The temporal impact of erythropoietin administration on mitochondrial function and dynamics in cardiac ischemia/reperfusion injury.

Erythropoietin (EPO) has been shown to provide protection against ischemia/reperfusion (I/R) injury in various experimental models. However, clinical trials revealed unsatisfactory results when EPO was given to patients with myocardial infarction following reperfusion. The timing of EPO administration and its relation to mitochondrial function may largely involve in this controversy. We hypothesized that EPO given at different time points exert varying cardioprotective effects in terms of myocardial infarct size, left ventricular (LV) function, arrhythmia, apoptosis, mitochondrial function, and mitochondrial dynamics in rats with cardiac I/R injury. Male Wistar rats were subjected to either sham (n = 6) or cardiac I/R operation (n = 48). Rats undergoing cardiac I/R operation (30-min ischemia, followed by 120-min reperfusion) were allotted into 4 subgroups (n = 12/group): vehicle, EPO pretreatment, EPO given during ischemia, and EPO given at reperfusion. EPO was administered intravenously at 5000 unit/kg. Arrhythmia and LV function were monitored throughout the protocol. Next, the hearts were collected to determine infarct size, mitochondrial function, mitochondrial dynamics, gap junction protein, and apoptosis. Cardiac I/R promoted arrhythmias, LV dysfunction, infarct size expansion, apoptosis, mitochondrial dysfunction and increased mitochondrial fission. EPO given either before or during ischemia, but not at reperfusion, attenuated arrhythmia scores, LV dysfunction, infarct size, and apoptosis. Only EPO given either before or during ischemia alleviated mitochondrial swelling, mitochondrial depolarization, and reduced the levels of p-Drp1ser616/Drp1. Data from in vitro study also confirmed that EPO directly attenuated mitochondrial dysfunction in H9c2 cells subjected to hypoxia/reoxygenation. In conclusion, the EPO administration, either before or during ischemia, exerted cardioprotection against I/R injury by attenuating mitochondrial dynamic imbalance, mitochondrial dysfunction, and apoptosis, leading to reduced infarct size and improved LV function.

Acetylcholine exerts cytoprotection against hypoxia/reoxygenation-induced apoptosis, autophagy and mitochondrial impairment through both muscarinic and nicotinic receptors.

Acetylcholine (ACh) has been shown to exert cardioprotection against myocardial ischemia/reperfusion (I/R) injury. However, whether ACh exerts its cardioprotection predominantly through the activation of muscarinic or nicotinic ACh receptors is not fully understood. We investigated the effects of hypoxia/reoxygenation (H/R) in the presence or absence of ACh receptor agonists in H9c2 cells. Cells (2.5 × 105 cells/well) were incubated in the hypoxic chamber with the ischemic solution (30 min) followed by reoxygenation (120 min) with the normal media. ACh or nicotinic ACh receptor agonist (GTS21) was applied 5 min prior to hypoxia, during hypoxia or at reoxygenation onset. Cell viability, apoptosis, ER stress, mitochondrial dynamics and biogenesis were determined. H/R significantly decreased cell viability and mitochondrial biogenesis and increased apoptosis, ER stress, mitochondrial fission and autophagic flux compared with the control. ACh and GTS21 significantly increased cell viability via reducing apoptosis, autophagy, and ER stress. However, ACh and GTS21 increased mitochondrial fusion when applied before or during hypoxia. During reoxygenation onset, only ACh increased mitochondrial biogenesis. Co-treatment with atropine reversed the beneficial effects of ACh and GTS21. Our findings demonstrated that ACh exerted cytoprotection against H/R-induced apoptosis, autophagy and mitochondrial impairment through the activation of both muscarinic and nicotinic receptors.

Revisiting the Cardioprotective Effects of Acetylcholine Receptor Activation against Myocardial Ischemia/Reperfusion Injury.

