modulator of vasodilation. However, the role of endothelial ENaCs in salt‐sensitive hypertension remains unclear. Here, we have investigated how endothelial ENaCs in Sprague‐Dawley (SD) rats respond to high‐salt (HS) challenge.

Experimental Approach

BP and plasma aldosterone levels were measured. We used patch‐clamp technique to record ENaC activity in split‐open mesenteric arteries (MAs). Western blot and Griess assay were used to detect expression of α‐ENaCs, eNOS and NO. Vasorelaxation in second‐order MAs was measured with wire myograph assays.

Key Results

Functional ENaCs were observed in endothelial cells and their activity was significantly decreased after 1 week of HS diet. After 3 weeks of HS diet, ENaC expression was also reduced. When either ENaC activity or expression was reduced, endothelium‐dependent relaxation (EDR) of MAs, in response to ACh, was enhanced. This enhancement of EDR was mimicked by amiloride, a blocker of ENaCs. By contrast, HS diet significantly increased contractility of MAs, accompanied by decreased eNOS activity and NO levels. However, ACh‐induced release of NO was much higher in MAs isolated from HS rats than those from NS rats.

Conclusions and Implications

HS intake increased the BP of SD rats, but simultaneously enhanced EDR by reducing ENaC activity and expression due to feedback inhibition. Therefore, ENaCs may play an important role in endothelial cells allowing the vasculature to adapt to HS conditions.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ENaC

epithelial sodium channel

HS

high‐salt

NS

normal‐salt

SD rat

Sprague‐Dawley rat

P_O

open probability

MAECs

mesenteric artery endothelial cells

EDR

endothelium‐dependent relaxation

SMC

smooth muscle cell

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Regulator of G‐protein signalling 5 protects against atherosclerosis in apolipoprotein E‐deficient mice

Background and Purpose

Atherosclerosis is a chronic inflammatory disease, in which ‘vulnerable plaques’ have been recognized as the underlying risk factor for coronary disease. Regulator of G‐protein signalling (RGS) 5 controls endothelial cell function and inflammation. In this study, we explored the effect of RGS5 on atherosclerosis and the potential underlying mechanisms.

Experimental Approach

RGS5^−/− apolipoprotein E (ApoE)^−/− and ApoE ^−/− littermates were fed a high‐fat diet for 28 weeks. Total aorta surface and lipid accumulation were measured by Oil Red O staining and haematoxylin–eosin staining was used to analyse the morphology of atherosclerotic lesions. Inflammatory cell infiltration and general inflammatory mediators were examined by immunofluorescence staining. Apoptotic endothelial cells and macrophages were assayed with TUNEL. Expression of RGS5and adhesion molecules, and ERK1/2 phosphorylation were evaluated by co‐staining with CD31. Expression of mRNA and protein were determined by quantitative real‐time PCR and Western blotting respectively.

Key Results

Atherosclerotic phenotypes were significantly accelerated in RGS5^−/− ApoE ^−/− mice, as indicated by increased inflammatory mediator expression and apoptosis of endothelial cells and macrophages. RGS5 deficiency enhanced instability of vulnerable plaques by increasing infiltration of macrophages in parallel with the accumulation of lipids, and decreased smooth muscle cell and collagen content. Mechanistically, increased activation of NF‐κB and MAPK/ERK 1/2 could be responsible for the accelerated development of atherosclerosis in RGS5‐deficient mice.

Conclusions and Implications

RGS5 deletion accelerated development of atherosclerosis and decreased the stability of atherosclerotic plaques partly through activating NF‐κB and the MEK‐ERK1/2 signalling pathways.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ApoE

apolipoprotein E

CHD

coronary heart disease

H&E

haematoxylin‐eosin

ICAM‐1

intercellular adhesion molecue‐1

LDL

low‐density lipoprotein

MEK

MAPK/ERK kinase

RGS

regulator of G‐protein signalling

SMC

smooth muscle cell

VCAM‐1

vascular cell adhesion molecule‐1

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Cryptotanshinone,an orally bioactive herbal compound from Danshen,attenuates atherosclerosis in apolipoprotein E‐deficient mice: role of lectin‐like oxidized LDL receptor‐1 (LOX‐1)

Background and Purpose

Cryptotanshinone (CTS) is a major bioactive diterpenoid isolated from Danshen, an eminent medicinal herb that is used to treat cardiovascular disorders in Asian medicine. However, it is not known whether CTS can prevent experimental atherosclerosis. The present study was designed to investigate the protective effects of CTS on atherosclerosis and its molecular mechanisms of action.

