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Keywords:

  • Ischemic heart disease;
  • SDF-1;
  • ischemia reperfusion injury;
  • myocardial infarction

SUMMARY

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Recent studies have shown that stromal cell derived factor-1 (SDF-1), first known as a cytokine involved in recruiting stem cells into injured organs, confers myocardial protection in myocardial infarction, which is not dependent on stem cell recruitment but related with modulation of ischemia-reperfusion (I/R) injury. However, the effect of SDF has been studied only in a preischemic exposure model, which is not clinically relevant if SDF is to be used as a therapeutic agent. Our study was aimed at evaluating whether or not SDF-1 confers cardioprotection during the reperfusion period. Hearts from SD rats were isolated and perfused with the Langendorff system. Proximal left coronary artery ligation, reperfusion, and SDF perfusion in KH buffer was done according to study protocol. Area of necrosis (AN) relative to area at risk (AR) was the primary endpoint of the study. Significant reduction of AN/AR by SDF in an almost dose-dependent manner was noted during both the preischemic exposure and reperfusion periods. In particular, infusion of a high concentration of SDF (25 nM/L) resulted in a dramatic reduction of infarct size, which was greater than that achieved with ischemic pre- or postconditioning. SDF perfusion during reperfusion was associated with a similar significant reduction of infarct size as preischemic SDF exposure. Further studies are warranted to assess the potential of SDF as a therapeutic agent for reducing I/R injury in clinical practice.


Introduction

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Ischemia reperfusion (I/R) injury refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia. The absence of oxygen and nutrients from the blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function. Studies in animal models of acute myocardial infarction (MI) suggest that lethal reperfusion injury accounts for up to 50% of the final size of a MI, and in these models a number of strategies have been shown to ameliorate lethal reperfusion injury [1].

MI remains a major threatening event to public human health. While the previous enthusiasm for stem cell therapy in myocardial repair has to be reevaluated because of the “only modest effect” of the current approach [2], efforts to reduce myocardial injury through modulation of ischemia-reperfusion injury (I/R injury) is regaining clinical interest [3, 4]. Stromal cell derived factor-1 (SDF-1) is a cytokine known to plays a key role in recruiting stem cells into injured organs [5, 6]. Its action is triggered by binding to its cognate receptor (CXCR4) on target cell membranes. Although most studies on the role of SDF-1 have been in the context of stem cell treatment [5–10], several recent studies have shown that SDF-1 confers myocardial protection in MI, which is not dependent on stem cell recruitment but related to modulation of the I/R injury mechanism [11–13]. It is noteworthy that such studies used SDF-1 “before ischemia” in a mouse model of infarction [11] or “during hypoxia” in cell culture [8, 12] or via direct intramyocardial injection [13]. Preischemic exposure of SDF-1 to myocardium is less clinically relevant unless SDF-1-induced myocardial protection is planned in advance; same reasoning applies to intramyocardial injection. Therefore, our study was designed to determine whether the cardio-protective effect of SDF-1 is maintained under the condition of postischemic exposure, that is, reperfusion targeting.

Materials and Methods

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

The experimental procedures and protocols used in this study were reviewed and approved by our Institutional Animal Care and Use Committee.

SDF-1 Synthesis and Chemicals

Because our study required a large amount of SDF-1, recombinant SDF was produced by the laboratory of Biomedical Science, Postec, Republic of Korea.

SDF-1 Preparation

The sdf-1 gene was cloned into the pET28a vector and expressed in E.coli strain BL21(DE3). The recombinant SDF-1 was refolded and purified using an on-column refolding method [14, 15].

Chemotaxis Assay of Recombinant SDF-1

The migration assay was performed in a 48-well ChemoTx chamber (Neuroprobe, Gaithersburg, MD, USA). A total of 27 μL of SDF-1 (200 nM) was applied to the bottom well of the chamber and 50 μL of a suspension of CCRF-CEM cells (4 × 106 cells/mL) were then added to the upper wells. After assembly, the chamber was incubated for 3 h at 37 °C with 5% CO2. The cells that had migrated into the bottom wells were then counted. This experiment was performed in triplicate.

