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Cytochrome c release in acute myocardial infarction predicts poor prognosis and myocardial reperfusion on contrast-enhanced magnetic resonance imaging

Liu, Zhen-Binga; Fu, Xiang-Huaa; Wei, Genga; Gao, Jun-Lingb

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doi: 10.1097/MCA.0000000000000040
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Abstract

Urgent reperfusion by a primary percutaneous coronary intervention (PPCI) has become the standard treatment of patients with ST-segment elevation myocardial infarction (STEMI) 1. However, a considerable proportion of patients do not achieve myocardial reperfusion, also known as no-reflow, 2 and have a poor outcome 3,4. Distal embolization, ischemic injury 5,6, and attempts at reperfusion can cause no-reflow, which induces cardiomyocyte apoptosis and loss 7. Long-lasting permeability transition pore opening induced by reperfusion gives rise to matrix swelling and mitochondrial membrane rupture, promoting additional release of proapoptotic molecules into the cytosol. One of these molecules is cytochrome c, which triggers the apoptotic process 8,9. Cytochrome c can also be released into the circulating blood by cell rupture. Marenzi et al. 10 reported that cytochrome c was detectable in the circulating blood during the early stages of STEMI. Peak values of cytochrome c were correlated negatively with myocardial blush grade (MBG). Thus, cytochrome c might be an apoptotic factor associated with myocardial reperfusion that is readily detectable in the serum. This creates opportunities for new therapeutic measures to prevent or minimize myocardial reperfusion injury and improve the prognosis after STEMI. Cardiovascular magnetic resonance imaging (CMR) is a standard method used to identify and quantify microvascular obstruction (MO) 11,12. We hypothesized that cytochrome c levels in the serum can correlate with no-reflow observed by CMR. In this study, we evaluated the relationship between the peak level of cytochrome c and the occurrence of no-reflow and MO on CMR in patients with STEMI.

Methods

Patient population

One hundred and sixty consecutive patients admitted with first STEMI for PPCI were enrolled from 1 June 2010 to 31 May 2011 at the Department of Cardiology, Second Hospital of Hebei Medical University (Shijiazhuang, China). Inclusion criteria were persistent symptoms for at least 30 min, ST-segment elevation of greater than 1 mm in two or more contiguous leads within 12 h of symptoms, age at least 18 years, and written informed consent. Exclusion criteria were severe valvular heart disease, thrombolysis, severe complications, left bundle branch block, cardiac pacing, ventricular pre-excitation syndrome, and contraindications to CMR.

The study was approved by the ethics committee of the Second Hospital of Hebei Medical University. All patients provided their written informed consent.

Primary percutaneous coronary intervention

PPCI was performed using standard guide catheters (6 Fr), guide wires, and balloon catheters through a radial or a femoral approach. The interventional cardiologist placed bare-metal or drug-eluting stents according to their clinical judgment. Aspiration thrombectomy was performed when possible. All patients were pretreated with oral aspirin (300 mg loading dose), oral clopidogrel (300–600 mg loading dose), and intravenous unfractionated heparin (70–100 IU/kg). Patients were treated with intravenous tirofiban (loading dose of 10 μg/kg) during stenting, unless contraindicated. After stenting, patients were placed on lifelong aspirin (75–100 mg/day), clopidogrel (75 mg/day for ≥12 months), and tirofiban (0.15 μg/kg/min for 24 h). Other medications included β-blockers, lipid-lowering agents, angiotensin-converting enzyme inhibitors, and angiotensin II receptor antagonists, as indicated 13.

Angiographic and electrocardiographic analysis

Coronary angiography including the target vessel was performed, as described previously 14. Thrombolysis in myocardial infarction (TIMI) flow grading before and after PPCI and the final MBG were assessed as described previously 15,16. Angiographic assessment was performed offline by two experienced observers blinded to the cytochrome c levels, electrocardiographic data, and the CMR imaging results. Disagreements between the two observers were resolved by joint evaluation of the data and discussion.

All patients had 12-lead electrocardiography performed before treatment and 90 min after revascularization. Electrocardiographic measurements were performed by two experienced observers blinded to the cytochrome c levels, electrocardiographic data, and CMR imaging results 14. ST-segment resolution after the procedure was categorized as complete (≥70%), partial (70–30%), or no improvement (<30%).

