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

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.

Fibroblast-seeded collagen gels in response to dynamic equibiaxial mechanical stimuli: A biomechanical study.

The remodeling of fibroblast-seeded collagen gels in response to dynamic mechanical stimuli was investigated by using a newly developed biaxial culture system capable of cyclically stretching planar soft tissues. Fibroblast-seeded collagen gels were subjected to three distinct dynamic mechanical conditions for six days: Cyclic Equibiaxial Stretching at two constant strain magnitudes (CES-7% and CES-20%), and Cyclic Equibiaxial Stretching with incrementally Increasing stain magnitude (ICES, 7% → 15% → 20% each for two days). The frequency of cyclic stretching was set at 1 Hz. At the end of culture, mechanical properties of the gels were examined by biaxial mechanical testing and checked again upon the removal of seeded cells. Collagen microstructure within the gels was illustrated by multiphoton microscopy. The mRNA levels of collagen type I and type III and fibronectin in the cells were examined by reverse transcription PCR. The protein expression of α-smooth muscle actin was detected by immunohistochemistry. We found that the gels cultured under cyclic stretching were stiffer than those cultured under static stretching. Particularly, the stiffness appeared to be significantly enhanced when the ICES was employed. The enhancement of mechanical properties by cyclic stretching appeared to persist upon cell removal, suggesting an irreversible remodeling of extracellular matrix. Second harmonic generation images showed that collagen fibers became thicker and more compact in the gels cultured under cyclic stretching, which may explain the mechanical findings. The mRNA expression of collagen type I in the cells of the ICES was significantly greater than that of the other groups except for the CES-20%. This study suggests that when cyclic stretching is to be used in engineering soft tissues, incrementally increasing strain magnitude may prove useful in the development of the tissue.

Development of fibroblast-seeded collagen gels under planar biaxial mechanical constraints: a biomechanical study.

Prior studies indicated that mechanical loading influences cell turnover and matrix remodeling in tissues, suggesting that mechanical stimuli can play an active role in engineering artificial tissues. While most tissue culture studies focus on influence of uniaxial loading or constraints, effects of multi-axial loading or constraints on tissue development are far from clear. In this study, we examined the biaxial mechanical properties of fibroblast-seeded collagen gels cultured under four different mechanical constraints for 6 days: free-floating, equibiaxial stretching (with three different stretch ratios), strip-biaxial stretching, and uniaxial stretching. Passive mechanical behavior of the cell-seeded gels was also examined after decellularization. A continuum-based two-dimensional Fung model was used to quantify the mechanical behavior of the gel. Based on the model, the value of stored strain energy and the ratio of stiffness in the stretching directions were calculated at prescribed strains for each gel, and statistical comparisons were made among the gels cultured under the various mechanical constraints. Results showed that gels cultured under the free-floating and equibiaxial stretching conditions exhibited a nearly isotropic mechanical behavior, while gels cultured under the strip-biaxial and uniaxial stretching conditions developed a significant degree of mechanical anisotropy. In particular, gels cultured under the equibiaxial stretching condition with a greater stretch ratio appeared to be stiffer than those with a smaller stretch ratio. Also, a decellularized gel was stiffer than its non-decellularized counterpart. Finally, the retained mechanical anisotropy in gels cultured under the strip-biaxial stretching and uniaxial stretching conditions after cell removal reflected an irreversible matrix remodeling.

The mechanisms of propofol-induced block on ion currents in differentiated H9c2 cardiac cells.

General anesthetic propofol (2,6-bis(isopropyl)-phenol) possess a chemical structure unrelated to other anesthetic drugs. It has been known to block a variety of ion currents. This study is designed to determine the effect of this drug on ion currents in differentiated H9c2 cardiac cells. The effects of propofol, an intravenous anesthetic agent with a distinct chemical structure, on ion currents of differentiated clonal cardiac (H9c2) cells were investigated in this study. Propofol (10-300 microM) suppressed the amplitude of delayed rectifier K(+) current (I(K(DR))) in a concentration-dependent manner with an IC(50) value of 36 microM. This compound reduced activation time constant and increased current inactivation, although no voltage dependency of propofol-induced block of I(K(DR)) can demonstrated. Neither diazoxide, pinacidil, nor caffeic acid phenethyl ester had any effect on propofol-induced block of I(K(DR)). Propofol (30 microM) had no effect on erg-mediated K(+) current in these cells; however, it suppressed L-type Ca(2+) current (I(Ca,L)) of cardiac and skeletal types to a similar extent. Intracellular dialysis with propofol (100 microM) had no effects on I(K(DR)) or I(Ca,L). Numerical simulations of I(K(DR)) based on a Markovian model reproduce the experimental results and show that propofol-induced blockade of I(K(DR)) is associated with an decrease in forward rate of the activation process and an increase in transitional rate into the inactivated state. Propofol can suppress I(K(DR)) in differentiated H9c2 cardiac cells in a concentration- and state-dependent manner. These effects can significantly contribute its action on functional activity of heart cells.