Remote Ischemic Preconditioning of Human Myocardium
Condition: Coronary Artery Bypass Graft Triple Vessel; Apoptotic DNA Damage
MeSH terms: Ischemia
Study type: Interventional
Phase: Phase 2

Summary: 
      The investigators aim to definitively show if human myocardium can be remotely
      preconditioned. In the writings there are no experiments that would prove that the remote
      preconditioning protocol effects on the higher protection of the human myocardial cells,
      against the ischemia-reperfusion injury.

      120 patients referred for coronary artery bypass grafting (CABG) procedure with use of
      cardiopulmonary bypass are planned will be included to the research. Patients will be
      randomized (1:1) to one of two groups: remote preconditioning or "placebo" intervention. On
      the day of surgery, after induction of anesthesia remote preconditioning will be elicited by
      3 cycles of 5min inflation (ischemia) and 5 min deflation (reperfusion) of blood pressure
      cuff on the right arm. In the control group the blood pressure cuff is going to be placed on
      the upper limb but the preconditioning protocol will not be carried out. On cannulation for
      CPB, right atrial appendage and myocardial biopsies of the left ventricular will be
      harvested. The investigators will study: (1) resistance of myocardium to hypoxia/reperfusion
      injury in in vitro experiments, assessed in isolated right atrial pectinate muscle trabeculae
      (2) induction of apoptosis and status of mitochondria in myocardium after the period of
      ischemia, and reperfusion in vitro (3) amount of myocardial necrosis in-vivo induced by
      period of ischemia and reperfusion during CABG as assessed by postoperative myocardial
      necrosis markers release (4) the systolic function of the myocardium at the postoperative and
      the kidney function in the postoperative period evaluated by the creatinine clearance; (5)
      induction of apoptosis and status of mitochondria in myocardium after the period of ischemia,
      and reperfusion during coronary artery bypass grafting, assessed in myocardial.

      There is going to be an ability to define does the remote preconditioning influence on the
      occurrence of apoptosis in the human myocardium in the in vivo conditions and does it
      influence on the postoperative course in patients undergoing cardiac surgery procedures. The
      investigators will try to study if remote preconditioning modify induction of apoptosis and
      its structure in response to injury. 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.
    

Detailed description: 
      The investigators will study:

        1. Resistance of isolated right atrial pectinate muscle trabeculae to simulated
           hypoxia/reperfusion in functional organ bath model

        2. Resistance of isolated right atrial pectinate muscle trabeculae to induction of
           apoptosis by simulated hypoxia/reperfusion

        3. Resistance of mitochondria in isolated right atrial pectinate muscle trabeculae to
           changes induced by simulated hypoxia/reperfusion

      Simultaneously we will assess:

        1. Amount of myocardial necrosis in vivo induced by period of ischemia and reperfusion
           during coronary artery bypass grafting (CABG) as assessed by postoperative myocardial
           necrosis markers release profile

        2. Myocardial function in vivo after the period of ischemia and reperfusion during CABG as
           assessed by hemodynamic measurements (thermodilution method), oxygen supply/consumption
           and inotropic support requirements

        3. Induction of apoptosis and status of mitochondria after the period of ischemia, and
           reperfusion during CABG as assessed in left ventricular myocardial biopsies The
           investigators will try correlate the in vitro and in vivo findings from the same
           patients.

      Methodology The study will be conducted both in vivo and ex-vivo. Patients referred for CABG
      for stable coronary artery disease will be recruited and randomized (1:1) by random digit
      generator to one of two groups: remote preconditioning or "placebo" intervention. Only
      patients in who at least 3 coronary artery bypass grafts with use of cardiopulmonary bypass
      (CPB) are planned will be included. On the day of surgery, after induction of anesthesia and
      before the skin incision remote preconditioning will be elicited. The "placebo" group will
      have the pressure cuff placed on the right arm but no inflations will be performed. To obtain
      blinding, the inflations will occur under surgical drapes, and will be performed by the same
      person every time, who will be involved in random sequence generation, and remote
      preconditioning application, but not in the care of the patient, or other research related
      tasks.

      All consented patients will have preoperative echocardiography, electrocardiography and blood
      tests including fool blood count, creatinine, BUN, and liver function test performed as part
      of their routine preoperative care. Additionally troponin T and CK-MB levels will be
      assessed. Furthermore patients will have performed electrocardiography on 1st day after
      operation.