Biochemical analysis

Peripheral venous blood was drawn at hospital presentation and once every 6 h until the peak plasma creatine kinase-MB isoenzyme (CK-MB) and serum cytochrome c levels were identified. The plasma was separated by centrifugation at 2500g for 10 min. Aliquots were stored at −80°C until assayed. The CK-MB mass was measured using electrochemiluminescence immunoassays on the Elecsys 2010 analyzer (Roche Diagnostics, Indianapolis, Indiana, USA). Human cytochrome c levels were measured using the commercially available enzyme-linked immunosorbent assay, QuantiGlo (R&D System Inc., Minneapolis, Minnesota, USA). A 100 μl sample was placed on the enzyme-linked immunosorbent assay plate without performing the cell lysis procedure according to the manufacturer’s instruction. Results were compared with standard curves. The lower detection limit was 0.08 ng/ml.

Cardiovascular magnetic resonance imaging

CMR using a 1.5 Tesla (T) MR scanner (Avanto-Siemens, Erlangen, Germany) was performed at days 3–7 after STEMI. The CMR protocol has been described in detail previously 17. Left ventricular function was assessed using the standard steady-state free precession technique. End-diastolic volume, end-systolic volume, left ventricular ejection fraction (LVEF), and myocardial mass were obtained using short-axis cine-MR images acquired using breath-hold and edited using retrospective ECG-triggered true-FISP. First-pass perfusion was evaluated in three short-axis sections centered on the mid-papillary muscle using an electrocardiograph-triggered T1-weighted inversion-recovery true-FISP sequence. Three short-axis slices (basal, mid cavity, and apical levels) were obtained after injecting a 0.1 mmol/kg gadolinium contrast bolus (Gadovist, Schering, Germany) at 5 ml/s, followed by a 20 ml saline flush in the right antecubital vein 18.

Cardiovascular magnetic resonance imaging analysis

LVEF, end-diastolic volume, end-systolic volume, and LV mass were calculated from the short-axis views. Infarct size and early MO were quantified by manually drawing the regions of hypointensity and the regions of hyperintensity on short-axis slices of the first-pass perfusion images, expressed as percentage of LV volume. Two fully blinded operators, using an offline dedicated workstation, performed all measurements. The early MO was located in the infarcted area and the area of MO was assessed separately (Fig. 1).

Fig. 1
Fig. 1:
Early enhancement cardiovascular magnetic resonance imaging in a patient with an at least mean peak level of cytochromec shows microvascular obstruction in the apical and mid-cavity slices (black arrows).

Statistical analysis

Patients were divided into a less than mean peak level of cytochrome c group (CC<mean) and an at least mean peak level of cytochrome c group (CC≥mean). Continuous variables were expressed as the mean±SD. Differences between CC<mean and CC≥mean groups were assessed using the t-test for independent samples. Variables that were not normally distributed (as evaluated by Kolmogorov–Smirnov tests) were presented as the median and interquartile range and were compared using the Wilcoxon rank-sum test. Categorical data were presented as percentages and compared using the χ2-test or Fisher’s exact test, as appropriate. Otherwise, the nonparametric Wilcoxon rank-sum test was used. The Kaplan–Meier method was used to show the timing of events during follow-up in relation to cytochrome c peak level groups, and the log-rank test was used to perform statistical assessment. All statistical tests were performed using the SPSS software, version 17.0 (SPSS Inc., Chicago, Illinois, USA). A two-tailed P value less than 0.05 was considered statistically significant.

End points

Follow-up was performed once every 3 months for 1 year through telephone using a standard questionnaire. The clinical end point of the present study was the occurrence of major adverse cardiovascular events (MACEs), defined as cardiac death, reinfarction, or new congestive heart failure. Clinical events were verified by hospital charts and direct contact with the treating physician. Reinfarction and new congestive heart failure were diagnosed as described previously 14. The composite MACE end point (death>re-MI>congestive heart failure) was counted only once to avoid double counting of patients with more than one event.

No extramural funding agency played a role in the study design, collection, analysis, interpretation of data, writing of the report, or the decision to submit the report for publication.

