Cardiomyopathy and Worsened Ischemic Heart Failure in SM22-α Cre-Mediated Neuropilin-1 Null Mice: Dysregulation of PGC1α and Mitochondrial Homeostasis

Abstract

Objective: Neuropilin-1 (NRP-1) is a multidomain membrane receptor involved in angiogenesis and development of neuronal circuits, however, the role of NRP-1 in cardiovascular pathophysiology remains elusive.

Approach and results: In this study, we first observed that deletion of NRP-1 induced peroxisome proliferator-activated receptor γ coactivator 1α in cardiomyocytes and vascular smooth muscle cells, which was accompanied by dysregulated cardiac mitochondrial accumulation and induction of cardiac hypertrophy- and stress-related markers. To investigate the role of NRP-1 in vivo, we generated mice lacking Nrp-1 in cardiomyocytes and vascular smooth muscle cells (SM22-α-Nrp-1 KO), which exhibited decreased survival rates, developed cardiomyopathy, and aggravated ischemia-induced heart failure. Mechanistically, we found that NRP-1 specifically controls peroxisome proliferator-activated receptor γ coactivator 1 α and peroxisome proliferator-activated receptor γ in cardiomyocytes through crosstalk with Notch1 and Smad2 signaling pathways, respectively. Moreover, SM22-α-Nrp-1 KO mice exhibited impaired physical activities and altered metabolite levels in serum, liver, and adipose tissues, as demonstrated by global metabolic profiling analysis.

Conclusions: Our findings provide new insights into the cardioprotective role of NRP-1 and its influence on global metabolism.

Keywords: cardiomyopathies; metabolomics; mitochondria; myocardial infarction; myocytes, cardiac; neuropilin-1.

Figure 1. Knockout of NRP-1 increased PGC1α in CMs and VSMCs

(A–B). Neonatal CMs were isolated from NRP-1^f/f mice and then infected with adenovirus Cre or adenovirus GFP as control. Expressions of NRP-1 and PGC1α were validated with qPCR (A) and Western blotting (B). (C). VSMCs were isolated from NRP-1^f/f mice and then infected with adenovirus Cre or adenovirus GFP as control. Expressions of NRP-1 and PGC1α were validated with qPCR. (D–F). qPCR was performed in neonatal CMs isolated from NRP-1^f/f mice and infected with adenovirus Cre or adenovirus GFP as control. (G). Frozen heart sections of WT and SM22-α-Nrp-1 KO mice were stained with antibodies against NRP-1 (red) and α-MHC (green). Nuclei were counterstained with DAPI. Results are representative of three independent experiments. (H). qPCR was performed in neonatal CMs of WT mice and SM22-α-Nrp-1 KO mice. (I). Total cellular DNA was extracted from neonatal CMs of control mice and SM22-α-Nrp-1 KO mice. Mitochondrial cytochrome oxidase 1 (CO1) and lipoprotein lipase (LPL) were analyzed with qPCR and expressed as a ratio of mtDNA and nuclear DNA. (J). Oxygen consumption of control and Nrp-1 KO MEFs were measured using O2k oxygraph. ROUNTINE was measured with normal serum-free culture medium. LEAK respiration was measured after oligomycin-induced inhibition of ATP synthesis. ETS, electron transfer system, was measured with the addition of FCCP. N=3 for each group. (K). Heart tissues of 1 year old WT mice and SM22-α-Nrp-1 KO mice were subjected to transmission electron microscopy. Mitochondrial area was measured and analyzed. Results are representative from 10 sections of one heart each group. (L). qPCR was performed with heart tissues of WT mice and SM22-α-Nrp-1 KO mice. N=3 for each group. (M). Kaplan-Meier survival curves of WT mice and SM22-α-Nrp-1 KO mice. In A, C–F, the ad GFP group was normalized to 1, and the ad Cre group was expressed as relative fold of control group. *, p<0.05, **, p<0.01, ***, p<0.001, compared with control group.

