Left ventricular response in the transition from hypertrophy to failure recapitulates distinct roles of Akt, β-arrestin-2, and CaMKII in mice with aortic regurgitation

Figures

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  Figure 1. Creation and evaluation of aortic regurgitation (AR) in mice. (A) The plastic catheter and the metal wire used in AR surgery. Scale bar =1 cm; (B) echocardiographic imaging of the region of ascending aorta and aortic valves which contains the plastic catheter and the metal wire, as labeled, just prior to aortic valve disruption; (C) schematic of the surgery procedure. From left to right, before, during and after aortic valves puncture; (D) color Doppler images of aortic arch flow during cardiac systole and diastole from sham-operated and AR mice 2 weeks after surgery. Red indicates flow toward, and blue indicates flow away from the transducer; (E) pulsed wave Doppler recordings of aortic arch flow during cardiac systole and diastole from sham-operated and AR mice. In this view, the systolic flow is downwards, and the diastolic flow is upwards (regurgitant). The scale in the panel is in units of meters/second (m/sec).

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  Figure 2. Pulse wave Doppler flow spectra and M-mode echocardiography in mice before aortic regurgitation AR (0W), and either 1, 2, 4, or 8 weeks after AR. (A) Doppler aortic arch flow velocity for mice at the indicated time points. The systolic flow is downwards, and the diastolic flow is upwards (regurgitant). Notice the appearance of backward flow following AR. Scale bar =1 m/s; (B) M-mode images of the left ventricle for the indicated time points demonstrating progressive LV dilatation following AR. Scale bar =2 mm; (C) mitral Doppler spectra. As discussed in the Methods Section, E waves (i.e., Doppler flow signals associated with early ventricular filling) and A waves (flow signals associated with atrial contraction) could be consistently identified in normal mice and 1 week after AR, but these signals merged at later stages after AR. Scale bar =1 m/s; (D) basal (without adenosine) coronary flow velocity waveforms. Scale bar =200 mm/s; (E) D/S VTIa, diastolic/systolic velocity-time integral of aortic arch flow; (F) LVEDD, left ventricular end-diastolic dimension measured using M-mode; (G) LVESD, left ventricular end-systolic dimension; (H) SV, stroke volume; (I) LVPWTd, left…

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  Figure 3. Invasive left ventricular hemodynamics in mice before aortic regurgitation AR (0W), and at 1, 2, 4, and 8 weeks after AR. (A) Left ventricular pressure (unit: mmHg); (B) left ventricular contraction velocity and relaxation velocity (unit: mmHg/s); (C) LVESP; (D) LVEDP; (E) maximal +dp/dt; (F) minimal −dp/dt. *, P<0.05 vs. sham; †, P<0.05 vs. 0W; ‡, P<0.05 vs. 1W; §, P<0.05 vs. 2W. LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure.

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  Figure 4. Gross samples and histological analyses of mouse hearts before aortic regurgitation AR (0W), and at 1, 2, 4, and 8 weeks after AR. (A) Representative pictures of whole heart (n=15–28 per group). Scale bar =1 mm; (B) HE-stained heart sections (n=8–14 per group). Scale bar =1 mm; (C) HE-stained cardiomyocytes (n=200–350 cells per group). Scale bar =20 µm; (D) Masson-stained interstitial fibrosis (n=32–56 slides per group). Scale bar =50 µm; (E) Masson-stained perivascular fibrosis (n=8–14 slides per group). Scale bar =50 µm; (F) quantification of CSA of cardiomyocytes; (G) quantification of interstitial fibrosis. *, P<0.05 vs. sham; †, P<0.05 vs. 0W; ‡, P<0.05 vs. 1W, §, P<0.05 vs. 2W; ||, P<0.05 vs. 4W.

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  Figure 5. The sequential changes in Nppa and Nppb before aortic regurgitation AR (0W), and at 1, 2, 4, and 8 weeks after AR. (A) Nppa; (B) Nppb. †, P<0.05 vs. 0W; ‡, P<0.05 vs. 1W; §, P<0.05 vs. 2W; ||, P<0.05 vs. 4W.

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  Figure 6. Time course changes in load-related signaling pathways of CaMKII (A), β-arrestin-2 (B), and Akt (C) before aortic regurgitation AR (0W), and at 1, 2, 4, and 8 weeks after AR. Original immunoblots are shown below the graphs. p, phosphorylated protein; t, total protein; p/r, phosphorylation ratio; †, P<0.05 vs. 0W; ‡, P<0.05 vs. 1W; §, P<0.05 vs. 2W; ||, P<0.05 vs. 4W.

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  Figure 7. Effects of the intervention of Akt, β-arrestin-2, and CaMKII on cardiac structure and function at 4 weeks after AR. (A) The mRNA levels of Nppa after wortmannin, KN-93, and β-arrestin-2 AAV9 treatment, respectively; (B) the mRNA levels of Nppb after wortmannin, KN-93, and β-arrestin-2 AAV9 treatment, respectively; (C) M-mode images of the left ventricle (unit: mm). Scale bar =2 mm; (D) left ventricular pressure (unit: mmHg); (E) ventricular contraction velocity and relaxation velocity (unit: mmHg/s); (F) HW/BW, heart weight-to-body weight ratio; (G) LVPWTd; (H) LVPWTs; (I) LVEDD; (J) LVESD; (K) LVEF; (L) LVFS; (M) LVESP; (N) LVEDP; (O) maximal +dp/dt. (P) Minimal −dp/dt. *, P<0.05 vs. Sham; †, P<0.05 vs. control; ‡, P<0.05 vs. KN93; §, P<0.05 vs. β-arrestin-2. LVPWTd, left ventricular posterior wall end-diastolic thickness; LVPWT, left ventricular posterior wall end-systolic thickness; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure.

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  Figure S1. Representative images of Masson-stained perivascular fibrosis in mice subjected to 4-week AR, TAC, or corresponding sham operations (scale bar, 50 µm). Perivascular fibrosis is more conspicuous in TAC mice than in AR mice. TAC, transverse aortic constriction. AR, aortic regurgitation.

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  Figure S2. The intervention of Akt, β-arrestin-2, and CaMKII in the hearts of AR mice. (A) The protein level of CaMKII after KN-93 treatment; (B) the protein level of β-arrestin-2 after β-arrestin-2 AAV9 treatment; (C) the protein level of Akt after wortmannin treatment. *, P<0.05 vs. Sham; †, P<0.05 vs. control.