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ISSN: 0022-9032 e-ISSN: 1897-4279

Remote Ischemic PrEconditioning of Human Myocardium (RIPE): study protocol for a double-blinded randomized controlled trial

Authors: Marek A. Deja, Ryszard Wiaderkiewicz, Piotr Czekaj, Ewa Czech, Marcin Malinowski, Leszek Machej, Andrzej Węglarzy, Adam Kowalówka, Magda Piekarska, Bartosz Szurlej, Tomasz Latusek

DOI: 10.5603/KP.a2017.0180 Article type: Original articles Submitted: 2017-07-21 Accepted: 2017-08-02 Published online: 2017-09-20

This article has been peer reviewed and published immediately upon acceptance. It is an open access article, which means that it can be downloaded, printed, and distributed freely, provided the work is properly cited. Articles in "Polish Heart Journal" are listed in PubMed.

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Remote Ischemic PrEconditioning of Human Myocardium (RIPE): study protocol for a double-blinded randomized controlled trial Marek A. Deja1, 2, Ryszard Wiaderkiewicz3, Piotr Czekaj3, Ewa Czech3, Marcin Malinowski1, 2

, Leszek Machej4, 5, Andrzej Węglarzy5, Adam Kowalówka1, 2, Magda Piekarska1, 2, Bartosz

Szurlej1, Tomasz Latusek1 1

Department of Cardiac Surgery, School of Medicine in Katowice, Medical University of

Silesia, Katowice, Poland 2

Department of Cardiac Surgery, Upper-Silesian Heart Center, Katowice, Poland

3

Chair of Histology and Embryology, School of Medicine in Katowice, Medical University of

Silesia, Katowice, Poland 4

Department of Anesthesia and Intensive Care Nursing, School of Health Sciences, Medical

University of Silesia, Katowice, Poland 5

Department of Cardiac Anesthesia, Upper-Silesian Heart Center, Katowice, Poland

Address for correspondence: Ryszard Wiaderkiewicz, Chair of Histology and Embryology, School of Medicine in Katowice, Medical University of Silesia, ul. Medyków 18, 40–752, Katowice, Poland, e-mail: [email protected]

Abstract Background: Remote preconditioning was showed to be a potent protective phenomenon in many animals. Several studies aimed to demonstrate it was feasible in humans by trying to show its protective effect during cardiac surgery. Of these studies some small studies and one bigger trial were positive while two other bigger studies showed no effectiveness of remote preconditioning as assessed by levels of postoperatively released cardiac markers. Recently two large clinical trials also failed to prove the benefit of remote preconditioning in cardiac surgery. No study showed that remote preconditioning actually increases resistance of human myocardium to standardized ischemic and reperfusion stimulus in experimental settings. In animal studies remote preconditioning was showed to improve mitochondrial function and structure however such data on human myocardium are scarce.

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Aim: The aim of the study is to determine whether remote preconditioning protects human myocardium against ischemia-reperfusion injury in both in vivo and in vitro conditions. Methods: The trial is designed as a single center, double-blinded, sham-controlled trial of 120 patients. We randomize (1:1) patients referred for coronary artery bypass grafting for stable coronary artery disease to remote preconditioning or “sham” intervention. The remote preconditioning is obtained by 3 cycles of 5min inflation and 5 min deflation of blood pressure cuff on the right arm. Postoperative course including myocardial enzymes profile will be analyzed. Moreover, in the in-vitro arm the clinically preconditioned myocardium will be assessed for function, mitochondria structure and mitochondria dependent apoptosis. The informed consent of all patients is obtained before enrollment into the study by the investigator. The study conforms to the spirit and the letter of the declaration of Helsinki. Conclusions: In case the effect of remote preconditioning is not measurable in ex-vivo assessment, the future attempt at implementing this phenomenon in clinical practice may be futile and should not be continued until the effect can be confirmed in controlled experimental setting. The study might therefore indicate future directions in trials of clinical implementation of remote preconditioning. Trial Registration: Clinical Trials Register (Clinicaltrials.gov) identifier: NCT01994707. The study was approved by Institutional Review Board of Medical University of Silesia (KNW/0022/KB1/160/12). Key words: remote ischemic preconditioning; coronary artery bypass graft surgery, troponin T, apoptosis, cardioprotection

