Figure 1.
PAP and its interaction with Pak1.
A: Pak1 is divided into N-terminal and C-terminal halves. The N-terminal half contains p21 binding domain (PBD) followed by a kinase inhibitory domain, which overlap with each other. The proline rich motifs interact with different cellular proteins including Nck, Grb2 and Pix, etc. PAP is derived from the autoinhibitory domain linked to a TAT sequence. PAP binds to Pak1 and may activate Pak1 through attenuation of Pak1 autoinhibition in a similar way as Cdc42 and Rac1 do. B: We examined the effects of the PAP on Pak1 phosphorylation in cultured neonatal rat ventricular myocytes (NRVMs) that were treated with PAP (20 µg/ml) for 2 hours. Immunoblotting analyses of Pak1 phosphorylation indicate that Pak1 activation was induced by angiotensin II (Ang II), in NRVMs (n = 3 independent experiments).
Figure 2.
PAP abrogates Ang-II induced hypertrophy in in vitro and in vivo models.
A: NRVMs were treated with Ang II (500 nM) with or without co-treatment of PAP (20 µg/ml) for 48 h, followed by α-actinin immunostaining. Cell size was measured and presented as the bar graphs (upper panel, 450 cells from three independent experiments). Representative images of double staining of NRVMs are shown in lower panel (green staining for α-actin; blue for DAPI, scale bar: 20 µm). B: Mean of cross-sectional areas measurements (upper panel); HE staining of heart cross-sections (lower panel, scale bar: 20 µm, n = 4). C: HW/TL ratios of mice before and after Ang II or Ang II+PAP treatments (n = 4).
Figure 3.
PAP protects heart from ventricular arrhythmia associated with hypertrophy.
A: Comparison of in vivo electrocardiographic parameters between before and after treatment of Ang II only or Ang II plus PAP treatment for 7 days, (n = 4–6 per group. B: Representative recordings of typical in vivo ECG recordings from anesthetized mice before and after Ang II treatment (1 mg/kg/day) with or without co-treatment of PAP (1 mg/kg/day) for 7 days.
Figure 4.
Ventricular epicardial electrical mapping with a multi-electrode array (MEA).
A: Representative activation maps of five successive cardiac cycles under sinus rhythm obtained from the hearts from the control group and Ang II+PAP group showed a general pattern of sequential activation, whereas the hearts from Ang II group often showed a disordered pattern with beat-to-beat variations (n = 6), inserted arrows indicate the conduction direction. B: Pacing induced activation maps generated by pacing in the center of array on the epicardium of left ventricle from mice without any treatment and treated with Ang II or Ang II + PAP (n = 6 for each group). C: Comparison of left ventricular conduction velocity (n = 6 for each group).
Figure 5.
Conduction velocity under PES condition.
A: Ventricular fibrillation threshold of the heart from mice treated with Ang II only and Ang II + PAP. B: Representative examples of ECG recordings from mice subjected to ex-vivo S1S1 ventricular pacing at 6.5 mA amplitude. Episodes of arrhythmias were showed in mice treated with Ang II only (n = 4 per group).
Figure 6.
PAP treatment ameliorated Ang ll-induced decrement and spatially heterogeneous distribution of Cx43.
A: Representative images of Cx43 staining. Thick arrows point to diffuse Cx43 labeling in the cytoplasm, whereas thin arrows show Cx43 distributed in intercalated discs. B: Immunostaining of Cx 43 was performed on sections of the heart from mice treated with Ang II or Ang II + PAP. The number of Cx43-positive clusters of Cx43 labeling are quantified, as shown in the bar graphs.(n = 4 per group,Scale bar, 20 um)
Figure 7.
PAP reduced Ang II induced increase in frequencies of calcium sparks and waves in ventricular myocytes.
A: The frequencies of calcium sparks (left panel) and waves (right panel) were measured from single ventricular myocytes isolated from mice administered with Ang II (10 mg/kg/day) with or without co-treatment of PAP (1 mg/kg/day) delivered by minipump for 7 days. The measurements were presented as means ± S.E.M (Control: n = 13; Ang II: n = 15; Ang II+PAP: n = 18). B: The 2D and 3D representative images showing calcium sparks and waves. Note: 2D Images were adjusted to enhance the contrast.
Figure 8.
PAP restored the Ang II induced reduction of amplitudes and prolongation in peak-plateau of calcium transients in ventricular myocytes.
A: The amplitude of the peak of calcium transients (upper panel) was measured by ΔF/F0. The duration of peak-plateau phase of the calcium transients (lower panel) was measured as the time interval between the upstroke of the fluorescence signal (measured at 80% of the maximum value) and the corresponding point on the decay (also measured at 80% of the maximum value). Both were presented as mean ± S.E.M (Control: n = 14; Ang II: n = 18; Ang II+PAP: n = 11). B: The representative traces showing the calcium transients of each group. C: The 2D and 3D representative images showing calcium transients.
Figure 9.
Regulation of cardiac excitation and hypertrophy by Pak1.
Pak1 regulates the activities of ion channels, Ca2+ handling and myofilament proteins through PP2A. Cardiac hypertrophy induced under pathological conditions is suppressed by Pak1 through JNK signaling and regulation of Cx43 expression and distribution.