Experimental Approach

Apolipoprotein E‐deficient (ApoE ^−/−) mice, fed an atherogenic diet, were dosed daily with CTS (15, 45 mg kg⁻¹ day⁻¹) by oral gavage. In vitro studies were carried out in oxidized LDL (oxLDL)‐stimulated HUVECs treated with or without CTS.

Key Results

CTS significantly attenuated atherosclerotic plaque formation and enhanced plaque stability in ApoE ^−/− mice by inhibiting the expression of lectin‐like oxLDL receptor‐1 (LOX‐1) and MMP‐9, as well as inhibiting reactive oxygen species (ROS) generation and NF‐κB activation. CTS treatment significantly decreased the levels of serum pro‐inflammatory mediators without altering the serum lipid profile. In vitro, CTS decreased oxLDL‐induced LOX‐1 mRNA and protein expression and, thereby, inhibited LOX‐1‐mediated adhesion of monocytes to HUVECs, by reducing the expression of adhesion molecules (intracellular adhesion molecule 1 and vascular cellular adhesion molecule 1). Furthermore, CTS inhibited NADPH oxidase subunit 4 (NOX4)‐mediated ROS generation and consequent activation of NF‐κB in HUVECs.

Conclusions and Implications

CTS was shown to have anti‐atherosclerotic activity, which was mediated through inhibition of the LOX‐1‐mediated signalling pathway. This suggests that CTS is a vasculoprotective drug that has potential therapeutic value for the clinical treatment of atherosclerotic cardiovascular diseases.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ApoE^−/−

apolipoprotein E deficient

CD36

cluster of differentiation 36

CTS

cryptotanshinone

HCD

high cholesterol diet

ICAM‐1

intracellular adhesion molecule 1

LOX‐1

lectin‐like oxidized low‐density lipoprotein receptor‐1

NOX4

NADPH oxidase subunit 4

oxLDL

oxidized low‐density lipoprotein

ROS

reactive oxygen species

SMCs

smooth muscle cells

SR‐A

scavenger receptor‐A

VCAM‐1

vascular cellular adhesion molecule 1

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Activation of TRPV1 attenuates high salt‐induced cardiac hypertrophy through improvement of mitochondrial function

Background and Purpose

High‐salt diet induces cardiac remodelling and leads to heart failure, which is closely related to cardiac mitochondrial dysfunction. Transient receptor potential (TRP) channels are implicated in the pathogenesis of cardiac dysfunction. We investigated whether activation of TRP vanilloid (subtype 1) (TRPV1) channels by dietary capsaicin can, by ameliorating cardiac mitochondrial dysfunction, prevent high‐salt diet‐induced cardiac hypertrophy.

Experimental Approach

Male wild‐type (WT) and TRPV1 ^−/− mice were fed a normal or high‐salt diet with or without capsaicin for 6 months. Their cardiac parameters and endurance capacity were assessed. Mitochondrial respiration and oxygen consumption were measured using high‐resolution respirometry. The expression levels of TRPV1, sirtuin 3 and NDUFA9 were detected in cardiac cells and tissues.

Key Results

Chronic high‐salt diet caused cardiac hypertrophy and reduced physical activity in mice; both effects were ameliorated by capsaicin intake in WT but not in TRPV1 ^−/− mice. TRPV1 knockout or high‐salt diet significantly jeopardized the proficiency of mitochondrial Complex I oxidative phosphorylation (OXPHOS) and reduced Complex I enzyme activity. Chronic dietary capsaicin increased cardiac mitochondrial sirtuin 3 expression, the proficiency of Complex I OXPHOS, ATP production and Complex I enzyme activity in a TRPV1‐dependent manner.

Conclusions and Implications

TRPV1 activation by dietary capsaicin can antagonize high‐salt diet‐mediated cardiac lesions by ameliorating its deleterious effect on the proficiency of Complex I OXPHOS. TRPV1‐mediated amendment of mitochondrial dysfunction may represent a novel target for management of early cardiac dysfunction.

