Calcitonin Gene-Related Peptide Inhibits Cardiomyocyte Injury Induced by Hypoxia / Reoxygenation via Restoration of Cytosolic and Mitochondrial Calcium

This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. From the Department of Anesthesia, Shanxi Medical University, Taiyuan, China; Department of Anesthesia, Second Hospital of Shanxi Medical University, Taiyuan, China; Key Laboratory of Cellular Physiology at Shanxi Medical University, National Education Commission, Taiyuan, China.

I schemia/reperfusion injury (IRI) is defined as the damage to cardiac tissue when blood supply returns after a period of ischemia (1).Among the therapeutic strategies for IRI, ischemic preand post-conditionings (2, 3) are effective in promotion of survival of myocytes (4), via activation of intrinsic cardioprotective mechanisms, in which many factors and molecules participate (2,5).Calcitonin gene-related peptide (CGRP), a principle transmitter of capsaicin-sensitive sensory nerves, plays an important role in the mediation of the cardioprotection afforded by ischemic pre-and post-conditions (6).However, the precise mechanism underlying the cardioprotective effects of CGRP is still unclear.Overload of cytosolic and mitochondrial calcium was found to play a pivotal role in the IRI (7,8), which is a consequence of the insults by pro-injury Volume 3 May, 2016 Number 3 factors (9).Previous reports from other and our groups demonstrated a dose-dependent inhibitory effects of CGRP on inward calcium currents in isolated myocytes (10,11).However, whether there is an association between CGRP produced cardioprotection and modulating the calcium inside the myocytes is still unknown.
In this study, we tested the hypothesis that the fact CGRP protects the cardiomyocyte from injury induced by H/R is associated with modulation of cytosolic and mitochondrial calcium, using cultured neonatal myocytes of rats.The setting facilitates the clarification of a direct activity of CGRP on the myocytes.

Ethics Statement
The study was reviewed and approved by the Institutional Animal Care and Use Committee of Shanxi Medical University and conducted in accordance with the guidelines for the care and use of laboratory animals (National Institute of Health Guide for the Care and Use of Laboratory Animals, NIH Publications No. 80-23, revised 1996, http://grants.nih.gov/86grants/olaw/ olaw.htm).

Cardiomyocyte Culture and Induction of Cell Injury
Myocyte cultures were prepared and the cardiomyocytes were identified as we previously reported (12).Briefly, the hearts were collected from the neonates of Sprague-Dawley rats (Shanxi Medical University Experimental Animal Laboratory), within 72 hours of birth, after euthanasia with ether.Then the tissues of the ventricles were minced and digested in 2 ml of solution containing collagenase type Ⅱ (1 mg/ml, Invitrogen Corporation, Carlsbad, CA, USA) for 3-5 minutes at 37℃.The cardiomyocytes were centrifuged at 1000 rpm for 5 minutes.The cells were collected and re-suspended in 12 ml Dulbecco's modified eagle medium (DMEM) in a 90 mm culture dish, and then incubated for 60 minutes at 37℃ in a carbon dioxide (CO2) incubator (Thermo, Hudson, NH, USA) with a gas phase of 5% CO2 in humidified air.The floating cardiomyocytes were collected and cell numbers were adjusted to 5 × 10 5 cells/ml in DMEM supplement-ed with 10% fetal bovine serum and 0.1 μmol/L of 5-bromo-2' -deoxyuridine (Sigma-Aldrich, St. Louis, MO, USA) to prevent non-myocardial cells proliferation.Then, 3 ml of the cell suspension were added into each well of the six-well cell culture clusters (Corning Gilbert Inc., Glendale, AZ, USA).For analysis of cell apoptosis with terminal deoxyuridine triphosphate (dUTP) deoxynucleotidyltransferase nick end-labeling (TUNEL) staining and the mitochondrion and cytosolic calcium assay, the myocytes were cultured on cover slips (pretreated with polylysine).
After 72 hours of incubation, the hypoxia/reoxygenation (H/R) of the cultured myocytes was carried out as follows.The cell culture clusters were transferred into a hermetic chamber (5, 000 cm 3 ) with circulating nitrogen (99.9% of N2) at rate of circulation of 0.5 l/minute, at 37℃ .The culture medium was replaced by the solution containing NaHCO3 (6.0 mmol/l), Na-Cl (98.5 mmol/l), KCl (10.0 mmol/l), CaCl2 (1.0 mmol/l), sodium lactate (40 mmol/l), MgSO4 (1.2 mmol/l) and HEPES (20 mmol/l) (13), which had been saturated with 99.9% of N2 for 2 hours (at a rate of 0.5 l/minute).After 3 hours of incubation in the oxygen-deprived culture (partial pressure of oxygen [PO2] <10 mm Hg), the medium was replaced by standard DMEM (PO2>100 mm Hg) and the cells were incubated in it for 2 hours.

