Lydia Lacerda

Ischaemic postconditioning protects against reperfusion injury via the SAFE pathway

AIMS Ischaemic postconditioning (IPostC) is a powerful protective phenomenon that activates prosurvival intrinsic signalling cascades to limit reperfusion injury. We propose that IPostC confers its infarct-sparing effect via activation of the newly described prosurvival Survivor Activating Factor Enhancement (SAFE) pathway, which involves the activation of the cytokine tumour necrosis factor alpha (TNFalpha) and signal transducer and activator of transcription-3 (STAT-3). METHODS AND RESULTS Isolated ischaemic/reperfused hearts from TNF knockout, TNF receptor-1 knockout, TNF receptor-2 knockout, cardiomyocyte-specific STAT-3-deficient mice or their respective wild-type, (TNF-WT) or (STAT-3-WT), were postconditioned by ischaemic episodes (IPostC) or with exogenous TNFalpha (0.5 microg/L) (TNF-PostC) at the onset of reperfusion. IPostC reduced infarct size (IS) in TNF-WT and TNFR1(-/-) hearts (by 33 and 27%, respectively, P < 0.05), whereas hearts from TNF(-/-) or TNFR2(-/-) failed to be postconditioned. TNF-PostC reduced IS by 37% (P < 0.05) in STAT-3-WT hearts but failed to protect cardiac-specific STAT-3(-/-) hearts. Administration of wortmannin, an inhibitor of PI-3 kinase/Akt, or PD98059, an inhibitor of extracellular regulated kinase 1/2 (Erk1/2), during the postconditioning stimulus did not abolish the infarct-sparing effect of TNF-PostC. AG490, an inhibitor of STAT-3, abrogated the protective effect of TNFalpha. Western blot analysis did not demonstrate the involvement of Akt or Erk1/2 in TNF-PostC, whereas STAT-3 phosphorylation was increased in both IPostC and TNF-PostC. CONCLUSION The protective effect of the SAFE pathway is shown in IPostC, with the activation of TNFalpha, its receptor type 2, and STAT-3. This signalling cascade is activated independently of the well-known Reperfusion Injury Salvage Kinases (RISK) pathway, which involves the kinases Akt and Erk1/2.

Pharmacological Preconditioning With Tumor Necrosis Factor-α Activates Signal Transducer and Activator of Transcription-3 at Reperfusion Without Involving Classic Prosurvival Kinases (Akt and Extracellular Signal–Regulated Kinase)

Background— We previously reported that tumor necrosis-factor-α (TNF-α) can mimic classic ischemic preconditioning (IPC) in a dose- and time-dependent manner. Because TNF-α activates the signal transducer and activator of transcription-3 (STAT-3), we hypothesized that TNF-α–induced preconditioning requires phosphorylation of STAT-3 rather than involving the classic prosurvival kinases, Akt and extracellular signal–regulated kinase (Erk) 1/2, during early reperfusion. Methods and Results— Isolated, ischemic/reperfused rat hearts were preconditioned by either IPC or low-dose TNF-α (0.5 ng/mL). Western blot analysis confirmed that IPC phosphorylated Akt and Erk 1/2 after 5 minutes of reperfusion (Akt increased by 34±6% and Erk, by 105±28% versus control; P<0.01). Phosphatidylinositol 3-kinase/Akt inhibition (wortmannin) or mitogen-activated protein kinase–Erk 1/2 kinase inhibition (PD-98059) during early reperfusion abolished the infarct-sparing effect of IPC. In contrast, TNF-α preconditioning did not phosphorylate these kinases (Akt increased by 7±7% and Erk, by 17±14% versus control; P=NS). Neither wortmannin nor PD-98059 inhibited TNF-α–mediated cardioprotection. However, TNF-α and IPC both phosphorylated STAT-3 and the proapoptotic protein Bcl-2 antagonist of cell death (BAD) (STAT-3 increased by 58±17% with TNF-α or by 68±12% with IPC; BAD increased by 75±8% with TNF-α or by 205±20% with IPC; P<0.01 versus control), thereby activating the former and inactivating the latter. The STAT-3 inhibitor AG 490 abolished cardioprotection and BAD phosphorylation with both preconditioning stimuli. Conclusions— Activation of the classic prosurvival kinases (Akt and Erk 1/2) is not essential for TNF-α–induced preconditioning in the early reperfusion phase. We show the existence of an alternative protective pathway that involves STAT-3 activation specifically at reperfusion in response to both TNF-α and classic IPC. This novel prosurvival pathway may have potential therapeutic significance.