Figure 2. Cardiomyopathy in SM22-α-Nrp-1 KO mice

(A–B). Representative morphology and Masson Trichrome’s staining of hearts of WT mice and SM22-α-Nrp-1 KO mice at 3 weeks, 12 weeks, 8 months and 1 year old. N=5 for each group. (C). Thicknesses of right ventricles and left ventricles were analyzed from the images of Masson Trichrome’s staining and expressed as a ratio of RV/LV. N=5 for each group. (D). Averaged CM cross-sectional areas of WT and SM22-α-Nrp-1 KO mice at 3 weeks old were measured with Alexa Fluor® 594 labeled wheat germ agglutinin (WGA). Nuclei were counterstained with DAPI. Two hundred to three hundred CMs were blindly counted per heart. N=7 for each group. (E) Representative micrographs of TUNEL staining of Heart sections of WT mice and SM22-α-Nrp-1 KO mice at 3 weeks old. Nuclei were counterstained with DAPI. Total TUNEL positive cells were counted blindly per heart section. N=7 for each group. *, p<0.05, ***, p<0.001, compared with WT group.

Figure 3. Evaluation of cardiac function of SM22-α-Nrp-1 KO mice

(A). Representative M-mode/2-dimenational (D)/3-D echocardiography in 1-year-old wild-type and SM22-α-Nrp-1 KO (NRP-1 KO) animals. Dimension by M-mode (B–D), area by 2-D (E–G), volume by 3-D (H), LV contractility (I) and mass (J) were measured. BW, body weight; LV, left ventricle; RV, right ventricle; 2-D/3-D, 2-/3- dimensions. N=5 for each group. (I). Blood pressure was measured by tail cuffed method in male WT mice and SM22-α-Nrp-1 KO mice at 3 weeks old and 8 months old. N=8 for WT mice at 3 weeks old, n=7 for SM22-α-Nrp-1 KO mice at 3 weeks old. N=7 for each group at 8 months old. (J). Masson's trichrome staining was performed in lungs of WT mice and SM22-α-Nrp-1 KO mice at 4 months and 8 months old. Results are representative from experiments with 3 mice of each group. *, p<0.05.

Figure 4. SM22-α-Nrp-1 KO mice exhibited deleterious effect in ischemic cardiomyopathy

(A). Kaplan-Meier survival curves in WT and SM22-α-Nrp-1 KO mice up to 4 weeks after myocardial infarction (N = 15 for WT mice, n=13 for SM22-α-Nrp-1 KO mice). (B). Bar graphs represent quantitative data of ejection fraction and fractional shortening from WT and SM22-α-Nrp-1 KO mice at 3 days after LAD occlusion (N=6 for WT mice, n=9 for SM22-α-Nrp-1 KO mice). (C). Histological assessment on day 4 post-LAD ligation in ventricle infarct areas. Panels represent standard hematoxylin and eosin (H&E) as well as immunohistochemistry for capillary density (Cav-1 staining) in the central infarct myocardium of WT mice and SM22-α-Nrp-1 KO hearts. Results are representative from experiments with 3 mice of each group. Areas of vascular dilation and interstitial hemorrhage are indicated (>). Magnification of all images is 200×. ***, p<0.001.

Figure 5. Involvement of Smad2 and Notch1 in NRP-1-mediated regulation of PPARγ and PGC1α

(A–B). Control MEFs and Nrp-1 KO MEFs were stimulated with TGF-β (10 ng/mL) for 24 h. PPARγ and PGC1α mRNA levels were analyzed with qPCR and expressed as relative fold of control group. (C) Heart lysates from WT and SM22-α-Nrp-1 KO mice were subjected to Western blotting with the indicated antibodies. (D–E). WT MEFs, Smad2 KO MEFs, and Smad3 KO MEFs were serum-starved overnight and then stimulated with TGFβ for 24 h. Total lysates (D), nuclear protein and cytoplasmic protein (E) were subjected to Western blotting with the indicated antibodies. (F–H). CMs isolated from neonatal NRP-1^f/f mice were infected with adenovirus Cre or adenovirus GFP as control for 48 h, and then transfected with either Smad2 expressing plasmids (F) or Nothc1-FLAG expressing plasmid (H) for 24 h. Total lysates were collected and subjected to Western blotting. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 6. Metabolic profile change in SM22-α-Nrp-1 KO mice

(A–B). Relative levels of 1,5-AG, glycerate, ribose, xylitol (A), and stearate, nonadecanoate, arachidonate (B) in the serum of WT and SM22-α-Nrp-1 KO mice. (C). Relative levels of 12,13-DHOME and 9,10-DHOME in the adipose tissues of WT and SM22-α-Nrp-1 KO mice. N=6 for each group. *, p<0.05, **, p<0.01, ***, p<0.001.