Langendorff Isolated Heart Perfusion Preparation

Male Sprague-Dawley rats, weighing 280–330 g obtained from KOATECH Co., Cheongwon-gun, Republic of Korea, were used. They received 100 mg/kg of pentobarbital sodium (Entobar®, Yongin-si, Republic of Korea) and 300 IU of heparin intraperitoneally. Hearts were isolated and perfused with modified Krebs-Henseleit (KH) solution containing (in mM) 118.5 NaCl, 4.7 KCl, 1.2 MgSO4, 1.8 CaCl2, 24.8 NaHCO3, 1.2 KH2PO4, and 10 glucose as described previously [16]. To induce regional ischemia, the proximal portion of left coronary artery (LCA) was first localized between the left atrial appendage and the right ventricular outflow tract. This was then followed by the passage of a 6–0 polypropylene suture around the major trunk of the LCA or its prominent branches. The ends of the thread were passed through a small piece of PE50 tube to form a snare. All hearts were then allowed to stabilize for at least 20 min. Ischemia was induced by pulling the snare and then fixing it by clamping the tubing with a small hemostat and was confirmed by regional cyanosis, a substantial decrease in left ventricular developed pressure (LVDP), or a fall in coronary flow. Reperfusion was initiated by releasing the snare. Hearts experiencing ventricular fibrillation (VF) usually revert spontaneously to sinus rhythm. VF lasting more than 45 seconds was treated with finger flick cardioversion until a perfusing rhythm was obtained. No pharmacological agents were used for defibrillation.

Assessment of Cardiac Function

To compare functional recovery, an air-bubble free, KH buffer-filled elastic balloon made of polyethylene plastic connected to a pressure transducer with tubing was inserted into the left ventricle (LV) through the left atrial appendage. The balloon was coupled to a graded threaded micro-syringe and balloon volume was adjusted to give a left ventricular end-diastolic pressure (LVEDP) of 5–10 mmHg at the beginning of the experiment. LVDP was calculated as the difference between the left ventricular systolic pressure (LVSP) and LVEDP. The rate-pressure product (RPP) was calculated as LVDP × heart rate. Cardiodynamic data, including heart rate, LVSP, LVEDP, and the maximum of first derivative of left ventricular pressure (+dP/dtmax), were continuously recorded with the BIOPAC system (BIOPAC Systems Inc., CA, USA), and analyzed using analysis software (BSL v3.7.3.).

Experimental Protocol

All hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. To determine the infarct limitation effect by SDF-1 targeting reperfusion, hearts were randomly assigned to SDF-1 untreated control hearts (CON) and pharmacological postconditioning with five different concentrations of SDF-1. SDF-1 was diluted with KH solution to the required final concentrations on the day of each experiment and was infused for 40 min, from 10 min before reperfusion to 30 min after reperfusion. The other groups of hearts were perfused with 1 μM of a CXCR4 receptor antagonist AMD3100 octahydrochloride (Toctirs bioscience, Ellisville, MO, USA) from 20 min before reperfusion to 30 min of reperfusion in SDF-1 treated hearts (SDF+AMD) or control hearts (AMD). As comparison, other groups of hearts were induced: SDF-1-induced preconditioning (3 cycles of 5 min administration of SDF-1 interspersed with 5 min of SDF-1 free periods, SDF-Pre), direct intracoronary injection of SDF-1 (3 mL of 2.5 nM SDF-1) immediately before reperfusion via aortic root over 1 min, ischemic preconditioning (3 cycles of 5 min ischemia and 5 min reperfusion before index ischemia, I-Pre), and ischemic postconditioning (6 cycles of 10 second reperfusion and 10 second global ischemia, I-Post) (Figure 1).

image

Figure 1. Study protocol. Isolated rat hearts were subjected to 30 min of regional ischemia followed by 2 h of reperfusion. Stromal cell derived factor-1 (SDF-1) is denoted by hatched rectangles. CON, untreated control hearts;, D-SDF, direct injection of SDF-1 through aortic root (3 mL of 2.5 nM SDF-1); SDF-Pre, SDF preconditioning; I-Pre, ischemic preconditioning; I-Post, ischemic postconditioning; Isc, ischemia; Rep, reperfusion.

