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

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: Ank GAG 1D4 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 Ank GAG 1D4 would potentially enhance the Ank GAG 1D4-mediated antiviral activity. Objective: To augment the affinity of Ank GAG 1D4 scaffold towards its CA target, through computational predictions and experimental designs. Method: Three dimensional structure of the binary complex formed by Ank GAG 1D4 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 Ank GAG 1D4 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 Ank GAG 1D4 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 Ank GAG 1D4-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 Ank GAG 1D4 ankyrin for its CA target. Ank GAG 1D4-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