Cardiac Preconditioning Research Articles

Abstract 107: The Innate Immune NALP3 Receptor Mediates Cardiac Ischemic Preconditioning in the Isolated Heart

Objective: Recent studies indicate that the innate immune system is not only activated by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As activation triggers (cellular K+ extrusion, ROS production) for the innate immune NALP3 inflammasome protein complex overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NALP3 inflammasome is involved in IPC and acute I/R injury of the heart. Methods & Results: Baseline performance and I/R injury was investigated in isolated, Langendorff-perfused hearts from wild-type, ASC-/- and NALP3-/- mice. Baseline cardiac performance was unaffected by genetic deletion of NALP3 inflammasome components. Deletion of the ASC and NALP3 component exacerbated IR injury on mechanical parameters of the heart. However, deficiency in ASC or NALP3 was without effect on cell death (LDH release during reperfusion). In a separate series we subjected these same hearts to IPC, consisting of 3 x 5 min I/R preceding the 35 min I. IPC protected WT and ASC-/- hearts against IR injury (improved function and less cell death). However, no IPC effects on function or cell death were observed in NALP3-/- hearts. NALP3-/- hearts had significantly decreased cardiac IL-6 levels with a trend towards lower Il-1β levels at end reperfusion, suggesting abrogating of IPC through diminished IL-6 and/or Il-1β signalling. However, subsequent experiments showed that inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) was without effect on IPC in WT hearts. In contrast, neutralising IL-6 using an antibody against IL-6 in WT hearts abrogated IPC in NALP3-/- hearts. Conclusions: The data suggest that the innate immune NALP3 protein, in an NALP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6 dependent mechanism

Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

ObjectiveRecent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart.Principal FindingsBaseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC−/− and NLRP3−/− mice. Deletion of NLRP3 inflammasome components ASC−/− or NLRP3−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3−/− hearts against I/R injury. NLRP3−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3−/− hearts when compared to WT hearts.ConclusionsThe data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.

Mitochondrial ROMK Channel Is a Molecular Component of MitoK ATP

Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP). Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death. The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.

Mitochondrial K+ channels are involved in ischemic postconditioning in rat hearts

The mitochondrial calcium-activated potassium channel (mitoK(Ca)) and the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) are both involved in cardiac preconditioning. Here, we examined whether these two channels are also involved in ischemic or pharmacological postconditioning. Using Langendorff perfusion, rat hearts were made hypoxic for 45 min and then reoxygenated for 30 min. Ischemic postconditioning (IPT) was achieved through application of 3 cycles of 10 s of reperfusion and 10 s of ischemia before reoxygenation, with and without paxilline (Pax; a mitoK(Ca) blocker) or 5-hydroxydecanoate (5-HD; a mitoK(ATP) blocker). Pharmacological postconditioning was carried out for 5 min at the onset of reoxygenation using NS1619 (a mitoK(Ca) opener) or diazoxide (Dia; a mitoK(ATP) opener). Pax and 5-HD abolished IPT-induced cardioprotection from reoxygenation injury, whereas administration of NS1619 or Dia significantly improved cardiac contractile activity and reduced aspartate aminotransferase (an index of myocyte injury) release following reoxygenation. In addition, isolated rat myocytes were loaded with tetramethylrhodamine methyl ester (TMRE; fluorescent mitochondrial membrane potential indicator) and 2',7'-dichlorofluorescein [DCFH; fluorescent reactive oxygen species (ROS) indicator] or Fluo-4-acetoxymethyl ester (Fluo-4-AM; fluorescent calcium indicator). When TMRE-loaded myocytes were laser illuminated, the DCFH and Fluo-4 fluorescence increased, and TMRE fluorescence decreased. These effects were significantly inhibited by NS1619 and Dia. We therefore conclude that IPT may protect the heart through activation of mitoK(ATP) and mitoK(Ca) channels, and that opening of these channels at the onset of reoxygenation protects the heart from reoxygenation injury, most likely by reducing excess generation of ROS and the resultant Ca(2+) overload.

