Toll-interacting protein (Tollip) is a critical regulator of the Toll-like receptor-mediated signalling pathway. However, the role of Tollip in chronic pressure overload-induced cardiac hypertrophy remains unclear. This study aimed to determine the functional significance of Tollip in the regulation of aortic banding-induced cardiac remodelling and its underlying mechanisms.
Methods and results
First, we observed that Tollip was down-regulated in human failing hearts and murine hypertrophic hearts, as determined by western blotting and RT–PCR. Using cultured neonatal rat cardiomyocytes, we found that adenovirus vector-mediated overexpression of Tollip limited angiotensin II-induced cell hypertrophy; whereas knockdown of Tollip by shRNA exhibited the opposite effects. We then generated a transgenic (TG) mouse model with cardiac specific-overexpression of Tollip and subjected them to aortic banding (AB) for 8 weeks. When compared with AB-treated wild-type mouse hearts, Tollip-TGs showed a significant attenuation of cardiac hypertrophy, fibrosis, and dysfunction, as measured by echocardiography, immune-staining, and molecular/biochemical analysis. Conversely, a global Tollip-knockout mouse model revealed an aggravated cardiac hypertrophy and accelerated maladaptation to chronic pressure overloading. Mechanistically, we discovered that Tollip interacted with AKT and suppressed its downstream signalling pathway. Pre-activation of AKT in cardiomyocytes largely offset the Tollip-elicited anti-hypertrophic effects.
Our results provide the first evidence that Tollip serves as a negative regulator of pathological cardiac hypertrophy by blocking the AKT signalling pathway.
Tollip; Cardiac remodelling; Pressure overload; AKT; Cardiomyocyte hypertrophy
Congenital coronary artery anomalies produce serious events that include syncope, arrhythmias, myocardial infarction, or sudden death. Studying the mechanism of coronary development will contribute to the understanding of the disease and help design new diagnostic or therapeutic strategies. Here, we characterized a new calcineurin–NFAT signalling which specifically functions in the epicardium to regulate the development of smooth muscle wall of the coronary arteries.
Methods and results
Using tissue-specific gene deletion, we found that calcineurin–NFAT signals in the embryonic epicardium to direct coronary smooth muscle cell development. The smooth muscle wall of coronary arteries fails to mature in mice with epicardial deletion of calcineurin B1 (Cnb1), and accordingly these mutant mice develop cardiac dysfunction with reduced exercise capacity. Inhibition of calcineurin at various developmental windows shows that calcineurin–NFAT signals within a narrow time window at embryonic Day 12.5–13.5 to regulate coronary smooth muscle cell development. Within the epicardium, NFAT transcriptionally activates the expression of Smad2, whose gene product is critical for transducing transforming growth factor β (TGFβ)–Alk5 signalling to control coronary development.
Our findings demonstrate new spatiotemporal and molecular actions of calcineurin–NFAT that dictate coronary arterial wall development and a new mechanism by which calcineurin–NFAT integrates with TGFβ signalling during embryonic development.
Epicardial; Calcineurin–NFAT; Coronary artery; Smooth muscle cell Differentiation; Smad2
Nitrite (NO2–), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored.
Methods and results
Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R.
These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.
Ischaemia; Protein kinase A; Pre-conditioning; Nitrite/nitrate; Mitochondria
Macrophage apoptosis plays a determinant role in progression of atherosclerotic lesions. An important goal in atherosclerosis research is to identify new components of macrophage apoptosis that can eventually be exploited as molecular targets in strategies aimed at manipulating macrophage function in the lesion. In the previous work from our laboratory, we have shown that transient receptor potential canonical 3 (TRPC3) channel is an obligatory component of survival mechanisms in human and murine macrophages and that TRPC3-deficient non-polarized bone marrow-derived macrophages exhibit increased apoptosis, suggesting that in vivo TRPC3 might influence lesion development. In the present work, we used a bone marrow transplantation strategy as a first approach to examine the impact of macrophage deficiency of TRPC3 on early and advanced atherosclerotic lesions of Apoe−/− mice.
