Calcineurin splicing variant calcineurin Aß1 improves cardiac function after myocardial infarction without inducing hypertrophy.

BACKGROUND: Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin Aß (CnAß) gene generates the CnAß1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAß1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown. METHODS AND RESULTS: We generated transgenic mice overexpressing CnAß1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAß1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnAß1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnAß1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation. CONCLUSIONS: Calcineurin Aß1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAß1 an attractive candidate for the development of future therapeutic approaches.

Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea.

The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac mesodermal induction in undifferentiated cells independently of cell proliferation. This analysis suggests that IGF-1Ea may be a good candidate to improve both in vitro production of cardiomyocytes from pluripotent stem cells and in vivo activation of the differentiation program of cardiac progenitor cells.

Heparan sulfate proteoglycans are receptors for the cell-surface trafficking and biological activity of transglutaminase-2.

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.

Characterization of ERK1/2 signalling pathways induced by adenosine receptor subtypes in newborn rat cardiomyocytes.

1. Adenosine A(1), A(2A), and A(3) receptors (ARs) and extracellular signal-regulated kinase 1/2 (ERK1/2) play a major role in myocardium protection from ischaemic injury. In this study, we have characterized the adenosine receptor subtypes involved in ERK1/2 activation in newborn rat cardiomyocytes. 2. Adenosine (nonselective agonist), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)), all increased ERK1/2 phosphorylation in a time- and dose-dependent manner. The combined maximal response of the selective agonists was similar to adenosine alone. Theophylline (nonselective antagonist) inhibited completely adenosine-mediated ERK1/2 activation, whereas a partial inhibition was obtained with DPCPX (A(1)), ZM 241385 (A(2A)), and MRS 1220 (A(3)). 3. PD 98059 (MEK1; ERK kinase inhibitor) abolished all agonist-mediated ERK1/2 phosphorylation. Pertussis toxin (PTX, G(i/o) blocker) inhibited completely CPA- and partially adenosine- and Cl-IB-MECA-induced ERK1/2 activation. Genistein (tyrosine kinase inhibitor) and Ro 318220 (protein kinase C, PKC inhibitor) partially reduced adenosine, CPA and Cl-IB-MECA responses, without any effect on CGS 21680-induced ERK1/2 phosphorylation. H89 (protein kinase A, PKA inhibitor) abolished completely CGS 21680 and partially adenosine and Cl-IB-MECA responses, without any effect on CPA response. 4. Cl-IB-MECA-mediated increases in cAMP accumulation suggest that A(3)AR-induced ERK1/2 phosphorylation involves adenylyl cyclase activation via phospholipase C (PLC) and PKC stimulation. 5. In summary, we have shown that ERK1/2 activation by adenosine in cardiomyocytes results from an additive stimulation of A(1), A(2A), and A(3)ARs, which involves G(i/o) proteins, PKC, and tyrosine kinase for A(1) and A(3)ARs, and Gs and PKA for A(2A)ARs. Moreover, the A(3)AR response also involves a cAMP/PKA pathway via PKC activation.

Heat shock protein-27 delays acute rejection after cardiac transplantation: an experimental model.

