Effect of SOD-1 over-expression on myocardial function during resuscitated murine
septic shock
Katja Baumgart1,a, Vladislava Simkova1,2,a, Florian Wagner1,a, Sandra Weber1, Michael
Georgieff1, Peter Radermacher1, Gerd Albuszies1, Eberhard Barth1
1
Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung,
2
Anesteziologicko-resuscitacni klinika, FN u sv. Anny, Brno, Czech Republic,
a
K.B., V.S. and F.W. equally contributed to this study
Institution at which the work was performed: Animal research laboratory of the
Department of Anesthesia at the University Medical School
Financial support: Supported by the Research Award of European Society of
Anaesthesiology 2005 (Eberhard Barth)
Financial disclosure: The authors have not disclosed any potential conflicts of interest.
Address for correspondence:
Peter Radermacher
Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung
Universitätsklinikum
Parkstrasse 11
D-89073 Ulm
Telephone: 49 731 500 60160
Fax: 49 731 500 60162
e-mail: peter.radermacher@uni-ulm.deAnesthesia, cecal ligation and puncture (CLP)
Electronic Supplement
Material and Methods:
Wild type and both heterozygous and homozygous SOD-1 over-expressing C57BL/6TG(SOD1)3Cje/J mice (Jackson Laboratories, Bar Harbour, ME, age 10–16 wks, body weight
25–33 g) of either gender were randomly allocated to sham operation (controls: wild type: 5
males, 4 females; heterozygous: 6 males, 6 females; homozygous: 4 males, 3 female) and
animals rendered septic (wild type: 4 males, 3 females; heterozygous: 5 males, 4 females;
homozygous: 4 males, 2 females) [1]).
A pressure-conductance catheter was introduced into the carotid artery and advanced into the
heart across the aortic valve under the guidance of the online pressure signal in order to assess
left heart pressure-volume relationships together with systemic hemodynamics as described in
detail previously [2]. The catheter was interfaced with a pressure-volume analog signal
amplifier. Real time conductance and pressure data were collected with an analogue-to-digital
converter. In brief, changes in the electric field generated by one pair of electrodes during
chamber filling and ejection are sensed as a change in voltage in the other pair of sensing
electrodes. Due to conductive tissues and fluids surrounding the left ventricular cavity (e.g.
the myocardial wall), the measured conductance and therefore the conductance-derived
volume differ from the true left ventricular volume. To account for this component of the
conductance signal that is not dependent on chamber dimension and “called parallel
conductance”, 10 μL of 10 % saline were given i.v. via central venous catheter before the first
measuring time point. The highly conductive saline transiently changes the conductivity of the
blood, practically without affecting parallel conductance. This change in conductivity allows
separation of the left ventricular blood volume from the parallel conductance. In addition to
determining parallel conductance, we calibrated and analyzed the measured conductance
signals via generating a regression equation for conductance as a function of a defined volume
over a range of different hematocrit values using an in vitro volumetric model [3]. This
volumetric model contains six cylindrical holes ranging from 13.2 to 172.3 μL, which were
filled with fresh heparinized mouse blood diluted with the hydroxyethylstarch solution to
obtain the same range of hematocrit values (21 – 38 %) as measured in vivo. The conductance
of each cylinder containing the known volume was measured with the conductance catheter at
37°C. From the measured raw conductance signals, the relationship of the known cylinder
volumes as a function of the conductance was plotted, and the regression equation for the
known volume of the calibrate cuvette V was calculated: The regression equation for the mean
hematocrit of 0.29 was V = 4.7·G 19, where G is the measured signal of conductance. Before
implantation, a strict setup and calibration procedure of the catheter was performed to prevent
influences of ambient conditions such as temperature, composition, and viscosity of
surrounding fluids. The catheter was presoaked for 30 min in 37° C warm saline, and
immediately before implantation the bridge amplifier was balanced and calibrated using the
built-in electronic calibration feature of the Millar control unit [4].
After the surgical instrumentation, a recovery period of one hour was allowed to establish
steady-state conditions. Thereafter, the following hemodynamic variables were recorded over
a period of 6 hours: heart rate, blood pressure, stroke volume, cardiac output, ejection
fraction, left ventricular end-diastolic and –systolic pressure and volume (i.e. the maximum
and minimum volumes, respectively [5]), maximal contraction (dp/dtmax) and relaxation
(dp/dtmin) as pressure development during isovolumetric contraction and relaxation, τWeiss as
an index for left ventricular relaxation [6]. Based on the median of end-systolic and enddiastolic pressure and volume relationships left ventricular pressure volume loops were
calculated. Our closed-chest approach, constructing pressure–volume loops based on
subsequent made preload-controlled measurements impossible. Consequently, single-beat
analyses were used assuming linear end-systolic and end-diastolic pressure-volume relations
with a fixed zero-volume and –pressure intercept [7].