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This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

AngII

angiotensin II

CI

complex I

CII

complex II

CS

citrate synthase

EF

ejection fraction

ETC

electronic transport chain

FS

shortening fraction

HS

high‐salt diet

iRTX

5′‐iodoresiniferatoxin

LVH

left ventricular hypertrophy

LVID

left ventricular internal diameter

LVPW

left ventricular posterior wall

ND

normal‐salt diet

OXPHOS

oxidative phosphorylation

RER

respiration exchange ratio

RES

resveratrol

ROS

reactive oxygen species

TRPCs

transient potential receptor channels

TRPV1

transient receptor potential vanilloid subfamily member 1

TRPV1^−/−

TRPV1 knockout

WT

wild‐type

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Nuciferine relaxes rat mesenteric arteries through endothelium‐dependent and ‐independent mechanisms

Background and Purpose

Nuciferine, a constituent of lotus leaf, is an aromatic ring‐containing alkaloid, with antioxidative properties. We hypothesize nuciferine might affect vascular reactivity. This study aimed at determining the effects of nuciferine on vasomotor tone and the underlying mechanism

Experimental Approach

Nuciferine‐induced relaxations in rings of rat main mesenteric arteries were measured by wire myographs. Endothelial NOS (eNOS) was determined by immunoblotting. Intracellular NO production in HUVECs and Ca²⁺ level in both HUVECs and vascular smooth muscle cells (VSMCs) from rat mesenteric arteries were assessed by fluorescence imaging.

Key Results

Nuciferine induced relaxations in arterial segments pre‐contracted by KCl or phenylephrine. Nuciferine‐elicited arterial relaxations were reduced by removal of endothelium or by pretreatment with the eNOS inhibitor L‐NAME or the NO‐sensitive guanylyl cyclase inhibitor ODQ. In HUVECs, the phosphorylation of eNOS at Ser¹¹⁷⁷ and increase in cytosolic NO level induced by nuciferine were mediated by extracellular Ca²⁺ influx. Under endothelium‐free conditions, nuciferine attenuated CaCl₂‐induced contraction in Ca²⁺‐free depolarizing medium. In the absence of extracellular calcium, nuciferine relieved the vasoconstriction induced by phenylephrine and the addition of CaCl₂. Nuciferine also suppressed Ca²⁺ influx in Ca²⁺‐free K⁺‐containing solution in VSMCs.

Conclusions and Implications

Nuciferine has a vasorelaxant effect via both endothelium‐dependent and ‐independent mechanisms. These results suggest that nuciferine may have a therapeutic effect on vascular diseases associated with aberrant vasoconstriction.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

[Ca²⁺]_i

intracellular calcium

[NO]_i

intracellular NO

DAF‐FM DA

4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate

EDHF

endothelium‐derived hyperpolarizing factor

eNOS

endothelial NOS

Fluo‐4 AM

fluo‐4‐acetoxymethylester

iNOS

inducible NOS

L‐NAME

Nω‐nitro‐L‐arginine methyl ester

ODQ

1H‐[1,2,4]oxadizolo[4,3‐a]quinoxalin‐1‐one

VDCCs

voltage‐dependent Ca²⁺ channels

VSMCs

vascular smooth muscle cells

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The role of hexokinase in cardioprotection – mechanism and potential for translation

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose‐6‐phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre‐ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B‐cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target.

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This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐8

Abbreviations

Akt

also known as PKB

AMPK

AMP‐activated PK

ANT

adenine nucleotide translocase

Bad

Bcl‐2‐associated death promoter

Bak

Bcl‐2 homologous antagonist/killer

Bax

Bcl‐2‐like protein 4

Bcl‐2

B‐cell lymphoma 2

Bcl‐xL

Bcl‐2‐extra large

CK

creatine kinase

CsA

cyclosporine A

CyP‐D

cyclophilin D

Drp1

dynamin‐related protein 1

G‐6‐P

glucose‐6‐phosphate

GSK3β

glycogen synthase kinase 3β

HK

hexokinase

I/R

ischaemia/reperfusion

IF1

ATP synthase inhibitor factor 1

IMM

inner mitochondrial membrane

IP

ischaemic preconditioning

mPTP

mitochondrial permeability transition pore

OMM

outer mitochondrial membrane

Opa‐1

optic athrophy 1

PCr

phosphocreatine

PPIase

peptidylprolyl isomerase

ROS

reactive oxygen species

SR

sarcoplasmic reticulum

T₀

time of ischaemic rigor start

TAT‐HK2

cell‐permeable peptide of HK2 binding domain

TP

temperature preconditioning

TSPO

translocator protein of the outer membrane

VDAC

voltage‐dependent anion channel

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Aspirin enhances protective effect of fish oil against thrombosis and injury‐induced vascular remodelling