Acute myocardial infarction (AMI) is the most common cause of acute myocardial injury and its most clinically significant form. The most effective treatment for AMI is to restore an adequate coronary blood flow to the ischemic myocardium as quickly as possible. However, reperfusion of an ischemic region can induce cardiomyocyte death, a phenomenon termed "myocardial ischemia/reperfusion (I/R) injury". Disruption of cardiac parasympathetic (vagal) activity is a common hallmark of a variety of cardiovascular diseases including AMI. Experimental studies have shown that increased vagal activity exerts cardioprotective effects against myocardial I/R injury. In addition, acetylcholine (ACh), the principle cardiac vagal neurotransmitter, has been shown to replicate the cardioprotective effects of cardiac ischemic conditioning. Moreover, studies have shown that cardiomyocytes can synthesize and secrete ACh, which gives further evidence concerning the importance of the non-neuronal cholinergic signaling cascades. This suggests that the activation of ACh receptors is involved in cardioprotection against myocardial I/R injury. There are two types of ACh receptors (AChRs), namely muscarinic and nicotinic receptors (mAChRs and nAChRs, respectively). However, the effects of AChRs activation in cardioprotection during myocardial I/R are still not fully understood. In this review, we summarize the evidence suggesting the association between AChRs activation with both electrical and pharmacological interventions and the cardioprotection during myocardial I/R, as well as outline potential mechanisms underlying these cardioprotective effects.

Vagus nerve stimulation exerts cardioprotection against myocardial ischemia/reperfusion injury predominantly through its efferent vagal fibers.

Vagus nerve stimulation (VNS) has been shown to exert cardioprotection against myocardial ischemia/reperfusion (I/R) injury. However, whether the cardioprotection of VNS is mainly due to direct activation through its ipsilateral efferent fibers (motor) rather than indirect effects mediated by the afferent fibers (sensory) have not been clearly understood. We hypothesized that VNS exerts cardioprotection predominantly through its efferent vagal fibers. Thirty swine (30-35 kg) were randomized into five groups: I/R no VNS (I/R), and left mid-cervical VNS with both vagal trunks intact (LC-VNS), with left vagus nerve transection (LtVNX), with right vagus nerve transection (RtVNX) and with atropine pretreatment (Atropine), respectively. VNS was applied at the onset of ischemia (60 min) and continued until the end of reperfusion (120 min). Cardiac function, infarct size, arrhythmia score, myocardial connexin43 expression, apoptotic markers, oxidative stress markers, inflammatory markers (TNF-α and IL-10) and cardiac mitochondrial function, dynamics and fatty acid oxidation (MFN2, OPA1, DRP1, PGC1α and CPT1) were determined. LC-VNS exerted cardioprotection against myocardial I/R injury via improvement of mitochondrial function and dynamics and shifted cardiac fatty acid metabolism toward beta oxidation. However, LC-VNS and LtVNX, both efferent vagal fibers are intact, produced more profound cardioprotection, particularly infarct size reduction, decreased arrhythmia score, oxidative stress and apoptosis and attenuated mitochondrial dysfunction compared to RtVNX. These beneficial effects of VNS were abolished by atropine. Our findings suggest that selective efferent VNS may potentially be effective in attenuating myocardial I/R injury. Moreover, VNS required the contralateral efferent vagal activities to fully provide its cardioprotection.

Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1.

BACKGROUND: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity. OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs. METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI). RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity. CONCLUSIONS: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

Enhanced production of functional extracellular single chain variable fragment against HIV-1 matrix protein from Escherichia coli by sequential simplex optimization.

The optimal culture condition for extracellular recombinant single chain variable fragment anti HIV-1 p17 protein (scFv anti-p17) production in Escherichia coli HB2151 was investigated by the sequential simplex optimization (SS) method. Five variable parameters were submitted in the fermentation process. The most favorable condition obtained from 19 independent experiments was as followed: 58 µM of IPTG induction to 1.7 OD600 nm at 25.5°C for 16 h with 202 rpm agitation rate. The amount of secreted scFv anti-p17 at the optimal condition was 38% higher than under the control condition. The binding activity of soluble extracellular scFv anti-p17 protein increased 95.5% and 73.2% in comparison with the control condition and non-optimized condition respectively. The soluble scFv anti-p17 from crude HB2151 lysated was subsequently purified by immobilized metal ion affinity chromatography (IMAC) with His-tag. The purified scFv anti-p17 was intact and retained its antigen-binding affinity against HIV-1 p17. We demonstrated that the sequential simplex optimization method was a key for exertion of high yield with fewer experimental requirements for acquiring of large scale secretory protein production.