Methods and results
After 3 weeks of high-fat diet, lesions in mice transplanted with bone marrow from Trpc3−/− donors were smaller and with reduced cellularity than controls. Advanced lesions from these mice exhibited reduced necrotic core, less apoptotic macrophages, and increased collagen content and cap thickness. In vitro, TRPC3-deficient macrophages polarized to the M1 phenotype showed reduced apoptosis, whereas both M1 and M2 macrophages had increased efferocytic capacity.
Bone marrow deficiency of TRPC3 has a dual beneficial effect on lesion progression by reducing cellularity at early stages and necrosis in the advanced plaques. Our findings represent the first evidence for a role of a member of the TRPC family of cation channels in mechanisms associated with atherosclerosis.
TRPC3 channel; Macrophage apoptosis; Atherosclerosis; Calcium channels
Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca2+ handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined.
Methods and results
We examined the effects of digitoxin (DGT) on Ca2+ handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca2+ waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated.
These results suggest that the arrhythmogenic adverse effects of CGs on Ca2+ handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
Calcium; Reactive oxygen species; NADPH oxidase; Mitochondria; CaMKII
The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle.
Methods and results
Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation.
Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.
GIRK channel; Acetylcholine; Adenosine; Action potential; Repolarization
β2-Spectrin is an actin-binding protein that plays an important role in membrane integrity and the transforming growth factor (TGF)-β signalling pathway as an adaptor for Smads. Loss of β2-spectrin in mice (Spnb2−/−) results in embryonic lethality with gastrointestinal, liver, neural, and heart abnormalities that are similar to those in Smad2+/−Smad3+/− mice. However, to date, the role of β2-spectrin in embryogenesis, particularly in heart development, has been poorly delineated. Here, we demonstrated that β2-spectrin is required for the survival and differentiation of cardiomyocytes, and its loss resulted in defects in heart development with failure of ventricular wall thickening.
Methods and results
Disruption of β2-spectrin in primary muscle cells not only inhibited TGF-β/Smad signalling, but also reduced the expression of the cardiomyocyte differentiation markers Nkx2.5, dystrophin, and α-smooth muscle actin (α-SMA). Furthermore, cytoskeletal networks of dystrophin, F-actin, and α-SMA in cardiomyocytes were disorganized upon loss of β2-spectrin. In addition, deletion of β2-spectrin in mice (Spnb2tm1a/tm1a) prevented proper development of the heart in association with disintegration of dystrophin structure and markedly reduced survival.
These data suggest that β2-spectrin deficiency leads to inactivation of TGF-β/Smad signalling and contributes to dysregulation of the cell cycle, proliferation, differentiation, and the cytoskeletal network, and it leads to defective heart development. Our data demonstrate that β2-spectrin is required for proper development of the heart and that disruption of β2-spectrin is a potential underlying cause of congenital heart defects.
β2-Spectrin; TGF-β; Cytoskeleton; Cardiogenesis
Contraction of the heart is regulated by electrically evoked Ca2+ transients (CaTs). H+ ions, the end products of metabolism, modulate CaTs through direct interactions with Ca2+-handling proteins and via Na+-mediated coupling between acid-extruding proteins (e.g. Na+/H+ exchange, NHE1) and Na+/Ca2+ exchange. Restricted H+ diffusivity in cytoplasm predisposes pH-sensitive Ca2+ signalling to becoming non-uniform, but the involvement of readily diffusible intracellular Na+ ions may provide a means for combatting this.
