Postischemic Contractile Function Research Articles

Glucagon and exenatide improve contractile recovery following ischaemia/reperfusion in the isolated perfused rat heart.

The inotropic effects of glucagon have been recognized for many years, but it has remained unclear whether glucagon signaling is beneficial to cardiac function. We evaluated the effects of glucagon alone and in combination with the glucagon-like peptide 1 (GLP-1) receptor agonist exenatide in the isolated perfused rat heart. The isolated perfused rat heart was used to investigate the initial inotropic and chronotropic effects of glucagon and exenatide during aerobic perfusion, and recovery of contractile function following ischaemia/reperfusion. Glucagon, but not exenatide, elicited an acute chronotropic and inotropic response during aerobic perfusion of the rat heart. Compared with control, glucagon improved recovery of left ventricular developed pressure (LVDP) by 33% (p< 0.05) and rate-pressure product (RPP) by 66% (p< 0.001) following ischaemia/reperfusion and amplified the mild recovery enhancement elicited by exenatide in a dose-dependent manner. Glucagon shows inotropic properties in the isolated perfused rat heart and improves contractile recovery following ischaemia/reperfusion, both alone and when co-administered with a GLP-1 receptor agonist. Glucagon and exenatide, a GLP-1 receptor agonist, combine to stimulate greater recovery of postischaemic contractile function in the Langendorff heart. Glucagon was inotropic and chronotropic, yet this initial effect decreased over time and did not account for the increased contractility observed postischaemia/reperfusion.

Notch1 Mediates Preconditioning Protection Induced by GPER in Normotensive and Hypertensive Female Rat Hearts.

G protein-coupled estrogen receptor (GPER) is an estrogen receptor expressed in the cardiovascular system. G1, a selective GPER ligand, exerts cardiovascular effects through activation of the PI3K-Akt pathway and Notch signaling in normotensive animals. Here, we investigated whether the G1/GPER interaction is involved in the limitation of infarct size, and improvement of post-ischemic contractile function in female spontaneous hypertensive rat (SHR) hearts. In this model, we also studied Notch signaling and key components of survival pathway, namely PI3K-Akt, nitric oxide synthase (NOS) and mitochondrial K+-ATP (MitoKATP) channels. Rat hearts isolated from female SHR underwent 30 min of global, normothermic ischemia and 120 min of reperfusion. G1 (10 nM) alone or specific inhibitors of GPER, PI3K/NOS and MitoKATP channels co-infused with G1, just before I/R, were studied. The involvement of Notch1 was studied by Western blotting. Infarct size and left ventricular pressure were measured. To confirm endothelial-independent G1-induced protection by Notch signaling, H9c2 cells were studied with specific inhibitor, N-[N-(3,5 difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 5 μM), of this signaling. Using DAPT, we confirmed the involvement of G1/Notch signaling in limiting infarct size in heart of normotensive animals. In the hypertensive model, G1-induced reduction in infarct size and improvement of cardiac function were prevented by the inhibition of GPER, PI3K/NOS, and MitoKATP channels. The involvement of Notch was confirmed by western blot in the hypertensive model and by the specific inhibitor in the normotensive model and cardiac cell line. Our results suggest that GPERs play a pivotal role in mediating preconditioning cardioprotection in normotensive and hypertensive conditions. The G1-induced protection involves Notch1 and is able to activate the survival pathway in the presence of comorbidity. Several pathological conditions, including hypertension, reduce the efficacy of ischemic conditioning strategies. However, G1-induced protection can result in significant reduction of I/R injury also female in hypertensive animals. Further studies may ascertain the clinical translation of the present results.

Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury.

