Abstract
No experimental study has shown that the myocardium of a remotely preconditioned patient is more resistant to a standardized ischaemic/hypoxic insult.
This was a single-centre randomized (1:1), double-blinded, sham-controlled, parallel-group study. Patients referred for elective coronary bypass surgery were allocated to either remote ischaemic preconditioning (3 cycles of 5-min ischaemia/5-min reperfusion of the right arm using a blood pressure cuff inflated to 200 mmHg) or sham intervention. One hundred and thirty-four patients were recruited, of whom 10 dropped out, and 4 were excluded from the per-protocol analysis. The right atrial trabecula harvested on cannulation for cardiopulmonary bypass was subjected to 60 min of simulated ischaemia and 120 min of reoxygenation in an isolated organ experiment. Postoperative troponin T release and haemodynamics were assessed in an in vivo study.
The atrial trabeculae obtained from remotely preconditioned patients recovered 41.9% (36.3–48.3) of the initial contraction force, whereas those from non-preconditioned patients recovered 45.9% (39.1–53.7) (P = 0.399). Overall, the content of cleaved poly (ADP ribose) polymerase in the right atrial muscle increased from 9.4% (6.0–13.5) to 19.1% (13.2–23.8) (P < 0.001) after 1 h of ischaemia and 2 h of reperfusion in vitro. The amount of activated Caspase 3 and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells also significantly increased. No difference was observed between the remotely preconditioned and sham-treated myocardium. In the in vivo trial, the area under the curve for postoperative concentration of troponin T over 72 h was 16.4 ng⋅h/ml (95% confidence interval 14.2–18.9) for the remote ischaemic preconditioning and 15.5 ng⋅h/ml (13.4–17.9) for the control group in the intention-to-treat analysis. This translated into an area under the curve ratio of 1.06 (0.86–1.30; P = 0.586).
Remote ischaemic preconditioning with 3 cycles of 5-min ischaemia/reperfusion of the upper limb before cardiac surgery does not make human myocardium more resistant to ischaemia/reperfusion injury.
NCT01994707.
INTRODUCTION
Remote ischaemic preconditioning (RIPC) is the phenomenon of short periods of ischaemia and reperfusion in an organ to protect a distant organ from the effects of a prolonged period of ischaemia and subsequent reperfusion [1]. RIPC has been shown in multiple species to be a universal phenomenon with systemic protective effects [1]. Although no mechanism has been defined, it resembles local ischaemic preconditioning at the target organ level, with the same changes in mitochondrial function and kinase expression [1]. One obvious potential clinical application of RIPC is in cardiac surgery, which requires a planned period of myocardial ischaemia and reperfusion. It has been postulated that several short periods of upper limb ischaemia and reperfusion administered via intermittent blood pressure cuff inflation prior to the operative intervention may enhance the protection of the myocardium and other organs during surgery.
Following mixed reports, either confirming or disproving the ability of RIPC to enhance myocardial protection during cardiac surgery [2–8], 2 large randomized multicentre clinical trials revealed that RIPC did not improve clinical outcomes in patients undergoing elective on-pump cardiopulmonary bypass grafting with or without valve surgery [9, 10].
Furthermore, no study has ever truly shown that remote preconditioning of human myocardium is feasible at all. There are many studies on human myocardium showing that ischaemic preconditioning can be elicited under experimental conditions and protects against a standardized ischaemic and reperfusion insult [11, 12]. Similar results can also be obtained with various forms of pharmacological preconditioning [11, 13–16]. However, although some studies have shown increased preservation of mitochondrial respiration and protective changes in cellular signalling in the human myocardium after RIPC [1, 17, 18], no experimental study has shown that the myocardium of a remotely preconditioned patient is more resistant to standardized ischaemic/hypoxic insult. We attempted to verify this by remotely preconditioning our patients and studying their intraoperatively obtained myocardium ex vivo.
We tested whether or not right arm ischaemia and reperfusion before coronary artery bypass grafting (CABG) elicited by 3 cycles of 5 min inflation and 5 min deflation of the blood pressure cuff up to 200 mmHg (i) renders the right atrial pectinate muscle trabeculae more resistant to simulated 60-min ischaemia and 120-min reperfusion in vitro and (ii) decreases the myocardial damage caused by ischaemia and reperfusion during surgical coronary revascularization performed with the use of cardiopulmonary bypass and warm blood cardioplegia. We hypothesized that the myocardium of the remotely preconditioned patient will be more resistant to ischaemic/reperfusion insult ex vivo even if the clinical effect of the enhanced myocardial protection was not detectable.
