Myocardial remodelling in c-kit deficient mice – quantitative assessment by

Magnetic Resonance Imaging and Mass Spectrometry Imaging

Work package - 1: Assessment of myocardial edema and permeability in c-kit deficient

mice versus mice treated with c-kit+ bone marrow cells

Experiment 1: Establishment of the optimal albumin-affine MR agent

n = myocardial infarction in WBB6F1-KitW/KitW-v mice(C-kit deficiency)

(A) albumin-affine gadofosveset trisodium (n=6 mice) (compared to B)

(B) Galbumin (n=6 mice)

(C) Evans Blue as the gold standard (n=6 mice) (validated A & B)

 Wild type mice - 6 (C57BL/6J mice) will serve as control

 KitW/KitW-v mice - 18

Experiment 2: Quantitative time course of micro vascular permeability and edema in c-kit
deficient mice

Based on WP1 results applying either Galbumin or gadofosveset trisodium, as determined

above experiment 2

vascular permeability and edema formation will be investigated in Kit W/Kit W-v mice by
dedicated small animal MRI dynamically over time point at day 0 (prior to MI), day1, day 7
and day 28 after myocardial infarction

One group (n=6 mice) will be investigated serially by MRI over time and sacrificed after the
final imaging on day 28.

A second group of (n=12 mice) will be investigated, where n=3 mice are sacrificed at each
time point immediately after imaging for ex vivo analysis by histology and Mass
Spectrometry Imaging (LA-ICP-MS) to determine the actual Gadolinium concentration in
tissue.

 KitW/KitW-v mice - 18
 Wild type mice - 18 (C57BL/6J mice) will serve as control

Experiment 3: Edema and Permeability in the c-kit mice Bone marrow stem cells treatment
C-kit deficiency is thought to increase edema and vascular permeability, while treatment
with c-kit+ BMC will attenuate both, leading to improved heart function. Here the main aim
to provide quantitative and dynamic MRI assessment of c-kit dependent myocardial edema
and vascular permeability, two hallmarks of early myocardial injury.

Evaluation of hypothesis that BMC treatment attenuates myocardial edema and

microvascular permeability, which will be assessed by our combined MRI-MSI approach
(again, n=3 mice will be sacrificed at each time point (day 0, 1, 7 and 28) immediately after
imaging).

Additional to investigating myocardial edema and permeability, CINE imaging will be

performed at each of the time points (day 0, 1, 7 and 28) to correlate the findings with the
heart function.

 KitW/KitW-v mice - 12

Aim of the experiments 1, 2, 3: C-kit deficiency is thought to increase edema and vascular
permeability, while treatment with c-kit+ BMC will attenuate both, leading to improved
heart function. We aim to provide quantitative and dynamic MRI assessment of c-kit
dependent myocardial edema and vascular permeability, two hallmarks of early myocardial
injury.

Total animals required for work package -1

No.of No. of
WP Experiment No Technics Wild Mice KitW/KitW-v
S.N (C57BL/6J) mice
o
1 WP-1/ Exp -1 MRI, MSI, IHC 6 18
Exp - 2 MRI, MSI, IHC 18 18
Exp - 3 MRI, MSI, IHC - 12
Total animals for work package -1 24 48
10% for drop-out compensation 2 4
Donor mice for c-kit+ BMS cells 12
Total animals 38 52
Work package 2: Assessment of the impact of c-kit on the synthesis of extracellular matrix
and elastin fibers by T1 mapping of molecular gadolinium probes.

Experiment 4: Quantitative time course of extra-cellular matrix synthesis in c-kit deficient

mice

Evaluation of Gadofluorine M and P (again in comparison to unspecific Gd-DTPA), which both

have been shown to exhibit high affinity to ECM proteins such as collagen. Preliminary
results suggest that CNR values for Gadofluorine P increase over time within the infarct,
which is accompanied by increased extracellular fibers shown by Elastica van Gieson staining

Investigation of the accumulation of Gadolfuorine P and Gd-ESMA serially over time on day
0, 1, 7 and 28 after LAD ligation, with sacrifice of the mice after the last imaging.