Background and Purpose

Although aspirin (acetylsalicylic acid) is commonly used to prevent ischaemic events in patients with coronary artery disease, many patients fail to respond to aspirin treatment. Dietary fish oil (FO), containing ω3 polyunsaturated fatty acids (PUFAs), has anti‐inflammatory and cardio‐protective properties, such as lowering cholesterol and modulating platelet activity. The objective of the present study was to investigate the potential additional effects of aspirin and FO on platelet activity and vascular response to injury.

Experimental Approach

Femoral arterial remodelling was induced by wire injury in mice. Platelet aggregation, and photochemical‐ and ferric chloride‐induced carotid artery thrombosis were employed to evaluate platelet function.

Key Results

FO treatment increased membrane ω3 PUFA incorporation, lowered plasma triglyceride and cholesterol levels, and reduced systolic BP in mice. FO or aspirin alone inhibited platelet aggregation; however, when combined, they exhibited synergistic suppression of platelet activity in mice, independent of COX‐1 inhibition. FO alone, but not aspirin, attenuated arterial neointimal growth in response to injury. Strikingly, a combination of FO and aspirin synergistically inhibited injury‐induced neointimal hyperplasia and reduced perivascular inflammatory reactions. Moreover, co‐administration of FO and aspirin decreased the expression of pro‐inflammatory cytokines and adhesion molecules in inflammatory cells. Consistently, a pro‐resolution lipid mediator‐Resolvin E1, was significantly elevated in plasma in FO/aspirin‐treated mice.

Conclusions and Implications

Co‐administration of FO and low‐dose aspirin may act synergistically to protect against thrombosis and injury‐induced vascular remodelling in mice. Our results support further investigation of adjuvant FO supplementation for patients with stable coronary artery disease.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

AA

arachidonic acid

CO

coconut oil

CVD

cardiovascular disease

DHA

docosahexaenoic acid

EPA

eicosapentaenoic acid

FO

fish oil

HDLC

high‐density lipoprotein cholesterol

LDLC

low‐density lipoprotein cholesterol

PCI

percutaneous coronary intervention

PUFA

polyunsaturated fatty acid; TG, triglyceride

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Advances in exploring the role of microRNAs in the pathogenesis,diagnosis and therapy of cardiac diseases in China

Cardiovascular disease has become the most serious health threat and represents the major cause of morbidity and mortality in China, as in other industrialized nations. During the past few decades, China''s economic boom has tremendously improved people''s standard of living but has also changed their lifestyle, increasing the prevalence of cardiovascular disease, the so‐called ‘disease of modern civilization’. This new trend has attracted a significant amount of research. Many of the studies conducted by Chinese investigators are orientated towards understanding the molecular mechanisms of cardiovascular disease. At the molecular level, the long‐standing consensus is that cardiovascular disease is associated with a sequence mutation (genetic anomaly) and expression deregulation (epigenetic disorder) of protein‐coding genes. However, new research data have established the non‐protein‐coding genes microRNAs (miRNAs) as a central regulator of the pathogenesis of cardiac disease and a potential new therapeutic target for cardiovascular disease. These small non‐coding RNAs have also been subjected to extensive, rigorous investigations by Chinese researchers. Over the years, a large body of studies on miRNAs in cardiovascular disease has been conducted by Chinese investigators, yielding fruitful research results and a better understanding of miRNAs as a new level of molecular mechanisms for the pathogenesis of cardiac disease. In this review, we briefly summarize the current status of research in the field of miRNAs and cardiovascular disease in China, highlighting the advances made in elucidating the role of miRNAs in various cardiac conditions, including cardiac arrhythmia, myocardial ischaemia, cardiac hypertrophy and heart failure. We have also examined the potential of miRNAs as novel diagnostic biomarkers and therapeutic targets.