MATERIALS AND METHODS
Study design
This was a single-centre randomized (1:1), double-blinded, sham-controlled, parallel-group study. The study was approved by the Institutional Review Board of the Medical University of Silesia, Katowice, Poland (KNW/0022/KB1/160/12). The study protocol has been published elsewhere [19].
We recruited patients aged 18–80 years referred for coronary artery bypass surgery for stable coronary artery disease with at least 3 distal anastomoses planned. The exclusion criteria are listed in the Supplementary Material, Materials and Methods. All participants provided informed consent prior to randomization.
The study was performed at the Department of Cardiac Surgery Medical University of Silesia, Katowice, Poland. The first patient was recruited on 23 October 2013 and the last patient on 27 April 2016.
Randomization and masking
Patients were allocated to either remote preconditioning or a sham intervention group by a random digit generator, using sealed envelopes to implement the allocation sequence. The randomization was performed in separate blocks for Western blotting and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique (see below). The person who generated the sequence and provided the envelopes was not involved in the trial. The remote preconditioning was elicited by 3 cycles of 5-min inflation to 200 mmHg (ischaemia) and 5-min deflation (reperfusion) of a blood pressure cuff on the right arm after the induction of anaesthesia and before the skin incision. The sham group had the blood pressure cuff placed on the arm for 30 min of ‘idle’ time between induction of anaesthesia and skin incision, but no inflations were performed. To ensure masking, the cuff was placed under surgical drapes and remained on the arm until the end of surgery. All envelopes were opened and preconditioning delivered (where indicated) by the same individual, who was not involved in the care of the patient or other trial-related tasks.
The ex vivo trial
On cannulation for cardiopulmonary bypass, the right atrial appendage was harvested for the ex vivo trial in all patients. It was immediately transferred to the isolated organ laboratory, in ice-cold Krebs–Henseleit solution. One pectinate muscle trabecula was harvested for baseline assessment of apoptosis with either Western blotting or immunohistochemistry (see below). Another single trabecula was subjected to simulated 60-min ischaemia and 120-min reperfusion in the functional experiment (see below) and studied for apoptosis afterwards.
At the end of the operation, just before closing the chest but not less than 40 min after removing the aortic cross-clamp, a 16-G needle true-cut biopsy of the left ventricular (LV) myocardium was obtained from the apex in all patients, to be assessed for apoptosis (see below).
Functional in vitro experiment
The electrically driven right atrial trabecula measuring less than 1 mm in diameter was studied under isometric conditions in the organ bath chamber containing the Krebs–Henseleit solution oxygenated with carbogen (95% oxygen and 5% carbon dioxide). We simulated 60 min of ischaemia by substituting oxygen with argon in carbogen (95% argon and 5% carbon dioxide) and replacing the Krebs–Henseleit solution with one containing no glucose or pyruvate. On reoxygenation, the carbogen was again added, and the tissue bath solution was replaced with the one that was initially used. The tissue was washed several times and left for a 120-min period of reoxygenation with washout every 15 min. Continuously recorded contractility was expressed as a percentage of the initial contraction force (before ischaemia and after the stabilization period) for a given preparation (for details see Supplementary Material, Functional In Vitro Experiment).
This functional model of ischaemia/reperfusion has been used in our laboratory and previously described in detail [12, 14, 16]. It has worked well to show myocardial protection by ischaemic and pharmacological preconditioning.
Myocardial apoptosis
Two atrial trabeculae from the same appendage, 1 harvested at baseline and another after the functional experiment (60-min ischaemia + 120-min reperfusion), were studied for markers of apoptosis (Western blotting and immunohistochemistry). The LV biopsies harvested before sternal closure, after at least 40 min of reperfusion, were assessed in a similar way. Half of the myocardial biopsy specimens and the right atrial trabeculae were harvested on liquid nitrogen to assess the expression of Caspase 3, cleaved Caspase 3, poly(ADP-ribose) polymerase (PARP) and cleaved PARP with Western blotting. Other specimens were harvested on 4% buffered paraformaldehyde (phosphate buffer) to look for TUNEL-positive cells. (For details on the Western blot immunoassay and TUNEL-staining methodology see the Supplementary Material, The Ex vivo trial).
The in vivo study
Anaesthesia was standardized with no anaesthetic gases allowed. Full haemodynamic monitoring was utilized using a Swan–Ganz catheter. After the remote preconditioning or sham procedure was applied, the operation was performed with the use of cardiopulmonary bypass in normothermia by an experienced cardiac surgeon. Intermittent warm-blood antegrade cardioplegia was used.
