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.

Intermittent insulin treatment mimics ischemic postconditioning via MitoKATP channels, ROS, and RISK.

OBJECTIVES: It has previously been demonstrated that 15-min continuous insulin infusion at immediate reperfusion affords cardioprotection. This study sought to reduce the treatment time of insulin and test if intermittent insulin infusions can mimic ischemic postconditioning. DESIGN: In a Langendorff perfused rat heart model of regional ischemia, hearts were at the onset of reperfusion subjected to either 5- or 1-min continuous insulin infusion or 3 × 30 s intermittent insulin infusions (InsPost); with or without inhibitors of Akt (SH-6), p70s6-kinase (rapamycin), mitochondrial ATP-sensitive potassium channels (5-hydroxydecanoic acid [5-HD]), or a scavenger of reactive oxygen species (ROS; 2-mercaptopropionyl glycine [MPG]). Infarct size is expressed as percent of area at risk and presented as mean ± standard error of the mean or s.e.m. RESULTS: Only InsPost was able to reduce infarct size compared with controls (InsPost 33 ± 6% vs. Ctr 52 ± 4%, p < 0.05.). This cardioprotection was abrogated by co-administering SH-6, rapamycin, 5-HD, or MPG. (InsPost + SH-6 56 ± 9%, InsPost + Rapa 55 ± 8%, InsPost + 5-HD 56 ± 7%, InsPost + MPG 60 ± 3% vs. InsPost 33 ± 6% p < 0.05). These results were corroborated by a significant increase in phosphorylated Akt and p70s6k in the InsPost group compared with controls. CONCLUSION: Short intermittent insulin infusions can mimic ischemic postconditioning and reduce myocardial infarct size via Akt/p70s6k and mKATP channels/ROS-dependent signaling.

Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart.

Since normalization strategies plays a pivotal role for obtaining reliable results when performing quantitative PCR (qPCR) analyses, this study investigated several miRNA normalization candidates in regards to their efficiency as normalization standards in the ischemic reperfused ex vivo rat heart, with special reference to regulation of the miRNAs miR-1 and miR-101b. The possibility of including primers for several miRNAs in one reverse transcription (RT) reaction was also investigated. Langendorff perfused rat hearts were subjected to 30 min regional ischemia and 0, 1, 5, 15, or 120 min reperfusion. Total RNA was isolated and reverse transcribed for miRNA qPCR analysis. Normalization candidates were evaluated by the NormFinder and geNorm algorithms and the following stability expression rank order was obtained: sno202 < U6B < U87 < snoRNA < 4.5S RNA A < Y1 < 4.5S RNA B < GAPDH. Applying U6B as a normalizer it was found that miR-1 and miR-101b was downregulated in the ischemic reperfused myocardium. Furthermore, up to three primers could be included in one RT reaction by replacing RNase-free water with two supplemental sets of primers in the TaqMan MicroRNA assay protocol. This study demonstrates the importance of validating normalization standards when performing miRNA expression analyses by qPCR, and that miR-1 and miR-101b may play an important role during early reperfusion of the ischemic rat heart.

Exploring the human plasma proteome for humoral mediators of remote ischemic preconditioning--a word of caution.

Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where scientists can mine for novel potential targets.

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.

Human catestatin peptides differentially regulate infarct size in the ischemic-reperfused rat heart.

In acute myocardial infarction increased plasma levels of chromogranin A are correlated with decreased survival. At the human chromogranin A gene locus there are two naturally occurring amino acid substitution variants within the catestatin region, i.e. Gly³64Ser and Pro³7°Leu, displaying differential potencies towards inhibition of nicotinic cholinergic agonist-evoked catecholamine secretion from sympathochromaffin cells and different degrees of processing from the prohormone. Here, we examine whether two of the variants and the wild type catestatin may affect the development of infarct size during ischemic reperfusion in the Langendorff rat heart model. The hearts were subjected to regional ischemia followed by reperfusion in the presence or absence of synthetic variants of human catestatin. Compared to the Gly³64Ser variant both the wild type and Pro³7°Leu variants increased infarct size while decreasing the cardiac levels of phosphorylated Akt and two of its downstream targets, FoxO1 and BAD. In conclusion, these findings suggest that, in contrast to the Gly³64Ser variant, wild type catestatin and the Pro³7°Leu variant (allele frequency ~0.3%) increased myocardial infarct size via a mechanism involving dephosphorylation of Akt and the two downstream targets during ischemic reperfusion in the isolated rat heart.