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					      Effetti intracoronarici del levosimendan nel maiale
                         anestetizzato


          E. Grossini1, PP Caimmi1,2, C Molinari1, G Teodori2, G Vacca1


1Dipartimento   di Medicina Clinica e Sperimentale, Università del Piemonte Orientale “A. Avogadro,”
                                               Novara

          2 Dipartimento   di Chirurgia Cardiaca, Ospedale Maggiore della Carità, Novara
                        LEVOSIMENDAN
              Calcium-sensitizing drug that has been shown:

• to elicit positive chronotropic and inotropic effects and increase coronary
blood flow in Langendorff-perfused guinea pig hearts

Kaheinen P et al. J Cardiovasc Pharmacol 2001;37: 367-374

• to improve the hemodynamics both in healthy volunteers and in patients
with congestive heart failure and acute MI

Lilleberg JM et al. Eur J Clin Pharmacol 1994;47: 267-274; Slawsky MT et al.
Circulation 2000;102: 2222-2227

• to increase the myocardial contractility and coronary blood flow in animal
models of global ischemia, stunned myocardium or heart failure

Figgitt DP et al. Drugs 2001;61: 613-627; Jamali IN et al. Anesth Analg
1997;85: 23-29

• to elicit opposite effects in animal models of regional ischemia

Tassani et al. Cardiovasc Drugs Ther 2002;16:435-441
                          MECHANISM OF ACTION



• Calcium sensitization of troponin C. No changes of the intracellular calcium
  levels

• Phosphodiesterase III (PDEIII) inhibition (higher doses)

• KATP channels opening in both myocardial and smooth muscle cells

• Role of autonomic nervous system ?

• Role of NO ?

                                SIDE EFFECT

                                HYPOTENSION

           Tassani P et al. Cardiovasc Drugs Ther 2002;16:435-441
        AIM OF THE FIRST PART OF THE STUDY


ANALYSIS OF THE HEMODYNAMIC EFFECTS OF THE INTRACORONARY (IC)

   INFUSION OF LEVOSIMENDAN AND OF THE RELATED ROLE OF THE

            AUTONOMIC NERVOUS SYSTEM AND OF NO
IC Levosimendan: Hemodynamic effects



                            EXPERIMENTAL PROTOCOL




Hemodynamic effects of the ic infusion of levosimendan at doses corresponding

to the bolus doses of 6, 12 and 24 µg/kg (12 pigs; 3 groups of 4 pigs each)




Hemodynamic effects of the ic infusion of levosimendan after the blockade of

the autonomic nervous system (ANS; 6 pigs) or the nitric oxide synthase (NOS; 3

pigs)
IC Levosimendan: Hemodynamic effects


                             EXPERIMENTAL SETUP




                        HR: heart rate; ABP: arterial blood pressure; LVP: left
                         ventricular pressure; ±dP/dtmax: max and min rate of
                        change of LVP; RAP=CVP: central venous pressure;
                            CBF: coronary blood flow (mean and phasic)
               Hemodynamic effects of the ic levosimendan in 12 pigs
              HR            ABP           RAP          LVEDP          +dP/dtmax           -dP/dtmax          CBF             CVR
           beats/min        mmHg         mmHg            mmHg             mmHg/s             mmHg/s         ml/min        mmHg/ml/min



Control    111 ± 10.9      90 ± 10.7     2.9 ± 0.3      5.2 ± 1.3      2337 ± 577.2      -2239.7 ± 610.5     48 ± 6.7       1.12 ± 0.3
           (96-120)       (78-104)      (2.4 -3.2)     (4.3-7.1)      (1984-3200 )     (-3150 a- 1843)      (40-54)        (0.8-1.4)


0.084 μg   110.2 ± 9.4     91 ± 9.8      2.9 ± 0.2      5.2 ± 1.1     2361.2 ± 594.2     -2248.7 ± 604.8    48.3 ± 6.3      1.13 ± 0.3
           (97-118)       (80-104)      (2.5-3.1)      (4.4-6.9)      (2000-3250)      (-3150 a-1850 )     (40.5-54)       (0.8-1.4)
ml/min