Serum concentrations of cardiac troponin T (electrochemiluminescence ‘ECLIA’, Roche) and creatine kinase isoenzyme myocardial band (CK-MB) (enzymatic assay, Roche) were measured preoperatively and 6 h, 12 h, 18 h, 24 h, 36 h, 48 h and 72 h after the removal of the aortic cross-clamp.
A full haemodynamic assessment using the Swan–Ganz catheter and the oxygen metabolism status based on arterial and mixed venous gas analysis was performed preoperatively and 1 h, 3 h, 6 h, 12 h, 18 h, 24 h, 36 h and 48 h after the removal of the aortic cross-clamp (for details see Supplementary Material, In vivo trial).
Outcomes
The primary outcome of the ex vivo trial was the functional recovery of a right atrial trabecula subjected to 60 min of simulated ischaemia and 120 min of reoxygenation in an isolated organ experiment. We assessed the recovery (as the percentage of the initial contraction force) at its maximum, after 30 min and 120 min of reoxygenation and after the final stimulation with 10−5 mol/l norepinephrine. Secondary outcomes were expression of activated Caspase 3 and cleaved PARP in the right atrial trabeculae and LV biopsies at the end of the reperfusion period. We also assessed the number of TUNEL-positive cells in both the atrial trabeculae and LV biopsies at the end of the reoxygenation/reperfusion period.
The primary outcome of the in vivo trial was the magnitude of myocardial injury as reflected by the area under the curve (AUC) for postoperative concentration of cardiac troponin T over the first 72 h after surgery. Additionally, we analysed the postoperative release of CK-MB, postoperative cardiac index, oxygen extraction ratio and the need for inotropic support as assessed by the inotropic index.
Statistical analysis
The details of the study group size and power calculation are presented in the Supplementary Material, Statistical metods. The missing values were below 5% in all variables tested. They were confirmed to be missing completely at random with the Little’s MCAR test and replaced with the expectation-maximization method. Descriptive statistics were summarized as means and standard deviation and frequencies (%) as appropriate. Categorical data were compared between the groups using the Fisher’s exact test. Continuous variables were compared using the Student’s t-test if normally or log-normally distributed (log transformed as appropriate), otherwise the Mann–Whitney test was used. The outcome data were presented as an arithmetic mean with 95% confidence interval (CI) if normally distributed and a geometric mean with 95% CI if log-normally distributed. The results were compared using a t-test or presented as the median with quartiles and compared using the Mann–Whitney test.
The AUC of troponin T concentration in serum was calculated according to the trapezoid rule. The results were log transformed and compared using a one-way analysis of variance. The ratios (with 95% CI) of RIPC to control were obtained by back transformation of the results of the analysis of variance.
The statistical analysis was performed using the IBM SPSS Statistics 22 software.
RESULTS
Of the 2413 patients screened, 134 were enrolled and assigned to RIPC (n = 66) or a sham procedure (n = 68) group (Fig. 1). The right atrial tissue for functional study and histological study was available in 125 patients (61 and 64, respectively) (Fig. 1). The baseline characteristics of the patients in the trial are presented in Table 1. The per-protocol analysis is presented in the Supplementary Material, Results.
