Calcineurin splicing variant calcineurin Aβ1 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 Aβ (CnAβ) gene generates the CnAβ1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAβ1 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 CnAβ1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAβ1 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 CnAβ1 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 CnAβ1 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 Aβ1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAβ1 an attractive candidate for the development of future therapeutic approaches.British Heart Foundation [PG/07/020/22503]; Spanish Ministry of Science and Innovation [BFU2009-10016]; National Institutes of Health Research, Cardiovascular Biomedical Research Unit at the Royal Brompton; Hare-field NHS Foundation Trust; Imperial College; Spanish Fondo Nacional de Investigaciones Sanitarias [EIF-040545, ERG-239158, CP08/00144

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

International audienceTransglutaminase 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

β-Adrenoceptor subtype expression and function in rat white adipocytes

1. The pharmacological features of rat white adipocyte β-adrenoceptor subtypes were investigated by saturation and β-agonist competition studies with [(3)H]-CGP 12177 and by lipolysis induced by β-agonists as well as their inhibition by CGP 20712A (selective β(1)-antagonist) and ICI 118551 (selective β(2)-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of β-adrenoceptor subtypes. 2. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22±0.07 nM) and the other with low affinity (23±7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3. The competition curves, with 15 nM [(3)H]-CGP 12177, for the β-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective β(3)-agonist, BRL 37344 (BRL), were clearly biphasic (P<0.001). The rank orders of agonist potency (pK(i)) in competing for [(3)H]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28±0.24)>NA (8.90±0.12)>Ad (8.65±0.12)>>BRL (4.53±0.17) and BRL (7.38±0.19)>>Iso (2.96±0.26)⩾NA (2.80±0.17)>Ad (2.10±0.11) indicating the expression of β(1)- and β(3)-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective β(1)-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81±9 fmol mg(−1)) and high pK(i) value (7.23±0.26) confirming the presence of β(1)-adrenoceptors. 4. To assess a possible contribution of the β(2)-subtype, procaterol (Proc), a selective β(2)-agonist, was used to compete with 2 nM [(3)H]-CGP 12177. A single low affinity (4.61±0.07) population of binding sites was identified. The density of these sites (71±12 fmol mg(−1)) was similar to the one obtained with Xam, suggesting that Proc displaced [(3)H]-CGP 12177 from the β(1)-subtype. 5. The functional potency (pD(2)) order with BRL (9.07±0.20) and catecholamines (Iso: 7.26±0.06, NA: 6.89±0.02 and Ad: 6.32±0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31±0.06 and 5.66±0.10, respectively. Proc stimulated lipolysis with a low potency (5.59±0.21). 6. The lipolytic response to 0.001 μM BRL was inhibited by both, selective β(1)- and β(2)-antagonist, in a monophasic manner with low potencies (CGP 20712A pK(i): <4.5 and ICI 118551 pK(i): 5.57±0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (>10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 μM Iso or 0.13 μM NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pK(i) values for the first sites were 8.41±0.09 and 8.58±0.17, respectively, and for the second population of sites 4.73±0.22 and 4.27±0.27, respectively. The proportion of the first sites was low: 19±4 and 22±5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 μM Proc (CGP 20712A: pK(i)1: 8.17±0.08, site1: 23±6%, pK(i)2: 4.77±0.14; ICI 118551: pK(i)1: 7.78±0.03, site1: 37±2%, pK(i)2: 5.35±0.25). 7. Our results show that the radioligand [(3)H]-CGP 12177 allows the characterization of β(1)- and β(3)-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on β(1)- and β(3)-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the β(3)-subtype