BACKGROUND: Rejection is the major obstacle to survival after cardiac transplantation. We investigated whether overexpression of heat shock protein (Hsp)-27 in mouse hearts protects against acute rejection and the mechanisms of such protection. METHODS: Hearts from B10.A mice overexpressing human Hsp-27 (Hsp-27tg), or Hsp-27-negative hearts from littermate controls (LCs) were transplanted into allogeneic C57BL/6 mice. The immune response to B10.A hearts was investigated using quantitative polymerase chain reaction for CD3+, CD4+, CD8+ T cells, and CD14+ monocytes and cytokines (interferon-γ, interleukin [IL]-2, tumor necrosis factor-α, IL-1ß, IL-4, IL-5, IL-10, transforming growth factor-ß) in allografts at days 2, 5, and 12 after transplantation. The effect of Hsp-27 on ischemia-induced caspase activation and immune activation was investigated. RESULTS: Survival of Hsp-27tg hearts (35±10.37 days, n=10) was significantly prolonged compared with LCs (13.6±3.06 days, n=10, P=0.0004). Hsp-27tg hearts expressed significantly more messenger RNA (mRNA) markers of CD14+ monocytes at day 2 and less mRNA markers of CD3+ and CD8+T cells at day 5 compared with LCs. There was more IL-4 mRNA in Hsp-27tg hearts at day 2 and less interferon-γ mRNA at day 5 compared with LCs. Heat shock protein-27tg hearts subjected to ischemia or to 24 hr ischemia-reperfusion injury demonstrated significantly less apoptosis and activation of caspases 3, 9, and 1 than LCs. T cells removed from C57BL/6 recipients of Hsp-27tg hearts produced a vigorous memory response to B10.A antigens, suggesting immune activation was not inhibited by Hsp-27. CONCLUSION: Heat shock protein-27 delays allograft rejection, by inhibiting tissue damage, through probably an antiapoptotic pathway. It may also promote an anti-inflammatory subset of monocytes.

Cross-regulation between beta 1- and beta 3-adrenoceptors following chronic beta-adrenergic stimulation in neonatal rat cardiomyocytes.

BACKGROUND AND PURPOSE: We have previously shown that beta-adrenoceptors continuously stimulated with noradrenaline induces an increase in beta(3)-adrenoceptors (G alpha(i)PCRs) and a decrease in beta(1)-adrenoceptors (G alpha(s)PCRs) at functional, genomic and protein levels. This compensatory modification induced by noradrenaline is probably one of the consequences of cardiac depression observed in heart disease. Therefore, we investigated further the interaction between beta(1)- and beta(3)-adrenoceptors in neonatal rat cardiomyocytes. EXPERIMENTAL APPROACH: Functional studies were performed by cyclic adenosine monophosphate (cAMP) accumulation assays in cells untreated or treated with dobutamine and ICI 118551 (beta(1)-adrenoceptor) or CL-3162436243 (beta(3)-adrenoceptor) for 24 h in the presence or absence of protein kinase inhibitors. Beta-adrenoceptor and protein kinase expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by Western blotting, respectively. KEY RESULTS: Chronic beta(1)- or beta(3)-adrenoceptor stimulation reduced beta(1)-adrenoceptor-mediated cAMP accumulation in association with a decrease in beta(1)-adrenoceptor mRNA and protein levels through protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38MAPK) activation. In contrast, both treatments induced an increase in beta(3)-adrenoceptor expression and beta(3)-adrenoceptor-inhibited forskolin response through PKC, extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK phosphorylation, although no beta(3)-adrenoceptor response was observed in untreated cells. ERK1/2 and p38MAPK were activated by both treatments. The modulation of beta(1)- or beta(3)-adrenoceptor function did not require stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) although chronic beta(1)-adrenoceptor stimulation activated SAPK/JNK. Beta(3)-adrenoceptor treatment activated Akt although PI3K was not involved in beta(3)-adrenoceptor up-regulation. CONCLUSION AND IMPLICATIONS: We show for the first time that chronic beta(1)- or beta(3)-adrenoceptor stimulation leads to the modulation of beta(1)- and beta(3)-adrenoceptors by a cross-regulation involving PKC, PI3K p38MAPK and MEK/ERK1/2 pathway, and through protein kinase A when beta(1)-adrenoceptors are chronically activated.

Characterization of P2Y receptor subtypes functionally expressed on neonatal rat cardiac myofibroblasts.