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This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

AF

atrial fibrillation

AMI

acute myocardial infarction

APD

action potential duration

Cx43

connexin 43 (also known as GJA1, gap junction protein, α1)

Drp1

dynamin‐related protein‐1

E‐C

excitation‐contraction

HSP60

heat shock protein 60

I_CaL

L type calcium current

JP2

junctophilin‐2

KCNJ2

gene for potassium inwardly rectifying channel, subfamily J, member 2 also known as K_ir2.1, inwardly rectifying potassium channel 2.1

LNA

locked nucleic acid

MI

myocardial infarct

miRNAs

microRNAs

NFAT

nuclear factor of activated T‐cells

SERCA2a

sarcoplasmic reticulum calcium ATPase

K_Ca2.3 (also known as SK3)

small conductance calcium‐activated potassium channels 3

TGF‐β1

transforming growth factor‐β1

TGFBR2

transforming growth factor‐β receptor II

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Advances in receptor conformation research: the quest for functionally selective conformations focusing on the β 2‐adrenoceptor

Seven‐transmembrane receptors, also called GPCRs, represent the largest class of drug targets. Upon ligand binding, a GPCR undergoes conformational rearrangement and thereby changes its interaction with effector proteins including the cognate G‐proteins and the multifunctional adaptor proteins, β‐arrestins. These proteins, by initiating distinct signal transduction mechanisms, mediate one or several functional responses. Recently, the concept of ligand‐directed GPCR signalling, also called functional selectivity or biased agonism, has been proposed to explain the phenomenon that chemically diverse ligands exhibit different efficacies towards the different signalling pathways of a single GPCR, and thereby act as functionally selective or ‘biased’ ligands. Current concepts support the notion that ligand‐specific GPCR conformations are the basis of ligand‐directed signalling. Multiple studies using fluorescence spectroscopy, X‐ray crystallography, mass spectroscopy, nuclear magnetic resonance spectroscopy, single‐molecule force spectroscopy and other techniques have provided the evidence to support this notion. It is anticipated that these techniques will ultimately help elucidate the structural basis of ligand‐directed GPCR signalling at a precision meaningful for structure‐based drug design and how a specific ligand molecular structure induces a unique receptor conformation leading to biased signalling. In this review, we will summarize recent advances in experimental techniques applied in the study of functionally selective GPCR conformations and breakthrough data obtained in these studies particularly those of the β₂‐adrenoceptor.

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This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

G_i

inhibitory G‐protein

GRK

GPCR kinase

G_s

stimulatory G‐protein

HDX‐MS

hydrogen‐deuterium exchange coupled to MS

pdb

Protein Data Bank

SMFS

single‐molecule force spectroscopy

TM

transmembrane

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Protective effect of 23‐hydroxybetulinic acid on doxorubicin‐induced cardiotoxicity: a correlation with the inhibition of carbonyl reductase‐mediated metabolism

Background and Purpose

The clinical use of doxorubicin, an effective anticancer drug, is severely hampered by its cardiotoxicity. 23‐Hydroxybetulinic acid (23‐HBA), isolated from P ulsatilla chinensis, enhances the anticancer effect of doxorubicin while simultaneously reducing its cardiac toxicity, but does not affect the concentration of doxorubicin in the plasma and heart. As the metabolite doxorubicinol is more potent than doxorubicin at inducing cardiac toxicity, in the present study we aimed to clarify the role of doxorubicinol in the protective effect of 23‐HBA.

Experimental Approach

Doxorubicin was administered to mice for two weeks in the presence or absence of 23‐HBA. The heart pathology, function, myocardial enzymes and accumulation of doxorubicin and doxorubicinol were then analysed. A cellular pharmacokinetic study of doxorubicin and doxorubicinol, carbonyl reductase 1 (CBR1) interference and molecular docking was performed in vitro.

Key Results

23‐HBA alleviated the doxorubicin‐induced cardiotoxicity in mice, and this was accompanied by inhibition of the metabolism of doxorubicin and reduced accumulation of doxorubicinol selectively in hearts. In H9c2 cells, the protective effect of 23‐HBA was shown to be closely associated with a decreased rate and extent of accumulation of doxorubicinol in mitochondria and nuclei. siRNA and docking analysis demonstrated that CBR1 has a crucial role in doxorubicin‐mediated cardiotoxicity and 23‐HBA inhibits this metabolic pathway.