      Anesthesia will be standardized and consist of midazolam 15mg orally 1h before surgery,
      etomidate 0.2mg/kg, fentanyl 5g/kg and pancuronium 0.1mg/kg iv for anesthesia induction,
      propofol 0.5-1.0 mg/kg/h and fentanyl 4g/kg/h infusion for anesthesia maintenance. No
      anesthetic gases will be used. Full hemodynamic monitoring will be used with Swan-Ganz
      catheter (not routine in CABG patients). First hemodynamic measurements and oxygen
      supply/consumption calculations will be performed preoperatively.

      Functional in vitro assessment. On cannulation for CPB, right atrial appendage, which is
      routinely removed and discarded for venous cannula placement will be harvested in all
      patients. The tissue will be was transferred in ice cold Krebs-Henseleit solution to the
      isolated organ laboratory in our department. One pectinate muscle trabecula will be harvested
      for baseline assessment of apoptosis or mitochondria (see below). Another single trabecula
      less than 1mm in diameter will be mounted in the organ chamber - Schuler Organbath (Hugo
      Sachs Elektronik, March-Hugstetten, Germany (HSE)) containing Krebs-Henseleit solution. It
      will be oxygenated via glass frit with carbogen (95% oxygen, 5% carbon dioxide) and
      maintained at 37°C. The trabecula will be driven with 1Hz 50ms square stimuli using platinum
      field electrodes and the potential of 150% of the threshold for given preparation. The
      stimulator Type 215 (HSE) will be used. The contraction force will be measured with F30
      isometric force transducer Type 372 (HSE). The signal will be enhanced with TAM A PlagSYS
      transducer amplifier module Type705 (HSE) and recorded using PowerLab/4SP system and Chart
      software (AD Instruments).

      The trabecula will be gradually stretched to 90% of optimal tension according to
      Frank-Starling relationship and left for 30min of stabilization and washout.

      60min ischemia will be simulated by substituting oxygen in carbogen with argon (95% argon, 5%
      carbon dioxide) and replacing Krebs-Henseleit solution with one containing no glucose or
      pyruvate. On reoxygenation the carbogen will be added again and the tissue bath solution will
      be replaced with one used initially. The tissue will be was washed several times and left for
      120min of reoxygenation period with washout every 15 min. This functional model of hypoxia
      reoxygenation has been used in our laboratory before [12-14]. Replacement of oxygen with
      argon results in the drop of tissue bath oxygen partial pressure from 475±52mmHg to
      51±1.8mmHg (p<0.001) [37-39]. It is accompanied by significant and rapid decline in isometric
      contraction force. During reoxygenation the contraction force returns initially and next we
      observe slow decline of muscle inotropism which we interpret as development of reoxygenation
      injury. At the end, we will use 10-4 M norepinephrine ((-)-Arterenol Bitartrate) to test for
      stunning.

      The contraction force will be recorded continuously. Contractility will be expressed in
      percent of the initial contraction force for given preparation. The investigators will
      compare the maximal recovered contraction force, the contraction force after 30min and 120min
      of reoxygenation and the contraction force produced by adding 10-4M norepinephrine at the end
      of reoxygenation.

      The investigators will also look for the signs of ischemic contracture development defined as
      an increase of resting tension of the trabecula. This increase, if present, starts shortly
      after the onset of hypoxia and continues steadily throughout the whole hypoxia period. The
      investigators will compare the increase in resting tension (in mN/mg tissue mass) at the end
      of hypoxia, that is at the time of maximal contracture.

      All measurements, and in particular recovery of function will be compared between trabeculae
      from remotely preconditioned and "placebo" patients.

      Two atrial trabeculae from the same appendage, one harvested at baseline, and another
      subjected to functional experiment (60 min hypoxia + 120 min reoxygenation) will be studied
      each time for:

        -  apoptosis induction - Caspase 3 and cleaved Caspase 3, PARP and cleaved PARP expression
           measured with Western-Blot, or

        -  apoptosis induction - assessed with TUNEL and immunohistochemistry staining for Caspase
           3, cleaved Caspase 3, PARP and cleaved PARP, or

        -  state of mitochondria (electron microscopy) As we plan to randomize 120 patients (see
           below) the material for apoptosis and mitochondria studies will be randomly taken from
           60 patients (30:30) for Western Blot, 40 patients (20:20) for immunohistochemistry and
           TUNEL, and 20 patients (10:10) for electron microscopy.

      Western Blot (2 trabeculae, baseline and after hypoxia/reoxygenation, from 30 remotely
      preconditioned and 30 "placebo" patients).

      For Western Blot immunoassay the trabeculae will be placed in liquid nitrogen
      Immunohistochemistry (2 trabeculae, baseline and after hypoxia/reoxygenation, from 20
      remotely preconditioned and 20 "placebo" patients).