 Gadolfuorine P, n=6 KitW/KitW-v mice & Gd-ESMA, n=6 KitW/KitW-v mice

 Wild type mice (n=6 for Gadolfuorine P & n=6 for Gd-ESMA) will serve as control

Experiment 5: Quantitative time course of elastin fiber synthesis in c-kit deficient mice

Investigation of (n=3) animals being sacrificed immediately after completion at each imaging
time point and the hearts being immediately excised for ex vivo analysis.

Histology (Hematoxilin&Eosin, Elastica van Gieson, collagen and Elastin/Tropoelastin

staining), Western Blotting (for collagen type I, Elastin, Tropoelastin) and LA-ICP-MS (for
determination of actual Gadolinium concentration)

 Gadolfuorine P, n=12 KitW/KitW-v mice & Gd-ESMA, n=12 KitW/KitW-v mice

 Wild type mice (n=12 for Gadolfuorine P & n=12 for Gd-ESMA) will serve as control

Experiment 6: ECM and elastin fiber synthesis in the BMC treatment group

To investigate the treated with a single-shot treatment of c-kit+ BMC at the time of
myocardial infarction to test the hypothesis if c-kit is able to increase ECM and elastin fiber
synthesis within the ischemic scar. N=3 of the treated mice will be sacrificed at each time
point (day 0, 1, 7 and 28) to permit detailed quantification of gadolinium tissue
concentrations by LA-ICP-MS

 Gadolfuorine P, n=12 KitW/KitW-v mice & Gd-ESMA, n=12 KitW/KitW-v mice

 Wild type mice (n=12 for Gadolfuorine P & n=12 for Gd-ESMA) will serve as control

Aim of the experiments 4, 5, 6: To prove a beneficial effect of c-kit+ BMC on ECM formation
and elastin fiber synthesis after myocardial infarction by novel quantitative imaging
biomarkers, obtained by combined MRI-MSI.
Total animals required for work package - 2

No.of No. of
WP Experiment No Technics Wild Mice KitW/KitW-v
S.N (C57BL/6J) mice
o
2 WP-2/ Exp - 4 MRI, MSI, IHC 12 12
Exp - 5 MRI, MSI, IHC, Western Blotting 24 24
Exp - 6 MRI, MSI, IHC, Western Blotting 24 24
Total animals for work package -2 60 60
10% for drop-out compensation 6 6
Donor mice for c-kit+ BMS cells 12
Total animals 78 66
Work Package 3: Therapeutic effects of targeting extracellular RNA complementary to c-
kit on myocardial permeability/edema as well as synthesis of extracellular matrix and elastin
fibers.

Experiment 7: Quantitative time course of permeability and edema in RNase treated mice

RNase I as described by Stieger et al (100µg/kg RNase I intravenously at 30 minutes, 3 and 6

hours after LAD ligation). Edema imaging and assessment of vessel permeability will be
performed as described above on day 0 (prior to MI), 1, 7 and 28 after myocardial infarction,
at each time point n=3 mice from each group will be sacrificed for ex vivo analysis by
histology and MSI

 n=12 KitW/KitW-v mice (100µg/kg RNase I intravenously at 30 minutes, 3 and 6 hours

after LAD ligation)
 Wild type mice (n=12 for) will serve as control

Experiment 8: Quantitative time course of ECM and elastin fiber synthesis in RNase treated
mice

C-kit deficient mice will be treated with RNase I and will be dynamically assessed over time
by ECM- and Elastin-targeted MRI using the techniques.