A longevity gene, Sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress, and exercise. The objective of this study is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults. Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. The cardiac functions by echocardiography show no difference in young wild-type C57BL/6 J (WT, 4-6 months), aged WT C57BL/6 J (24-26 months), and young inducible cardiomyocyte-specific SIRT1 knockout (icSIRT1 KO) (4-6 months) mice under physiological conditions. However, after 45 mins ischaemia and 24-h reperfusion, the ejection fraction of aged WT and icSIRT1 KO mice was impaired. The aged WT and icSIRT1 KO hearts vs. young WT hearts also show an impaired post-ischemic contractile function in a Langendorff perfusion system. The infarct size of aged WT and icSIRT1 KO hearts was larger than that of young WT hearts. The immunoblotting data demonstrated that aged WT and icSIRT1 KO hearts vs. young WT hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischaemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged WT and icSIRT1 KO hearts; this could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impaired substrate metabolism in the hearts; fatty acid oxidation is augmented and glucose oxidation is blunted during ischaemia and reperfusion. Adeno-associated virus (AAV9)-Sirt1 was delivered into the aged hearts via a coronary delivery approach, which significantly rescued the protein level of SIRT1 and the ischemic tolerance of aged hearts. Furthermore, AMPK agonist can rescue the tolerance of aged heart and icSIRT1 KO heart to ischemic insults. Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation, and AMPK modulates SIRT1 activity via regulation of NAD+ level during ischaemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice

Steroid hormone receptors including estrogen receptors (ER) classically function as ligand-regulated transcription factors. However, estrogens also elicit cellular effects through binding to extra-nuclear ER (ERα, ERβ, and G protein-coupled ER or GPER) that are coupled to kinases. How extra-nuclear ER actions impact cardiac ischemia-reperfusion (I/R) injury is unknown. We treated ovariectomized wild-type female mice with estradiol or an estrogen-dendrimer conjugate (EDC), which selectively activates extra-nuclear ER, or vehicle interventions for two weeks. I/R injury was then evaluated in isolated Langendorff perfused hearts. Two weeks of treatment with estradiol significantly decreased infarct size and improved post-ischemic contractile function. Similarly, EDC treatment significantly decreased infarct size and increased post-ischemic functional recovery compared to vehicle-treated hearts. EDC also caused an increase in myocardial protein S-nitrosylation, consistent with previous studies showing a role for this post-translational modification in cardioprotection. In further support of a role for S-nitrosylation, inhibition of nitric oxide synthase, but not soluble guanylyl cyclase blocked the EDC mediated protection. The administration of ICI182,780, which is an agonist of G-protein coupled estrogen receptor (GPER) and an antagonist of ERα and ERβ, did not result in protection; however, ICI182,780 significantly blocked EDC-mediated cardioprotection, indicating participation of ERα and/or ERβ. In studies determining the specific ER subtype and cellular target involved, EDC decreased infarct size and improved functional recovery in mice lacking ERα in cardiomyocytes. In contrast, protection was lost in mice deficient in endothelial cell ERα. Thus, extra-nuclear ERα activation in endothelium reduces cardiac I/R injury in mice, and this likely entails increased protein S-nitrosylation. Since EDC does not stimulate uterine growth, in the clinical setting EDC-like compounds may provide myocardial protection without undesired uterotrophic and cancer-promoting effects.

Abstract 12050: Cardiomyocyte Specific Deletion of Sirt1 Gene Alters Substrate Metabolism and Sensitizes Myocardium to Ischemia and Reperfusion Injury

Introduction: A longevity gene, sirtuin 1 (SIRT1) is a conserved NAD + -dependent protein deacetylase. AMP-activated protein kinase (AMPK) is an energy sensor. AMPK and SIRT1 signaling pathways have common activators such as caloric restriction, oxidative stress and exercise. Hypothesis: Aging-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults are due to a decreased SIRT1 levels in aging. Methods: Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. Results: The cardiac functions by echocardiography show no difference in young (SIRT1 flox/flox ), aged (SIRT1 flox/flox ) and young inducible cardiomyocyte specific SIRT1 knockout (icSIRT1 KO) mice under physiological conditions, but after 45 mins ischemia and 24 hours reperfusion, the ejection fraction of aged and icSIRT1 KO mice were impaired. The aged and icSIRT1 KO hearts versus young hearts also show an impaired post-ischemic contractile function in Langendorff perfusion system. The infarct size of aged and icSIRT1 KO hearts was larger than that of young hearts (p&lt;0.05). The immunoblotting data demonstrated that aged and icSIRT1 KO hearts versus young hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged and icSIRT1 KO hearts that could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impair substrate metabolism in the hearts, fatty acid oxidation is augmented and glucose oxidation is blunted during ischemia and reperfusion. Aged and icSIRT1 KO hearts versus young hearts demonstrated significant cardiac fibrosis and macrophage infiltration during ischemia and reperfusion. Furthermore, AMPK agonist A769662 treatment can rescue the tolerance of aged heart but not icSIRT1 KO hearts to ischemic insults. Conclusions: Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation during ischemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