Methods and results
CaTs were imaged in fluo3-loaded rat ventricular myocytes paced at 2 Hz. Cytoplasmic [Na+] ([Na+]i) was imaged using SBFI. Intracellular acidification by acetate exposure raised diastolic and systolic [Ca2+] (also observed with acid-loading by ammonium prepulse or CO2 exposure). The systolic [Ca2+] response correlated with a rise in [Na+]i and sarcoplasmic reticulum Ca2+ load, and was blocked by the NHE1 inhibitor cariporide (CO2/HCO3−-free media). Exposure of one half of a myocyte to acetate using dual microperfusion (CO2/HCO3−-free media) raised diastolic [Ca2+] locally in the acidified region. Systolic [Ca2+] and CaT amplitude increased more uniformly along the length of the cell, but only when NHE1 was functional. Cytoplasmic Na+ diffusivity (DNa) was measured in quiescent cells, with strophanthidin present to inhibit the Na+/K+ pump. With regional acetate exposure to activate a local NHE-driven Na+-influx, DNa was found to be sufficiently fast (680 µm2/s) for transmitting the pH–systolic Ca2+ interaction over long distances.
Na+ ions are rapidly diffusible messengers that expand the spatial scale of cytoplasmic pH–CaT interactions, helping to co-ordinate global Ca2+ signalling during conditions of intracellular pH non-uniformity.
Acidosis; Calcium; Diffusion; E–C coupling; Na+–H+ exchange
Circulating endothelium-derived extracellular vesicles (EV) levels are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. Previously, we found that lung-derived EVs (LEVs) induce bone marrow-derived progenitor cells to express lung-specific mRNA and protein. In this study, we sought to determine whether LEV or plasma-derived EV (PEV) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodelling.
Methods and results
LEV, PEV isolated from monocrotaline (MCT-EV)- or vehicle-treated mice (vehicle-EV) were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodelling were assessed by RV-to-body weight (RV/BW) and blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT- vs. vehicle-injected mice (1.99 ± 0.09 vs. 1.04 ± 0.09 mg/g; 0.159 ± 0.002 vs. 0.062 ± 0.009%). RV/BW, WT/D ratios were higher in mice injected with MCT-EV vs. mice injected with vehicle-EV (1.63 ± 0.09 vs. 1.08 ± 0.09 mg/g; 0.113 ± 0.02 vs. 0.056 ± 0.01%). Lineage-depleted bone marrow cells incubated with MCT-EV and marrow cells isolated from mice infused with MCT-EV had greater expression of endothelial progenitor cell mRNAs and mRNAs abnormally expressed in PAH than cells incubated with vehicle-EV or isolated from vehicle-EV infused mice. MCT-EV induced an apoptosis-resistant phenotype in murine pulmonary endothelial cells and lineage-depleted bone marrow cells incubated with MCT-EV induced pulmonary hypertension when injected into healthy mice.
EV from MCT-injured mice contribute to the development of MCT-induced pulmonary hypertension. This effect may be mediated directly by EV on the pulmonary vasculature or by differentiation of bone marrow cells to endothelial progenitor cells that induce pulmonary vascular remodelling.
Pulmonary hypertension; Endothelium; Microparticles; Bone marrow stem/progenitor cells; Monocrotaline
Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, is expressed in vascular smooth muscle cells (VSMCs) of blood vessels and functions to repress VSMC migration and vascular remodelling. The goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration.
Methods and results
Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 associated with the actin cytoskeleton. In response to chemoattractant stimulation, Csrp2 (mouse CRP2 gene symbol)-deficient (Csrp2−/−) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2−/− VSMCs following chemoattractant stimulation. Mammalian 2-hybrid and co-immunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibres in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs, whereas CRP2 was restricted to FAs and stress fibres. Furthermore, we demonstrated that p130Cas expression and phosphorylation promote neointima formation following arterial injury.
These studies demonstrate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.
Vascular smooth muscle cells; Migration; Cysteine-rich protein 2; P130Cas
Calcium fluctuations and cardiac hypertrophy occur during pregnancy, but the role of the well-studied calcium-activated phosphatase, calcineurin, has not been studied in this setting. The purpose of this study was to determine whether calcineurin signalling is required for cardiac remodelling during pregnancy in mice.
