C

Merged

α-SMA

Vimentin

A

B

Figure S1: Representative immunocytochemistry images of cardiac myofibroblasts from adult rats examined by fluorescence microscopy. Vimentin staining (A )with α-smoothmuscle actin (α-SMA) staining (B ). Magnification x40. C: Merged

Figure S2: Cardiac protein levels of collagen III (col III). Quantification of band intensities was measured by densitometry and normalized to respective α-tubulin. Values are mean±SEM of 6 animals. *p<0.05.

Col III

α-Tubulin

CT HFD0

1

2 *

Col

III p

rote

in le

vels

(fold

of c

ontr

ol)

Figure S3: Effect of different doses of leptin on the proliferation in cardiac myofibroblasts at 24 hours. Cell proliferation was determined by an MTT assay. Data are expressed as percent of controls (vehicle, v). Values are mean±SEM of four assays.

0

60

120%

(vs

CT)

V 10 50 100 Leptin (ng/ml)

Figure S4: The effect of cells stimulated with leptin (100ng/ml) in presence or absence of melatonin (10-3 mmol/L) and rapamycin (10-4 mmol/L) on metalloproteinase (MMP) 2 activity in cardiac myofibroblasts

* * *

V Leptin Leptin Leptin + Mel + Rap

V Leptin Leptin Leptin + Mel + Rap

Act

ivity

MM

P-2

0

0.5

1

1.5

Figure S5: Superoxide anion production in cells stimulated with leptin (100 ng/ml) at different times in presence or absence of rapamycin (10-4 mmol/L). Values are mean±SEM of four assays. *p<0.001 vs vehicle (v, control conditions); † p<0.001 vs leptin.

0

50

100

150

Fluo

resc

ence

(A.U

.)* * * *

** † † † Leptin + Rap

VehicleLeptin

V 1 2 4 (hours)

Figure S6: Scheme illustrating the possible mechanism involved in the fibrotic effect induced by leptin in cardiac myofibroblasts.. Leptin could favour collagen deposition by modulating both its synthesis and also its degradation. Leptin induced collagen synthesis by increasing ROS (reactive oxygen species) production directly or through the activation of mTOR pathway. Galectin-3 and growth factors such as TGF-β and CTGF seem to be the final mediators of the collagen synthesis induced by leptin. The reduction in MMP-2 (metalloproteinase-2) activity induced by leptin seems to involve an oxidative stress- and mTOR pathway-independent mechanism.

LEPTIN

EXTRACELLULAR MATRIX

MMP-2activity

DegradationCollagen I

ROS mTOR activation

Galectin-3TGF-β

CTGF

SynthesisCollagen I

Cardiac myofibroblasts