Nix-mediated apoptosis links myocardial fibrosis, cardiac remodeling, and hypertrophy decompensation

Abstract

Background: Pathological cardiac hypertrophy inevitably remodels, leading to functional decompensation. Although modulation of apoptosis-regulating genes occurs in cardiac hypertrophy, a causal role for programmed cardiomyocyte death in left ventricular (LV) remodeling has not been established.

Methods and results: We targeted the gene for proapoptotic Nix, which is transcriptionally upregulated in pressure overload and Gq-dependent hypertrophies, in the mouse germ line or specifically in cardiomyocytes (knockout [KO]) and conditionally overexpressed it in the heart (transgenic [TG]). Conditional forced Nix expression acted synergistically with the prohypertrophic Gq transgene to increase cardiomyocyte apoptosis (0.8+/-0.1% in GqTG versus 7.8+/-0.6% in GqTG+NixTG; P<0.001), causing lethal cardiomyopathy with LV dilation and depressed systolic function (percent fractional shortening, 39+/-4 versus 23+/-4; P=0.042). In the reciprocal experiment, germ-line Nix ablation significantly reduced cardiomyocyte apoptosis (4.8+/-0.2% in GqTG+NixKO versus 8.4+/-0.5% in GqTG; P=0.001), which improved percent fractional shortening (43+/-3% versus 27+/-3%; P=0.017), attenuated LV remodeling, and largely prevented lethality in the Gq peripartum model of apoptotic cardiomyopathy. Cardiac-specific (Nkx2.5-Cre) Nix KO mice subjected to transverse aortic constriction developed significantly less LV dilation by echocardiography and magnetic resonance imaging, maintained concentric remodeling, and exhibited preserved LV ejection fraction (61+/-2% in transverse aortic constriction cardiac Nix KO versus 36+/-6% in transverse aortic constriction wild-type mice; P=0.003) at 9 weeks, with reduced cardiomyocyte apoptosis at day 4 (1.70+/-0.21% versus 2.73+/-0.35%; P=0.032).

Conclusions: Nix-induced cardiomyocyte apoptosis is a major determinant of adverse remodeling in pathological hypertrophies, a finding that suggests therapeutic value for apoptosis inhibition to prevent cardiomyopathic decompensation.

Figure 1. Apoptotic synergy between cardiac-expressed Nix and Gq in neonatal mice

A. Representative left ventricular (LV) M-mode echocardiograms in short axis view at 6 weeks age. B. Survival curves (P value by log-rank test). C. Echocardiographic left ventricular fractional shortening (black, %FS) and remodeling (LV radius/wall thickness, white, r/h; n=4-8/group). D. Representative western blot for Nix (inset) and apoptotic indices by TUNEL analysis (n=3/group). *= P<0.05 vs control, ^#=P<0.05 for Nix/Gq vs Gq by post-hoc test after one-way ANOVA.

Figure 2. Nix gene ablation diminishes apoptosis in Gq-mediated peripartum cardiomyopathy, improving function and minimizing death

A. Representative non-pregnant (baseline) and post-partum 14 day hearts. B. Representative LV M-mode echocardiograms. C. Echocardiographic LV fractional shortening (%FS) and D. remodeling (LV radius/wall thickness, r/h; n=4-6/group). E. Kaplan Meier survival curves for peripartum Gq expressors, with and without Nix (P value by log-rank test). F. Apoptotic indices at 1 day post-partum (n=4/group), *= P<0.05 vs WT, ^#=P<0.05 for NixKO+Gq vs Gq by post-hoc test after one-way ANOVA.

Figure 3. Cardiac-specific Nix ablation prevents functional decompensation after acute transverse aortic coarctation (TAC)

A. Cardiac-specific Nix ablation strategy. B. qPCR of Nix mRNA expression after TAC (n=3/group). *= P<0.05 vs non-operated by t-test. C. Schematic of TAC modeling studies. D. Representative invasively determined hemodynamic tracings after TAC (ΔP:transcoarctation gradient, LVEDP:LV end-diastolic pressure, HR: heart rate). E. Transcoarctation gradient in cardiac Nix del mice (n=11) and controls (n=13). P value reported is by t-test. G. Peak positive dP/dt and H. dP/dt at 40 mm Hg LV pressure in cardiac Nix del mice and Nix floxed controls subjected to TAC (n=4/non-operated group; n=11-13/TAC group; P values reported are by t-test for comparision of TAC groups.) Non operated mice are shown for comparision. H. Change in ANF, SERCA2a, and α-skeletal actin mRNA levels four days after TAC in WT (white bars) and cardiac Nix del (black bars).

Figure 4. Cardiac-specific Nix ablation prevents structural remodeling and functional decompensation after TAC

Time-dependent echocardiographic outcomes after TAC in control (Nix floxed, white circles, n=15) and cardiac Nix del (black squares, n=13) mice. A. LV mass. B. LV end-diastolic dimension (LVEDD). C. Ratio of LV radius to wall thickness (r/h). D. LV fractional shortening (%FS). E. Vcf: Velocity of circumferential shortening. F. Vcfc: Vcf corrected for heart rate. P values by post-hoc test vs WT TAC after two-way repeated measures ANOVA.

Figure 5. Cardiac-specific Nix ablation abrogates LV remodeling after TAC

A. Representative Masson's trichrome stained coronal sections. B. Representative magnetic resonance images at 9 weeks after TAC.

Figure 6. Cardiac-specific Nix ablation prevents cardiomyocyte apoptosis and myocardial fibrosis after TAC

Representative myocardial sections showing: A. Cardiomyocyte cross section area (FITC tagged wheat germ agglutinin labeling (200X); B. TUNEL positivity (1000X); C. Cleaved caspase 3 (brown staining; left panels (400X) and immunoblot (right) showing cleaved PARP (top) with Ponceau red loading control (bottom). D. Myocardial fibrosis (Masson's trichrome, (200X) nine weeks after TAC in cardiac Nix del mice and Nix floxed controls. Group data are in bar graphs to the right (n=4/non-operated group and n=6-12/TAC group). P values by t-test (TAC WT vs TAC Nix del). Non operated mice are shown for comparison.