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Determination of Area at Risk and Infarct Size

At the end of each experiment, the area at risk (AR) and area at necrosis (AN) were measured as described in our previous study [17]. In brief, LCA perfusion circuit was reoccluded, and diluted fluorescent polymer microspheres 2–9 μm in diameter (Duke Scientific Corp. Palo Alto, CA, USA) were infused to demarcate the AR. The hearts were weighed, frozen at –20°C for 1–3 h, and cut into 2 mm thick transverse slices using a rat heart slice matrix (Zivic Instruments, Pittsburgh, PA, USA). The slices were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich Chemical., St. Louis, MO, USA) in sodium phosphate buffer (pH 7.4) at 37°C for 20 min. The slices were immersed in 10% formalin to enhance the contrast between viable (stained) and necrotic (unstained) tissue. LV was removed from the remaining tissue and then the LV slices were compressed to a uniform 2 mm thickness by placing them (basal side) between two glass plates separated by a 2 mm space. The AR of myocardium was identified by illuminating the slices with UV light as the tissue without fluorescence. The AN (unstained by TTC) and AR (no fluorescence) zone regions were traced on a clear acetate transparent sheet and quantified with the Image Tool (UTHSCSA Image Tool, version 3.0). The areas were converted into volumes by multiplying the areas by slice thickness. The volume of AN was expressed as a percentage of the AR volume. All measurements were performed in a blinded fashion (Figure 2).

image

Figure 2. Upper panel: Images of infarct myocardium by TTC staining (unstained by TTC) in control heart and SDF postconditioned heart (25 nM). Lower panel: The area of risk (AR) of myocardium was identified by illuminating the slices with UV light as the tissue without fluorescence (upper half of myocardium in A). The area of necrosis (AN) was identified by unstained region by TTC (closed circle in B) in AR zone.

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Tissue Lysate Preparation and Western Blot Analysis

Additional group of hearts (each n = 8) were randomly assigned into a control group and a SDF-1 postconditioning group to determine the involvement of the extracellular signal regulated kinase 1/2 (ERK1/2) and Akt (protein kinase B) in SDF-1 postconditioning. Hearts were subjected to 30 min of regional ischemia followed by 5 min of reperfusion. SDF-1 (2.5 nM concentration) postconditioning was induced. Samples were homogenized in ice-cold lysis buffer. Equal amount of protein (50 μg/lane) were loaded and electrophoresed on SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane. Membranes were blocked with nonfat milk and then incubated with the primary antibodies (Cell Signaling Technology Inc., Beverly, MA, USA) that recognize phosphorylation of ERK1/2 and Akt at 4°C overnight. The primary antibody bindings were detected with a secondary anti-rabbit antibody and visualized by the enhanced chemiluminescence method. Data for total- and phospho-ERK1/2 represent the sum of the 42- and 44-kDa bands.

Statistical Analysis

Data are expressed as means ± SEM. Data analysis was performed with a personal computer statistical software package (SPSS for Windows, Release 17.0; SPSS Inc, Chicago, IL, USA). Data were analyzed using t-test and one-way analysis of variance (ANOVA) with Bonferroni post-hoc testing. Attempts to determine a 50% effective dose (ED50) of intracoronary concentration of SDF were made by using probit analysis. Differences were considered to be statistically significant when P values were less than 0.05.

Results

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

A total of 112 rat hearts were used for infarct size measurement. Four hearts were excluded because of an LVDP < 80 mmHg (n = 2) and sustained arrhythmia (n = 2) during the stabilization. We therefore report the data for 108 successfully completed infarct experiments (each n = 9).

