Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results Positron Emission Tomography (PET) studies revealed that transgenic mice with cardiac-specific overexpression of GRK2 negatively impacted cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes HF development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 (IRS1) in cardiomyocytes causing insulin-dependent negative signaling feedback including inhibition of membrane translocation of the glucose transporter, GLUT4. This identifies IRS1 as a novel non-receptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents post-ischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of HF. Conclusions Our data provide novel insight into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through βARs and metabolism through the insulin receptor.

Rationale GRK2 is abundantly expressed in the heart and its expression and activity is increased in injured or stressed myocardium. This up-regulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes and its inhibition by a peptide comprised of the last 194 amino acids of GRK2 (known as βARKct) is cardioprotective. Objective The aim of this study was to elucidate the signaling mechanism that accounts for the pro-death signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the βARKct. Methods and Results Using in vivo mouse models of ischemic injury and also cultured myocytes we found that GRK2 localizes to mitochondria providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced pro-death signaling. Localization of GRK2 to mitochondria was dependent upon phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases (ERKs), resulting in enhanced GRK2 binding to heat shock protein 90 (Hsp90), which chaperoned GRK2 to mitochondria. Mechanistic studies invivo and invitro showed that ERK regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent pro-death signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions We identify GRK2 as a pro-death kinase in the heart acting in a novel manner through mitochondrial localization via ERK regulation.

We tested the hypothesis that the high-conductance calciumactivated potassium (K Ca ) channel is involved in the cardioprotection of preconditioning with ischemic insults. In the isolated perfused rat heart subjected to ischemia/reperfusion, effects of ischemic preconditioning (IPC) on infarct size and lactate dehydrogenase (LDH) release were abolished by 1 M paxilline (Pax), an inhibitor of the K Ca channel, administered 30 min before, but not during, ischemia. In isolated ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), preconditioning with MI/A increased their viability, and the effect was abolished by administering Pax before MI/A. Like IPC, 10 M NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-trifluoromethyl-2Hbenzimidazol-2-one; NS), an opener of K Ca channels, reduced infarct size and LDH release, effects attenuated by Pax. The harmful and protective effects of blockade and activation of the K Ca channel were accompanied by impaired and improved left ventricular contractile functions, respectively. In addition, the effect of NS was not altered by 100 M 5-hydroxydecanoate, an inhibitor of the K ATP channel. Neither was the effect of 100 M diazoxide, an activator of the K ATP channel, altered by Pax. Furthermore, opening of the mitochondrial permeability transition pore (mPTP) with 20 M atractyloside abolished the beneficial effects of IPC or NS in the isolated rat heart and myocyte. Inhibition of mPTP opening with 0.2 M cyclosporin A decreased the infarct size and LDH release and improved the contractile function, effects not attenuated by Pax. In conclusion, the study provides evidence that the K Ca channel triggers cardioprotection of IPC, which involves mPTP.

The results show a significant dependence of the skin surface topography on the age of the volunteer and the body site measured. Particularly the waviness, the Fourier analysis and the furrow profiles reflect such dependencies. The system used also fulfils the requirements for the testing of therapies, as was demonstrated by the reduction of mimic wrinkles after treatment with botulinum Toxin A.

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To test the hypothesis that estrogen confers cardioprotection by suppressing the expression of ␤-adrenoceptor (␤-AR), we first correlated the infarct size in response to ischemic insult and ␤-AR stimulation with the expression of ␤ 1 -AR in sham, ovariectomized (Ovx) and estrogen replaced (Ovx ϩ E 2 ) rats. When ␤-AR is being activated during ischemia, the infarct size was significantly greater in Ovx than in the sham and Ovx ϩ E 2 rats. There is a negative correlation between the infarct size and the expression level of ␤ 1 -AR as revealed by Western blotting and supported by binding analysis. Incubation of ventricular myocytes from Ovx rats with estrogen at 10 Ϫ9

Cardiac delta- and kappa- but not mu-ORs mediate the cardioprotection produced by RPC. Both protein kinase C and the mitochondrial KATP channel were involved in this effect.