Experimental Protocol
As shown by figure 1, the cultured cells were randomly divided into four groups.The control group, the cells were kept in normoxic culture for 5 hours, without any treatment.The H/R group, the cells were treated with the hypoxia for 3 hours followed by the reoxygenation for 2 hours, without any other treatment.The CGRP postconditioning group (H/R + CGRP), the cells were treated with the H/R and CGRP, given at 10 -8 mol/L (the final concentration, Tocris, UK) immediately before the start of the reoxygenation.The CGRP8-37 antagonizing group (H/R + CGRP8-37 + CGRP), CGRP8-37 (Tocris, UK), a specific antagonist of CGRP receptor, was administrated (at the final concentration of 10 -7 mol/L), 1 hour before the induction of cell-hypoxia, then the treatments of H/R and CGRP (10 -8 mol/ L) were followed as scheduled.The doses of CGRP and CGRP8-37 used in this study were determined according to our previous studies (11,14).The cell injury was evaluated by examinations of the cell apoptosis and the alterations of lactate dehydrogenase (LDH) and caspase-3.

Determination of Cardiomyocyte Apoptosis
The TUNEL assay was performed to analyze the cell apoptosis as previously reported (11,14).Briefly, at the end of the reoxygenation, the cells were fixed with 4% paraformaldehyde for 1 hour at room temperature and then the cells were processed as the instruction for TUNEL assay.The apoptotic cells were visualized by stain with an in situ cell death detection kit (Roche, Switzerland).Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) and visualized by microscopy.Apoptotic index was calculated as the percentage of apoptotic cells and total number of cells.

Measurements of LDH and Caspase-3
The changes of LDH and caspase-3 were determined by enzyme-linked immunosorbent assay (ELISA), as we previously reported (15,16).Briefly, at the end of the experiments, 100 μl of culture medium collected from each well for the assessment of LDH and then the cultured myocytes in each well were rinsed with phosphatebuffered saline (PBS) and scraped from the culture cluster with a cell lifter (Corning Gilbert Inc., Glendale, AZ, USA), then loaded into a 1.5 ml EP tube.Then 100 μl of cold lysis buffer were added and the cells were incubated for 60 minutes on ice.The lysate from cell extracts was centrifuged at 14,000 × g for 10 minutes at 4℃ .The supernatant was collected.The bicinchoninic acid assay (BCA) method was used to determine the protein content extracted from the cultured cells and the culture medium collected from each well, respectively, with Pierce BCA Protein Assay Kit (Thermo Scientific, Hudson, NH, USA).Then LDH and caspase-3 were determined using a rat LDH kit (ScienCell, Carlsbad, CA, USA) and a rat caspase-3 kit (R&D Systems Inc., Minneapolis, MN, USA) respectively.The results were produced by using a microplate reader (Thermo Multiskan Ascent microplate spectrophotometer, Thermo electron corporation, USA) and expressed as fold of control.

Determination of Cytosolic and Mitochondrial Calcium
Cytosolic and mitochondrial calcium was analyzed by co-incubating the cells with a cell-permeant but mitochondria-impermeant calcium fluorophore, Fluo-3 AM (5 μmol/l, Beyotime, China) and a mitochondria-permeant calcium fluorophore, Rhod-2 AM (5 μmol/l, Sigma-Aldrich, St. Louis, MO, USA), respectively, as reported (17).Cells were visualized by microscopy and photographed.Quantitative analysis of the images was carried out using ImageJ, a NIH software.The calcium concentration was expressed as proportion of the fluorescence intensity (FI).