Protein Refolding and Purification

We obtained approximately 0.5 mg/L of protein from the E. coli cells and, according to SDS-PAGE analysis, the purity of the recombinant SDF-1 was 95%. The purified recombinant SDF-1 had three extra N-terminal amino acids (Ser–Gly–Ser) compared with native SDF-1. The N-terminal Ser residue came from the TEV cleavage site and the Gly–Ser residue came from the BamH1 restriction enzyme site codon. In MALDI-TOF mass spectrometry, a peak corresponding to recombinant SDF-1 appears around m/z = 8194. This result indicates that the recombinant molecular weight is higher than commercially available SDF-1(MW = 7964) because it has an extra three amino acids (Ser–Gly–Ser MW = 230) (Figure 3).

image

Figure 3. Recombinant SDF-1. (A) The purified recombinant SDF-1 had three extra N-terminal amino acids (Ser–Gly–Ser) compared with native SDF-1 (B) The activity of recombinant SDF-1 was not different from that of the commercially available SDF-1.

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Chemotaxis Assay for Commercially Available SDF-1 and Recombinant SDF-1

To validate the activity of the recombinant SDF-1, we investigated the interactions of recombinant SDF-1 with the receptor using a CCRF-CEM cell line that naturally expresses CXCR4. The activity of recombinant SDF-1 was not different from that of the commercially available SDF-1(Figure 3).

Effects of SDF-1 Pre- and Postconditioning on Myocardial Infarction

There were no significant group differences with respect to body weight, heart weight, LV volume, AR volume, and AR/LV among groups (Table 1). The AR ranged from 54.0% to 58.0% of the LV volume, which indicates that an equivalent degree of regional ischemia was induced among the groups (P > 0.05). The infarct size in control hearts was 34.4 ± 3.9% of the AR. In SDF-1 postconditioning groups, an SDF-1 concentration of 25 nM in KH buffer was used, because there is evidence that this concentration of SDF-1 maximally induces ERK1/2 phosphorylation in rat myocytes [11]. As shown in Figure 2, 25 nM of SDF-1 postconditioning dramatically reduced AN/AR (2.6 ± 0.6%, P < 0.001) compared to the control group. We further tested the infarct limitation effect by SDF-1 postconditioning with the four different concentrations, 10 nM, 2.5 nM, 1 nM, and 250 pM. The AN/AR ratios were also significantly decreased compared to the control group in a dose dependent manner: 5.4 ± 0.8% in 10 nM, 8.7 ± 1.9% in 2.5 nM, 9.4 ± 0.9% in 1 nM, and 15.1 ± 1.5% in 250 pM SDF-1, respectively (P < 0.05 vs. control group). Direct intracoronary injection of SDF-1 (18.6 ± 2.9%) also significantly decreased AN/AR compared to control hearts (P < 0.05). The AN/AR by 25 nM SDF-1 postconditioning was significantly lower than that of 250 pM SDF-1 postconditioning, I-Pre (16.4 ± 2.6%), and I-Post (13.8 ± 2.5%, P < 0.05), respectively. The infarct sparing effect by SDF-1 postconditioning (25 nM) was totally blocked by a CXCR4 receptor antagonist AMD3100 (29.3 ± 2.5%) at a dose that had no effect on infarct size (P < 0.05). SDF-1 preconditioning (2.5 nM) also reduced the AN/AR (11.9 ± 2.8%) compared to control group (P < 0.05). And, there was no significant difference in reducing infarction between SDF-1 preconditioning and other groups of SDF infusions during reperfusion (Figure 4). A 50% effective dose (ED50) of intracoronary concentration of SDF by using probit analysis was 0.57 pM/L (Figure 5).

Table 1. Morphometric data
GroupBW (gm)HW (gm)LV (cm3)AR (cm3)AR/LV (%)
  1. Values are mean ± SEM. SDF-0, untreated control hearts; SDF, stromal cell derived factor-1; SDF-Pre, SDF-1 preconditioning; D-SDF, direct intracoronary SDF-1 injection; AMD, CXCR4 receptor antagonist AMD3100; I-Pre, ischemic preconditioning; I-Post, ischemic postconditioning; BW, body weight; HW, heart weight; LV, left ventricle; AR, area at risk. There were no significant differences among the groups.

