b -Adrenergic Receptor ( b -AR) Subtypes Have Opposing Effects on Survival

1. FIGURE 3. At 6 mos. LV %FS was decreased and LVEDD was increased in MLP-/- and MLP-/-/b1+/- mice but not in MLP-/-/b2-/- mice. METHODS • b1-/- or b2-/- 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 * * * * * * RESULTS * * FIGURE 1. Ablation of b1 vsb2-ARs had opposite effects on early (2 wks) and late (6 mos) survival. 97% of MLP-/-/b1-/- mice died between ED10-15. WT MLP-/- MLP-/-/b1+/- MLP-/-/b2-/- MLP-/-/b1+/- MLP-/-/b2-/- FIGURE 4. At 6 mos. MLP-/- and MLP-/-/b1+/- developed cardiomegaly, whereas MLP-/-/b2-/- did not. FIGURE 2. At 6 mos. MLP-/- and MLP-/-/b1+/- had decreased exercise capacity whereas MLP-/-/b2-/- mice had normal exercise capacity. CONCLUSIONS • b-AR subtypes play critical but differential roles during the development of a genetic (MLP) cardiomyopathy. • The b1-AR positively modulates survival and cardiac function whereas the b2-AR has the opposite effect. • Total ablation of the b1-AR is embryonic lethal in the absence of MLP. • b-AR modulation of phospholamban may play a role in the severity of heart failure in MLP-/- mice. * * b-Adrenergic Receptor (b-AR) Subtypes Have Opposing Effects on Survival and Cardiac Function in MLP Cardiomyopathy Mingming Zhao, Giovanni Fajardo and Daniel Bernstein Department of Pediatrics, Stanford University, Stanford, CA ABSTRACT FIGURE 5. At 6 mos. HW/BW and LW/BW ratios were increased in MLP-/- and MLP-/-/b1+/- mice, but not in MLP-/-/b2-/- mice. Alterations in b-AR signaling have been shown to modulate severity of a genetic cardiomyopathy (Muscle LIM Protein, MLP-/- mice). b2-AR overexpression increases mortality, whereas bARKct partially and phospholamban ablation fully rescues MLP-/- mice. To dissect the role ofb1 vsb2 -ARs in modulating MLP cardiomyopathy, we crossbred MLP-/- with b1-/- or b2-/- mice. Ablation of b1 vsb2 -AR had opposite effects on early survival: MLP-/-/ b2-/- (91.2%, n=57), MLP-/- (74.0%, n=27), MLP-/-/ b1 +/- (62.3%, n=53), MLP-/-/b1-/- (3%, n=31). Nearly all MLP-/-/ b1-/- mice died in utero between ED10 and ED15. MLP-/-/b2-/- rescued LV function in 80% of MLP-/-/b2-/- 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-/-/b1+/- (F/S 22.9±9.9%*, LVEDD 5.8±1.3mm*, n=11). MLP-/-/b2-/- mice ran longer (22.7±3.0min) on a graded treadmill protocol vs. MLP-/- (14.6±2.0 min*) and MLP-/-/b1+/- (13.3±5.2 min*). MLP-/-/b2-/- mice had normal heart size (HW/BW 4.7±1.0) vs MLP-/- (7.6±2.5*) and MLP-/-/b1+/- (8.3±3.4*). In conclusion, b-AR subtypes play critical but differential roles during the development of a genetic (MLP) cardiomyopathy. The b1-AR positively modulates survival and cardiac function whereas the b2-AR has an opposite effect. Total ablation of the b1-AR is embryonic lethal in the absence of MLP. FIGURE 6. Phospholamban phosphorylation was decreased in MLP-/- and MLP-/-/b1+/- mice but was normal in MLP-/-/b2-/- mice. INTRODUCTION • 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 b2-AR overexpression increases mortality, whereas the bARK1 inhibitor (bARKct) 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). PURPOSE To further investigate the role of b-AR subtypes in the development of MLP cardiomyopathy. * p< 0.05 by ANOVA