The Role of MicroRNA in Anesthetic‐Induced Cardiac Preconditioning

Anesthetic preconditioning has been reported to decrease myocardial infarct size after ischemia‐reperfusion injury. However, the underlying mechanisms are not fully understood. MicroRNAs (miRNAs) are short nucleotide sequences that have been implicated in many different cell signaling pathways via suppression of messenger RNA. In this study, we investigated the effect of anesthetic exposure on miRNA expression in the left ventricle of the rat heart. Male Wistar rats were exposed to 30 min of one minimum alveolar concentration of isoflurane. Total RNA was extracted from the left ventricle of the heart and converted to cDNA. Using real‐time PCR, microarray‐based analyses of 88 miRNAs were performed, revealing twenty upregulated miRNAs. One microRNA, miR‐21, has consistently shown a twofold upregulation in hearts exposed to anesthetic. This microRNA has been demonstrated to decrease infarct size in hearts exposed to ischemic preconditioning via its action on heat shock protein 70, endothelial nitric oxide synthase, and inducible nitric oxide synthase. Further investigation into the isoflurane‐induced upregulation of miR‐21 may reveal that miR‐21‐mediated pathways contribute to the preconditioning effect of anesthetics. This work is supported by NIH Grants R01HL034708 and P01GM066730 for Z. Bosnjak.

Stepwise Ischemic Preconditioning Mitigates Prolonged Ischemia/Reperfusion injury

The current cardiac ischemic preconditioning (IPC) protocol may not be clinically beneficial due to its deleterious effects on myocardium. This study attempted to assess the effects of a novel stepwise IPC on cardiac performance, infarct size and arrhythmia in a rabbit model of prolonged I/R. Anesthetized open chest rabbits were subjected to 60 min occlusion of the first left antero‐lateral coronary branch followed by 180 min reperfusion. Animals were divided into five groups (n=6 each). All animals except for control group were subjected to four IPC groups as follows: 1) short IPC group (three cycles of 2min I/R); 2) standard IPC group (three cycles of 5 min I/R); 3) prolonged IPC group (three cycles of 10 min I/R); 4) stepwise IPC group (2, 5, 10 min I/R). Compared to control group, short, standard, and prolonged IPC decreased arrhythmia score by 16%, 67% and 33%, respectively; while stepwise IPC resulted in a 78% reduction of arrhythmias. The stepwise IPC showed the most marked reduction of LV infarct size, compared to the short, standard and prolonged IPC (8.6% vs. 22%, 10.3% and 15.7%). These results suggest that the stepwise IPC has better cardioprotective effects against prolonged I/R injury and may serve as an acceptable approach to heart surgeries such as angioplasty on a beating heart.

Cardiac cell therapy: pre-conditioning effects in cell-delivery strategies

Stem-cell therapy holds great promise for the treatment of ischemic heart disease. However, the benefit of cardiac cell therapy has not yet been proven in long-term clinical trials. Poor engraftment and survival of transplanted cells is one of the major concerns for the successful application of stem cells in cardiac cell therapy. Cell and cardiac pre-conditioning are now being explored as new approaches to support cell survival and enhance the therapeutic efficacy. In this paper, we summarize the state-of-the-art methods of cell delivery and cell survival post-delivery, with a focus on the pre-conditioning approaches that have been attempted to support the survival of transplanted cells.

Prolonged Oral Administration of Oleuropein Might Protect Heart against Aconitine-induced Arrhythmia.

In this study, it was surveyed to know whether an oral single dose of oleuropein could mimic the cardiac preconditioning in rats' hearts or whether its prolonged oral administration could protect the heart against the aconitine-induced arrhythmia in rats. Eighty male Wistar rats were divided into two series (n = 8 in each group). In the first series, all groups (except the control (Con) group) were given a single oral dose of oleuropein (20 mg/Kg) 1, 3, 24 and 48 h before the infusion of aconitine. In the second series, except the Con group, the other four groups were given oral oleuropein (20 mg/Kg/day) for 3, 7, 14 and 28 days, before the infusion of aconitine. Electrocardiogram was recorded to monitor arrhythmia. Data of the first series showed that the initiation time of arrhythmia, the initiation of ventricular tachycardia (VT), the numbers of reversible ventricular fibrillation (VF) and the death time had no significant difference compared with Con group. In the second series, a significant protection was occurred only in the 28 days group that was evident with increased initiation time of arrhythmia, increased initiation time of VT, and increased the number of reversible VF and death time in compared to the Con group. The findings of this study show that the oral administration of a single dose of oleuropein could not mimic the preconditioning effects in rat hearts, but the prolonged administration of oleuropein for about four weeks could protect the heart against aconitine-induced arrhythmia.