SDF-0323.3 ± 6.71.54 ± 0.050.732 ± 0.0180.421 ± 0.02057.5 ± 2.2
SDF-25nM310.6 ± 5.61.52 ± 0.030.734 ± 0.0420.409 ± 0.01456.6 ± 2.4
SDF-10nM317.2 ± 3.91.53 ± 0.030.726 ± 0.0270.409 ± 0.01557.0 ± 3.0
SDF-2.5nM325.0 ± 5.71.59 ± 0.060.726 ± 0.0270.409 ± 0.01554.0 ± 3.0
SDF-1nM323.3 ± 6.91.60 ± 0.050.748 ± 0.0260.383 ± 0.01456.7 ± 0.7
SDF-250pM322.2 ± 3.31.60 ± 0.020.748 ± 0.0210.438 ± 0.02957.9 ± 3.3
SDF-Pre318.7 ± 5.81.59 ± 0.020.749 ± 0.0260.419 ± 0.01456.0 ± 1.1
D-SDF316.0 ± 4.61.60 ± 0.030.734 ± 0.0290.409 ± 0.01356.9 ± 2.3
SDF+AMD318.8 ± 3.71.60 ± 0.040.750 ± 0.0300.428 ± 0.01558.0 ± 1.1
AMD324.3 ± 3.61.54 ± 0.030.743 ± 0.0330.400 ± 0.02453.4 ± 1.6
I-Pre316.7 ± 6.01.59 ± 0.040.722 ± 0.0160.392 ± 0.02354.3 ± 2.7
I-Post314.4 ± 7.21.51 ± 0.030.728 ± 0.0240.392 ± 0.01754.2 ± 2.7
image

Figure 4. Area at necrosis (AN) relative to percentage of area at risk (AR) as evaluated by triphenyltetrazolium chloride (TTC) staining following 30 min occlusion and 2 h reperfusion in an isolated rat heart model. Values are means ± SEM. SDF 0; untreated control hearts (CON). D-SDF; direct injection of SDF-1 (3 mL of 2.5 nM SDF-1) through aortic rout. For SDF-Pre, I-Pre, and I-Post see the text. *P < 0.05 vs. CON, †P < 0.05 vs. SDF of 25 nM.

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image

Figure 5. Curve illustrating the relationship between AN/AR and the dose of SDF-1. ED50 is 0.57 pM; sigmoid equation Y =ϕ(–1.278–0.39χ).

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Effects of SDF-1 Pre- and Postconditioning on Hemodynamics

There were no significant differences in baseline cardiodynamic variables among the groups (Table 2). Heart rate (HR) was significantly decreased in SDF-1 groups compared with control, which was also attributed to significant lower RPP in SDF groups. However, there were no significant differences in either LVDP or dP/dtmax between control and SDF-1 groups (P > 0.05). This SDF effect of lowering both HR and RPP was completely reversed by pretreatment of SDF-1 antagonist (AMD3100, Table 2).

Table 2. Changes in cardiodynamics in isolated rat hearts (each n = 9)
GroupHRLVDPRPP+dP/dtmax
BaselineR-30R-120BaselineR-30R-120BaselineR-30R-120BaselineR-30R-120
  1. Values are mean ± SEM. SDF-0, untreated control hearts; SDF, stromal cell derived factor-1; SDF-Pre, SDF-1 preconditioning; D-SDF, direct intracoronary SDF-1 injection; AMD, CXCR4 receptor antagonist AMD3100; HR, heart rate (beats/min); LVDP, left ventricular developed pressure (mmHg); RPP, rate-pressure product (bpm·mmHg/103); +dP/dtmax, maximum positive left ventricular pressure derivative (mmHg/s/103); R-30, 30 min after reperfusion; R-120, 120 min after reperfusion. Numbers in parentheses are percentage recovery from the baseline levels.

  2. *P < 0.05 vs. baseline.