Immunofluorescence of CGRP Receptor
After 72 hours of incubation, CGRP receptor was detected using immunofluorescence assay with antibodies against calcitonin receptor-like receptor (CRLR, 1:50, Santa Cruz, Dallas TX, USA) and receptor-activity-modifying proteins 1 (RAMP-1), 1:50, Santa Cruz, USA).The cells were divided into two groups (N=3), dyed with CRLR antibody and RAMP-1 antibody respectively.Cells were visualized by microscopy and photographed.formed by one-way analysis of variance (ANO -VA) and by the Tukey's test.A value of P<0.05 was considered to be statistically significant.All data analysis was conducted with the SPSS 13.0 software package.

Cardiomyocyte Apoptosis
As shown in figure 2, a significant increase of apoptotic ratio of the myocytes was detected in H/R group, by 80% , compared to the control group (P<0.001).Treatment with CGRP significantly attenuated the cell apoptosis in the H/R group, by 36%, when compared to that in the H/ R group (P<0.001).CGRP8-37, a specific antagonist of CGRP receptor, effectively reversed the effect of CGRP, by 85% (P=0.001).

Caspase-3 and LDH
As the apoptosis ratio, greater levels of caspase-

Cytosolic and Mitochondrial Calcium
As shown in figure 4, significantly higher concentrations of cytosolic and mitochondrial calcium were observed from the myocytes in H/R group, compared to those of controls (cytosolic, P=0.006; mitochondrial, P=0.003).Exogenously administrated CGRP (at 10 -8 mol/l) significantly inhibited both of the increases of the concentrations of the cytosolic and mitochondrial calcium (cytosolic, P=0.006; mitochondrial, P= 0.034).The effects of CGRP on calcium were blocked by CGRP8-37, indicating the effects mediated by the specific receptor of CGRP (cytosolic, P=0.014; mitochondrial, P=0.043).

CGRP Recepters
As shown in figure 5, the green fluorescence showed CRLR (Figure 5A) and RAMP-1 (Figure 5B), respectively.CGRP receptors in the cardiomyocytes were distributed in the cytoplasm and nucleus.