Reduced cardiac CapZ protein protects hearts against acute ischemia–reperfusion injury and enhances preconditioning

The Z-disc protein CapZ has historically been classified as a structural element, anchoring sarcomeric actin. Our previous work expanded its role to include signal transduction, as CapZ transgenic myofilaments are less sensitive to protein kinase C (PKC). Myocardial PKC has paradoxical effects, mediating both preconditioning and ischemia–reperfusion (IR) injury. Our objective was to determine how decreased CapZ affects IR injury and cardiac preconditioning. Mouse hearts were subjected to 20min global ischemia and 60min reperfusion. Some hearts were preconditioned with intermittent IR (IPC). Left ventricular function was assessed and myocardial tissue collected post-IR for molecular analysis and tissue staining. Post-ischemic function was significantly better and infarct size smaller in CapZ transgenic hearts, as compared to wildtype. IPC decreased IR damage in both wildtype and CapZ transgenic hearts, although CapZ transgenic hearts performed significantly better than wildtype. Immunoblotting revealed increased myofilament-associated PKC-α and -ε following IR in wildtype hearts, but no change in PKC-δ or -ζ. By contrast, post-IR myofilament-associated PKC-α was significantly higher in CapZ transgenic mice but the rise in PKC-ε was attenuated. Both PKC-δ and PKC-ζ decreased in CapZ transgenic myofilaments following IR. IPC increased myofilament-associated PKC-α and -ε, while decreasing PKC-δ in wildtype hearts. Preconditioned CapZ IPC hearts showed attenuated increases in myofilament PKC-α and -ε, but also a significant decrease in myofilament PKC-δ and -ζ. These data demonstrate significant differences in post-IR myofilament PKC in untreated and preconditioned CapZ transgenic mice. CapZ reduction did not dramatically affect post-IR myofilament function, nor did preconditioning. These results demonstrate that CapZ deficiency decreases IR injury, while providing enhanced cardioprotection with IPC. The cardioprotected phenotype of CapZ transgenic mice is associated with an altered translocation of PKC-isoforms to cardiac myofilaments.

Age-related loss of cardiac preconditioning: Impact of protein kinase A

Helium induces preconditioning (He-PC) by mitochondrial calcium-sensitive potassium (mK Ca) channel-activation, but this effect is lost in the aged myocardium. Both, the upstream signalling pathway of He-PC and the underlying mechanisms for an age-related loss of preconditioning are unknown. A possible candidate as upstream regulator of mK Ca channels is protein kinase A (PKA). We investigated whether 1) regulation of PKA is involved in He-PC and 2) regulation of PKA is age-dependent. Young (2–3 months) and aged (22–24 months) Wistar rats were randomised to eight groups (each n = 8). All animals underwent 25 min regional myocardial ischemia and 120 min reperfusion. Control (Con, Age Con) animals were not further treated. Young rats inhaled 70% helium for 3 × 5min (He-PC). The PKA-blocker H-89 (10 μg/kg) was administered with and without helium (He-PC + H-89, H-89). Furthermore, we tested the effect of direct activation of mK Ca channels with NS1619. The adenylyl cyclase activator forskolin (For) was administered in young (300 μg/kg) and aged animals (300 and 1000 μg/kg). He-PC reduced infarct size from 60 ± 4% (Con) to 37 ± 10% ( p < 0.05). Infarct size reduction was completely abolished by H-89 (58 ± 5%; p < 0.05), but H-89 alone had no effect (57 ± 2%). NS1619 reduced infarct size in the same concentration in both, young and aged rats (35 ± 6%; p < 0.05 vs. Con and 34 ± 8%; p < 0.05 vs. Age Con). Forskolin in a concentration of 300 μg/kg reduced infarct size in young (37 ± 6%; p < 0.05) but not in aged rats (48 ± 13%; n.s.). In contrast, 1000 μg/kg Forskolin reduced infarct size also in aged rats (28 ± 3%; p < 0.05). He-PC is mediated by activation of PKA. Alterations in PKA regulation might be an underlying mechanism for the age-dependent loss of preconditioning.