  3. P < 0.05 vs. SDF-0.

SDF-0280.5 ± 11.8253.6 ± 11.4278.3 ± 12.8118.0 ± 6.975.5 ± 7.1*43.9 ± 5.8*33.1 ± 2.519.0 ± 1.8*12.4 ± 2.0*3.1 ± 0.41.6 ± 0.2*1.1 ± 0.1*
SDF-25nM275.0 ± 8.1228.6 ± 11.6*212.1 ± 8.4*,†113.0 ± 4.371.8 ± 6.9*29.4 ± 4.7*30.9 ± 0.916.5 ± 1.7*6.3 ± 1.1*,†3.1 ± 0.11.6 ± 0.2*1.0 ± 0.1*
SDF-10nM285.1 ± 5.7221.8 ± 8.1*193.8 ± 14.6*,†111.4 ± 7.172.9 ± 7.6*32.1 ± 6.1*31.7 ± 1.916.1 ± 1.5*6.1 ± 1.3*,†3.0 ± 0.21.5 ± 0.1*0.9 ± 0. 1*
SDF-2.5nM280.8 ± 1.0216.5 ± 17.3*180.4 ± 15.6*,†112.7 ± 3.068.5 ± 10.4*30.9 ± 3.8*31.6 ± 0.815.8 ± 0.7*5.9 ± 1.2*,†3.3 ± 0.11.8 ± 0.2*1.0 ± 0.1*
SDF-1nM289.1 ± 6.0229.4 ± 13.0*158.4 ± 17.0*,†112.4 ± 8.269.4 ± 8.2*29.9 ± 2.7*32.4 ± 2.215.6 ± 1.8*5.0 ± 0.8*,†3.0 ± 0.21.6 ± 0.2*0.8 ± 0.1*
SDF-250pM283.3 ± 8.5237.8 ± 9.9*174.8 ± 17.2*,†113.8 ± 5.068.5 ± 5.5*31.5 ± 3.3*32.2 ± 1.816.6 ± 1.9*5.7 ± 1.1*,†3.1 ± 0.11.8 ± 0.2*1.0 ± 0.1*
SDF-Pre276.3 ± 16.3191.9 ± 25.7*,†150.3 ± 14.7*,†116.7 ± 5.764.4 ± 8.2*34.1 ± 3.7*32.1 ± 2.113.4 ± 2.8*5.0 ± 0.7*,†3.2 ± 0.31.7 ± 0.3*0.9 ± 0.1*
D-SDF270.7 ± 18.6222.1 ± 19.6*223.6 ± 15.9*113.7 ± 6.662.5 ± 7.3*35.1 ± 5.0*31.6 ± 3.214.5 ± 2.4*7.9 ± 1.3*3.2 ± 0.31.6 ± 0.3*1.0 ± 0.1*
SDF+AMD288.0 ± 13.7231.8 ± 18.5*249.3 ± 22.2118.4 ± 10.067.6 ± 7.2*40.7 ± 5.5*33.5 ± 2.316.1 ± 3.1*9.9 ± 1.8*3.1 ± 0.21.7 ± 0.1*1.0 ± 0.2*
AMD277.4 ± 11.4222.8 ± 22.9*236.3 ± 21.8115.0 ± 6.869.6 ± 9.6*36.9 ± 5.9*31.9 ± 2.116.9 ± 3.7*9.1 ± 2.0*3.0 ± 0.41.8 ± 0.3*1.1 ± 0.2*
Evidence of Involvement of ERK1/2 in SDF-1 Induced Postconditioning

To determine the involvement of the ERK1/2 and Akt in the cardioprotective effect of SDF-1 postconditioning, ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylations were measured at 20 min after reperfusion and 5 min after reperfusion in perfused rat hearts. SDF-1 treated hearts (377.8 ± 40.0%) had significant increases in ERK1/2 phosphorylation compared to the control group (208.3 ± 33.9%, P < 0.05 vs. SDF), suggesting the involvement of ERK1/2 signaling pathway in cardioprotection by SDF-1 induced postconditioning (Figure 6). However, there was no significant difference in Akt phosphorylation between the control (117.0 ± 17.3%) and SDF postconditioned hearts (110.3 ± 15.6%; P > 0.05).

image

Figure 6. (A) Western blotting of phospho-ERK1/2 and total-ERK1/2 (Thr202/Tyr204) in isolated rat hearts. Left, representative Western blot of myocardial samples acquired from the risk zone at 20 min of ischemia (I 20’, n = 6) and 5 min after reperfusion (R 5’, n = 8). Right, percentage changein ERK1/2 phosphorylation at 5 min of reperfusion relative to the value of I 20’. CON, untreated control hearts; SDF, stromal cell derived factor postconditioning. Each bar is the mean ± SEM. *P < 0.05 vs. CON 6-B. Western blotting of phospho-Akt and total-Akt (Ser473) in isolated rat hearts. Left, representative Western blot of myocardial samples acquired from the risk zone at 20 min of ischemia (I 20’, n = 6) and 5 min after reperfusion (R 5’, n = 8). Right, percentage change in Akt phosphorylation at 5 min of reperfusion relative to the value of I 20’. CON, untreated control hearts; SDF, stromal cell derived factor postconditioning. Each bar is the mean ± SEM.