Methods and results
We first examined calcineurin expression in the heart of mice during pregnancy. We found both calcineurin levels and activity were significantly increased in early-pregnancy and decreased in late-pregnancy. Since progesterone levels start to rise in early-pregnancy, we investigated whether progesterone alone was sufficient to modulate calcineurin levels in vivo. After implantation of progesterone pellets in non-pregnant female mice, cardiac mass increased, whereas cardiac function was maintained. In addition, calcineurin levels increased, which is also consistent with early-pregnancy. To determine whether these effects were occurring in the cardiac myocytes, we treated neonatal rat ventricular myocytes (NRVMs) with pregnancy-associated sex hormones. We found that progesterone treatment, but not oestradiol, increased calcineurin levels. To obtain a functional read-out of increased calcineurin activity, we measured the activity of the transcription factor NFAT, a downstream target of calcineurin. Progesterone treatment significantly increased NFAT activity in NRVMs, and this was blocked by the calcineurin inhibitor cyclosporine A (CsA), showing that the progesterone-mediated increase in NFAT activity requires calcineurin activity. Importantly, CsA treatment of mice completely blocked pregnancy-induced cardiac hypertrophy.
Our results show that calcineurin is required for pregnancy-induced cardiac hypertrophy, and that calcineurin activity in early-pregnancy is due at least in part to increased progesterone.
Calcineurin; Cardiac hypertrophy; Pregnancy; Progesterone; NFAT
Angiogenic sprouts arise from microvessels formed by endothelial cells (ECs) invested by pericytes (PCs). The aim of this study was to examine the role of PCs in angiogenic sprouting, an understudied phenomenon.
Methods and results
We adapted a human EC spheroid model to examine PC effects on vascular endothelial growth factor-A-induced EC sprouting in vitro by using Bcl-2-transduced human umbilical vein ECs to reduce apoptosis in collagen gels. Human placental PCs, separated from endothelial spheroids by a transwell, or addition of PC-conditioned media increased EC sprouting primarily through hepatocyte growth factor (HGF). Mixed endothelial–PC spheroids formed similar numbers of endothelial sprouts as endothelial spheroids but the sprouts from mixed spheroids were invested by PCs within 24 h. PCs were recruited to the sprouts by platelet-derived growth factor (PDGF)-BB; inhibition of PDGF signalling reduced PC coverage and increased EC sprouting. Transplanted endothelial spheroids give rise to sprouts in vivo that evolve into perfused microvessels. Mixed endothelial–PC spheroids form similar numbers of microvessels as endothelial-only spheroids, but acquire human PC investment and have reduced average lumen diameter.
PCs promote endothelial sprouting by elaborating HGF, but when recruited to invest endothelial sprouts by PDGF-BB, limit the extent of sprouting in vitro and lumen diameter in vivo.
Endothelial cells; Pericytes; Bcl-2; Platelet-derived growth factor; Hepatocyte growth factor
Signalling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (Trpc) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signalling. We tested this concept by investigating the role of cGMP/cGKI signalling on vascular tone and peripheral resistance using Trpc6−/−, Trpc3−/−, Trpc3−/−/6−/−, Trpc1−/−/3−/−/6−/−, and SM-specific cGKI−/− (sm-cGKI−/−) mice.
Methods and results
α-Adrenergic stimulation induced similar contractions in L-NG-nitroarginine methyl ester (l-NAME)-treated aorta and comparably increased peripheral pressure in hind limbs from all mouse lines investigated. After α-adrenergic stimulation, 8-Br-cGMP diminished similarly aortic tone and peripheral pressure in control, Trpc6−/−, Trpc3−/−, Trpc3−/−/6−/−, and Trpc1−/−/3−/−/6−/− mice but not in sm-cGKI−/− mice. In untreated aorta, α-adrenergic stimulation induced similar contractions in the aorta from control and Trpc3−/− mice but larger contractions in sm-cGKI−/−, Trpc6−/−, Trpc3−/−/6−/−, and Trpc1−/−/3−/−/6−/− mice, indicating a functional link between cGKI and Trpc6 channels. Trpc3 channels were detected by immunocytochemistry in both isolated aortic smooth muscle cells (SMCs) and aortic endothelial cells (ECs), whereas Trpc6 channels were detected only in ECs. Phenylephrine-stimulated Ca2+ levels were similar in SMCs from control (Ctr) and Trpc6−/− mice. Carbachol-stimulated Ca2+ levels were reduced in ECs from Trpc6−/− mice. Stimulated Ca2+ levels were lowered by 8-Br-cGMP in Ctr but not in Trpc6−/− ECs.
