Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish

Abstract

In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.

Exploiting the Freshwater Shrimp Neocaridina denticulata as Aquatic Invertebrate Model to Evaluate Nontargeted Pesticide Induced Toxicity by Investigating Physiologic and Biochemical Parameters

“…The heartbeat and maxilliped movement were observed in the following groups: no starvation (control), starvation, and starvation with 1 ppm (IMI treatment). The Poincaré plot is a scattergram generated by plotting each R-R interval towards the previous one, based on our previous methods [ 38 ]. R-R interval is the interval between successive beats defined by heartbeats and gill ventilation.…”

Measurement of Multiple Cardiac Performance Endpoints in Daphnia and Zebrafish by Kymograph

“…End diastolic diameter (EDD) 1 for short axis, EDD 2 for long axis, end systolic diameter (ESD) 1 from short axis, and ESD 2 for long axis can be manually measured using the produced kymograph and adaptation from the method used by Hsiao et al [12] as can be seen on Figure 2. The fractional shortening (FS) which is a measurement of ventricular contractility, can be calculated using the following formula [32]:…”

“…End diastolic diameter (EDD) 1 for short axis, EDD 2 for long axis, end systolic diameter (ESD) 1 from short axis, and ESD 2 for long axis can be manually measured using the produced kymograph and adaptation from the method used by Hsiao et al [12] as can be seen on Figure 2. The fractional shortening (FS) which is a measurement of ventricular contractility, can be calculated using the following formula [32]: By assuming the ventricle is spheroid and the length of unknown diameter is the same as the shorter diameter, end diastolic volume (EDV) can be calculated using the following equation: By assuming the ventricle is spheroid and the length of unknown diameter is the same as the shorter diameter, end diastolic volume (EDV) can be calculated using the following equation:…”

“…Any significant changes within systolic interval and diastolic interval can be considered as heartbeat irregularity. Finally, by combining heart rate data with stroke volume (SV), cardiac output (CO) can be calculated, as previously mentioned by Hsiao et al [12].…”

“…In order to reduce data variation, the current method for heartbeat measurement in zebrafish and Daphnia involved a recording of the region of interest (ROI) and observation of dynamic pixel changes using a computer which was previously published by Sampurna et al [11] using changes of pixel intensity due to blood cell movement during heart contraction. To further improve cardiac measurement endpoint, there are some other parameters such as stroke volume and cardiac output [12] which were not mentioned in previous studies. Currently, there are also several methods using manual counting [10,13,14], MATLAB [15][16][17][18], commercialized software [19][20][21], deep learning [22], and free software [11,23,24] already published for cardiac performance detection; however, these methods might be expensive, require some coding skills, or have limited measurement endpoints (summarized in Table 1).…”