Abstract 16115: Rapamycin Protects Diabetic Heart Against Ischemia-reperfusion Injury by Inhibiting JNK and STAT4

Background: Persistent activation of the mammalian target of rapamycin (mTOR) is implicated in diabetic complications and plays a critical role in myocardial injury following ischemia/reperfusion. Recently we reported that mTOR inhibition with rapamycin improves cardiac function in type 2 diabetic (T2D) mice through attenuation of oxidative stress and alteration of antioxidants and contractile protein expression. Since increased JNK and STAT4 activities have been strongly associated with T2D-induced cardiovascular complications, we hypothesized that rapamycin would reduce myocardial infarction (MI) in T2D mice through inhibition of JNK and STAT4. Methods and Results: Adult male wild type (C57) or db/db T2D mice were treated daily for 28 days with rapamycin (0.25 mg/kg, i.p.). Following treatment, the hearts were subjected to global I (30 min) and R (60 min) in Langendorff mode. In addition, cardiomyocytes were isolated from treated mice and subjected to 40 min simulated ischemia (SI) and 1 hr or 18 hr reoxygenation (RO) to assess necrosis (by trypan blue staining) or apoptosis (by TUNEL assay), respectively. Diabetes exacerbated ischemic infarct size (measured by tetrazolium chloride staining) in db/db mice as compared to C57 mice (Fig. A), and also enhanced cardiomyocyte necrosis (Fig. B) and apoptosis (Fig. C). Rapamycin treatment reduced infarct size following I/R and protected adult cardiomyocytes against necrosis and apoptosis following SI/RO. Rapamycin also improved post-ischemic contractile function in db/db mice (Fig. D). Western blot analysis revealed a marked increase in phosphorylation of JNK and STAT4 in diabetic hearts, which were reduced in rapamycin-treated diabetic hearts (Fig. E). Conclusion: Rapamycin treatment reduces MI possibly by inhibiting JNK and STAT4 in T2D heart. We propose that inhibition of JNK and STAT4 with rapamycin therapy may be an attractive strategy to improve prognosis in diabetics following MI.

Abstract 405: Inhibition of Class I Histone Deacetylases at Reperfusion Attenuates Ischemia-reperfusion Injury and Modifies Mitochondrial Acetylation

Although rapid reperfusion of ischemic tissue is the treatment of choice for myocardial infarction, much of the resultant damage occurs as a consequence of reperfusion itself. Previously, we have shown that pretreatment with MS-275, a selective class I histone deacetylase (HDAC) inhibitor, preserves left-ventricular (LV) function and substantially reduces the area of infarcted tissue in isolated rat hearts subjected to ischemia-reperfusion (IR) injury. Here, we tested the hypothesis that MS-275 treatment at reperfusion reduces LV tissue damage and improves post-ischemic LV contractile function. To do this, hearts from male Sprague-Dawley rats were isolated and perfused ex vivo on a Langendorff perfusion apparatus. A saline-filled balloon was inserted into the left ventricle of the heart to monitor ventricular pressure development throughout the experiment. Hearts were subjected to 30 minutes of ischemia, followed by 60 minutes of reperfusion. MS-275 was administered during the entire reperfusion phase, and resultant functional data were compared to untreated hearts. There was no difference in any metric of pre-ischemic contractile function between groups. 10nM MS-275 administered at reperfusion significantly improved multiple measures of LV function, including dP/dtmax, -dP/dtmax, developed pressure and rate pressure product. We also observed a significant reduction in infarct area of treated hearts compared to control, as measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Unexpectedly, mass spectrometry analysis revealed significant changes in acetylation state of multiple mitochondrial enzymes. Administration of MS-275 during the reperfusion phase of IR is sufficient to partially rescue LV function from reperfusion-induced damage. This study emphasizes the importance of exploring class I HDAC inhibitors for protection against ischemia-reperfusion.