BACKGROUND AND PURPOSE: Little is known about P2Y receptors in cardiac fibroblasts, which represent the predominant cell type in the heart and differentiate into myofibroblasts under certain conditions. Therefore, we have characterized the phenotype of the cells and the different P2Y receptors at the expression and functional levels in neonatal rat non-cardiomyocytes. EXPERIMENTAL APPROACH: Non-cardiomyocyte phenotype was determined by confocal microscopy by using discoidin domain receptor 2, alpha-actin and desmin antibodies. P2Y receptor expression was investigated by reverse transcription-polymerase chain reaction and immunocytochemistry, and receptor function by cAMP and inositol phosphate (IP) accumulation induced by adenine or uracil nucleotides in the presence or absence of selective antagonists of P2Y(1) (MRS 2179, 2-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt), P2Y(6) (MRS 2578) and P2Y(11) (NF 157, 8,8'-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalene trisulphonic acid hexasodium salt) receptors. G(i/o) and G(q/11) pathways were evaluated by using Pertussis toxin and YM-254890 respectively. KEY RESULTS: The cells (>95%) were alpha-actin and discoidin domain receptor 2-positive and desmin-negative. P2Y(1), P2Y(2), P2Y(4), P2Y(6) were detected by reverse transcription-polymerase chain reaction and immunocytochemistry, and P2Y(11)-like receptors at protein level. All di- or tri-phosphate nucleotides stimulated IP production in an YM-254890-sensitive manner. AMP, ADPbetaS, ATP and ATPgammaS increased cAMP accumulation, whereas UDP and UTP inhibited cAMP response, which was abolished by Pertussis toxin. MRS 2179 and NF 157 inhibited ADPbetaS-induced IP production. MRS 2578 blocked UDP- and UTP-mediated IP responses. CONCLUSION AND IMPLICATIONS: P2Y(1)-, P2Y(2)-, P2Y(4)-, P2Y(6)-, P2Y(11)-like receptors were co-expressed and induced function through G(q/11) protein coupling in myofibroblasts. Furthermore, P2Y(2) and P2Y(4) receptor subtypes were also coupled to G(i/o). The G(s) response to adenine nucleotides suggests a possible expression of a new P2Y receptor subtype.

Induction of beta3-adrenergic receptor functional expression following chronic stimulation with noradrenaline in neonatal rat cardiomyocytes.

This study aimed to characterize beta(3)-adrenergic receptors (ARs) in rat neonatal cardiomyocytes using the noradrenaline (NOR) properties to modulate the expression and function of the three beta-ARs. We assessed the effect of NOR (physiological nonselective agonist), isoprenaline (ISO, beta-nonselective agonist), dobutamine (DOB, beta(1)-selective agonist), and procaterol (PROC, beta(2)-selective agonist) on cAMP accumulation using cardiomyocytes untreated or treated with 100 microM NOR for 24 h. The inhibition of forskolin-stimulated cAMP accumulation was determined using NOR, isoprenaline, and the beta(3)-selective agonists 4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344) and 5-[-2-([-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243). The experiments were performed in the absence or presence of propranolol or 2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP 20712A) and/or 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118551) to inhibit beta(1)- and beta(2)-AR stimulation and 1-(2-ethylphenoxy)-3-[[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino-(2S)-2-propanol hydrochloride (SR 59230A) (beta(3)-selective antagonist). In addition, the level of the three subtypes was determined by reverse transcription polymerase chain reaction and Western blotting. NOR pretreatment decreased the activation of cAMP induced by NOR, isoprenaline, and DOB, whereas PROC response was abolished. The inhibition of NOR response by CGP 20712A or ICI 118551 demonstrated that beta(1)- and beta(2)-ARs are down-regulated and that beta(2)-AR functional activity was also abolished in cardiomyocytes exposed to chronic stimulation. beta(3)-AR function was observed with NOR and ISO when beta(1)-/beta(2)-ARs were blocked and with both beta(3)-selective agonists in NOR-treated cells only. This response was completely inhibited by SR 59230A and involved G(i) protein. Furthermore, the results from functional studies agree well with those from expression experiments. In conclusion, these data provide strong evidence that beta(3)-ARs are functionally up-regulated and coupled to G(i) protein in rat neonatal cardiomyocytes following chronic exposure to NOR when beta(1)- and beta(2)-ARs are down-regulated.