The results suggest that cGKI and Trpc1,3,6 channels are not functionally coupled in vascular SM. Deletion of Trpc6 channels impaired endothelial cGKI signalling and vasodilator tone in the aorta.
Phenylephrine; Smooth muscle; Relaxation; Nitric oxide; Endothelium
Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (∼200–300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components ‘of the desmosome’ and ‘of the gap junction’, and defined their spatial organization.
Methods and results
We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at ∼20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of ‘the desmosomal protein’ plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte–Carlo simulations indicating an attraction between PKP2 and Cx43.
(i) Super-resolution fluorescence microscopy, complemented with Monte–Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.
Connexin43; Plakophilin-2; Ankyrin-G
In this study, we investigated whether pre-conditioning (PC) by electrical stimulation (EleS) induces cytoprotective effect on cardiac stem cells (CSCs) and determined its underlying molecular mechanisms.
Methods and results
Sca-1+ CSCs were isolated from male C57BL6 mice (12 weeks) hearts. PC of CSCs with EleS (EleSCSCs) was carried out for 3 h at 1.5 V followed by exposure to 300 µM H2O2 for 5 h. Cytoprotective effects and cell adhesion ability were significantly increased by EleS as evaluated by transferase-mediated dUTP nick-end labelling (TUNEL), lactate dehydrogenase (LDH) release assay, and adhesion assay. EleS increased phosphorylation of AKT, focal adhesion kinase (FAK), and glycogen synthase kinase (GSK3β), as well as decreased caspase-3 cleavage. Interestingly, inhibition of AKT or FAK abolished the pro-survival effects of EleS. We found that connective tissue growth factor (Ctgf) was responsible for EleS-induced CSC survival and adhesion.The survival rate of EleSCSCs after transplantation in the infarcted myocardium was significantly increased together with improvement in cardiac function. Importantly, knockdown of Ctgf abolished EleS-induced cytoprotective effects and recovery of cardiac function. Furthermore, we identified miR-378 as a potential Ctgf regulator in EleSCSCs.
EleS enhanced CSC survival in vitro and in vivo as well as functional recovery of the ischaemic heart through an AKT/FAK/CTGF signalling pathway. It is suggested that Ctgf and miR-378 are novel therapeutic targets for stem cell-based therapy.
Electrical stimulation; Survival; Ischaemic heart; CTGF; miR-378
Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca2+)-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca2+ entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation.
Methods and results
We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca2+ mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca2+ concentration ([Ca2+]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd3+-sensitive current with similar features of the Ca2+ release-activated Ca2+ current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca2+/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca2+/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca2+ influx, CREB activation and VSMCs proliferation.
Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.
Smooth muscle; Proliferation; Ion channels; EGFR
Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2–4) in cardiac excitation–contraction coupling (ECC).
Methods and results
Sarcomere shortening and Ca2+ transients were recorded simultaneously in adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca2+ transients, sarcomere shortening, and troponin I phosphorylation, whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3, or 4 increased phosphorylation of the myosin-binding protein C, but neither had an effect on L-type Ca2+ channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared with individual PDE inhibition, but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal β-adrenergic receptor stimulation increased ECC, and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4 = PDE3 > PDE2. Identical results were obtained on ECC protein phosphorylation.
Our results demonstrate that PDE2, PDE3, and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca2+ transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance, upon β-adrenergic stimulation or PDE3 inhibition.
Phosphodiesterase; Excitation–contraction coupling; cAMP; Protein phosphorylation
Dissection and rupture of the ascending aorta are life-threatening conditions resulting in 80% mortality. Ascending aortic replacement in patients presenting with thoracic aortic aneurysm (TAA) is determined by metric measurement. However, a significant number of dissections occur outside of the parameters suggested by the current guidelines. We investigate the correlation among altered haemodynamic condition, oxidative stress, and vascular smooth muscle cell (VSMC) phenotype in controlling tissue homoeostasis.