Sestrin2 promotes LKB1-mediated AMPK activation in the ischemic heart.

The regulation of AMPK in the ischemic heart remains incompletely understood. Recent evidence implicates the role of Sestrin2 in the AMPK signaling pathway, and it is hypothesized that Sestrin2 plays an influential role during myocardial ischemia to promote AMPK activation. Sestrin2 protein was found to be expressed in adult cardiomyocytes and accumulated in the heart during ischemic conditions. Sestrin2 knockout (KO) mice were used to determine the importance of Sestrin2 during ischemia and reperfusion (I/R) injury. When wild-type (WT) and Sestrin2 KO mice were subjected to in vivo I/R, myocardial infarct size was significantly greater in Sestrin2 KO compared with WT hearts. Similarly, Langendorff perfused hearts indicated exacerbated postischemic contractile function in Sestrin2 KO hearts compared with WT. Ischemic AMPK activation was found to be impaired in the Sestrin2 KO hearts. Immunoprecipitation of Sestrin2 demonstrated an association with AMPK. Moreover, liver kinase B1 (LKB1), a major AMPK upstream kinase, was associated with the Sestrin2-AMPK complex in a time-dependent manner during ischemia, whereas this interaction was nearly abolished in Sestrin2 KO hearts. Thus, Sestrin2 plays an important role in cardioprotection against I/R injury, serving as an LKB1-AMPK scaffold to initiate AMPK activation during ischemic insults.

Abstract 87: Sestrin2 Promotes LKB1-Mediated AMPK Activation in the Ischemic Heart

AMP-activated protein kinase (AMPK) has emerged as a pertinent stress-activated protein kinase shown to have substantial cardioprotective capabilities against myocardial ischemia/reperfusion injury. When activated during ischemia, AMPK produces effects that have been demonstrated to be beneficial to the myocardium by means of increasing GLUT4 translocation and glucose uptake, activating eNOS, decreasing apoptosis, and increasing autophagic flux. However, the molecular basis behind the regulation of AMPK activity in the ischemic and reperfused heart remains incompletely understood. Recent evidence implicates the role of Sestrin2 in the AMPK signaling pathway and it is hypothesized that Sestrin2 plays an influential role during myocardial ischemia in order to promote AMPK activation. Sestrin2 protein was found to be expressed in adult cardiomyocytes and accumulated in the heart during ischemic conditions. Sesn2 KO mice were used to determine the importance of Sestrin2 during I/R injury. Sesn2 KO cardiac phenotype analysis indicated no significant difference in left ventricular function as measured by a pressure-volume loop catheter. When wild type (WT) and Sesn2 KO mice were subjected to in vivo I/R, myocardial infarct size was significantly greater in Sesn2 KO compared to WT hearts. Similarly, Langendorff perfused hearts indicated exacerbated post-ischemic contractile function in Sesn2 KO hearts when compared to WT. Ischemic AMPK activation was found to be impaired in the Sesn2 KO hearts. Immunoprecipitation of Sestrin2 demonstrated an association with AMPK. Moreover, LKB1, a major AMPK upstream kinase was associated with the Sestrin2-AMPK complex in a time-dependent manner during ischemia while this interaction was nearly abolished in Sesn2 KO hearts. Thus, Sestrin2 plays an important role in cardioprotection against I/R injury by acting as an LKB1-AMPK scaffold to initiate AMPK activation during ischemic insults.

Activation of α1B-adrenoceptors contributes to intermittent hypobaric hypoxia-improved postischemic myocardial performance via inhibiting MMP-2 activation