DISCUSSION
The important findings of this study were the cardioprotective effects of CGRP and its association with homeostasis of cytosolic and mitochondrial calcium.Although CGRP has shown cardioprotective properties, reduced infarct size (16,18) and improved cardiac performance (19), few underlying mechanisms are known.Previous reports from other and our groups demonstrated that CGRP attenuated the increases in L-type calcium current of cardiomyocytes (10,11) and apoptosis of cultured cardiomyocytes (12,14), arising a potential effect of the peptide on calcium modulation.Here in this study, we tested the hypothesis that CGRP may protect cardiomyocytes from the injury induced by hypoxia and reoxygenation, which may be associated with modulation of cytosolic and mitochondrial calcium.Firstly, the experimental setting of H/R in this study induced the significant and reproductive cell injury in the cultured cardiomyocytes, shown as increase of LDH, caspase-3 and cell apoptosis.The experimental setting partially mimicked the environment of cardiomyocytes in acute ischemia and reperfusion, which is characterized by lower PO2 followed by reoxygenation and the adaptive responses of the cardiomyocytes to the changes of oxygen in the surrounding matrix, but without neural and paracrine humoral modulations.Secondly, the distribution of CGRP receptors were localized in the myocytes, which is an important base on which the pharmacological experiments with CGRP and CGRP8-37 can be possibly carried out in this study.This experimental model was applicable to test the protective effect of CGRP on cardiomyocytes.
Apoptosis is closely associated with many cardiovascular diseases (20).Caspase-3 stands at the junction of pathways mediated by other caspases in the cell, activation of which results in an increase of cell apoptosis, an enlargement of the infarct size and impairment of cardiac functions (21,22).Increase of the amount of caspase-3 observed in this study served as an indicator of activation of the pro-apoptotic pathways by the H/R, as it went up well with the increases of the myocyte apoptosis, which also paralleled well with the increases of cytosolic and mitochondrial calcium, presenting an association of the up-grading of the calcium concentration with the activation of caspase-3 and increase in apoptosis of the cardiomyocytes.Furthermore, down-regulating the calcium concentrations by CGRP, in this study, went well with the reduction of caspase-3 and an attenuation of apoptosis of the cardiomyocytes, clearly presenting the association of homeostasis of cytosolic and mitochondrial calcium with increase of cell survival.The findings may suggest that the cardioprotective effect of CGRP may be associated with securing the homeostasis of cytosolic and mitochondrial calcium.
The release of LDH to the surrounding medium is a reliable marker of myocyte injury that re-  lates to cell death (23,24).Therefore, increased level of LDH by H/R and inhibition of the elevation of LDH by CGRP served as one of the supporting quantitative parameters presenting the cell injury and the effect on cell protection from the injury, respectively, in this study.The results, obtained in this study on the alteration of apoptosis and LDH with the changes in caspase-3 and the calcium, without and with CGRP, clearly demonstrated a cardioprotective effect of CGRP and its association with modulation of the cytosolic and mitochondrial calcium.The calcium signaling system is highly complex and intimately related with excitation-contraction coupling mechanism, gene expression, enzyme functions and cardiomyocyte injury (25,26).Myocardial ischemia and reperfusion induces increase of calcium in the cardiomyocytes (25,26), which is responsible for cell death and postischemic contractile dysfunction (27,28).An increase in cytosolic calcium will stimulate calcium-dependent phospholipases, inducing the breakdown of cell membranes and release of toxic productions (25).It is well known, that calcium overload may disturb the cell metabolism, via causing dysfunction of mitochondria (29), which plays a crucial role in cell apoptosis (25), by an interaction of the endoplasmic reticulum and the mitochondria, upon pro-apoptotic stimulus, and induces the release of calcium from en-doplasmic reticulum and calcium overload in mitochondria (30).Mitochondrial uptake of calcium is a crucial process in cell physiology (19,31).Overload of mitochondrial calcium induces the opening of the MPTP (8,32), which may lead to the release of cytochrome C and other pro-apoptotic molecules that initiate the apoptotic cascade (32).Elevation of the levels of calcium also increases calcium-ATPase activity and results in enhancement of consumption of ATP, which may lead to cell death (25) and alleviation of cytosolic and mitochondrial calcium overload is an effective way to facilitate cell survival (25,26).The effects of CGRP observed in this study were similar to the effects of diltiazem, a calcium blocker, on calcium elevation and cell apoptosis.
The findings of this study expended current knowledge on the mechanism of CGRP on cardioprotection.However, the authors of this article would state that there were no groups of calcium ionophore and calcium antagonist.We would further investigate and elucidate the cellular and molecular mechanisms of cardioprotection induced by CGRP and its relationship with calcium modulation.

CONCLUSION
It could be concluded that CGRP produces cardioprotection, which may be associated with securing homeostasis of cytosolic and mitochondrial calcium.

Figure 3 .
Figure 3. Changes of LDH in the Culture Medium and Caspase-3 in the Myocytes.This figure presented changes of LDH (A) in the culture medium and caspase-3 (B) in the myocytes in the groups of control (Ct), hypoxia/ reoxygenation (H/R), CGRP treatment (H/R+CGRP) and CGRP8-37 antagonizing (H/R + CGRP + C8-37).* P<0.05 compared to the control; $ P<0.05 compared to the H/R group; # P<0.05 compared to the H/R + CGRP group.

Figure 4 .
Figure 4. Changes of Concentration of Cytosolic Calcium (A-D) and Mitochondrial Calcium (E-H).A and E. Representative images for control groups (Ct); B and F. for hypoxia/reoxygenation groups (H/R); C and G. for CGRP treatment groups (H/R +CGRP); D and H. for CGRP8-37 antagonizing groups (H/ R + CGRP + C8-37); I. Summary of the observations.Scale bars were 50 μm.* P<0.05 compared to the control; $ P<0.05 compared to the H/ R group; # P<0.05 compared to the H/R+CGRP group.