Baseline characteristics (intention-to-treat)
| RIPC (n = 66) | Sham (n = 68) | |
|---|---|---|
| Demographics | ||
| Age (years) | 63.8 ± 6.2 | 62.4 ± 7.8 |
| Weight (kg) | 80.8 ± 13.3 | 82.2 ± 14.0 |
| BMI (kg/m2) | 27.1 (24.9–29.8) | 27.8 (26.0–30.6) |
| BSA (m2) | 1.92 (1.81–2.03) | 1.98 (1.81–2.08) |
| Male gender | 53 (83) | 50 (74) |
| Laboratory tests | ||
| Troponin T (ng/ml) | 0.008 ± 0.003 | 0.008 ± 0.004 |
| CK-MB (U/l) | 12 (10–16) | 12 (11–17) |
| Glucose (mg/dl) | 105 (97–116) | 105 (95–114) |
| Creatinine (mg/dl) | 0.9 ± 0.1 | 0.9 ± 0.2 |
| ALT (U/l) | 24.5 (19–35) | 28 (20–42) |
| AST(U/l) | 22 (18–25) | 23 (19–32) |
| Bilirubin (mg/dl) | 0.5 (0.3–0.7) | 0.5 (0.3–0.7) |
| Cardiac status | ||
| EF (%) | 55 (50–60) | 55 (50–60) |
| CCS score class | ||
| III | 7 (11) | 9 (13) |
| IV | 0 | 0 |
| NYHA class | ||
| I | 29 (44) | 27 (40) |
| II | 36 (55) | 38 (56) |
| III | 1 (1.5) | 3 (4.4) |
| Previous MI | 25 (38) | 25 (37) |
| Previous PCI | 22 (33) | 21 (31) |
| Smokers | 7 (11) | 10 (15) |
| Past smokers | 44 (67) | 43 (63) |
| Medications at the time of randomization | ||
| ACE inhibitors/ARBs | 56 (85) | 60 (88) |
| β-blockers | 60 (90.9) | 66 (97.1) |
| CCB | 16 (24) | 13 (19) |
| Loop diuretic | 5 (7.6) | 8 (12) |
| Spironolactone | 8 (12) | 6 (8.8) |
| Statins | 65 (98.5) | 64 (94.1) |
| Nitrate | 13 (20) | 10 (15) |
| EuroSCORE II (%) | 0.95 (0.72–1.18) | 0.93 (0.69–1.51) |
| RIPC (n = 66) | Sham (n = 68) | |
|---|---|---|
| Demographics | ||
| Age (years) | 63.8 ± 6.2 | 62.4 ± 7.8 |
| Weight (kg) | 80.8 ± 13.3 | 82.2 ± 14.0 |
| BMI (kg/m2) | 27.1 (24.9–29.8) | 27.8 (26.0–30.6) |
| BSA (m2) | 1.92 (1.81–2.03) | 1.98 (1.81–2.08) |
| Male gender | 53 (83) | 50 (74) |
| Laboratory tests | ||
| Troponin T (ng/ml) | 0.008 ± 0.003 | 0.008 ± 0.004 |
| CK-MB (U/l) | 12 (10–16) | 12 (11–17) |
| Glucose (mg/dl) | 105 (97–116) | 105 (95–114) |
| Creatinine (mg/dl) | 0.9 ± 0.1 | 0.9 ± 0.2 |
| ALT (U/l) | 24.5 (19–35) | 28 (20–42) |
| AST(U/l) | 22 (18–25) | 23 (19–32) |
| Bilirubin (mg/dl) | 0.5 (0.3–0.7) | 0.5 (0.3–0.7) |
| Cardiac status | ||
| EF (%) | 55 (50–60) | 55 (50–60) |
| CCS score class | ||
| III | 7 (11) | 9 (13) |
| IV | 0 | 0 |
| NYHA class | ||
| I | 29 (44) | 27 (40) |
| II | 36 (55) | 38 (56) |
| III | 1 (1.5) | 3 (4.4) |
| Previous MI | 25 (38) | 25 (37) |
| Previous PCI | 22 (33) | 21 (31) |
| Smokers | 7 (11) | 10 (15) |
| Past smokers | 44 (67) | 43 (63) |
| Medications at the time of randomization | ||
| ACE inhibitors/ARBs | 56 (85) | 60 (88) |
| β-blockers | 60 (90.9) | 66 (97.1) |
| CCB | 16 (24) | 13 (19) |
| Loop diuretic | 5 (7.6) | 8 (12) |
| Spironolactone | 8 (12) | 6 (8.8) |
| Statins | 65 (98.5) | 64 (94.1) |
| Nitrate | 13 (20) | 10 (15) |
| EuroSCORE II (%) | 0.95 (0.72–1.18) | 0.93 (0.69–1.51) |
Data are mean ± SD or median with interquartile range or n (%).
ACE: angiotensin-converting enzyme; ALT: alanine transaminase; ARBs: angiotensin-II-receptor blockers; AST: aspartate transaminase; BMI: body mass index; BSA: body surface area; CCB: calcium channel blockers; CCS: Canadian Cardiovascular Society; CK-MB: creatine kinase isoenzyme myocardial band; EF: ejection fraction; EuroSCORE: European system for cardiac operative risk evaluation; MI: myocardial infarction; NYHA: New York Heart Association; PCI: percutaneous coronary intervention; RIPC: remote ischaemic preconditioning, sham control group; SD: standard deviation.