Inhibition of matrix metalloproteinases-2 (MMP-2) activation renders cardioprotection from ischemia/reperfusion (I/R) injury; however, the signaling pathways involved have not been fully understood. Intermittent hypobaric hypoxia (IHH) has been shown to enhance myocardial tolerance to I/R injury via triggering intrinsic adaptive responses. Here we investigated whether IHH protects the heart against I/R injury via the regulation of MMP-2 and how the MMP-2 is regulated. IHH (Po2 = 84 mmHg, 4-h/day, 4 wk) improved postischemic myocardial contractile performance, lactate dehydrogenase (LDH) release, and infarct size in isolated perfused rat hearts. Moreover, IHH reversed I/R-induced MMP-2 activation and release, disorders in the levels of MMP-2 regulators, peroxynitrite (ONOO(-)) and tissue inhibitor of metalloproteinase-4 (TIMP-4), and loss of the MMP-2 targets α-actinin and troponin I. This protection was mimicked, but not augmented, by a MMP inhibitor doxycycline and lost by the α1-adrenoceptor (AR) antagonist prazosin. Furthermore, IHH increased myocardial α1A-AR and α1B-AR density but not α1D-AR after I/R. Concomitantly, IHH further enhanced the translocation of PKC epsilon (PKCε) and decreased the release of mitochondrial cytochrome c due to I/R via the activation of α1B-AR but not α1A-AR or α1D-AR. IHH-conferred cardioprotection in the postischemic contractile function, LDH release, MMP-2 activation, and nitrotyrosine as well as TIMP-4 contents were mimicked but not additive by α1-AR stimulation with phenylephrine and were abolished by an α1B-AR antagonist chloroethylclonidine and a PKCε inhibitor PKCε V1-2. These findings demonstrate that IHH exerts cardioprotection through attenuating excess ONOO(-) biosynthesis and TIMP-4 loss and sequential MMP-2 activation via the activation of α1B-AR/PKCε pathway.

Phenylephrine postconditioning increases myocardial injury: are alpha-1 sympathomimetic agonist cardioprotective?

We studied effects of phenylephrine (PHE) on postischemic functional recovery and myocardial injury in an ischemia-reperfusion (I-R) experimental model. Rat hearts were Langendorff-perfused and subjected to 30 min zero-flow ischemia (I) and 60 min reperfusion (R). During R PHE was added at doses of 1 μM (n = 10) and 50 μM (n = 12). Hearts (n = 14) subjected to 30 and 60 min of I-R served as controls. Contractile function was assessed by left ventricular developed pressure (LVDP) and the rate of increase and decrease of LVDP; apoptosis by fluorescent imaging targeting activated caspase-3, while myocardial injury by lactate dehydrogenase (LDH) released during R. Activation of kinases was measured at 5, 15, and 60 min of R using western blotting. PHE did not improve postischemic contractile function. PHE increased LDH release (IU/g); 102 ± 10.4 (Mean ± standard error of mean) control versus 148 ± 14.8 PHE (1), and 145.3 ± 11 PHE (50) hearts, (P < 0.05). PHE markedly increased apoptosis. Molecular analysis showed no effect of PHE on the activation of proapoptotic c-Jun N-terminal kinase signaling; a differential pattern of p38 mitogen activated protein kinase (MAPK) activation was found depending on the PHE dose used. With 1 μM PHE, p-p38/total-p38 MAPK levels at R were markedly increased, indicating its detrimental effect. With PHE 50 μM, no further changes in p38 MAPK were seen. Activation of Akt kinase was decreased implying involvement of different mechanisms in this response. PHE administration during reperfusion does not improve postischemic recovery due to exacerbation of myocardial necrosis and apoptosis. This finding may be of clinical and therapeutic relevance.

Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death

While Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5μmol/dm(3)) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIIδ and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function.

Impaired Cardiac SIRT1 Activity by Carbonyl Stress Contributes to Aging-Related Ischemic Intolerance

Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1) is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R) injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2), SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05). Exogenous toxic aldehydes (4-HNE) exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R) injury, which could all be rescued by Alda-1 (ALDH2 activator) (all P<0.05). However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05). The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1+/− knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1+/− hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.