Conclusions and Implications

Inhibition of CBR‐mediated doxorubicin metabolism might be one of the protective mechanisms of 23‐HBA against doxorubicin‐induced cardiotoxicity. The present study provides a new research strategy guided by pharmacokinetic theory to elucidate the mechanism of drugs with unknown targets.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

23‐HBA

23‐hydroxybetulinic acid

AST

aspartate aminotransferase

CBR1

carbonyl reductase 1

CMC

carboxymethyl cellulose

CK

creatine kinase

CK‐MD

creatine kinase isoenzyme

EF

ejection fraction

FS

fractional shortening

i.g

intragastrically

LV

left ventricle

MDR

multidrug resistance

TCM

traditional Chinese medicine

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Class A1 scavenger receptors in cardiovascular diseases

Class A1 scavenger receptors (SR‐A1) are membrane glycoproteins that can form homotrimers. This receptor was originally defined by its ability to mediate the accumulation of lipids in macrophages. Subsequent studies reveal that SR‐A1 plays critical roles in innate immunity, cell apoptosis and proliferation. This review highlights recent advances in understanding the structure, receptor pathway and regulation of SR‐A1. Although its role in atherosclerosis is disputable, recent discoveries suggest that SR‐A1 function in anti‐inflammatory responses by promoting an M2 macrophage phenotype in cardiovascular diseases. Therefore, SR‐A1 may be a potential target for therapeutic intervention of cardiovascular diseases.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

acLDL

acetylated low‐density lipoprotein

ER

endoplasmic reticulum

I/R

ischaemia/reperfusion

LTA

lipoteichoic acid

MI

myocardial infarction

mLDL

modified low‐density lipoprotein

oxLDL

oxidized low‐density lipoprotein

PRR

pattern recognition receptor

RAGE

receptor for advanced glycated end‐products

SR‐A1

class A1 scavenger receptor

TLR4

Toll‐like receptor 4

TRAF6

TNF receptor‐associated factor 6

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Role of the MPTP in conditioning the heart – translatability and mechanism

Mitochondria have long been known to be the gatekeepers of cell fate. This is particularly so in the response to acute ischaemia‐reperfusion injury (IRI). Following an acute episode of sustained myocardial ischaemia, the opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion, mediates cell death. Preventing MPTP opening at the onset of reperfusion using either pharmacological inhibitors [such as cyclosporin A (CsA) ] or genetic ablation has been reported to reduce myocardial infarct (MI) size in animal models of acute IRI. Interestingly, the endogenous cardioprotective intervention of ischaemic conditioning, in which the heart is protected against MI by applying cycles of brief ischaemia and reperfusion to either the heart itself or a remote organ or tissue, appears to be mediated through the inhibition of MPTP opening at reperfusion. Small proof‐of‐concept clinical studies have demonstrated the translatability of this therapeutic approach to target MPTP opening using CsA in clinical settings of acute myocardial IRI. However, given that CsA is a not a specific MPTP inhibitor, more novel and specific inhibitors of the MPTP need to be discovered – the molecular identification of the MPTP should facilitate this. In this paper, we review the role of the MPTP as a target for cardioprotection, the potential mechanisms underlying MPTP inhibition in the setting of ischaemic conditioning, and the translatability of MPTP inhibition as a therapeutic approach in the clinical setting.

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This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐8

Abbreviations

ANT

adenine nucleotide translocase

CABG

coronary artery bypass graft

CsA

cyclosporin A

CypD

cyclophilin D

Drp1

dynamin‐related protein 1

GSK

glycogen synthase kinase

IPC

ischaemic preconditioning

IPost

ischaemic postconditioning

IRI

ischaemia‐reperfusion injury

LV

left ventricular

MI

myocardial infarct

MitoK_ATP

mitochondrial ATP‐sensitive potassium channel

MPTP

mitochondrial permeability transition pore

OMM

outer mitochondrial membrane

OPA1

optic atrophy 1

PMI

perioperative myocardial injury

PPCI

primary percutaneous coronary intervention

RIC

remote ischaemic conditioning

RISK

reperfusion injury salvage kinase

ROS

reactive oxygen species

SAFE

survivor activating factor enhancement

STEMI

ST segment elevation myocardial infarction

VDAC

voltage‐dependent anion channel

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Biased β2‐adrenoceptor signalling in heart failure: pathophysiology and drug discovery