To assess myocardial SOD and catalase enzymatic activities, the heart was removed
immediately at the end of the observational period, frozen in liquid nitrogen and stored at –70
°C. A 100 mg tissue aliquot was homogenized in 500 μL of chilled sucrose puffer (0.25M
Sucrose, 10mM Tris, 1mM EDTA), centrifuged at 13,000 g for 5 minutes, and the SOD and
catalase activities were assessed in 100μL supernatant aliquots using commercial kits (SOD
Assay Kit – WST, Dojindo Molecular Technologies, Inc., Kumamoto, Japan, with Superoxide
Dismutase from bovine erythrocytes, 3K, Sigma as standard and BIOXYTECH® Catalase520TM, Oxis Research, Portland, OR). The enzyme activities are expressed as Umg-1 protein
(DC Protein Assay, Bio-Rad Laboratories, Hercules, CA).
Statistical analysis
All data are presented as median and range unless otherwise stated. After exclusion of normal
distribution using the Kolmogorov-Smirnov-test, time-dependent changes within each group
were tested using a Friedman ANOVA on ranks for repeated measurements and a subsequent
Dunn’s test for multiple comparisons. Differences between groups were analyzed by the
Mann-Whitney rank sum test for unpaired samples. In all cases, p<0.05 was regarded as
significant.
Results:
Table 1. Parameters of systemic hemodynamics and left heart function in the sham-operated
mice. dP/dtmax maximal systolic contraction, dP/dtmin maximal diastolic relaxation  TauWeiss
diastolic relaxation time. All data are median, range, # depicts p<0.05 vs. wild type controls,
* p<0.05 vs. 18 hours post sham-operation.
18h post laparotomy
21h post laparotomy
24h post laparotomy
Heart rate
Wild type
396
(341;555)
362 (325;396)
370
(183;554)
[min-1]
Heterozygous
410
(346;591)
386 (319;478)
392
(322;644)
Homozygous
426
(374;543)
408 (359;521)
392
(359;466)
Mean arterial
Wild type
78
(63;109)
70
(50;83)
63
(51;82) *
pressure
Heterozygous
83
(65;105)
72
(61;88)
68
(51;77) *
[mmHg]
Homozygous
84
(73;93)
74
(59;93)
65
(50;68) *
Stroke
Wild type
21
(12;30)
28
(23;44)
34
(29;47) *
volume
Heterozygous
23
(12;54)
34
(18;66)
44
(20;60) *
[µl]
Homozygous
27
(22;34)
37
(22;45)
40
(24;62) *
Cardiac
Wild type
7.6
(6.8;14.2)
9.7 (7.9;16.3)
12.3
(8.8;21.8) *
output
Heterozygous
9.6
(4.3;31.7)
13.5 (6.5;25.8)
18.0
(7.1;37.1) *
[mL·min-1]
Homozygous
14.0
(9.4;16.1)
16.3 (9.4;19.3)
15.4
(9.4;24.5)
Enddiastolic
Wild type
38
(28;62)
59
(50;91)
89
(78;106) *
volume
Heterozygous
45
(36;90)
71
(45;110)
108
(70;140) *
[µl]
Homozygous
63
(45;76) #
78
(69;101)
103
(84;125) *
Enddiastolic
Wild type
16
(9;18)
17
(10;20)
17
(9;19)
pressure
Heterozygous
15
(7;24)
16
(7;26)
15
(11;21)
[mmHg]
Homozygous
14
(8;19)
13
(7;17)
13
(8;18)
Endsystolic
Wild type
22
(16;33)
32
(24;57)
52
(49;66) *
volume
Heterozygous
23
(16;57)
34
(26;66)
57
(44;96) *
[µl]
Homozygous
44
(25;66) #
46
(30;58)
57
(45;87) *
Endsystolic
Wild type
114
(89;140)
93
(87;109) *
89
(75;107) *
pressure
Heterozygous
110
(87;132)
93
(73;114) *
92
(79;108) *
[mmHg]
Homozygous
113
(91;130)
92
(73;110) *
86
(66;102) *
Ejection
Wild type
48
(44;70)
47
(37;58)
40
(37;49)
fraction
Heterozygous
56
(32;70)
50
(35;66)
51
(23;60)
[%]
Homozygous
44
(34;57)
43
(32;60)
41
(29;58)
dP/dtmax
Wild type
8111
(6942;10437)
7000 (6000;7485) *
6879
(5000;8091) *
[mmHg·sec-1] Heterozygous
8315
(5682;10310)
7198 (5363;7709) *
6767
(5360;9624) *
Homozygous
8300
(6192;10154)
6655 (5139;7796) *
6001
(4867;8251) *
-7000 (-10358;-6304) -6192 (-7358;-5091)
-6240
(-7134;-5000)
[mmHg·sec-1] Heterozygous
-7645 (-9480;-4948)
-6001 (-7182;-4836)
-5841
(-7821;-4932)
Homozygous
-7820 (-8705;-5554)
-6193 (-7415;-4612)
-5617
(-6975;-4373)
dP/dtmin
Wild type

Wild type
6.8
(5.8;7.8)
7.2 (6.0;8.2)
7.2
(4.7;7.7)
[msec]
Heterozygous
6.8
(5.2;9.3)
6.9 (5.3;10.0)
7.0
(5.2;7.6)
Homozygous
5.9
(5.1;8.5)
6.5 (5.9;7.2)
6.8
(6.1;7.4)
Table 2. Parameters of systemic hemodynamics and left heart function in the CLP mice.
