-Adrenergic Receptor (-AR) Subtypes Have Opposing Effects on Survivaland Cardiac Function in MLP CardiomyopathyMingming Zhao, Giovanni Fajardo and Daniel Bernstein

Department of Pediatrics, Stanford University, Stanford, CA

Alterations in -AR signaling have been shown to modulate severity of a genetic cardiomyopathy (Muscle LIM Protein, MLP-/- mice). 2-AR overexpression increases mortality, whereas ARKct partially and phospholamban ablation fully rescues MLP-/- mice. To dissect the role of 1

vs 2 -ARs in modulating MLP cardiomyopathy, we crossbred MLP-/- with 1-/- or 2-/- mice. Ablation of 1 vs 2 -AR had opposite effects on early survival: MLP-/-/ 2-/- (91.2%, n=57), MLP-/- (74.0%, n=27), MLP-/-/ 1 +/- (62.3%, n=53), MLP-/-/1-/- (3%, n=31). Nearly all MLP-/-/ 1-/- mice died in utero between ED10 and ED15. MLP-/-/2-/- rescued LV function in 80% of MLP-/-/2-/- mice (F/S 39.6±12.1%, LVEDD 4.6±1.1mm, n=15 ) vs MLP-/- (F/S 18.3±4.7%*, LVEDD 5.7±0.7mm*, *:p<0.05, n=10) and MLP-/-/1+/- (F/S 22.9±9.9%*, LVEDD 5.8±1.3mm*, n=11). MLP-/-/2-/- mice ran longer (22.7±3.0min) on a graded treadmill protocol vs. MLP-/- (14.6±2.0 min*) and MLP-/-/1+/- (13.3±5.2 min*). MLP-/-/2-/- mice had normal heart size (HW/BW 4.7±1.0) vs MLP-/- (7.6±2.5*) and MLP-/-/1+/- (8.3±3.4*). In conclusion, -AR subtypes play critical but differential roles during the development of a genetic (MLP) cardiomyopathy. The 1-AR positively modulates survival and cardiac function whereas the 2-AR has an opposite effect. Total ablation of the 1-AR is embryonic lethal in the absence of MLP.

ABSTRACT

INTRODUCTION

CONCLUSIONS

• -AR subtypes play critical but differential roles during the

development of a genetic (MLP) cardiomyopathy.

• The -AR positively modulates survival and cardiac function

whereas the -AR has the opposite effect.

• Total ablation of the -AR is embryonic lethal in the absence

of MLP.• -AR modulation of phospholamban may play a role in the

severity of heart failure in MLP-/- mice.

• MLP (Muscle LIM Protein) is a regulator of myogenic differentiation and is involved in cytoarchitecture organization.

• MLP deficient mice develop dilated cardiomyopathy in two phases: heart failure occurs in 50-70% within 10d and in the remainder by 3-6 months (Arber et al. Cell 88:393, 1997).

• Previous studies have shown that 2-AR overexpression increases mortality, whereas the ARK1 inhibitor (ARKct) partially rescues MLP-/- mice (Rockman et al. PNAS 12:7000, 1998)..

• Ablation of phospholamban (PLB), an inhibitor of the SR Ca2+ ATPase (SERCA), rescues MLP-/- mice (Minamisawa et al. Cell 99:313, 1999).

To further investigate the role of -AR subtypes in the development of MLP cardiomyopathy.

FIGURE 5. At 6 mos. HW/BW and LW/BW ratios were increased in

MLP-/- and MLP-/-/1+/- mice, but not in MLP-/-/2-/- mice.

PURPOSE

FIGURE 6. Phospholamban phosphorylation was decreased in

MLP-/- and MLP-/-/1+/- mice but was normal in MLP-/-/2-/- mice.

0

0.5

1

1.5

2

2.5

3

Phospho-PhospholambanMLP-/-/-/- -/-MLP -/-MLP /+/- WT

WT MLP-/- MLP-/-/1+/- MLP-/-/2-/-

RESULTS

FIGURE 1. Ablation of 1 vs 2-ARs had opposite effects

on early (2 wks) and late (6 mos) survival.97% of MLP-/-/1-/- mice died between ED10-15.

METHODS

30

40

50

60

70

80

90

100

0 50 100 150 200

Postnatal Days

Percent Survival

MLP-/-/-/-

-/-MLP

-/-MLP /+/-

0 10 20 30

Running Time (Min)

MLP-/-/-/-

-/-MLP

-/-/MLP +/-

FIGURE 2. At 6 mos. MLP-/- and MLP-/-/1+/- had decreased exercise capacity whereas MLP-/-/2-/- mice had normal exercise capacity.

1-/- or 2-/- mice were mated to MLP-/- mice (courtesy Dr. K. Chien) and pre- and post-natal survival was recorded. Genotyping of newborn and adult mice was performed by PCR. Genotyping of mouse embryos was performed at ED10, ED15 and ED18. Cardiac function (%FS, LVEDD by echo) and treadmill exercise capacity were assessed at 6 months. Mice were sacrificed at 7 months and heart and lung weights were recorded and normalized to body weight. Western blots were performed on LV samples for PLB phosphorylation

*

*

FIGURE 3. At 6 mos. LV %FS was decreased and LVEDD was increased in MLP-/- and MLP-/-/1+/- mice but not in

MLP-/-/2-/- mice.

FIGURE 4. At 6 mos. MLP-/- and MLP-/-/1+/- developed

cardiomegaly, whereas MLP-/-/2-/- did not.

MLP-/-/+/- MLP-/-/-/-

0

10

20

30

40

50

60

LV Function (%FS)

MLP-/-/-/- -/-MLP -/-MLP /+/-

**

0

1

2

3

4

5

6

7

8

LVEDD (mm)

MLP-/-/-/- -/-MLP -/-/MLP +/-

**

0

2

4

6

8

10

12

HW/BW Ratio

MLP-/-/-/- -/-MLP -/-MLP /+/- WT

* *

0

1

2

3

4

5

6

7

8

LungW/BW Ratio

MLP-/-/-/- -/-MLP -/-MLP /+/- WT

**

* p< 0.05 by ANOVA