Hartowanie na odległość ludzkiej mięśniówki serca — protokół badania klinicznego

Streszczenie Wstęp: Hartowanie na odległość traktowane jest jako zjawisko mogące wywierać potencjalnie efekty ochronne u różnych zwierząt. Kilka badań zostało zaprojektowanych celem wykazania występowania tego efektu u człowieka podczas operacji kardiochirurgicznych. Wśród tych badań można znaleźć kilka małych badań i jedno większe, których wyniki były pozytywne, podczas gdy dwa inne większe badania wykazały brak skuteczności hartowania na odległość. Ich efekt oceniano poprzez stężenie enzymów martwicy mięśnia sercowego w okresie pooperacyjnym. W ostatnich dwóch dużych badaniach klinicznych z 2015r. nie wykazano zalet hartowania na odległość w przypadku 2

zabiegów kardiochirurgicznych. Dotychczas nie przeprowadzono badań wykazujących, że hartowanie na odległość rzeczywiście zwiększa odporność serca u ludzi na standaryzowane niedokrwienie i reperfuzję wywoływane w warunkach doświadczalnych. W badaniach na zwierzętach wykazano hartowanie na odległość mitochondriów i poprawę struktury mitochondriów, ale takie dane o ludzkich kardiomiocytach są rzadkie. Cel: Celem badania jest określenie czy hartowanie na odległość chroni ludzką mięśniówkę serca przed urazem niedokrwienno-reperfuzyjnym w warunkach in-vivo i in-vitro. Metody: Badanie zostało zaprojektowane jako jednoośrodkowe, podwójnie zaślepione, z grupą badaną i kontrolną obejmującą łącznie 120 pacjentów. Pacjentów zakwalifikowanych do zabiegu pomostowania aortalno-wieńcowego ze stabilną postacią choroby wieńcowej randomizowano (1:1) do grupy hartowania na odległość lub do grupy kontrolnej. Procedura hartowania na odległość obejmowała 3 cykle 5 minutowego napełnienia i 5 minutowego spuszenia powietrza z mankietu do mierzenia ciśnienia założonego na prawe ramię pacjenta. W okresie pooperacyjnym analizowano wartości enzymów martwicy mięśnia sercowego. Ponadto, w warunkach in-vitro analizowano funkcję hartowanego myocardium, struktura mitochondriów oraz stopień zachodzącej apoptozy. Uzyskanie pisemnej zgody od pacjentów było warunkiem włączenia do badania przez badaczy. Badanie realizowano zgodnie z zasadami deklaracji helsińskiej. Wyniki i Wnioski: W sytuacji, gdy efekt hartowania na odległość nie okazałby się być mierzalny w warunkach oceny ex-vivo, próby stosowania niniejszego zjawiska w praktyce klinicznej w przyszłości mogą być nieskuteczne i nie powinny być kontynuowane dopóki efekt hartowania na odległość nie zostanie potwierdzony w warunkach eksperymentalnych. Badanie może tym niemniej wskazać przyszłe kierunki badań klinicznych nad hartowaniem na odległość. Rejstracja badania: Clinical Trials Register (Clinicaltrials.gov) identyfikator: NCT01994707. Badanie uzyskało zgodę Komisji Bioetycznej Śląskiego Uniwersytetu Medycznego w Katowicach (KNW/0022/KB1/160/12). Słowa kluczowe: hartowanie na odległość, pomostowanie aortalno-wieńcowe, troponina T, apoptoza, kardioprotekcja