dP/dtmax maximal systolic contraction, dP/dtmin maximal diastolic relaxation  TauWeiss
diastolic relaxation time. All data are median, range, # depicts p<0.05 vs. wild type controls,
* p<0.05 vs. 18 hours post CLP, § p<0.05 vs sham-operated mice.
18h post CLP
21h post CLP
24h post CLP
Heart rate
Wild type
384 (366;560)
524 (405;584) §
492 (379;560) §
[min-1]
Heterozygous
445 (369;547)
539 (452;598) §
525 (398;550) §
Homozygous
543 (412;558)
498 (410;553)
518 (412;615) §
Mean arterial
Wild type
72 (62;93)
77 (68;97)
72 (60;88)
pressure
Heterozygous
72 (50;110)
83 (69;96)
67 (56;84)
[mmHg]
Homozygous
79 (62;116)
76 (64;91)
68 (65;76)
Stroke
Wild type
24 (18;31)
30 (18;58)
34 (24;68) *
volume
Heterozygous
24 (19;33)
37 (21;43)
29 (26;54) *
[µl]
Homozygous
25 (20;27)
29 (27;34)
38 (30;44) *
Cardiac
Wild type
9.3 (7.1;17.1)
15.4 (7.6;30.3)
16.7 (8.9;33.1) *
output
Heterozygous
10.9 (9.2;15.6)
22.0 (9.4;41.8)
14.5 (9.1;29.2) *
[mL·min-1]
Homozygous
10.1 (9.9;12.0) §
15.0 (11.1;18.4)
21.1 (12.5;23.5) *
Enddiastolic
Wild type
68 (35;90)
82 (36;115)
100 (58;133)*
volume
Heterozygous
56 (35;89)
83 (56;126)
104 (70;125)*
[µl]
Homozygous
66 (53;78)
63 (54;84)
92 (69;110)*
Enddiastolic
Wild type
18 (8;20)
15 (12;19)
15 (11;18)
pressure
Heterozygous
12 (9;19)
11 (8;23)
16 (9;22)
[mmHg]
Homozygous
12 (10;19)
10 (8;13)
13 (7;18)
Endsystolic
Wild type
43 (26;64) §
58 (43;76) §
73 (43;110)*
volume
Heterozygous
29 (16;61)
44 (23;75)
71 (41;90)*
[µl]
Homozygous
29 (20;50)
35 (26;52)
57 (30;72)
Endsystolic
Wild type
98 (86;108)
103 (90;132)
99 (90;125)
pressure
Heterozygous
103 (81;145)
111 (100;134) §
105 (97;115)
[mmHg]
Homozygous
109 (82;116)
110 (84;131)
104 (91;125) §
Ejection
Wild type
42 (29;64)
40 (31;50)
42 (18;48)
fraction
Heterozygous
47 (31;57)
49 (22;64)
36 (21;43)
[%]
Homozygous
45 (35;67)
45 (38;56)
38 (33;57)
dP/dtmax
Wild type
7622 (7083;8857)
[mmHg·sec-1] Heterozygous 7916 (5560;11555)
Homozygous 10677 (7000;12895)# §
dP/dtmin
Wild type
-7267 (-8522;6000)
[mmHg·sec-1] Heterozygous -7755 (-11443;-5347)
Homozygous -9480 (-12401;-6000)

Wild type
[msec]
8690 (7182;11443) §
8506 (5218;10501) §
10003 (9209;13677) # §
9863 (5277;10581) §
9623 (9432;9975) §
9400 (8857;10000) §
-8102 (-11235-;7000) §
-7918 (-10549;-5809) §
-9676 (-10693;-8398) §
-8491 (-9462;-6130) §
-8634 (-9640;-7309) §
-8000 (-8889;-5905) §
6.3 (5.3;6.8)
5.4 (4.4;5.8) §
5.3 (4.8;6.4) §
Heterozygous
5.7 (4.9;6.4) §
4.7 (4.3;5.7) §
5.0 (4.6;7.3) §
Homozygous
5.5 (5.1;6.5)
4.6 (4.4;5.4) §
4.8 (4.4;5.4) §
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