Change      -0.6 ± 1.3      1 ± 1.1       0 ± 0.1       -0.02 ± 0.4     24.2 ± 17.7          -9 ± 8.2       0.3 ± 0.4      0.005± 0.007
             (-2 -1)        (0-2)       (-0.1-0.1)     (-0.5-0.5)        (10-50)            (-20-0)          (0-1)         (0-0.015)


Control    113.2 ± 11.1   84.7 ± 12.4    2.6 ± 0.3      4.4 ± 0.6      2289 ± 361.9      -2173.2± 337.7      60 ± 9.5         1 ± 0.35
            (98-123)      (70-100)        (2.2-3)      (3.8-5.2)      (1946-2800)      (-2660 a-1888)       (50-70)         (0.79-1.53)


0.168 μg   114.7 ± 11.1    85 ± 10.6     2.7 ± 0.2       4.4 ± 0.4     2315 ± 351.4      -2166.2 ± 338.5   75.7 ± 12.4      0.8 ± 0.3
           (100-126)       (72-98)      (2.3-2.9)         (4-5)        (1980-2810)     (-2650 a-1870)       (65-88)       (0.53-1.24)
ml/min

Change      1.5 ± 1.2       0.2 ± 2      0.05 ± 0.1     -0.02 ± 0.2     26 ± 19.6            7 ± 9.6        15.7±4.6*       -0.2 ± 0.09*
             (0-3)          (-2-2)      (-0.1-0.1)     (-0.3-0.2)       (10-50)             (-5-18           (11-22)     (-0.29 a -0.09)


Control    105 ± 10.6       95 ± 8        3 ± 0.2        4.8 ± 0.5    2591.2 ± 251.3     -2476.5 ± 260.9   61.7 ± 6.8       1.17 ± 0.32
           (95-120)       (88-106)      (2.8-3.3)        (4-5.2)      (2306-2904)      (-2825 a-2206)      (54-68)         (0.85-1.5)


0.336 μg    107 ± 10.4     95.7 ± 8.4   2.9 ± 0.3        4.7 ± 0.6     2631.5 ± 264      -2458.7 ± 263.4   87.5 ± 14.8       0.8 ± 0.2
           (100-122)      (89-108       (2.6-3.4)      (3.8-5.2)      (2310-2950)      (-2800 a-2180       (68-104)         (0.58-1)
ml/min

Change       2 ± 2.4       0.7 ± 1.9     -0.07 ± 0.1   -0.07 ± 0.09     40.2 ± 31.1        17.7 ± 18.6      25.7±10*      -0.37 ± 0.11*
             (-1-5)        (-2-2)       (-0.2-0.1)      (-0.2-0)         (4-79)            (-10-30)          (14-36)      (-0.5 to -0.27)
IC Levosimendan: Hemodynamic effects


                     EXAMPLE OF EXPERIMENTAL
                           RECORDINGS




                                               HR: heart rate

                                               ABP: arterial blood     pressure

                                               LVP: left ventricular
                                               pressure

                                               ±dP/dtmax: max and min rate of
                                               change of LVP

                                               CVP: central venous pressure

                                               CBF: coronary blood flow
IC Levosimendan: Hemodynamic effects


                             CBF EFFECT OF LEVOSIMENDAN



              6 μg/kg                    12 μg/kg               24 μg/kg
               (n=4)                      (n=4)                  (n=4)