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

Endocannabinoids and the cardiovascular response to stress

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS), resulting in cardiovascular responses. The endocannabinoid system (ECS), a ubiquitously expressed lipid signalling system, modulates both HPA and SNS activity. The purpose of this review is to explore the possible involvement/role of the ECS in the cardiovascular response to stress. The ECS has numerous cardiovascular effects including modulation of blood pressure, heart rate, the baroreflex, and direct vascular actions. It is also involved in a protective manner in response to stressors in cardiac preconditioning, and various stressors (for example, pain, orthostasis and social stress) increase plasma levels of endocannabinoids. Given the multitude of vascular effects of endocannabinoids, this is bound to have consequences. Beneficial effects of ECS upregulation could include cardioprotection, vasodilatation, CB(2)-mediated anti-inflammatory effects and activation of peroxisome proliferator-activated receptors. Negative effects of endocannabinoids could include mediation of the effects of glucocorticoids, CB(1)-mediated metabolic changes, and metabolism to vasoconstrictor products. It is also likely that there is a central role for the ECS in modulating cardiovascular activity via the HPA and SNS. However, much more work is required to fully integrate the role of the ECS in mediating many of the physiological responses to stress, including cardiovascular responses.

Geranylgeranylacetone induced delayed cardiac preconditioning requires caveolin‐3

Geranylgeranylacetone (GGA) is a Japanese, anti-ulcer drug that has been shown to protect the myocardium against ischemia/reperfusion injury via upregulation of heat shock protein (HSP). Furthermore, caveolin-3 (Cav-3) is necessary in delayed anesthetic preconditioning. In this study we investigated the effect of Cav-3 and caveolae on GGA induced delayed cardiac preconditioning. We utilized an in vivo mouse model where mice were exposed to GGA via gavage and allowed to recover for 24 hrs. After 24 hrs of recovery, mice underwent 30-min coronary artery occlusion followed by 2 hrs of reperfusion at which time infarct size was determined. Infarct size as a percent of the area at risk was significantly reduced by GGA in control mice (22.3 ± 3.0% vs. 41.6 ± 3.0%). However, this protection was not observed in Caveolin-3 knockout mice. Furthermore, GGA administration increased caveolae formation and HSP70 expression in control mice, but was ineffective in Cav-3 knockout mice. In conclusion, GGA increases HSP 70 expression and caveolae formation which is dependent on Cav-3 expression.

Exercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart

The ability of exercise to protect the heart against ischemia-reperfusion (I/R) injury is well known in both human epidemiological studies and experimental animal models. In this review article, we describe what is currently known about the ability of exercise to precondition the heart against infarction. Just 1 day of exercise can protect the heart against ischemia/reperfusion damage, and this protection is upheld with months of exercise, making exercise one of the few sustainable preconditioning stimuli. Exercise preconditioning depends on the model and intensity of exercise, and appears to involve heightened oxidant buffering capacity, upregulated subunits of sarcolemmal ATP-sensitive potassium channels, and adaptations to cardiac mitochondria. We review the putative mechanisms involved in exercise preconditioning and point out many areas where future research is necessary to advance our understanding of how this stimulus confers resistance against I/R damage.

Site-Mapping of In Vitro S-nitrosation in Cardiac Mitochondria: Implications for Cardioprotection

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n=3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.