INTRODUCTION No experimental study showed that human myocardium can be remotely preconditioned against standardized ischemic/hypoxic insult. We aim to remove this major knowledge gap by 3

applying remote preconditioning to the patient and studying ex-vivo the myocardium obtained thereafter. We assume that we will be able to show that remote preconditioning by brief periods of ischemia of the arm, protects segments of human right atrial appendage myocardium subjected to simulated hypoxia and reoxygenation in-vitro. This proof of principle is crucial. In case the effect of remote preconditioning is not measurable in ex-vivo assessment, the future attempt at implementing this phenomenon in clinical practice may be futile and should not be continued until the effect can be confirmed in controlled experimental setting. On the other hand if we manage to proof that the remote preconditioning truly protects human myocardium, we will have clinical data, and the results from myocardial biopsies of the very same patients to correlate, and possibly reconcile any apparent discrepancy between ex-vivo and in vivo study. The study might therefore indicate future directions in trials of clinical implementation of remote preconditioning. Finally, it is believed that remote preconditioning acts through its influence on mitochondria [1, 2] similarly to ischemic preconditioning. Our study will be one of very few examining its influence on mitochondrial structure and induction of apoptosis and certainly the first studying this phenomenon in human myocardium.

METHODS Trial design The trial is a single center, randomized, double-blinded, sham-controlled trial of patients subjected to coronary artery bypass grafting for stable coronary artery disease. Due to the assumption that some patients will be excluded from the study because of intraoperative events (see below), we planned to recruit the patients to the study until we have enrolled 120 patients undergoing the surgery according to the protocol and not complicated by a perioperative myocardial infarction. Patients, surgeons, treatment team and data analysts are blinded to treatment allocation. The study is conducted both in-vivo and ex-vivo [Figure 1].

Inclusion and exclusion criteria The study was approved by Institutional Review Board of Medical University of Silesia (KNW/0022/KB1/160/12). The study conforms to the spirit and the letter of the declaration of Helsinki. The informed consent of all patients is obtained before enrollment into the study by the investigator. Patient inclusion and exclusion criteria are presented in the Table 1. 4

Exclusions after randomization include (1) perioperative myocardial infarction (MI), (2) perioperative technical difficulties possibly leading to myocardial damage and (3) breaches of protocol related to operative procedure (no operation performed, no cardiopulmonary bypass use, no aortic cross-clamp applied). The patients who develop postoperative MI and those in who technical problems during surgery might have led to myocardial damage will be excluded from the study. We believe that perioperative MI is predominantly caused by vessel occlusion (plaque rupture, microembolism, graft occlusion, surgical error) and should not influence the assessment of remote preconditioning protective effect against global myocardial ischemia/reperfusion injury. The perioperative MI (type 5) will be diagnosed based on the Third Universal Definition of Myocardial Infarction based on data collected with 2 days after surgery [3]. The diagnosis based on cardiac marker levels, EKG and echo results will be made by an independent cardiologist who will not have been involved in other study related tasks.

Primary and secondary endpoints The primary endpoint of the study is the postoperative release of cardiac troponin T. The area under the curve of the marker level over time will be compared between the groups. The secondary endpoints include: Creatine Kinase isoenzyme MB, hemodynamic assessment with oxygen metabolic assessment and creatinine clearance (CKD-Epi method) and the results from in vitro study: right atrial muscle inotropism, Western-blot, immunohistochemistry and electron microscopy.

Sample size calculation To calculate the size of the study group we used the troponin T level (area under the curve) as a primary endpoint similarly to Rahman et al. [4]. We estimated, based on the previous positive studies of remote preconditioning in coronary artery bypass grafting (CABG), that remote preconditioning should decrease the troponin T AUC by 40% (a standardized difference of 0·8). Thus, with the hypothetical standardized difference of 0.6, we need 120 patients to be able to show lower troponin T release in the remote preconditioning group with p