Imaging will be performed on day 0, 1, 7 and 28 following ischemic injury with n=3 animals
from each subgroup being sacrificed immediately after imaging. Full quantitative assessment
of Gadofluorine P and Gd-ESMA will be performed on n=3 of each subgroup by LA-ICP-MS
and results will be further corroborated by Western blotting and histology

 Gadolfuorine P, n=12 KitW/KitW-v mice & Gd-ESMA. n=12 KitW/KitW-v mice

 Wild type mice (n=12 for Gadolfuorine P & n=12 for Gd-ESMA) will serve as control
(Above groups are myocardial infarction and treated with RNase I)

Experiment 9: Complementary effect of RNase treatment to c-kit function on edema and

permeability

The therapeutic effects of complementary treatment with ckit+ BMC and targeting eRNA via
RNase I treatment. Therefore, infarct bearing Kit W/KitW-v mice and control wild-type mice will
both to be treated with a single-shot intravenous treatment of 10 6 c-kit+ BMC and RNase I
(100µg/kg RNase I)

 n=12 KitW/KitW-v mice (MI + BMC+ RNase I)

 Wild type mice (n=12 for) will serve as control (MI + BMC+ RNase I)
Experiment 10: Complementary effect of RNase treatment to c-kit function on ECM/elastin
fiber synthesis

KitW/KitW-v mice and wild type mice treated with RNase I formation of ECM (Gadofluorine P)
and elastin fibers (Gd-ESMA) will be investigated by combined MRI-MSI

 Gadolfuorine P, n=12 KitW/KitW-v mice & Gd-ESMA , n=12 KitW/KitW-v mice

 Wild type mice (n=12 for Gadolfuorine P & n=12 for Gd-ESMA) will serve as control

Aim of the experiments 7, 8, 9, 10: To prove the beneficial effects of targeting eRNA, either
alone or in conjunction with ckit+ BMC treatment, by combined MRI-MSI. Combined RNase
and c-kit+ BMC treatment will ameliorate myocardial edema and microvascular permeability
and improve the formation of extra-cellular matrix and elastin fibers and finally heart
function.

Total animals required for work package - 3

No.of No. of
WP Experiment No Technics Wild Mice KitW/KitW-v
S.N (C57BL/6J) mice
o
3 WP-3/ Exp -7 MRI, MSI, IHC 12 12
Exp - 8 MRI, MSI, IHC, Western Blotting 24 24
Exp - 9 MRI, MSI, IHC 12 12
Exp - 10 MRI, MSI, IHC, Western Blotting 24 24
Required for experiments 72 72
10% for drop-out compensation 7 7
Donor mice for c-kit+ BMS cells 12
Total animals 91 79
Total animals required for 3 work packages

No.of No. of
WP Experiment No Technics Wild Mice KitW/KitW-v
S.N (C57BL/6J) mice
o
1 WP-1/ Exp -1 MRI, MSI, IHC 6 18
Exp - 2 MRI, MSI, IHC 18 18
Exp - 3 MRI, MSI, IHC - 12
2 WP-2/ Exp - 4 MRI, MSI, IHC 12 12
Exp - 5 MRI, MSI, IHC, Western Blotting 24 24
Exp - 6 MRI, MSI, IHC, Western Blotting 24 24
3 WP-3/ Exp -7 MRI, MSI, IHC 12 12
Exp - 8 MRI, MSI, IHC, Western Blotting 24 24
Exp - 9 MRI, MSI, IHC 12 12
Exp - 10 MRI, MSI, IHC, Western Blotting 24 24
Required for experiments 156 180
10% for drop-out compensation 15 18
Donor mice for c-kit+ BMS cells 36
Total animals 207 198

Timeline of the Project

Work
package Experiments Timeline Total months
Surgery practice, MRI handling and experimental
preparation (May 2020) 1
1 Experiment - 1 June 2020 to August 2020 3
Experiment - 2 September 2020 to November 2020 3
Experiment - 3 December 2020 to March 2021 4
2 Experiment - 4 April 2021 to June 2021 3
Experiment - 5 July 2021 to September 2021 3
Experiment - 6 October 2021 to December 2021 4
3 Experiment - 7 January 2022 to February 2022 2
Experiment - 8 March 2022 to May 2022 3
Experiment - 9 June 2022 to July 2022 2
Experiment - 10 August 2022 to October 2022 3
Thesis writing November 2022 to March 2023 5
36