Baseline characteristics (intention-to-treat)
| RIPC (n = 66) | Sham (n = 68) | |
|---|---|---|
| Demographics | ||
| Age (years) | 63.8 ± 6.2 | 62.4 ± 7.8 |
| Weight (kg) | 80.8 ± 13.3 | 82.2 ± 14.0 |
| BMI (kg/m2) | 27.1 (24.9–29.8) | 27.8 (26.0–30.6) |
| BSA (m2) | 1.92 (1.81–2.03) | 1.98 (1.81–2.08) |
| Male gender | 53 (83) | 50 (74) |
| Laboratory tests | ||
| Troponin T (ng/ml) | 0.008 ± 0.003 | 0.008 ± 0.004 |
| CK-MB (U/l) | 12 (10–16) | 12 (11–17) |
| Glucose (mg/dl) | 105 (97–116) | 105 (95–114) |
| Creatinine (mg/dl) | 0.9 ± 0.1 | 0.9 ± 0.2 |
| ALT (U/l) | 24.5 (19–35) | 28 (20–42) |
| AST(U/l) | 22 (18–25) | 23 (19–32) |
| Bilirubin (mg/dl) | 0.5 (0.3–0.7) | 0.5 (0.3–0.7) |
| Cardiac status | ||
| EF (%) | 55 (50–60) | 55 (50–60) |
| CCS score class | ||
| III | 7 (11) | 9 (13) |
| IV | 0 | 0 |
| NYHA class | ||
| I | 29 (44) | 27 (40) |
| II | 36 (55) | 38 (56) |
| III | 1 (1.5) | 3 (4.4) |
| Previous MI | 25 (38) | 25 (37) |
| Previous PCI | 22 (33) | 21 (31) |
| Smokers | 7 (11) | 10 (15) |
| Past smokers | 44 (67) | 43 (63) |
| Medications at the time of randomization | ||
| ACE inhibitors/ARBs | 56 (85) | 60 (88) |
| β-blockers | 60 (90.9) | 66 (97.1) |
| CCB | 16 (24) | 13 (19) |
| Loop diuretic | 5 (7.6) | 8 (12) |
| Spironolactone | 8 (12) | 6 (8.8) |
| Statins | 65 (98.5) | 64 (94.1) |
| Nitrate | 13 (20) | 10 (15) |
| EuroSCORE II (%) | 0.95 (0.72–1.18) | 0.93 (0.69–1.51) |
| RIPC (n = 66) | Sham (n = 68) | |
|---|---|---|
| Demographics | ||
| Age (years) | 63.8 ± 6.2 | 62.4 ± 7.8 |
| Weight (kg) | 80.8 ± 13.3 | 82.2 ± 14.0 |
| BMI (kg/m2) | 27.1 (24.9–29.8) | 27.8 (26.0–30.6) |
| BSA (m2) | 1.92 (1.81–2.03) | 1.98 (1.81–2.08) |
| Male gender | 53 (83) | 50 (74) |
| Laboratory tests | ||
| Troponin T (ng/ml) | 0.008 ± 0.003 | 0.008 ± 0.004 |
| CK-MB (U/l) | 12 (10–16) | 12 (11–17) |
| Glucose (mg/dl) | 105 (97–116) | 105 (95–114) |
| Creatinine (mg/dl) | 0.9 ± 0.1 | 0.9 ± 0.2 |
| ALT (U/l) | 24.5 (19–35) | 28 (20–42) |
| AST(U/l) | 22 (18–25) | 23 (19–32) |
| Bilirubin (mg/dl) | 0.5 (0.3–0.7) | 0.5 (0.3–0.7) |
| Cardiac status | ||
| EF (%) | 55 (50–60) | 55 (50–60) |
| CCS score class | ||
| III | 7 (11) | 9 (13) |
| IV | 0 | 0 |
| NYHA class | ||
| I | 29 (44) | 27 (40) |
| II | 36 (55) | 38 (56) |
| III | 1 (1.5) | 3 (4.4) |
| Previous MI | 25 (38) | 25 (37) |
| Previous PCI | 22 (33) | 21 (31) |
| Smokers | 7 (11) | 10 (15) |
| Past smokers | 44 (67) | 43 (63) |
| Medications at the time of randomization | ||
| ACE inhibitors/ARBs | 56 (85) | 60 (88) |
| β-blockers | 60 (90.9) | 66 (97.1) |
| CCB | 16 (24) | 13 (19) |
| Loop diuretic | 5 (7.6) | 8 (12) |
| Spironolactone | 8 (12) | 6 (8.8) |
| Statins | 65 (98.5) | 64 (94.1) |
| Nitrate | 13 (20) | 10 (15) |
| EuroSCORE II (%) | 0.95 (0.72–1.18) | 0.93 (0.69–1.51) |
Data are mean ± SD or median with interquartile range or n (%).