The body is constantly faced with a dynamic requirement for blood flow. The heart is able to respond to these changing needs by adjusting cardiac output based on cues emitted by circulating catecholamine levels. Cardiac β‐adrenoceptors transduce the signal produced by catecholamine stimulation via G_s proteins to their downstream effectors to increase heart contractility. During heart failure, cardiac output is insufficient to meet the needs of the body; catecholamine levels are high and β‐adrenoceptors become hyperstimulated. The hyperstimulated β₁‐adrenoceptors induce a cardiotoxic effect, which could be counteracted by the cardioprotective effect of β₂‐adrenoceptor‐mediated G_i signalling. However, β₂‐adrenoceptor‐G_i signalling negates the stimulatory effect of the G_s signalling on cardiomyocyte contraction and further exacerbates cardiodepression. Here, further to the localization of β₁‐ and β₂‐adrenoceptors and β₂‐adrenoceptor‐mediated β‐arrestin signalling in cardiomyocytes, we discuss features of the dysregulation of β‐adrenoceptor subtype signalling in the failing heart, and conclude that G_i‐biased β₂‐adrenoceptor signalling is a pathogenic pathway in heart failure that plays a crucial role in cardiac remodelling. In contrast, β₂‐adrenoceptor‐G_s signalling increases cardiomyocyte contractility without causing cardiotoxicity. Finally, we discuss a novel therapeutic approach for heart failure using a G_s‐biased β₂‐adrenoceptor agonist and a β₁‐adrenoceptor antagonist in combination. This combination treatment normalizes the β‐adrenoceptor subtype signalling in the failing heart and produces therapeutic effects that outperform traditional heart failure therapies in animal models. The present review illustrates how the concept of biased signalling can be applied to increase our understanding of the pathophysiology of diseases and in the development of novel therapies.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ACEI

ACE inhibitors

CaMKII

Ca²⁺/calmodulin‐dependent kinase II

ct

carboxy terminus

EGFR

epidermal growth receptor

Epac

exchange protein directly activated by cAMP

G_i

inhibitory G protein

GRK

GPCR kinase

G_s

stimulatory G protein

HF

heart failure

PKA

cAMP‐dependent protein kinase

SNS

sympathetic nervous system

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Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt‐induced hypertension in mice

Background and Purpose

Inflammasomes are multimeric complexes that facilitate caspase‐1‐mediated processing of the pro‐inflammatory cytokines IL‐1β and IL‐18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL‐1β and IL‐18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis.

Experimental Approach

Wild‐type and inflammasome‐deficient ASC ^−/− mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real‐time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry.

Key Results

1K/DOCA/salt‐induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro‐caspase‐1, and the cytokine, pro‐IL‐1β, as well as protein levels of active caspase‐1 and mature IL‐1β. Following treatment with 1K/DOCA/salt, ASC ^−/− mice displayed blunted pressor responses and were also protected from increases in renal expression of IL‐6, IL‐17A, CCL2, ICAM‐1 and VCAM‐1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt‐treated mice.

Conclusions and Implications

Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL‐1β pathway as a potential therapeutic target in hypertension.

Linked Articles

This article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2016.173.issue-4

Abbreviations

AIM2

absent in melanoma 2

ASC

apoptosis‐associated speck‐like protein containing a carboxy‐terminal CARD

CARD

caspase activation and recruitment domain

CCL

C‐C motif chemokine ligand

DAMP

danger‐associated molecular pattern

DOCA

deoxycorticosterone acetate

HRP

horseradish peroxidase

ICAM‐1

intercellular adhesion molecule‐1

NLRC4 (IPAF)

NOD‐like receptor family CARD domain‐containing protein 4

NLRP

NOD‐like receptor family pyrin domain‐containing protein

PAMP

pathogen‐associated molecular pattern

VCAM‐1

vascular cell adhesion molecule‐1

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Meningeal blood flow is controlled by H2S‐NO crosstalk activating a HNO‐TRPA1‐CGRP signalling pathway

Background and Purpose

Meningeal blood flow is controlled by CGRP released from trigeminal afferents and NO mainly produced in arterial endothelium. The vasodilator effect of NO may be due to the NO–derived compound, nitroxyl (HNO), generated through reaction with endogenous H₂S. We investigated the involvement of HNO in CGRP release and meningeal blood flow.