Is red wine a SAFE sip away from cardioprotection? Mechanisms involved in resveratrol‐ and melatonin‐induced cardioprotection

Abstract:  Epidemiological studies suggest that regular moderate consumption of red wine confers cardioprotection but the mechanisms involved in this effect remain unclear. Recent studies demonstrate the presence of melatonin in wine. We propose that melatonin, at a concentration found in red wine, confers cardioprotection against ischemia–reperfusion injury. Furthermore, we investigated whether both melatonin and resveratrol protect via the activation of the newly discovered survivor activating factor enhancement (SAFE) prosurvival signaling pathway that involves the activation of tumor necrosis factor alpha (TNFα) and the signal transducer and activator of transcription 3 (STAT3). Isolated perfused male mouse (wild type, TNFα receptor 2 knockout mice, and cardiomyocyte‐specific STAT3‐deficient mice) or rat hearts (Wistars) were subjected to ischemia–reperfusion. Resveratrol (2.3 mg/L) or melatonin (75 ng/L) was perfused for 15 min with a 10‐min washout period prior to an ischemia–reperfusion insult. Infarct size was measured at the end of the protocol, and Western blot analysis was performed to evaluate STAT3 activation prior to the ischemic insult. Both resveratrol and melatonin, at concentrations found in red wine, significantly reduced infarct size compared with control hearts in wild‐type mouse hearts (25 ± 3% and 25 ± 3% respectively versus control 69 ± 3%, P < 0.001) but failed to protect in TNF receptor 2 knockout or STAT3‐deficient mice. Furthermore, perfusion with either melatonin or resveratrol increased STAT3 phosphorylation prior to ischemia by 79% and 50%, respectively (P < 0.001 versus control). Our data demonstrate that both melatonin and resveratrol, as found in red wine, protect the heart in an experimental model of myocardial infarction via the SAFE pathway.

Diazoxide-induced respiratory inhibition – a putative mitochondrial K ATP channel independent mechanism of pharmacological preconditioning

The ischemic preconditioning biological phenomenon has been explored to identify putative pharmacologic agents to mimic this cytoprotective program against cellular ischemic injury. Diazoxide administration confers this cytoprotection, however, whether this is via direct activation of the putative mitochondrial KATP (mKATP) channel which was originally proposed has been questioned. Here, we present data supporting an alternate hypothesis evoking mitochondrial respiratory inhibition rather than mKATP channel activation, as a mediating event in the diazoxide-activated cytoprotective program. Mitochondrial respiration and reactive oxygen species (ROS) production was measured in digitonin-permeabilized C2C12 myotubes, allowing for the modulation of mKATP conductance by changing the potassium concentration of the medium (0–130 mM). Diazoxide dose-dependently attenuated succinate-supported respiration, an effect that was independent of mKATP channel conductance. Similarly, 5-hydroxydecanoate (5-HD), a putative mKATP channel blocker, released diazoxide-induced respiratory inhibition independently of potassium concentration. Since diazoxide-induced cytoprotection and respiratory inhibition are both integrally linked to ROS generation we repeated above experiments following ROS generation using DCF fluorescence. Cytoprotective doses of diazoxide increased ROS generation independently of potassium concentration and 5-HD inhibited ROS production under the same conditions. Collectively these data support the hypothesis that diazoxide-mediated cytoprotection is independent of the conductance of the mKATP channel and rather implicate mitochondrial respiratory inhibition-triggered ROS signaling.