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Discussion

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

In this study, although cardiac function does not appear to be improved by SDF-1 as it is usually in ischemic pre- and postconditioned hearts, exogenously sustained SDF-1 perfusion targeting reperfusion significantly reduced MI in isolated rat hearts and its infarct limitation effect followed a dose dependent trend. The results of Western immunoblotting in this study suggest that ERK1/2 might be involved in the cardioprotection by SDF-1 postconditioning. One difference with previous studies showing a protective effect of SDF-1 on ischemic injury is that we specifically sought an “effective dose” based on dose–response relationship of SDF for reducing infarction, which actually required multiple testing of different SDF concentrations. Our study showed that the effective dose 50% (ED 50%) of SDF-1 was only 0.57 pmol/L, which means that SDF may start to play a role in reducing infarction at an extremely low concentration. Moreover, a dramatic effect of intracoronary injection of SDF-1 in reducing MI up to almost 90% was seen with a high concentration of SDF-1 (25 nM/L) and the antiinfarct effect actually exceeded that of I-Pre or I-Post.

Studies on the role of SDF-1, one of the most important chemotactic cytokines, first focused on stem cell recruitment in ischemic heart disease. Bone marrow-derived cells, which express the SDF-1 receptor CXCR4, have been shown to act as vascular stem/progenitor cells (including endothelial progenitor cells, EPCs) and to promote angiogenesis after MI [5–10]. In addition to this cell therapy-targeted effect, the direct role of SDF-1 in preventing I/R injury has also been reported recently [11–13]. Although stem cell recruitment is also partially associated with reducing I/R injury [18], most studies showed a straightforward action of SDF-1 independent of stem cell recruitment in reducing I/R injury [9–13, 19]. It has been proposed that SDF-1–CXCR4 is functional and couples to downstream MAPK and Akt intracellular signaling. Its activation results in protection not only against hypoxia/reoxygenation damage in vitro but also against myocardial I/R injury in vivo[11]. Our Western immunoblotting analysis revealed a significant increase in ERK1/2 phosphorylation in SDF-1 postconditioning, however, there was no increase in Akt phosphorylation in our experiment. The survival kinase cascades ERK1/2 and PI3K-Akt may exhibit “cross-talk” such that inhibition of one cascade may activate the other and vice versa [20]. However, it does not seem that the cross-talk always occurs. For instance, a recent study stressed the pivotal role of ERK1/2 but not PI3K/Akt in I-Post mediated cardioprotection [21]. In this regard, the relative importance of the two kinases in mediating the protection by SDF-1 postconditioning remains controversial. The results from our study suggest that ERK1/2 but not PI3K/Akt is the predominant mediator of SDF-1 induced postconditioning [20, 21].

Regarding the antiapoptosis role of SDF-1, a study suggested that PI3K/Akt/eNOS activation is required for the inhibitory effect of SDF-1 on EPC apoptosis in which the SDF-1/CXCR4 decreases EPC apoptosis in a dose dependent manner [10]. In our study, there appeared to be a dose dependent response in SDF-1 infusion during the early reperfusion period, and preischemic exposure of SDF-1 also showed reduction of I/R injury as was seen in Saxena's study [13]. Therefore, our study showed a consistent effect of SDF-1 in reducing I/R injury regardless of pre- or postischemic exposure. Interestingly, cognate receptor CXCR4 expression levels in the myocardium were reported to be increased more than 5-fold after induction of MI in animal experiments [22]. Taken together with the finding that plasma SDF-1 increases significantly after MI, it could be postulated that the axis of SDF-1–CXCR4 is one of the important self-defense mechanisms against tissue injury [19, 23].