Roles of the nitric oxide signaling pathway in cardiac ischemic preconditioning against myocardial ischemia-reperfusion injury

SummaryNitric oxide (NO), a vasoactive gas that can freely diffuse into the cell, has many physiological effects in various cell types. Since 1986, numerous studies of ischemic preconditioning against ischemia-reperfusion (I/R) injury have been undertaken and the roles of the NO signaling pathway in cardioprotection have been explored. Many studies have confirmed the effect of NO and that its relative signaling pathway is important for preconditioning of the cardioprotective effect. The NO signaling against I/R injury targeted on the mitochondria is believed to be the end-target for cardioprotection. If the NO signaling pathway is disrupted or inhibited, cardioprotection by preconditioning disappears. During preconditioning, signaling is initiated from the sarcolemmal membrane, and then spread into the cytoplasm via many series of enzymes, including nitric oxide synthase (NOS), the NO-producing enzyme, soluble guanylyl cyclase (sGC), and protein kinase G (PKG). Finally, the signal is transmitted into the mitochondria, where the cardioprotective effect occurs. It is now well established that mitochondria act to protect the heart against I/R injury via the opening of the mitochondrial ATP-sensitive K+ channel and the inhibition of mitochondrial permeability transition (MPT). This knowledge may be useful in developing novel strategies for clinical cardioprotection from I/R injury.

Remifentanil in critically ill cardiac patients

Remifentanil has a unique pharmacokinetic profile, with a rapid onset and offset of action and a plasmatic metabolism. Its use can be recommended even in patients with renal impairment, hepatic dysfunction or poor cardiovascular function. A potential protective cardiac preconditioning effect has been suggested. Drug-related adverse effects seem to be comparable with other opioids. In cardiac surgery, many randomized controlled trials demonstrated that the potential benefits of the use of remifentanil not only include a profound protection against intraoperative stressful stimuli, but also rapid postoperative recovery, early weaning from mechanical ventilation, and extubation. Remifentanil shows ideal properties of sedative agents being often employed for minimally invasive cardiologic techniques, such as transcatheter aortic valve implantation and radio frequency treatment of atrial flutter, or diagnostic procedures such as transesophageal echocardiography. In intensive care units remifentanil is associated with a reduction in the time to tracheal extubation after cessation of the continuous infusion; other advantages could be more evident in patients with organ dysfunction. Effective and safe analgesia can be provided in case of short and painful procedures (i.e. chest drain removal). In conclusion, thanks to its peculiar properties, remifentanil will probably play a major role in critically ill cardiac patients.

Role of microRNAs in cardiac preconditioning.

Preconditioning (PC) of the heart by sublethal ischemia, mild heat shock, or hypoxia has evolved as a powerful experimental tool to discover novel signaling mechanisms in cardioprotection. The ultimate goal is to determine novel therapeutic targets for potential application in humans to protect the heart against ischemia-related injuries. In recent years, there has been a tremendous interest in understanding the role of small noncoding RNAs, microRNAs (miRs), in cardiovascular diseases. miRs have been recognized as regulators of gene expression by destabilization and translational inhibition of target messenger RNAs. Studies have shown that several miRs, including miR-1, miR-133, miR-21, miR-126, miR-320, miR-92a, and miR-199a, are regulated after preconditioning and play an active role in protecting the heart against ischemia/reperfusion injury. These miRs also drive the synthesis of important cardioprotective proteins including heat shock protein (HSP)-70, endothelial nitric oxide synthase, inducible nitric oxide synthase, HSP-20, Sirt1, and hypoxia-inducible factor 1a. We believe that identification and targeted delivery of miR(s) in the heart could have an immense therapeutic potential in reducing myocardial infarction in patients suffering from heart disease.