ACE: angiotensin-converting enzyme; ALT: alanine transaminase; ARBs: angiotensin-II-receptor blockers; AST: aspartate transaminase; BMI: body mass index; BSA: body surface area; CCB: calcium channel blockers; CCS: Canadian Cardiovascular Society; CK-MB: creatine kinase isoenzyme myocardial band; EF: ejection fraction; EuroSCORE: European system for cardiac operative risk evaluation; MI: myocardial infarction; NYHA: New York Heart Association; PCI: percutaneous coronary intervention; RIPC: remote ischaemic preconditioning, sham control group; SD: standard deviation.
Trial profile. CABG: coronary artery bypass grafting; CCS: Canadian Cardiovascular Society; MI: myocardial infarction; OPCAB: off-pump coronary artery bypass grafting; PCI: percutaneous coronary intervention; Pre-op: preoperative; RA: right atrium; RIPC: remote ischaemic preconditioning; VT: ventricular tachycardia.
Ex vivo trial
The contraction force of the isolated right atrial trabecula decreased to 12.8% (95% CI 11.1–14.8) of the initial contraction force in the RIPC group and to 13.4% (11.5–15.6) (P = 0.670) of the initial contraction force in the control group as a result of the 60-min simulated ischaemia. Tissue from the remotely preconditioned patients recovered maximally 41.9% (36.3–48.3) of the initial contraction force, whereas the atrial trabeculae from not preconditioned patients recovered to 45.9% (39.1–53.7) (P = 0.399) during reperfusion (Fig. 2). Although the contraction force gradually decreased during 2 h of reperfusion to 27.6% (23.8–32.0) and 25.5% (21.6–30.1) (P = 0.482), respectively, the trabeculae responded equally well to 10−5 mol/l of norepinephrine in both groups: 55.1% (48.0–63.2) vs 53.2% (44.8–63.2) (P = 0.750).
The right atrial trabecula contractility in the isolated organ experiment. Data are geometric mean (95% confidence interval) relative to the initial isometric contraction force of the given trabecula. NE: Norepinephrine; RIPC: remote ischaemic preconditioning.
Although the content of the activated Caspase 3 (in relation to total Caspase 3) in the right atrial muscle increased over a period of simulated ischaemia and reperfusion from 8.1% (interquartile range 2.9–12.9) to 9.7% (3.4–20.3) (P = 0.001; n = 66), no difference was observed between the groups (Fig. 3). Similarly, the content of cleaved PARP in relation to total PARP increased in the right atrial tissue from 9.4% (6.0–13.5) directly after harvesting to 19.1% (13.2–23.8) after 1 h of simulated ischaemia and 2 h reperfusion (P < 0.001; n = 66); however, no difference was observed between the preconditioned and control tissue (Fig. 3). Finally, no differences were observed in the content of activated Caspase 3 and cleaved PARP between LV biopsies obtained after 40-min reperfusion from remotely preconditioned (n = 28) and sham-treated (n = 32) patients (Fig. 3).
Relative content of activated Caspase 3 and cleaved PARP in the right atrial trabecula before and after simulated ischaemia/reperfusion in vitro and in LV biopsy after 40 min from the removal of the aortic cross-clamp. Data are expressed as median with interquartile range; whiskers are 10th and 90th percentile. The panels are representative Western blots for Caspase 3 and PARP from 3 patients. Channels 1, 2 and 3 are the right atrium before ischaemia/reperfusion, the right atrium after simulated ischaemia/reperfusion ex vivo and LV biopsy after ischaemia/reperfusion in vivo, respectively. LV: left ventricular; OD: optic density; PARP: poly(ADP-ribose) polymerase; RA: right atrium; RIPC: remote ischaemic preconditioning.
The number of TUNEL-positive cells was negligible in the right atrial trabecula after harvesting—median 0% (0–0.1%). It significantly increased to 1.0% (0.5–1.7%) after 1 h of simulated ischaemia and 2 h of reperfusion (P < 0.001) (Fig. 4). No difference was observed between the remotely preconditioned and sham-treated myocardium. No significant number of TUNEL-positive cells was observed in the LV biopsy harvested at the end of the reperfusion period.