The cardiodynamic effect of SDF-1 in our study showed that HR and RPP were reduced to a greater extent than in the control group while infarct size was significantly reduced in SDF groups. Reduced HR and RPP in SDF-1 groups could contribute to reduce oxygen demand in ischemic myocardium. Our study result actually supports LaRocca's report that SDF-1 can act as a beta-adrenergic antagonist [24]. Beta blocking agent is the well-known drug to induce myocardial salvage in MI [25].

Indeed, there have been many studies to support the excellent therapeutic impact of delivery of SDF-1 or its analogue into the area of MI [9, 26]. However, those studies were largely done by less relevant clinical approaches such as exposure to SDF-1 prior to ischemia, intramyocardial injection, or gene delivery, among others. Furthermore, if the dramatic effect of SDF-1 in reducing I/R injury is to be translated into clinical therapeutics, the following factors should also be considered. First, there is a potential limitation to the use of the SDF-1α protein because of its sensitivity to cleavage by several proteases, including MMP-2 and CD26/dipeptidylpeptidase IV (DPP IV) [26]. Rapid inactivation of the chemokine in vivo may not allow its biological effect unless it is protected from specific proteinases until it works in target organs. Second, many of the mechanisms of reperfusion injury are activated within the first few minutes after reperfusion, although the processes may continue over hours, leading to progressive necrosis by a wave front of injury. To address these factors, we used intracoronary injection of SDF-1 at the time of initiation of reperfusion. Although the method of SDF-1 postconditioning in our study using Langendorff system is generally accepted as a conventional way of approach to test the effect of drug perfused at time of reperfusion, it could still have the possibility of exposure of the drug before beginning of reperfusion. To address this factor, we examined direct intracoronary injection effect of SDF-1 at the time of initiation of reperfusion. To do this, 3 mL of 2.5 nM SDF-1 was directly infused throughout 1 min via aortic root. The infarct size was significantly reduced compared to SDF-1 untreated hearts by direct coronary injection. However, the degree of infarct limitation effect was less than that of the same concentrations of continuous perfusion group. The reason for why is not clear, but the infused duration and/or total administered dosages of SDF-1 may account for the discrepancy. In groups of SDF-1 postconditioning, SDF-1 was perfused for 40 min, while only 1 min in direct intracoronary injection groups. In addition, total infused volume of SDF-1 in SDF-1 postconditioning groups were ranged from 200 mL to 440 mL, whereas only 3 mL of SDF-1 was infused in direct intracoronary injection group because of technical limitations.

Our study findings may cautiously suggest the therapeutic potential of SDF-1 to reduce ischemic damage of the heart, for instance, intracoronary injection of SDF-1 during primary PCI in ST-segment elevation MI. There have been many efforts to translate the benefit of reducing I/R injury that has been shown in animal models into clinical settings. However, only a few of these clinical efforts, such as ischemic postconditioning [27], administration of adenosine [28], inhibition of the mitochondrial permeability transition pore (MPTP) [4], and intracoronary delivery of supersaturated oxygen have resulted in beneficial therapeutics impact [29]. Although many factors are to be considered to explain the discrepancy between animal and clinical experience, SDF-1 might cautiously be considered a drug that can potentially overcome this discrepancy because SDF-1 action involves many mechanisms, such as stem cell recruitment to repair infarct [5, 6], late ischemic preconditioning [18], among others, leading to improvement of ischemic damage as well as reduction of I/R injury.

Our study has several limitations. First, the infarct model used in this study was an isolated heart model (ex-vivo) with the Langendorff system. An in-vivo study with an open chest heart model might have yielded different results from that of our study. Second, the recombinant SDF-1 used in this study is slightly different from wild (commercially available) SDF-1 in its molecular structure. However, the chemotaxis assay in our study showed that recombinant SDF-1 has the same biologic effect as that of commercially available SDF-1.

Acknowledgements

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

This study was supported by both (1) Medical Research Institute Grant (2004–41), Pusan National University Hospital and (2) KOSEF through Center for Electro-Photo Behaviors in Advanced Molecular Systems (2011–007166, CB).

References

  1. Top of page
  2. SUMMARY
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References
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