Ischaemic and morphine-induced post-conditioning: impact of mKCa channels

BackgroundMitochondrial calcium-sensitive potassium (mKCa) channels are involved in cardiac preconditioning. In the present study, we investigated whether also ischaemic-, morphine-induced post-conditioning, or both is mediated by the activation of mKCa channels in the rat heart in vitro. MethodsAnimals were treated in compliance with institutional and national guidelines. Male Wistar rats were randomly assigned to one of seven groups (each n=7). Control animals were not further treated. Post-conditioning was induced either by 3×30 s of ischaemia/reperfusion (I-PostC) or by administration of morphine (M-PostC, 1 µM) for 15 min at the onset of reperfusion. The mKCa-channel inhibitor paxilline (1 µM) was given with and without post-conditioning interventions (M-PostC+Pax, I-PostC+Pax, and Pax). As a positive control, we determined whether direct activation of mKCa channels with NS1619 (10 µM) induced cardiac post-conditioning (NS1619). Isolated hearts underwent 35 min ischaemia followed by 120 min reperfusion. At the end of reperfusion, infarct sizes were measured by triphenyltetrazolium chloride staining. ResultsIn the control group, infarct size was 53 (5)% of the area at risk. Morphine- and ischaemic post-conditioning reduced infarct size in the same range [M-PostC: 37 (4)%, I-PostC: 35 (5)%; each P<0.05 vs control]. The mKCa-channel inhibitor paxilline completely blocked post-conditioning [M-PostC+Pax: 47 (7)%, I-PostC+Pax: 51 (3)%; each P<0.05 vs M-PostC and I-PostC, respectively]. Paxilline itself had no effect on infarct size (NS vs control). NS1619 reduced infarct size to 33 (4)% (P<0.05 vs control). ConclusionsIschaemic- and morphine-induced post-conditioning is mediated by the activation of mKCa channels.

More Reasons to Say Goodbye to Glyburide

Sulfonylureas have been used to control hyperglycemia in type 2 diabetes longer than any class of agents except insulins. Their use early in the course of diabetes is supported by evidence of long-term medical benefit (1), and although available in generic form and thus not commercially promoted, they are widely prescribed (2). Given these facts, it is surprising that much remains unknown about these agents, notably how the various members of the class should be deployed in current treatment algorithms. An article by Zeller et al. in this issue of JCEM (3) sheds considerable light on this question. The authors report analyses of prospectively collected data from a French registry concerning experience of patients with type 2 diabetes who were hospitalized for myocardial infarction. The question posed was what association, if any, might exist between prior use of sulfonylureas and death and other in-hospital outcomes in these patients. Analyses included comparisons between the individual sulfonylureas used in this population: glyburide (called glibenclamide in Europe), glimepiride, and gliclazide (in most cases given as an extended-acting formulation). The rationale for the study came from the known adverse effect of glyburide on cardiac ischemic preconditioning, the clinical relevance of which has been uncertain. One key finding of the French study was that, among the 1310 acutely ill patients studied, unadjusted mortality rates were lower for those whose treatment on admission included a sulfonylurea (3.9%) than for those taking insulin (9.4%), those on no antihyperglycemic drugs (8.4%), and those on oral therapies other than sulfonylureas (6.4%). Another was that when patients taking glyburide were compared with those taking gliclazide or glimepiride, two sulfonylureas that appear to lack cardiac effects, early mortality was almost 3-fold higher (7.5 vs. 2.7%) with glyburide. It is well known that epidemiological analyses of “real-world” experiences with prescribed pharmacotherapy may be confounded by incomplete collection of information on actual drug usage, concomitant therapies, and medical outcomes, in addition to biases introduced by physicians’ tendencies to assign different treatments to different kinds of patients. Many of these problems were reduced or eliminated by the prospective design of this study, which included systematic recording of detailed clinical information on all patients in a narrow window of time by a structured team of investigators. The problem of allocation bias in assignment of treatments, however, remains in this study as in others. Hence, the authors used several statistical methods to adjust for this kind of confounding, including an extensive multivariable model, further adjustment using a propensity score for assignment to glyburide treatment, and analysis of findings with glyburide vs. the other sulfonylureas by subgroups. These statistical adjustments did not alter the associations of sulfonylurea use with lower mortality than other forms of therapy, and of glyburide use with higher mortality than seen with the other sulfonylureas. The excess of mortality with glyburide vs. other sulfonylureas showed no tendency toward heterogeneity between subgroups. Thus, several approaches to testing the validity of the observations were supportive, and the magnitude of the effects was substantial. After multivariable adjustment, the risk of death accompanyinguseofa sulfonylureawashalf thatwhena sulfonylurea was not used (odds ratio, 0.50; 95% confidence interval, 0.27–0.94; P 0.03). After similar adjustment for covariates, the risk of early mortality was 85% lower with glimepiride or gliclazide than with glyburide (odds ratio, 0.15; 95% confidence interval, 0.04–0.56; P 0.005), and with the propensity score included in the model, the risk was 87%lower(oddsratio,0.13;95%confidence interval,0.21–