Beneficial Effects of Mammalian Target of Rapamycin Inhibition on Left Ventricular Remodeling After Myocardial Infarction
Sebastian J. Buss, MD*,
Sebastian Muenz, MD*,
Johannes H. Riffel, MD*,
Pratima Malekar, PhD*,
Marco Hagenmueller, PhD*,
Celine S. Weiss, MD*,
Florian Bea, MD*,
Raffi Bekeredjian, MD*,
Martina Schinke-Braun, PhD ,
Seigo Izumo, MD, PhD,
Hugo A. Katus, MD* and
Stefan E. Hardt, MD*,*
* Department of Cardiology, University of Heidelberg, Heidelberg, Germany
Novartis Institutes of Biomedical Research, Cardiovascular Research, Cambridge, Massachusetts
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1 mTOR Inhibition Attenuates Myocardial Remodeling After MI
(A) Transthoracic echocardiographic M-Mode images recorded from the parasternal short axis on the level of the papillary muscles of the left ventricle (LV) in sham-, vehicle-, and everolimus (RAD)-treated (3.0 mg/kg/day) animals after 28 days. (B) Ejection fraction (EF) was significantly higher in the RAD-treatment group after 28 days as compared with vehicle. (C) End-diastolic diameter (EDD) and (D) end-systolic diameter (ESD) of the LV estimated from transthoracic echocardiographic M-Mode images. The EDD and ESD were significantly reduced in RAD-treated animals (3.0 mg/kg/day) after 28 days. *p < 0.05 versus sham; #p < 0.05 versus vehicle. Sham n = 6, vehicle n = 14, RAD n = 14. MI = myocardial infarction; mTOR = mammalian target of rapamycin.
|
|
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2 Representative Pressure-Volume Loops During Steady-State Conditions in Sham-, Vehicle-, and RAD-Treated Rats 28 Days After MI
A significant rightward shift of the pressure volume loops is observed in the vehicle-treated animals, which can be reduced ("leftwarded") by treatment with RAD 3.0 mg/kg/day. RVU = relative volume unit; other abbreviations as in Figure 1.
|
|
View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 3 Effects of RAD Treatment on Infarct Size and Myocardial Hypertrophy
(A) Quantitative measurement of infarct size by triphenyl tetrazolium chloride (TTC) staining. Infarct size was significantly smaller in RAD-treated animals as compared with vehicle-treated animals 28 days after MI. (B) Representative examples of Masson's trichrome staining of infarcted hearts. Smaller infarct size was confirmed by histology in the RAD-treatment group. (C) The increased ratio of heart weight (HW) to tibia length (TL) in vehicle-treated MI-animals was significantly reduced after RAD treatment. *p < 0.05 versus sham; #p < 0.05 versus vehicle. Abbreviations as in Figure 1.
|
|
View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 4 Effects of RAD Treatment on Cardiac Myocyte Size and ANF Expression
(A) Exemplary hematoxylin eosin stainings of a section of the remote area of the MI in sham, vehicle, and RAD-treated animals after 28 days. (B) Quantitative measurement of the myocyte size 28 days after MI. Myocyte size was significantly smaller in RAD-treated animals as compared with vehicle-treated animals. Sham n = 5, vehicle n = 5, RAD n = 13. (C) Gene expression of atrial natriuretic factor (ANF) normalized to the house-keeping gene HPRT was significantly increased in vehicle MI-animals but not in the RAD-treatment group. *p < 0.05 versus sham; #p < 0.05 versus vehicle. Abbreviations as in Figure 1.
|
|
View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 5 Protein Levels and Phosphorylation Levels of mTOR-Related Regulators of Protein Synthesis
(A) Western blot analysis revealed a significant decrease of phosphorylation of 4E-BP1 (Thr 70) after administration of RAD after MI. (B) Protein levels of phospho-p70/S6K after MI; p70/S6K, 1 of the main downstream targets of mTOR—which mediates cellular growth—displayed a significant decrease of phosphorylation after administration of RAD after MI. (C) Phospho ribosomal S6K is significantly reduced by mTOR-inhibition. Coomassie blue staining confirms similar loading of protein. Veh = vehicle; other abbreviations as in Figure 1.
|
|
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 7 Morphology and Echocardiography After Initiation of RAD Treatment 3 Days After MI
(A) The increased HW/TL ratio in vehicle-treated MI-animals was significantly reduced after RAD treatment (3.0 mg/kg/day) starting 3 days after infarction. (B) Quantitative measurement of the myocyte size 28 days after MI. Myocyte size was significantly smaller in RAD-treated animals as compared with vehicle-treated animals. (C to E) Transthoracic echocardiography. (C) The EF was significantly higher in the RAD-treatment group after 28 days as compared with vehicle. (D and E) The EDD and ESD of the LV showed a significant reduction due to RAD treatment compared with vehicle. #p < 0.05 versus vehicle. Abbreviations as in Figures 1 and 3.
|
|
View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 8 Autophagy and NF B Activation 28 Days After MI With RAD Treatment Starting 1 or 3 Days After MI
(A) LC3, a marker of autophagy, is still up-regulated in the remote area 28 days after MI in RAD-treated animals after 1 and 3 days (3.0 mg/kg/day). (B) The concomitant down-regulation of the ubiquitin proteasome system is still observed. (C and D) Electrophoretic mobility shift assay blot demonstrating reduced activation of NFB in MI animals with RAD treatment initiated 3 days after MI in the remote area of the myocardium. Densitometric quantification is shown in D. #p < 0.05 versus vehicle. Abbreviations as in Figures 1 and 6.
|
|
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 9 Long-Term Effects of RAD Treatment
The RAD treatment (starting on day 1 after MI) for 28 days led to sustained improvement of LV function 3 months after MI (squares, solid lines), whereas vehicle-treated animals display a progressive deterioration of LV ejection fraction (triangles, dashed lines). Data from serial echocardiography in the same animals are depicted. #p < 0.05 versus vehicle. Abbreviations as in Figure 1.
|
|
|