Abstract 19434: Early Ischemic Preconditioning Requires Akt- and PKA-Mediated Activation of eNOS via Serine 1176 Phosphorylation

Aims: The role of endothelial nitric oxide synthase (eNOS)/NO signaling is well-documented in late preconditioning or postconditioning; however, the role of eNOS and its activation in early IPC remains controversial and poorly defined. This study investigates the role of eNOS in early IPC and what signaling pathways and molecular interactions regulate eNOS activation during early IPC. Methods and Results: Rat hearts were subjected to 30-min global ischemia and reperfusion (I/R) with or without IPC (3 cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor, L-NAME, PI 3 K inhibitor, LY294002/LY, or PKA inhibitor, H89, during IPC induction. IPC improved postischemic contractile function and reduced infarction compared to I/R with this abrogated by NOS inhibition. eNOS Ser1176 , Akt Ser473 , and PKA Thr197 phosphorylation was increased following IPC. I/R decreased eNOS Ser1176 phosphorylation, while IPC increased it. Mass spectroscopy confirmed eNOS Ser1176 phosphorylation and quantative western blots showed that ∼24 % modification of eNOS Ser1176 was present following induction of early IPC. Immunoprecipitation demonstrated eNOS, Akt and PKA complexation. Immunohistology showed that IPC induced Akt and PKA phosphorylation in cardiomyocytes and endothelial cells. With eNOS activation, IPC increased NO production as measured by EPR spin trapping and fluorescence microscopy. LY or H89 not only greatly decreased Akt Ser473 or PKA Thr197 phosphorylation, respectively; but also abolished IPC-induced preservation of eNOS and eNOS Ser1176 phosphorylation as well as subsequent cardioprotection. Conclusion: Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection with eNOS-derived NO serving a critical role.

Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation

The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC. Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOS(Ser1176), Akt(Ser473), and PKA(Thr197) phosphorylation was increased following IPC. I/R decreased eNOS(Ser1176) phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOS(Ser1176) phosphorylation and quantitative Western blots showed ∼24% modification of eNOS(Ser1176) following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt(Ser473) or PKA(Thr197) phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOS(Ser1176) phosphorylation as well as cardioprotection. Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.

Myocardial protection with mild hypothermia

Mild hypothermia, 32-35° C, is very potent at reducing myocardial infarct size in rabbits, dogs, sheep, pigs, and rats. The benefit is directly related to reduction in normothermic ischaemic time, supporting the relevance of early and rapid cooling. The cardioprotective effect of mild hypothermia is not limited to its recognized reduction of infarct size, but also results in conservation of post-ischaemic contractile function, prevention of no-reflow or microvascular obstruction, and ultimately attenuation of left ventricular remodelling. The mechanism of the anti-infarct effect does not appear to be related to diminished energy utilization and metabolic preservation, but rather to survival signalling that involves either the extracellular signal-regulated kinases and/or the Akt/phosphoinositide 3-kinase/mammalian target of rapamycin pathways. Initial clinical trials of hypothermia in patients with ST-segment elevation myocardial infarction were disappointing, probably because cooling was too slow to shorten normothermic ischaemic time appreciably. New approaches to more rapid cooling have recently been described and may soon be available for clinical use. Alternatively, it may be possible to pharmacologically mimic the protection provided by cooling soon after the onset of ischaemia with an activator of mild hypothermia signalling, e.g. extracellular signal-regulated kinase activator, that could be given by emergency medical personnel. Finally, the protection afforded by cooling can be added to that of pre- and post-conditioning because their mechanisms differ. Thus, myocardial salvage might be greatly increased by rapidly cooling patients as soon as possible and then giving a pharmacological post-conditioning agent immediately prior to reperfusion.

Hydrogen Sulfide Regulates Na+/H+ Exchanger Activity via Stimulation of Phosphoinositide 3-Kinase/Akt and Protein Kinase G Pathways

Intracellular pH (pH(i)) is an important endogenous modulator of cardiac function. Inhibition of Na(+)/H(+) exchanger-1 (NHE-1) protects the heart by preventing Ca(2+) overload during ischemia/reperfusion. Hydrogen sulfide (H(2)S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H(2)S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 μM, produced sustained decreases in pH(i) in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl(4) prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester (KT5823) significantly attenuated NaHS-suppressed NHE-1 activity and/or NaHS-induced cardioprotection. Although KT5823 failed to affect NaHS-induced Akt phosphorylation, Akt inhibitor did attenuate NaHS-stimulated PKG activity. In conclusion, this work demonstrated for the first time that H(2)S produced cardioprotection via the suppression of NHE-1 activity involving a PI3K/Akt/PKG-dependent mechanism.