Methods and results
We demonstrate using finite element analysis (FEA) based on computed tomography geometries that TAA patients have higher wall stress in the ascending aorta than non-dilated patients. We also show that altered haemodynamic conditions are associated with increased levels of reactive oxygen species (ROS), direct regulators of the VSMC phenotype in the microregional area of the ascending aorta. Using in vitro and ex vivo studies on human tissues, we show that ROS accumulation correlates with media layer degeneration and increased connective tissue growth factor (CTGF) expression, which modulate the synthetic VSMC phenotype. Results were validated by a murine model of TAA (C57BL/6J) based on Angiotensin II infusion showing that medial thickening and luminal expansion of the proximal aorta is associated with the VSMC synthetic phenotype as seen in human specimens.
Increased peak wall stress correlates with change in VSMC towards a synthetic phenotype mediated by ROS accumulation via CTGF. Understanding the molecular mechanisms that regulate VSMC towards a synthetic phenotype could unveil new regulatory pathways of aortic homoeostasis and impact the risk-stratification tool for patients at risk of aortic dissection and rupture.
Thoracic aortic aneurysm; ROS; CTGF; VSMC phenotype
Ischaemic heart disease (IHD) remains the leading cause of death and disability worldwide. As a result, novel therapies are still needed to protect the heart from the detrimental effects of acute ischaemia–reperfusion injury, in order to improve clinical outcomes in IHD patients. In this regard, although a large number of novel cardioprotective therapies discovered in the research laboratory have been investigated in the clinical setting, only a few of these have been demonstrated to improve clinical outcomes. One potential reason for this lack of success may have been the failure to thoroughly assess the cardioprotective efficacy of these novel therapies in suitably designed preclinical experimental animal models. Therefore, the aim of this Position Paper by the European Society of Cardiology Working Group Cellular Biology of the Heart is to provide recommendations for improving the preclinical assessment of novel cardioprotective therapies discovered in the research laboratory, with the aim of increasing the likelihood of success in translating these new treatments into improved clinical outcomes.
Cardioprotection; Myocardial infarction; Animal models; Ischaemia; Reperfusion
The formyl peptide receptor (FPR) subtype FPR2/ALX transduces pro-inflammatory responses and participates in the resolution of inflammation depending on activation. The aim of the present study was to unravel the role of FPR2/ALX signalling in atherosclerosis.
Methods and results
Expression of FPR2/ALX was analysed in 127 human carotid atherosclerotic lesions and revealed that this receptor was expressed on macrophages, smooth muscle cells (SMCs), and endothelial cells. Furthermore, FPR2/ALX mRNA levels were significantly up-regulated in atherosclerotic lesions compared with healthy vessels. In multiple regression, age, creatinine, and clinical signs of increased cerebral ischaemia were independent predictors of FPR2/ALX expression. To provide mechanistic insights into these observations, we generated Ldlr−/−xFpr2−/− mice, which exhibited delayed atherosclerosis development and less macrophage infiltration compared with Ldlr−/−xFpr2+/+ mice. These findings were reproduced by transplantation of Fpr2−/− bone marrow into Ldlr−/− mice and further extended by in vitro experiments, demonstrating a lower inflammatory state in Fpr2−/− macrophages. FPR2/ALX expression correlated with chemo- and cytokines in human atherosclerotic lesions and leucocytes. Finally, atherosclerotic lesions in Ldlr−/−xFpr2−/− mice exhibited decreased collagen content, and Fpr2−/− SMCs exhibited a profile of increased collagenase and decreased collagen production pathways.
FPR2/ALX is proatherogenic due to effects on bone marrow-derived cells, but promoted a more stable plaque phenotype through effects on SMCs. Taken together, these results suggest a dual role of FPR2/ALX signalling in atherosclerosis by way of promoting disease progression and but increasing plaque stability.