Experimental Approach

Blood flow in exposed dura mater of rats was recorded by laser Doppler flowmetry. CGRP release from the dura mater in the hemisected rat head was quantified using an elisa. NO and H₂S were localized histochemically with specific sensors.

Key Results

Topical administration of the NO donor diethylamine‐NONOate increased meningeal blood flow by 30%. Pretreatment with oxamic acid, an inhibitor of H₂S synthesis, reduced this effect. Administration of Na₂S increased blood flow by 20%, an effect abolished by the CGRP receptor antagonist CGRP _8‐37 or the TRPA1 channel antagonist HC030031 and reduced when endogenous NO synthesis was blocked. Na₂S dose‐dependently increased CGRP release two‐ to threefold. Co‐administration of diethylamine‐NONOate facilitated CGRP release, while inhibition of endogenous NO or H₂S synthesis lowered basal CGRP release. NO and H₂S were mainly localized in arterial vessels, HNO additionally in nerve fibre bundles. HNO staining was lost after treatment with L‐NMMA and oxamic acid.

Conclusions and Implications

NO and H₂S cooperatively increased meningeal blood flow by forming HNO, which activated TRPA1 cation channels in trigeminal fibres, inducing CGRP release. This HNO‐TRPA1‐CGRP signalling pathway may be relevant to the pathophysiology of headaches.

Abbreviations

CBS

cystathionine β‐synthase

CSE

cystathionine γ‐lyase

Cy3

cyanine dye 3

DAF

4‐amino‐5‐methylamino‐2′,7′‐difluoresceine diacetate

HNO

nitroxyl

iNOS

inducible NOS

L‐NMMA

L‐NG‐monomethylarginine acetate

MMA

middle meningeal artery

MST

mercaptopyruvate sulfurtransferase

nNOS

neuronal NOS

NONOate

diethylamine‐NONOate, DEANONOate

ODQ

1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one

sGC

soluble GC

SIF

synthetic interstitial fluid

TRPA1

transient receptor potential ankyrin 1 channel

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Genistein alleviates pressure overload‐induced cardiac dysfunction and interstitial fibrosis in mice

Background and Purpose

Pressure overload‐induced cardiac interstitial fibrosis is viewed as a major cause of heart failure in patients with hypertension or aorta atherosclerosis. The purpose of this study was to investigate the effects and the underlying mechanisms of genistein, a natural phytoestrogen found in soy bean extract, on pressure overload‐induced cardiac fibrosis.

Experimental Approach

Genisten was administered to mice with pressure overload induced by transverse aortic constriction. Eight weeks later, its effects on cardiac dysfunction, hypertrophy and fibrosis were determined. Its effects on proliferation, collagen production and myofibroblast transformation of cardiac fibroblasts (CFs) and the signalling pathways were also assessed in vitro.

Key Results

Pressure overload‐induced cardiac dysfunction, hypertrophy and fibrosis were markedly attenuated by genistein. In cultured CFs, genistein inhibited TGFβ1‐induced proliferation, collagen production and myofibroblast transformation. Genistein suppressed TGFβ‐activated kinase 1 (TAK1) expression and produced anti‐fibrotic effects by blocking the TAK1/MKK4/JNK pathway. Further analysis indicated that it up‐regulated oestrogen‐dependent expression of metastasis‐associated gene 3 (MTA3), which was found to be a negative regulator of TAK1. Silencing MTA3 by siRNA, or inhibiting the activity of the MTA3‐NuRD complex with trichostatin A, abolished genistein''s anti‐fibrotic effects.

Conclusions and Implications

Genistein improved cardiac function and inhibited cardiac fibrosis in response to pressure overload. The underlying mechanism may involve regulation of the MTA3/TAK1/MKK4/JNK signalling pathway. Genistein may have potential as a novel agent for prevention and therapy of cardiac disorders associated with fibrosis.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

CFs

cardiac fibroblasts

CTGF

connective tissue growth factor

ECM

extracellular matrix

EdU

5‐ethynyl‐2′‐deoxyuridine

EF

ejection fraction

ER

oestrogen receptors

FS

fractional shortening

MTA3

metastasis‐associated gene 3

NuRD

nucleosome remodelling and deacetylase

TAC

transverse aortic constriction

TAK1

TGFβ‐activated kinase 1

TSA

trichostatin A

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