Ischemia/reperfusion injury is increased and cardioprotection by a postconditioning protocol is lost as cardiac hypertrophy develops in nandrolone treated rats

We hypothesized that nandrolone (ND)-abuse induces cardiac hypertrophy, increases myocardial susceptibility to ischemia/reperfusion (I/R) injury, and reduces responsiveness to postconditioning (PostC) cardioprotection. Wistar-rats were ND treated for 2 weeks (short_ND) or 10 weeks (long_ND). Vehicle-treated rats served as controls. Hearts were retrogradely perfused and left ventricular pressure (LVP) was measured before and after 30-min global ischemia. In subgroups of hearts, to induce cardioprotection a PostC protocol (five cycles of 10-s reperfusion and 10-s ischemia) was performed. β-adrenoreceptors, kinases (Akt and GSK-3β) and phosphatases (PP2A sub A and PP2A sub B) were examined by Western blot before and after ischemia. After 120-min reperfusion, infarct size was measured. Short_ND slightly increased cardiac/body weight ratio, but did not affect cardiac baseline nor post-ischemic contractile function or infarct size when compared to vehicle hearts. However, PostC limited cardiac dysfunction much more in short_ND hearts than the other groups. Although cardiac/body weight ratio markedly increased after long_ND, baseline LVP was not affected. Yet, post-ischemic contracture and infarct size were exacerbated and PostC was unable to reduce infarct size and ventricular dysfunction. While short_ND increased phosphatases, non-phosphorylated and phosphorylated Akt, long_ND reduced phosphatase-expression and Akt phosphorylation. Both short_ND and long_ND had no effect on the GSK-3β-phosphorylation but increased the expression of β(2)-adrenoreceptors. In reperfusion, PostC increased Akt phosphorylation regardless of protective effects, but reduced phosphatase-expression in protected hearts only. In conclusion, short_ND improves post-ischemic myocardial performance in postconditioned hearts. However, long_ND increases myocardial susceptibility to I/R injury and abolishes cardioprotection by PostC. This increased susceptibility might be related to steroid-induced hypertrophy and/or to altered enzyme expression/phosphorylation.

13 Distinct role of Ca2+–calmodulin-dependent protein kinase II in excitation–contraction coupling under certain conditions

Ca2+-homeostatic proteins play an important role in Ca2+excitation–contraction coupling (ECC); however, the involvement of Ca2+calmodulin-dependent protein kinase II delta (CaMKII) in this process is less known. It has been suggested...

Comparable Cardioprotection at Reperfusion by Magnesium Orotate and Cyclosporin A: A Study in Isolated Rat Hearts

Orotic acid and its salts has been previously shown to improve the energy status of cardiomyocytes and to protect myocardium following transplantation and cardioplegic arrest. The present study carried out in Langendorff perfused rat hearts was purported to compare cardioprotection elicited by CsA with the one afforded by magnesium orotate (Mg-O) when administrated at reperfusion. To this aim isolated rat hearts (n = 6-8/group) subjected to 30 min of global ischemia and 30 min of reperfusion were randomized to receive: (1) no intervention (Controls), (2) Mg-O (1 mM) and (3) CsA (0.2 μM) in the perfusion buffer throughout the reperfusion period. Recovery of post-ischemic ventricular function was assessed by the left ventricular developed pressure (LVDP), rate pressure product (RPP), and maximal and minimal first derivatives of left ventricular pressure (dLVP/dtmax and dLVP/dtmin) as indices of contractility and relaxation, respectively. All contractile parameters were expressed as percentage of their pre-ischemic values. At the end of the reperfusion period, both Mg-O and CsA induced a substantial recovery of contractile function parameters. Accordingly, LVDP was 59.4 ± 2.24% and 65.8 ± 1.56% in Mg-O and CsA groups, respectively vs. 36 ± 3.68% in Controls (p < 0.001). Similarly, both Mg-O and CsA administration improved contractility (59,4 ± 2.24% and 65.97 ± 1,6%, p=NS) and relaxation indices (67,6 ± 0,8% and 65,8 ± 1,5%, p=NS) when compared to Controls (p < 0.001). In isolated rat hearts, acute administration of Mg-O and CsA at reperfusion was associated with significant improvement of the postischemic contractile function.