Formyl peptide receptors; Inflammation; Lipid mediators; Smooth muscle cells
During cardiac hypertrophy, cardiomyocytes (CMs) increase in the size and expression of cytoskeletal proteins while reactivating a foetal gene programme. The process is proposed to be dependent on increased nuclear export and, since nuclear pore trafficking has limited capacity, a linked decrease in import. Our objective was to investigate the role of nuclear import and export in control of hypertrophy in rat and human heart failure (HF).
Methods and results
In myocardial tissue and isolated CMs from patients with dilated cardiomyopathy, nuclear size was increased; Nucleoporin p62, cytoplasmic RanBP1, and nuclear translocation of importins (α and β) were decreased while Exportin-1 was increased. CM from a rat HF model 16 weeks after myocardial infarction (MI) reproduced these nuclear changes. Nuclear import, determined by the rate of uptake of nuclear localization sequence (NLS)-tagged fluorescent substrate, was also decreased and this change was observed from 4 weeks after MI, before HF has developed. Treatment of isolated rat CMs with phenylephrine (PE) for 48 h produced similar cell and nuclear size increases, nuclear import and export protein rearrangement, and NLS substrate uptake decrease through p38 MAPK and HDAC-dependent pathways. The change in NLS substrate uptake occurred within 15 min of PE exposure. Inhibition of nuclear export with leptomycin B reversed established nuclear changes in PE-treated rat CMs and decreased NLS substrate uptake and cell/nuclear size in human CMs.
Nuclear transport changes related to increased export and decreased import are an early event in hypertrophic development. Hypertrophy can be prevented, or even reversed, by targeting import/export, which may open new therapeutic opportunities.
Myocytes; Hypertrophy; Myocardial infarction; Signal transduction; Nuclear transport
Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation–contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in cardiomyocytes.
Methods and results
Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; αMHC-shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by >70% in αMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes implicated in TT development remained mostly unchanged. At the same time, intracellular Ca2+ handling was disrupted in ventricular myocytes from αMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection fraction, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8.
Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.
Transverse tubules; E-C coupling; Junctophilin-2; Developmental biology
To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion.
Methods and results
Seven days post-permanent ligation, hearts from CD13 knockout (CD13KO) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13KO infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13KO mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13KO infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13KO infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling.
In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.
CD13; Myocardial infarction; Dendritic cells; Macrophages; Monocytes
Caveolae are membrane microdomains where important signalling pathways are assembled and molecular effects transduced. In this study, we hypothesized that shear stress-mediated vasodilation (SSD) of mouse small coronary arteries (MCA) is caveolae-dependent.
Methods and results
MCA (80–150 μm) isolated from wild-type (WT) and caveolin-1 null (Cav-1−/−) mice were subjected to physiological levels of shear stress (1–25 dynes/cm2) with and without pre-incubation of inhibitors of nitric oxide synthase (L-NAME), cyclooxygenase (indomethacin, INDO), or cytochrome P450 epoxygenase (SKF 525A). SSD was endothelium-dependent in WT and Cav-1−/− coronaries but that in Cav-1−/− was significantly diminished compared with WT. Pre-incubation with L-NAME, INDO, or SKF 525A significantly reduced SSD in WT but not in Cav-1−/− mice. Vessels from the soluble epoxide hydrolase null (Ephx2−/−) mice showed enhanced SSD, which was further augmented by the presence of arachidonic acid. In donor–detector-coupled vessel experiments, Cav-1−/− donor vessels produced diminished dilation in WT endothelium-denuded detector vessels compared with WT donor vessels. Shear stress elicited a robust intracellular Ca2+ increase in vascular endothelial cells isolated from WT but not those from Cav-1−/− mice.
Integrity of caveolae is critical for endothelium-dependent SSD in MCA. Cav-1−/− endothelium is deficient in shear stress-mediated generation of vasodilators including NO, prostaglandins, and epoxyeicosatrienoic acids. Caveolae plays a critical role in endothelial signal transduction from shear stress to vasodilator production and release.
Caveolae; Shear stress; Coronary artery; Soluble epoxide hydrolase; Calcium