Insulin and GSK3ß-inhibition abrogates the infarct sparing-effect of ischemic postconditioning in ex vivo rat hearts.

OBJECTIVES: Pharmacological treatment of reperfusion injury using insulin and GSK3ß inhibition has been shown to be cardioprotective, however, their interaction with the endogenous cardioprotective strategy, ischemic postconditioning, is not known. DESIGN: Langendorff perfused ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. For the first 15 min of reperfusion hearts received either vehicle (Ctr), insulin (Ins) or a GSK3ß inhibitor (SB415286; SB41), with or without interruption of ischemic postconditioning (IPost; 3 × 30 s of global ischemia). In addition, the combination of insulin and SB41 for 15 min was assessed. RESULTS: Insulin, SB41 or IPost significantly reduced infarct size versus vehicle treated controls (IPost 33.5 ± 3.3%, Ins 33.5 ± 3.4%, SB41 30.5 ± 3.0% vs. Ctr 54.7 ± 6.8%, p < 0.01). Combining insulin and SB415286 did not confer additional cardioprotection compared to the treatments given alone (SB41 + Ins 26.7 ± 3.5%, ns). Conversely, combining either of the pharmacological reperfusion treatments with IPost completely abrogated the cardioprotection afforded by the treatments separately (Ins + IPost 59.5 ± 3.4% vs. Ins 33.5 ± 3.4% and SB41 + IPost 50.2 ± 6.6% vs. SB41 30.5 ± 3.0%, both p < 0.01), and was associated with blunted Akt, GSK3ß and STAT3 phosphorylation. CONCLUSION: Pharmacological reperfusion treatment with insulin and SB41 interferes with the cardioprotection afforded by ischemic postconditioning.

Reperfusion therapy with low-dose insulin or insulin-like growth factor 2; myocardial function and infarct size in a porcine model of ischaemia and reperfusion.

In an open-chest porcine model, we examined whether myocardial pharmacological conditioning at the time of reperfusion with low-dose insulin or insulin-like growth factor 2 (IGF2), not affecting serum glucose levels, could reduce infarct size and improve functional recovery. Two groups of anaesthetized pigs with either 60 or 40 min. of left anterior descending artery occlusion (total n = 42) were randomized to receive either 0.9% saline, insulin or IGF2 infusion for 15 min., starting 5 min. before a 180-min. reperfusion period. Repeated fluorescent microsphere injections were used to confirm ischaemia and reperfusion. Area at risk and infarct size was determined with Evans blue and triphenyltetrazolium chloride staining. Local myocardial function was evaluated with multi-layer radial tissue Doppler strain and speckle-tracking strain from epicardial echocardiography. Western blotting and TUNEL staining were performed to explore apoptosis. Infarct size did not differ between treatment groups and was 56.7 ± 6.8%, 49.7 ± 9.6%, 56.2 ± 8.0% of area at risk for control, insulin and IGF2 group, respectively, in the 60-min. occlusion series. Corresponding values were 45.6 ± 6.0%, 48.4 ± 7.2% and 34.1 ± 5.8% after 40-min. occlusion. Global and local cardiac function did not differ between treatment groups. No differences related to treatment could be found in myocardial tissue cleaved caspase-3 content or the degree of TUNEL staining. Reperfusion therapy with low-dose insulin or with IGF2 neither reduced infarct size nor improved function in reperfused myocardium in this in vivo porcine model.

Activation of corticotropin releasing factor receptor type 2 in the heart by corticotropin releasing factor offers cytoprotection against ischemic injury via PKA and PKC dependent signaling.

Corticotrophin-releasing factor receptor 2ß (CRFR2ß) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.

Daunorubicin therapy is associated with upregulation of E3 ubiquitin ligases in the heart.

Daunorubicin (DNR) and doxorubicin (DOX) are two of the most effective anthracycline drugs known for the treatment of systemic neoplasms and solid tumors. However, their clinical use is hampered due to profound cardiotoxicity. The mechanism by which DNR injures the heart remains to be fully elucidated. Recent reports have indicated that DOX activates ubiquitin proteasome-mediated degradation of specific transcription factors; however, no reports exist on the effect of DNR on the E3 ubiquitin ligases, MURF-1 (muscle ring finger 1) and MAFbx (muscle atrophy F-box). The aim of this study was to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate some of the signalling mechanisms involved. Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze-clamped for biochemical analysis. DNR treatment significantly attenuated cardiac function and increased apoptosis in the heart. DNR-induced cardiac cytotoxicity was associated with upregulation of the E3 ligases, MURF-1 and MAFbx and also caused significant increases in two markers of autophagy, beclin-1 and LC3. These changes observed in the heart were also associated with attenuation of the phosphoinositide 3-kinase/Akt signalling pathway.