Results

Baseline characteristics

Cytochrome c levels were determined before and after reperfusion in all 160 patients. On admission, cytochrome c was detectable in the blood of 92 patients (57.5%; 1.16±0.44 ng/ml) and was not detectable in 68 patients. TIMI flow grade in patients with no detectable cytochrome c was less than that in patients with detectable cytochrome c (P=0.01; Table 1). The peak concentration of cytochrome c after symptom onset was 1.52±0.48 ng/ml (range 0.19–2.73). Demographic and clinical characteristics are shown in Table 2. The peak CK-MB level in the CC<mean group was significantly higher than that in the CC≥mean group (P=0.044). There were no other significant differences between the two groups. Almost all patients received aspirin, clopidogrel, ACE inhibitors or an AT-1 antagonist, and glycoprotein IIb/IIIa inhibitors (Table 3).

Table 1
Table 1:
Main patient characteristics
Table 2
Table 2:
Main patient characteristics
Table 3
Table 3:
Concomitant medication

No-reflow assessed by ST-segment resolution and TIMI flow/MBG

Nineteen (11.9%) patients had no ST-resolution (<30%), a condition consistent with no-reflow. Patients without ST-resolution had higher cytochrome c peak levels after reperfusion than patients with incomplete or complete ST-resolution (31–100%) (P=0.003; Fig. 2). After reperfusion, a total of 147 (91.9%) patients had TIMI flow 2–3 and 131 (81.9%) had a final MBG of 2–3. Patients with CC<mean had a higher incidence of final MBG 2–3 than patients with CC≥mean (P=0.017) (Table 2). There were significantly higher cytochrome c peak levels in patients with a final MBG of 0–1 after reperfusion (P=0.002) (Fig. 2).

Fig. 2
Fig. 2:
Cytochromec peak level according to myocardial blush grade (MBG), early microvascular obstruction (MO), and ST-resolution (STR) after reperfusion.

Cardiovascular magnetic resonance imaging evaluation

The median time between admission and CMR was 5 days (interquartile range 4–6 days) for both groups. LVEF in patients with CC<mean was higher than in patients with CC≥mean (P=0.029; Table 2). There were 103 (64.4%) patients who had early MO. Patients with CC≥mean had a significantly higher incidence of early MO (P=0.008) and a significantly larger extent of early MO (P=0.020) than patients with CC<mean (Table 2). There was no difference in infarct size in the two groups (Table 2). There were significantly higher cytochrome c peak levels in patients with early MO (P=0.025) after reperfusion compared with patients with no early MO (Fig. 2).

Clinical outcomes

All patients were followed for 1 year. There were seven deaths (4.4%) during follow-up. These patients had significantly higher mean cytochrome c peak values than surviving patients (2.05±0.34 vs. 1.52±0.43 ng/ml, P=0.003). At the 1-year follow-up, patients with CC≥mean had a higher incidence of cardiac death (log rank, P=0.029) than patients with CC<mean (Fig. 3). One (1.1%) patient had a nonfatal reinfarction and two (2.3%) patients had congestive heart failure in the CC≥mean group. Two (2.7%) patients had nonfatal reinfarctions and one patient (1.4%) had congestive heart failure in the CC<mean group. There was no difference in MACE in the two CC groups at 1 year (12.3 vs. 4.6%, P=0.071) (Fig. 4).

Fig. 3
Fig. 3:
Kaplan–Meier curve of the incidence of all-cause mortality for patients with cytochromec peak level less than mean and at least the mean.
Fig. 4
Fig. 4:
Kaplan–Meier curve of the cumulative incidence of death, reinfarction, and new congestive heart failure for patients with cytochromec level less than mean and at least the mean.

Discussion

In recent years, many treatments including a variety of medications and approaches, especially PPCI, have led to improved clinical outcomes in patients with STEMI 1. Myocardial ischemia and reperfusion injury in the process of STEMI triggered myocardial MO and necrotic or apoptotic processes. This can result in cardiac cell death and negate any clinical benefits 5–7. Although troponins and CK-MB have been used as biomarkers for myocardial cell lysis and to evaluate the extent of myocardial damage, no specific biomarker has been identified to assess reperfusion-associated apoptosis. There is no method to measure reperfusion-induced myocardial damage. The effects of PPCI on myocardium free from reperfusion-induced injury also cannot be reliably evaluated in a clinical trial. Cytochrome c is a proapoptotic signaling molecule 8 localized within the mitochondrial cristae. It is bound to the mitochondrial inner membrane and is not detectable in the blood of healthy humans 19. Ischemia–reperfusion injury can induce irreversible openings in mitochondrial permeability transition pores (mPTP) and mitochondrial swelling, resulting in mitochondrial release of cytochrome c into the cytosol 9,20,21. Nevertheless, cytochrome c release by mPTP-independent mechanisms still remains undefined and needs to be further explored. Thus, cytochrome c is detectable in the blood of patients with STEMI. Elevated levels of circulating cytochrome c have been reported in early-stage STEMI, similar to our findings 10.