HDL protects against ischemia reperfusion injury by preserving mitochondrial integrity.

OBJECTIVE High density lipoproteins (HDL) protect against ischemia reperfusion injury (IRI). However the precise mechanisms are not clearly understood. The novel intrinsic prosurvival signaling pathway named survivor activating factor enhancement (SAFE) path involves the activation of tumor necrosis factor (TNF) alpha and signal transducer and activator of transcription 3 (STAT3). SAFE plays a crucial role in cardioprotection against IRI. We propose that HDL protect against IRI via activation of the SAFE pathway and modulation of the mitochondrial permeability transition pore (mPTP) opening. METHODS AND RESULTS Isolated mouse hearts were subjected to global ischemia (35 min) followed by reperfusion (45 min). HDL were given during the first 7 min of reperfusion. In control hearts, the post-reperfusion infarct size was 41.3 ± 2.3%. Addition of HDL during reperfusion reduced the infarct size in a dose-dependent manner (HDL 200 μg protein/ml: 25.5 ± 1.6%, p < 0.001 vs. control). This protective effect was absent in TNF deficient mice (TNF-KO) or cardiomyocyte-STAT3 deficient mice (STAT3-KO). Similarly, HDL, given as a preconditioning stimulus, improved cell survival and inhibited mPTP opening in isolated cardiomyocytes subjected to simulated ischemia. These protective responses were inhibited in cardiomyocytes from TNF-KO and STAT3-KO mice. CONCLUSION Our data demonstrate that HDL protect against IRI by inhibition of mPTP opening, an effect mediated via activation of the SAFE pathway.

Relationship Between Left Ventricular Geometry and Soluble ST2 in a Cohort of Hypertensive Patients

Left ventricular (LV) hypertrophy (LVH) is classified according to geometric pattern into 4 types: concentric hypertrophy, eccentric hypertrophy, concentric remodeling, and normal geometry. Prevalence of death and cardiovascular complications associated with hypertension depend on the geometric pattern. Although soluble ST2 levels, a novel cardiac biomarker of mechanical strain is increased in hypertension, the relationship with hypertensive LV geometric patterns has not been studied. The authors investigated the relationship between soluble ST2 levels and LV geometric patterns in a cohort of hypertensive patients. LVH was considered present when echocardiographic LV mass index exceeded 49.2 g/m2.7 in men and 46.2 g/m2.7 in women. Patients with concentric hypertrophy had higher soluble ST2 levels compared with patients with normal geometry (20.4±8.4 ng/mL vs 14.3±5.4 ng/mL, P<.002). Therefore, soluble ST2 level is not only affected by hypertensive LV, but may be a future biomarker in differentiating concentric hypertrophy from normal geometry in hypertension.

The proposed role of plasma NT pro-brain natriuretic peptide in assessing cardiac remodelling in hypertensive African subjects : cardiovascular topic

Summary Aim Although plasma NT-proBNP differentiates hypertension (HT) with or without left ventricular hypertrophy (LVH) from hypertensive heart failure (HHF), most of the published data are based on studies in Western populations. Also, most previous studies did not consider left ventricular (LV) diastolic function and right ventricular (RV) function. We therefore examined the relation between NT-proBNP on LV and RV remodelling in an African hypertensive cohort. Methods Subjects were subdivided into three groups after echocardiography: hypertensives without LVH (HT) (n = 83); hypertensives with LVH (HT+LVH) (n = 50); and those with hypertensive heart failure (HHF) (n = 77). Results Subjects with HHF had significantly higher NT-proBNP levels compared to the HT+LVH group (p < 0.0002). NT-proBNP correlated positively with right atrial area, an indirect measure of RV function. Conclusions NT-proBNP is proposed as a useful biomarker in differentiating hypertension with or without LVH from hypertensive heart failure in black hypertensive subjects.