                                   CBF increase : 26.3%   CBF increase : 41.3%
IC Levosimendan: Hemodynamic effects



                      STUDY OF THE MECHANISM OF ACTION


•   Ic infusion of levosimendan (at doses corresponding to 12 and 24 µg/kg)
                                                                      g/kg

    in 2 groups of 3 pigs after the infusion of atropine, phentolamine and

    propranolol




•    Ic infusion of levosimendan (at doses corresponding to 12 and 24 µg/kg)

    after ic L-NAME (n=3)
IC Levosimendan: Hemodynamic effects




                         CBF EFFECT OF LEVOSIMENDAN
                           AFTER THE ANS BLOCKADE

                        12 μg/kg                 24 μg/kg




                   CBF increase : 25.2%     CBF increase : 40.8%
IC Levosimendan: Hemodynamic effects



                       CBF EFFECT OF LEVOSIMENDAN
                         AFTER THE NOS BLOCKADE




                           12 μg/kg           24 μg/kg
IC Levosimendan: Hemodynamic effects


                                       RESULTS


    • INCREASE OF CBF CAUSED BY IC INFUSION OF CORRESPONDING
      DOSES OF 12 AND 24 µg/kg OF LEVOSIMENDAN (26.3% AND 41.3 %)


    • DECREASE OF CVR CAUSED BY IC INFUSION OF CORRESPONDING
      DOSES OF 12 AND 24 µg/kg OF LEVOSIMENDAN (20.6% AND 31.2 %)


    • NO EFFECT ON THE OTHER HEMODYNAMIC VARIABLES


    • NO EFFECT ON QTC


    • NO EVIDENCED ROLE OF THE AUTONOMIC NERVOUS SYSTEM


    • ROLE OF ENDOTHELIAL NO
    AIM OF THE SECOND PART OF THE STUDY


CARDIOPROTECTION AGAINST REGIONAL MYOCARDIAL ISCHEMIA



          • Hemodynamic effects of ic levosimendan




            • Effects on apoptosis and autophagia
Cardioprotection against regional myocardial ischemia


                                   SETUP SPERIMENTALE




                                      Hemodynamic variables

HR= heart rate; ABP=arterial blood pressure; CBF= coronary blood flow; CO= cardiac output; LVP=left
ventricular pressure; RAP= right atrial pressure; dP/dtmax= max and min rate of change of LVP; %SS=
                                         segmental shortening
    Cardioprotection against regional myocardial ischemia



                                 EXPERIMENTAL PROTOCOL

•     Hemodynamic effects of the ic infusion of levosimendan (12 and 24 µg/kg in

      10 min; 1.5, 3, 6, 12 µg/min in 10 min) in the presence of regional myocardial
                                         min

      ischemia (15 pigs):

         -ABP
         -HR
         -RAP
         -LVP e dP/dtmax
         -CBF
          -%SS= EDL-EDS/EDLx100
          -CO


•     Apoptosis and autophagia induced by regional myocardial ischemia: role of ic

       levosimendan (12 µg/kg in 10 min)
                                    min
Cardioprotection against regional myocardial ischemia


                                         RESULTS




  •The ic administration of corresponding doses of 12 or 24 µg/kg in 10 min of
         levosimendan in the presence of regional myocardial ischemia
  does not cause any significant change in myocardial indexes of contractility




   • The ic administration of 12 or 24 µg/kg in 10 min of levosimendan causes
                                an increase of CBF
Cardioprotection against regional myocardial ischemia


    EFFECTS OF LEVOSIMENDAN ON APOPTOSIS AND AUTOPHAGY

               Myocardial biopsies performed before the induction of
          ischemia, at the end of ischemia (3.5 hours of ischemia) and after
                 levosimendan ic bolus administration of 12 µg/kg


      MARKERS OF APOPTOSIS                              MARKERS OF AUTOPHAGY



 FRAGMENTED NUCLEI (TUNEL                           BECLIN 1, A MAMMALIAN GENE
 ASSAY)                                             REQUIRED FOR THE ONSET OF
                                                    AUTOPHAGY
 BAX EXPRESSION, A PROAPOPTOTIC
 GENE                                                   MONODANSYLCADAVERINE (MDC)