      Expression of Caspase 3, cleaved Caspase 3, PARP and cleaved PARP proteins in tissue sections
      will be detected immunohistochemically. Trabeculae will be fixed overnight in 10%
      neutral-buffered formalin (phosphate buffer), subsequently passed through graded alcohol
      solutions, processed three times in xylene, and finally embedded in paraffin blocks. The
      documentation of immunohistochemical reactions will be performed with SSC-DC58AP camera
      (Sony) coupled with Nikon Eclipse E400 optical microscope.

      TUNEL (terminal deoxynucleotidyl transferase dUTP nick and labeling) (2 trabeculae, baseline
      and after hypoxia/reoxygenation, from 20 remotely preconditioned and 20 "placebo" patients).

      Tissue sections will be deparaffinized in 2 changes of xylene for 5 minutes each, and
      hydrated with 2 changes of 100% ethanol for 3 minutes each, and 95% ethanol for 1 minute. The
      documentation of immunohistochemical reactions will be performed with SSC-DC58AP camera
      (Sony) coupled with Nikon Eclipse E400 optical microscope.

      Electron microscopy (2 trabeculae, baseline and after hypoxia/reoxygenation, from 10 remotely
      preconditioned and 10 "placebo" patients).

      Trabeculae will be placed in cacodyl buffer with 2% glutaraldehyde. The mitochondria will be
      micrographed with JEOL-JEM 100CX transmission electron microscope (JEOL Inc, Peabody, Mass)
      with magnification x16000. The electron micrographs will next be saved and mitochondria size
      and structure will be analyzed using Image ProPlus software (Media Scanalytics).

      The in vivo trial The operation will be performed with the use of CPB in normothermia by
      experienced cardiac surgeon. Intermittent warm-blood (37°C) antegrade cardioplegia
      (miniplegia) will be used for myocardial protection. As only patients requiring at least 3
      coronary bypass grafts will be recruited, we expect aortic crossclamp time to last at least
      30 minutes. After cross clamp removal the proximal anastomoses will be performed using side
      biting clamp. The patient will be weaned off CPB, hemostasis will be secured and the chest
      will be closed over chest tubes. Just before closing the chest 16G needle-true cut biopsy of
      the left ventricular myocardium will be obtained from the apex area. The investigators expect
      the reperfusion time from the cross-clamp removal to obtaining the biopsy to last at least 40
      min. The exact ischemia (cross-clamp) time, and reperfusion time (until obtaining the biopsy)
      will be measured.

      The myocardial biopsies will be harvested and assessed in the same way as atrial trabecule.
      The investigators will randomly perform Western blotting in 60 (30:30) biopsies harvested on
      liquid nitrogen, immunohistochemistry including TUNEL in 40 (20:20) harvested on 10% neutral
      buffered formalin, and electron microscopy in 20 (10:10) harvested on cacodyl buffer with 2%
      glutaraldehyde. The methods of assessing Caspase3, cleaved Caspase 3, PARP, cleaved PARP,
      TUNEL and mitochondria have been described above.

      After the operation patient will be transferred to ICU and treated as per routine.

      The primary endpoint of clinical observation will be the postoperative release of cardiac
      troponin T.

      The investigators will measure serum concentration of cardiac Troponin T
      (electrochemiluminescence "ECLIA", Roche) preoperatively and next 72h from cross-clamp
      removal. At the same time points the level of creatine kinase isoenzyme MB will be assessed
      (enzymatic assay, Roche). The area under the curve of the marker level over time will be
      compared between the groups.

      To assess the myocardial function all patients will have pulmonary artery catheter (Swan Ganz
      catheter) inserted preoperatively. Full hemodynamic assessment (thermodilution method) as
      well as oxygen metabolism status based on arterial and mixed venous gas analysis will be
      performed preoperatively and next 48h after aortic cross-clamp removal.

      Apart from measuring cardiac index we will calculate left and right cardiac work indices. The
      oxygen delivery index and extraction ratio will be calculated. The lactate levels will also
      be measured. The investigators will also assess the need for inotropic support at the same
      time points using so called inotropic index.

      All patients will have postoperative electrocardiogram on day 2 and 4 as per postoperative
      care routine and additionally on 1st day after operation. Similarly other routine
      postoperative tests that are performed as part of the routine postoperative care will be
      performed, monitored and used to look for the differences in postoperative course. This
      includes echocardiography as well as creatinine (eGFR).
    

Intervention name(s): Remote ischemic preconditioning; Placebo
Intervention type(s): Procedure; Procedure
Intervention description(s): Blood pressure cuff inflation; Deflated cuff on arm for 30 minutes.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Basic Science
Keywords: Remote ischemic preconditioning; Coronary artery bypass graft surgery; Troponin T; Apoptosis; Experimental; Cardioprotection