Interaction of ß1-adrenoceptor with RAGE mediates cardiomyopathy via CaMKII signaling.

Stimulation of ß1-adrenergic receptor (ß1AR), a GPCR, and the receptor for advanced glycation end-products (RAGE), a pattern recognition receptor (PRR), have been independently implicated in the pathogenesis of cardiomyopathy caused by various etiologies, including myocardial infarction, ischemia/reperfusion injury, and metabolic stress. Here, we show that the two distinctly different receptors, ß1AR and RAGE, are mutually dependent in mediating myocardial injury and the sequelae of cardiomyopathy. Deficiency or inhibition of RAGE blocks ß1AR- and RAGE-mediated myocardial cell death and maladaptive remodeling. Ablation or blockade of ß1AR fully abolishes RAGE-induced detrimental effects. Mechanistically, RAGE and ß1AR form a complex, which in turn activates Ca2+/calmodulin-dependent kinase II (CaMKII), resulting in loss of cardiomyocytes and myocardial remodeling. These results indicate that RAGE and ß1AR not only physically crosstalk at the receptor level, but also functionally converge at the common mediator, CaMKII, highlighting a combined inhibition of RAGE and ß1AR as a more effective therapy to treat diverse cardiovascular diseases, such as myocardial infarction, ischemia/reperfusion injury, and diabetic cardiovascular complications.

Regulation of swelling-activated Cl(-) current by angiotensin II signalling and NADPH oxidase in rabbit ventricle.

AIMS: We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl(-) current (I Cl,swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit I(Cl,swell) upon mechanical stretch of beta(1D) integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced I Cl,swell. METHODS AND RESULTS: I Cl,swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced I Cl,swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT1) antagonists. AT1 stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both I Cl,swell and AngII-induced Cl(-) current, whereas exogenous EGF evoked a Cl(-) current that was suppressed by osmotic shrinkage. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase, and PI-3K inhibitors LY294002 and wortmannin blocked I Cl,swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, O2*. NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated I Cl,swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. O2* rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited I Cl,swell, and exogenous H2O2 overcame suppression of I Cl,swell by AT1 receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. CONCLUSION: Activation of I Cl,swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in I Cl,swell activation by integrin stretch. This in part explains why I Cl,swell is persistently activated in several models of cardiac disease.

EGFR kinase regulates volume-sensitive chloride current elicited by integrin stretch via PI-3K and NADPH oxidase in ventricular myocytes.

Stretch of beta1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl(-) SAC resembles the volume-sensitive Cl(-) current (I(Cl,swell)). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl(-) SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for beta1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl(-) SAC that was 1.13 +/- 0.10 pA/pF at +40 mV. AG1478 (10 muM), an EGFR kinase blocker, inhibited 93 +/- 13% of Cl(-) SAC, and intracellular pretreatment with 1 muM AG1478 markedly suppressed Cl(-) SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl(-) current (0.81 +/- 0.05 pA/pF at +40 mV) that was fully blocked by 10 muM tamoxifen, an I(Cl,swell) blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 microM), blockers of PI-3K, inhibited Cl(-) SAC by 67 +/- 6% and 91 +/- 25% respectively, and the EGF-induced Cl(-) current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl(-) current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl(-) SAC and EGF-induced Cl(-) current by 88 +/- 9% and 127 +/- 11%, respectively. These results suggest that beta1 integrin stretch activates Cl(-) SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl(-) SAC and the EGF-induced Cl(-) currents are likely to be the volume-sensitive Cl(-) current, I(Cl,swell).

Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes.

Direct stretch of beta1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl(-) SAC resemble those of the volume-sensitive Cl(-) current, I(Cl,swell). Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for beta1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 microM), an AT1R competitive antagonist, blocked Cl(-) SAC but did not significantly alter the background Cl(-) current in the absence of integrin stretch. AT1R signaling is mediated largely by H(2)O(2) produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 microM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl(-) SAC elicited by stretch and the background Cl(-) current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl(-) SAC as well as a large fraction of the background Cl(-) current. With continuing integrin stretch, Cl(-) SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl(-) current that was rapidly and completely blocked by DPI (60 microM). Moreover, exogenous H(2)O(2) (10, 100, and 500 microM), the eventual product of NADPH oxidase activity, also activated Cl(-) SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H(2)O(2) scavenger, attenuated the response to stretch. Application of H(2)O(2) during NADPH oxidase inhibition by either DPI (60 microM) or AEBSF (0.5 mM) did not fully reactivate Cl(-) SAC, however. These results suggest that stretch of beta1-integrin in cardiac myocytes elicits Cl(-) SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl(-) SAC, providing a second regulatory pathway.

Stretch of beta 1 integrin activates an outwardly rectifying chloride current via FAK and Src in rabbit ventricular myocytes.

Osmotic swelling of cardiac myocytes and other types of cells activates an outwardly rectifying, tamoxifen-sensitive Cl- current, ICl,swell, but it is unclear whether Cl- currents also are activated by direct mechanical stretch. We tested whether specific stretch of beta1-integrin activates a Cl- current in rabbit left ventricular myocytes. Paramagnetic beads (4.5-microm diameter) coated with mAb to beta1-integrin were applied to the surface of myocytes and pulled upward with an electromagnet while recording whole-cell current. In solutions designed to isolate anion currents, beta1-integrin stretch elicited an outwardly rectifying Cl- current with biophysical and pharmacological properties similar to those of ICl,swell. Stretch-activated Cl- current activated slowly (t1/2 = 3.5 +/- 0.1 min), partially inactivated at positive voltages, reversed near ECl, and was blocked by 10 microM tamoxifen. When stretch was terminated, 64 +/- 8% of the stretch-induced current reversed within 10 min. Mechanotransduction involved protein tyrosine kinase. Genistein (100 microM), a protein tyrosine kinase inhibitor previously shown to suppress ICl,swell in myocytes, inhibited stretch-activated Cl- current by 62 +/- 6% during continued stretch. Because focal adhesion kinase and Src are known to be activated by cell swelling, mechanical stretch, and clustering of integrins, we tested whether these tyrosine kinases mediated the response to beta1-integrin stretch. PP2 (10 microM), a selective blocker of focal adhesion kinase and Src, fully inhibited the stretch-activated Cl- current as well as part of the background Cl- current, whereas its inactive analogue PP3 (10 microM) had no significant effect. In addition to activating Cl- current, stretch of beta1-integrin also appeared to activate a nonselective cation current and to suppress IK1. Integrins are the primary mechanical link between the extracellular matrix and cytoskeleton. The present results suggest that integrin stretch may contribute to mechano-electric feedback in heart, modulate electrical activity, and influence the propensity for arrhythmogenesis.