Phosphatidylinositol 3-kinase and ERK1/2 are not involved in adenosine A1, A2A or A3 receptor-mediated preconditioning in rat ventricle strips.

Mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (PKB; also known as Akt) are important antiapoptotic signalling pathways which have recently been implicated in cardioprotection. However, at present the involvement of ERK1/2 and PI3-kinase/PKB in adenosine receptor-mediated cardioprotection is poorly understood. In this study we used isolated rat right ventricular strips, contracted by electrical-field stimulation, in order to investigate the role of ERK1/2 and PI3-kinase/PKB in adenosine receptor-induced cardioprotection. Ventricle strips were pretreated for 2 min with the agonists adenosine (non-selective), CPA (A1 selective), CGS 21680 (A2A selective) and Cl-IB-MECA (A3 selective) before 30 min hypoxia followed by 30 min reoxygenation. Each agonist significantly improved posthypoxic percentage contraction recovery compared to control strips. Similarly hypoxic preconditioning (10 min hypoxia followed by 20 min reoxygenation) significantly improved posthypoxic percentage contraction recovery compared to non-preconditioned strips. The selective adenosine receptor antagonists DPCPX (A1), ZM 241385 (A2A) and MRS 1220 (A3) attenuated cardioprotection induced by CPA, CGS 21680 and Cl-IB-MECA, respectively. Pre-incubation (30 min) of ventricle strips with the MEK1 inhibitor PD 98059 (50 microM) or the PI3-kinase inhibitor wortmannin (100 nM) significantly reduced posthypoxic percentage contraction recovery induced by hypoxic preconditioning. In contrast, PD 98059 and wortmannin had no significant effect on cardioprotection induced by CPA, Cl-IB-MECA or CGS 21680. Overall these data indicate that although selective A1, A2A and A3 adenosine receptor agonists induce preconditioning in rat right ventricular strips the effects are independent of ERK1/2- and PI3-kinase-dependent pathways. In contrast ERK1/2 and PI3-kinase-dependent pathways do appear to be involved in early hypoxic preconditioning.

Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes.

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A(1), A(2A) and A(3)) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A(1) selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A(2A) selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O(2)) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A(1) and A(3) receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G(i)/G(o)-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.

Cardiovascular vagosympathetic activity in rats with ventromedial hypothalamic obesity.

OBJECTIVE: Rats with ventromedial hypothalamic lesion (VMH) are massively obese with endogenous hyperinsulinemia, insulin resistance, low sympathetic activity, and high parasympathetic activity, which are likely to induce hypertension. The goal was to follow in this model the long-term hemodynamic changes and to investigate the role of autonomic nervous system and insulin resistance in these changes. RESEARCH METHODS AND PROCEDURES: Heart rate and blood pressure were monitored for 12 weeks after operation using a telemetric system in VMH and sham rats. Plasma catecholamines and heart beta-adrenoceptors were measured. Glucose tolerance was studied after an intravenous glucose injection and insulin sensitivity during a euglycemic hyperinsulinemic clamp test. RESULTS: A marked bradycardia and only a mild increase in blood pressure occurred in VMH rats compared with sham animals. Response to autonomic-acting drugs showed an increase in heart vagal tone and responsiveness to a beta-agonist drug. Plasma catecholamine levels were markedly increased, and the density and affinity of heart beta-adrenoceptors were similar in VMH, sham, and control rats. Muscle glucose use was reduced by 1 week after operation in VMH animals. DISCUSSION: These results show the following in this model of massively obese rats with sympathetic impairment: 1). adrenal medulla secretion is increased, probably as a result of hyperinsulinemia and increased vagal activity; 2). cardiac responsiveness to beta-agonist stimulation is increased; and 3). despite these changes and suspected resistance to the vasodilative effect of insulin, blood pressure does not increase. We conclude that high vagal activity may be protective against hypertension associated with obesity.