(A) The number of TUNEL-positive cells in the right atrial trabecula before and after simulated ischaemia/reperfusion in vitro and in LV biopsy after 40 min from the removal of the aortic cross-clamp. Data are expressed as median with interquartile range; whiskers are 10th and 90th percentile. (B) The representative immunohistochemical images with TUNEL staining in the right atrial trabecula before (RA before) and after simulated ischaemia/reperfusion ex vivo (RA after) and in LV biopsy 40 min after the removal of the aortic cross-clamp. TUNEL-positive cell nuclei are blue stained. Both negative (without TdT transferase) and positive (nuclease-treated cells) controls are included. Scale bar: 50 µm. LV: left ventricular; RA: right atrium; RIPC: remote ischaemic preconditioning; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.
In vivo trial
We performed 2.9 ± 0.7 grafts per patient in the RIPC group and 2.8 ± 0.5 in the sham group (P = 0.221) with an average aortic cross-clamp time of 40.6 ± 10.7 min and 39.8 ± 10.9 min (P = 0.648), respectively. Details of the operations are presented in the Supplementary Material, Table S2.
The preoperative troponin T level was below 0.014 ng/ml in all patients. The geometric mean troponin AUC over 72 h after the removal of the aortic cross-clamp was 16.4 ng⋅h/ml (95% CI 14.2–18.9) for the RIPC group and 15.5 ng⋅h/ml (13.4–17.9) for the control group in the intention-to-treat analysis (Fig. 5). This translates into an AUC ratio of 1.06 (0.86–1.30; P = 0.586). The postoperative CK-MB level increased similarly in both groups with an AUC over 72 h of 1743 IU⋅h/l (1546–1965) vs 1760 IU⋅h/l (1603–1932), respectively, in the intention-to-treat analysis (Fig. 5). The CK-MB AUC ratio was 0.99 (0.85–1.15; P = 0.899). The results were similar in the per-protocol analysis (Supplementary Material, Results).
Cardiac troponin T (A) and CK-MB (B) concentration in serum in the perioperative period—intention-to-treat analysis. Data are expressed as geometric mean (95% confidence interval). P-value is for comparison of area under the curve between the groups. CK-MB: creatine kinase isoenzyme myocardial band; RIPC: remote ischaemic preconditioning.
Cardiac function assessed using the Swan–Ganz catheter varied from cardiac index of 2.57 l/min/m2 (2.41–2.73) in the RIPC group vs 2.72 l/min/m2 (2.54–2.90) in the control group (P = 0.256) 1 h after the cross-clamp removal to 3.01 l/min/m2 (2.86–3.16) vs 3.06 l/min/m2 (2.92–3.19), respectively, (P = 0.640) 48 h later (Supplementary Material, Results). Again, the result remained similar in the per-protocol analysis (Supplementary Material, Results).
No benefit of RIPC was detectable for the left or right ventricular work, oxygen extraction ratio or requirement for inotropic support in both the intention-to-treat analysis and the per-protocol analysis. Similarly, the postoperative renal function did not differ between the groups in both types of analyses (Supplementary Material, Results).
DISCUSSION
We present the first trial showing that the myocardium of patients subjected to RIPC prior to surgery is not more resistant to standardized ischaemia/reperfusion in vitro. The right atrial trabeculae harvested from both groups showed the same level of contractile function recovery after simulated 1 h ischaemia, developed the same level of stunning over 2 h of simulated reperfusion and responded with similar inotropism to pharmacological stimulation. The myocardium of both groups presented similar levels of apoptosis, as assessed by the activation of Caspase 3, degradation of PARP and DNA fragmentation at the end of reperfusion in vitro.
Although others (Jensen et al.) have studied electrically driven isolated human myocardium in a similar ex vivo setup before, they assessed the influence of dialysate obtained from remotely preconditioned, not anaesthetized volunteers on the functional recovery of isolated human right atrial trabeculae after simulated ischaemia reperfusion [20]. The myocardium in their study was obtained from patients who did not undergo RIPC.
Furthermore, we found no protective effect of RIPC against ischaemia/reperfusion injury inflicted during cardiac surgery as assessed by the release of cardiac markers or postoperative cardiac function. The Cochrane Systematic Review [21] identified 29 studies involving 5392 patients and concluded that RIPC did not improve clinical outcome in people undergoing CABG with or without valve surgery and that there was moderate-quality evidence that RIPC reduced the postoperative cardiac troponin T release. Thus, it comes as no surprise that the cardiac markers and haemodynamics did not differ between the groups in our relatively small clinical study.
We used the experimental model that was standardized in our laboratory to show increased resistance of human myocardium against ischaemia/reperfusion after preconditioning with transient ischaemia or pharmacological agents [12, 14, 16]. We hoped that we would be able to show ex vivo that myocardium obtained from remotely preconditioned patients is more resilient, regardless of the observed clinical effect. However, our results argue against the ability to remotely precondition the human myocardium in the clinical settings.