We found that 82.4% of the patients that we evaluated with undetectable cytochrome c had a pre-PPCI TIMI 0–1 flow grade, whereas only 58.7% of patients with a detectable cytochrome c had a pre-PPCI TIMI 0–1 flow grade. This finding suggests that cytochrome c was not released into the blood of those patients in whom spontaneous myocardial reperfusion did not occur before PPCI, even though myocardial necrosis had occurred (as indicated by the increased CK-MB and troponin I values at hospital presentation) 10. In addition, 31.2% of our patients had an initial TIMI 2–3 flow grade. This was compatible with spontaneous ischemia–reperfusion injury. Thrombosis and clot lysis have been reported to occur simultaneously and cause intermittent flow obstruction and distal embolization 22.

CMR can accurately identify and quantify MO and infarct size 11,12. Moreover, early MO assessed by CMR is an independent long-term prognosticator for cardiovascular morbidity in patients with STEMI treated with PPCI 23. Hence, early MO was evaluated in our study. Although a higher cytochrome c peak value did not correlate with infarct size, there was a significant association between early MO and lower LVEF. We found that a higher cytochrome c peak value was significantly associated with a lower MBG, similar to previous reports 10. This suggests that poor myocardial reperfusion occurred more frequently in patients with higher cytochrome c peak values. Patients with higher cytochrome c peak values had higher CK-MB levels, suggesting a relationship with extension of the infarcted area 24. Patients with indicators of impaired myocardial reperfusion (STR, MBG, and early MO) had a greater cytochrome c peak level than patients without impaired perfusion. Patients with higher cytochrome c peak levels had a higher incidence of cardiac death after the 1-year follow-up and patients who died had the highest cytochrome c peak levels. Cytochrome c level was closely related to the incidence of cardiac death in patients with STEMI. These findings suggest that cytochrome c was released into the blood because of myocardial necrosis and reperfusion injury and was closely related to myocardial reperfusion and the clinical outcome of patients with STEMI. Our findings were consistent with several previous studies showing that the blood cytochrome c levels were associated with the intensity of apoptosis in different tissues and with mitochondrial injury 25,26. The underlying cellular mechanisms of how cytochrome c is released into the blood of patients with STEMI are not known and need further study.

Cytochrome c may be a novel biomarker for myocardial reperfusion and prognosis after STEMI. Our findings corroborate this possibility and further contribute toward the understanding of the pathophysiologic mechanism of STEMI. If our findings are verified in larger studies, cytochrome c may be used to evaluate new measures for cardioprotection. Preventing or reducing cytochrome c release into the bloodstream may decrease infarct size and improve clinical outcome in patients with STEMI.

The release of cytochrome c caused by ischemia–reperfusion injury with an irreversible opening of the mPTP can induce apoptotic cell death 8,21. Both pharmacological and genetic treatments have been reported to prevent mPTP opening before myocardial reperfusion and reduce myocardial infarct size 27–30. Cardioprotective pharmacological treatment 31 was performed with cyclosporine-A, a known mPTP inhibitor. Administration of cyclosporin-A just before PCI reduced myocardial infarct size by 30–40% in patients presenting with STEMI. As cytochrome c is an apoptotic factor, it could be used to assess the efficacy of antiapoptotic strategies and new methods to reduce myocardial apoptosis induced by ischemia–reperfusion injury during STEMI.

Although the results of our study supported our hypothesis, this study does have some limitations. This is a small single-center experience with limited follow-up to fully assess the longer term cardiac morbidity/mortality rates and cytochrome c levels. This study may have underestimated the size of MO as only basal, mid, and apical short-axis rest perfusion slices were used. A potential strength of the study is that cytochrome c levels were drawn at similar times before CMR imaging for both the high and the low cytochrome c groups. As infarct size and especially MO can vary with time after STEMI, imaging time after reperfusion is an important factor in evaluating the presence and extent of MO and infarct size. The two groups in this study had similar time delays before CMR imaging, limiting this potential bias.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

acute myocardial infarction; cytochrome c; magnetic resonance imaging; microvascular obstruction; no-flow

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