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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???