 DOWNREGULATION OF THE
 ANTIAPOPTOTIC GENE, BCL-2
Cardioprotection against regional myocardial ischemia


                                        TUNEL ASSAY




         CONTROL                                    %   100


                                                         80



               3,5         4          LEVO               60


                                                         40


     I                                                   20


                                                          0
                                                                    -                            -
                                                                            -LL   +L   1              -L
                                                                                                      L        ++ L
                                                                                                                L
    PI
                                                              3,5                 4        3,5             4          h


                                                                        I                            PI


 • INCREASE OF FRAGMENTED NUCLEI IN THE INFARCTED MYOCARDIUM (I) AND, AT HIGHER
   EXTENT, IN THE PERI-INFARCT TISSUE (PI)

 • IC LEVOSIMENDAN REDUCES THE FRAGMENTED NUCLEI
Cardioprotection against regional myocardial ischemia



                                  BAX EXPRESSION

                     I               PI


                                                        100


     3,5                                                 80


                                                         60


                                                         40
     4
                                                         20


                                                          0
                                                                    -L       +L               -L
                                                                                            -L ++ L
                                                                                                 L
  LEVO                                                              -L       +L   1


                                                              3,5        4            3,5    4        h


                                                                    I                       PI



 • BAX EXPRESSION ACTIVATION EVIDENT 3.5 h AND EVEN HIGHER 4 h AFTER ISCHEMIA

 • IC LEVOSIMENDAN REDUCES BAX EXPRESSION IN THE I TISSUE AND IN THE PI TISSUE
Cardioprotection against regional myocardial ischemia



                                  BCL-2 EXPRESSION


                      I               PI                %
                                                            100


      3,5
                                                             80



                                                             60


       4
                                                             40



                                                             20



                                                              0
   LEVO                                                                     -L       +L   1         -L        +L
                                                                            -L       +L              -L       +L


                                                                  3,5            4            3,5         4        h

                                                                        I                            PI




• DOWNREGULATION OF BCL-2 IS EVIDENT 3.5 h AND EVEN HIGHER 4 h AFTER ISCHEMIA

• IC LEVOSIMENDAN PREVENTS THE DOWNREGULATION OF BCL-2
Cardioprotection against regional myocardial ischemia


                                            MDC
                   I              PI




                                                        100



     3,5                                                 80



                                                         60




     4                                                   40



                                                         20



                                                          0
 LEVO                                                                                 1
                                                                        -L       +L              -L       +L


                                                              3,5            4            3,5         4        h


                                                                    I                           PI




 • THE NUMBER OF MDC POSITIVE CELLS IS HIGHER IN THE PI MYOCARDIUM THAN IN THE I AREA

 • IC LEVOSIMENDAN SIGNIFICANTLY INCREASES THE NUMBER OF MDC-POSITIVE CELLS
Cardioprotection against regional myocardial ischemia


                               BECLIN 1 EXPRESSION

                        I            PI


                    I                 PI
                                                        100

      3,5
                                                         80



                                                         60


      4                                                  40



                                                         20



                                                          0
   LEVO                                                                          1
                                                                    -L      +L             -L   +L


                                                              3,5       4            3,5   4         h



                                                                    I                      PI

• BECLIN 1 TRASLOCATES FROM NUCLEUS TO CYTOSOL IN I TISSUE AND, PARTICULARLY, IN THE PI
AREA 3.5 h AFTER STENOSIS, THEN CYTOSOLYC BECLIN 1 DECLINES

• IC LEVOSIMENDAN KEEPS THE BECLIN 1 EXPRESSION IN THE CYTOPLASM IN THE PI TISSUE
Hemodynamic effects of ic 1.5, 3, 6, 12 µg/min

       of levosimendan (10 min each)
       Hemodynamic effects of 1.5, 3, 6, 12 µg/min levosimendan in the presence of
                            regional myocardial ischemia
          HR            ABP          RAP         LVEDP       CBF             CO                dP/dtmax
                                                                                               dP/                %SS-
                                                                                                                  %SS-I           %SS-
                                                                                                                                  %SS-N