Some of the arguments used in clinical trials to explain the apparent lack of RIPC benefit were related to the fact that the mode of protection already available (e.g. cardioplegia) is strong enough and the ischaemic insult too small for any RIPC benefit to be registered in the study. This is why the laboratory setting with a standardized injuring stimulus and no additional protection was the only way to definitively prove or disprove whether RIPC could be achieved in patients. It has also been theorized that volatile anaesthetics or cardiopulmonary bypass might precondition the patients, and therefore, no extra preconditioning arising from RIPC is possible. In addition, some clinical scenarios such as recent angina (ischaemia) may precondition the patients, whereas others such as diabetes may make them resistant to preconditioning. Therefore, we did not use anaesthetic gases, we harvested the tissue for an ex vivo study before cardiopulmonary bypass, and we imposed multiple exclusion criteria, in an attempt to make the groups relatively homogenous, without confounding clinical characteristics.
One reason for not observing the benefit of RIPC in our study may be related to the use of propofol, which has been claimed to abolish remote preconditioning. The evidence is, however, based on 2 small clinical trials [22, 23], and the study describing the underlying mechanism lacks a positive control [24]. Although we cannot exclude the possibility that propofol abolished RIPC-derived benefit in our trial, its influence on RIPC is far from established, as underlined in the NEJM discussion related to the RIPHeart and ERRICA trials [25]. Additionally, some of the studies showing the benefit of RIPC were performed using propofol-based anaesthesia [3, 4, 8], whereas some studies using a propofol-free protocol have shown no benefit of RIPC [26]. Conversely, in other studies, propofol itself has been reported to provide cardioprotection [27, 28], whereas volatile anaesthetics have been claimed to attenuate RIPC [29, 30]. Nevertheless, to exclude the influence of propofol on our results, we would need to repeat our ex vivo protocol using myocardia harvested from patients anaesthetized in an alternate manner. Finally, beta-blockers, which are commonplace in cardiac surgical patients, can potentially also attenuate remote preconditioning benefits [30].
Another reason why the myocardia of remotely preconditioned patients were not more resistant to ischaemia reperfusion might be linked to the protocol of remote preconditioning itself. It may well be that the 3 cycles of 5-min upper limb ischaemia interspaced with 5-min reperfusion was too weak (or too strong) a stimulus to evoke the protection or that the time window between the arm ischaemia/reperfusion, and the heart ischaemia was inadequate. However, the protocol we used is a standard one, used in most studies of remote preconditioning that have claimed positive results [21]. In addition, the window of preconditioning appears similar in most trials of remote preconditioning in cardiac surgery, with the preconditioning procedure being performed between the induction of anaesthesia and skin incision.
Limitations
The most important limitation of our study is the propofol-based anaesthesia. However, one has to appreciate that this propofol-based anaesthesia is nowadays used in majority of cardiac surgical centres, and thus, the trial reflects the ‘real-world’ scenario. The use of propofol was discussed in detail above.
Applying strict exclusion criteria allowed for the recruitment of a relatively homogeneous and confounder-free study group, which inadvertently was rather low risk, and was subjected to short aortic cross-clamp times. Indeed, the postoperative troponin and CK-MB levels in our patients were lower than in most trials of RIPC [10, 21], making it less likely to observe a significant effect of RIPC on the release of myocardial necrosis markers. Still, the 2 large multicentre trials (ERICCA and RIPHeart) that failed to show reduction in clinical end points also assessed postoperative troponin release and found no difference between the RIPC and control groups [9, 10]. In addition, the severity of surgical ischaemic insult had no influence on the RIPC effect on the atrial myocardium, which was harvested before the cardiopulmonary bypass and subjected to significant ischaemia/reperfusion in vitro.
CONCLUSIONS
Our study suggests that RIPC obtained with a typical protocol of 3 cycles of 5-min ischaemia/reperfusion of the upper limb before cardiac surgical procedure does not make human myocardium more resistant to ischaemia/reperfusion. Further research should, therefore, concentrate on establishing a preconditioning protocol, which exploits conditions found to provide additional myocardial resilience in a standardized experimental setting. Only then can one expect clinical studies to provide definite answers.
ACKNOWLEDGEMENTS
The authors thank Piers Murphy for proofreading the draft.
Funding
The work was supported by the National Science Centre (Poland) [UMO-2012/07/B/NZ5/02549].
Conflict of interest: none declared.