C                                                                            5018.6 ± 194.4    2549.6 ± 202.7     15 ± 2.3        16.7 ± 4.1


I         97.4 ± 14     96.8 ± 9.7   2.3 ± 0.3   5.2 ± 0.5   52.2 ± 10.1     4049 ± 160.8      1713.2 ± 367.5     9.6 ± 2.3       12 ± 2.9


change                                                                       (-969.6 ± 49.1)   (-836.4 ± 335.6)   (-5.4 ± 1.6)    (-4.7 ± 1.6)


L1.5
L1.5      97.6 ± 13.7   97 ± 10.5    2.4 ± 0.4   5.2 ± 0.6   62.8 ± 9.7      4529.6 ± 189.3    1808.2 ± 381.5     9.9 ± 2.3       12.8 ± 2.9


change                                                       (10.6 ± 1.5)    (480.6 ± 34.2)    (95 ± 37.3)        (0.24 ± 0.16)   (0.78 ± 0.9)


L3        97.6 ± 13.7   97 ± 10.5    2.4 ± 0.4   5.2 ± 0.6   81.6 ± 20.1     4841 ± 197.3      1898.4 ± 437.8     10.7 ± 2.2      14 ± 3



change                                                       (29.4 ± 13.5)   (792 ± 38.8)      (185.2 ± 85.9)     (1.1 ± 0.86)    (2 ± 0.9)


L6        97.6 ± 13.7   97 ± 10.5    2.4 ± 0.4   5.2 ± 0.6   84.5 ± 23.1     5204.6 ± 210.1    2025.8 ± 421.2     12.6 ± 1.7      14.9 ± 4.2



change                                                       (32.3 ± 15.8)   (1155.6 ± 50.9)   (312.6 ± 56.1)     (3 ± 2.1)       (2.9 ± 1.9)


L12       97.6 ± 13.7   97 ± 10.5    2.4 ± 0.4   5.2 ± 0.6   83.7 ± 17.7     4984.8 ± 196.8    2097.2 ± 398.6     11.7 ± 2.9      13.3 ± 3.1


change                                                       (31.5 ± 10.5)   (935.8 ± 37.7)    (384 ± 54.3)       (2.1 ± 1.2)     (1.36 ± 1.2)
Cardioprotection against regional myocardial ischemia

                      EXAMPLE OF EXPERIMENTAL RECORDING
Cardioprotection against regional myocardial ischemia


                                               RESULTS




                                                              27.2%
                                           25.8%                                    23.7%
                                   22.2%                              23%

                                                                              18%

                             12%                         11.7%                          11.9%

                                                                            6.9%
                      5.3%
                                                         3%
Cardioprotection against regional myocardial ischemia


                                         RESULTS




The ic administration of 1.5, 3, 6, 12 µg/min levosimendan induces graded

   increases of dP/dtmax, CO, EF and CBF in the absence of changes of

  arterial blood pressure and heart rate. Levosimendan causes graded

     increases of %SS both in the ischemic and peri-ischemic area.
                                  CONCLUSIONS

        THE RESULTS OBTAINED IN THIS STUDY INDICATE THAT THE IC
      ADMINISTRATION OF LEVOSIMENDAN MAY EXERT CARDIOPROTECTION


          •     THROUGH THE REDUCTION OF THE APOPTOTIC CELL DEATH AND

              THE INDUCTION OF AUTOPHAGY AS A CELL SURVIVAL MECHANISM

                 at the corresponding doses commonly used in patients


         •     THROUGH POSITIVE DIRECT EFFECTS ON MYOCARDIAL FUNCTION

              AND PERFUSION IN THE ABSENCE OF CHANGES OF HEMODYNAMIC

                                       VARIABLES

                                    at higher doses


•   Effects of higher doses on apoptosis (autophagia???)

•   Mechanisms of actions???

				
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