P21‐Activated Kinase 2 as a Novel Target for Ventricular Tachyarrhythmias Associated with Cardiac Adrenergic Stress and Hypertrophy

Figures

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  Figure 1. Pak2 deficiency aggravates the inducibility of cardiac arrhythmias in mice challenged with acute isoproterenol (ISO) or chronic (5 weeks) TAC challenge in vivo. A) The protocol scheme applied to Pak2f/f and Pak2cko mice under acute isoproterenol (ISO) or chronic TAC challenge. B) Representative in vivo cardiac electrophysiological recordings from Pak2f/f and Pak2cko mice with different frequencies of burst stimulation. C) VT inducibility during in vivo electrocardiogram measurements in Pak2f/f and Pak2cko mice before and after isoproterenol challenge. D) Quantification of VT occurrence following the application of isoproterenol in Pak2f/f and Pak2cko mice at different frequencies of burst stimulation. E) Representative in vivo cardiac electrophysiological recordings from Pak2f/f and Pak2cko mice at 5 weeks after sham or TAC surgery. F) Occurrence of ventricular ectopic beats of Pak2f/f and Pak2cko mice at 5 weeks after sham or TAC surgery. G) Survival curves of four groups of mice after operation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 2. Pak2 deficiency aggravates the inducibility of VT and alternans in mice under chronic (5 weeks) TAC challenge in ex vivo hearts. A) Flow chart of optical mapping scheme. B) VT inducibility in ex vivo Langendorff hearts after TAC challenge in Pak2f/f and Pak2cko mice. C) Heat maps (indicating the spectral intensity differences of Ca2+ transients) showing frequency‐dependent CaT alternans during progressive decrements in cycle length between 100–70 ms. The corresponding CaT traces are also exhibited from the selected region within the boxes. D) Calculation of CaT alternans ratio for each group (n = 6 hearts for each group). Large alternans ratio (>20%) are counted for evaluation of the severity of CaT alternans. E) Typical AP traces during VT induced by progressive pacing; the activation map shows that the initiation positions for ectopy and reentry overlap with regions harboring the most severe AP alternans. F) Typical consecutive phase maps during VT in a Pak2cko mouse heart, showing the fast re‐entry rotors meandering on the epicardium without losing the coupling of AP and CaT, along with the corresponding AP and CaT traces. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 3. Cardiac specific Pak2 overexpression attenuates isoproterenol or TAC‐induced cardiac arrhythmias and Ca2+ alternans. A) The protocol scheme for Pak2WT (Rosa26CAG‐LSL‐Pak2) and Pak2ctg mice with acute isoproterenol (ISO) or chronic TAC challenge. B) VT inducibility during in vivo electrocardiogram measurement in Pak2WT and Pak2ctg mice before and after isoproterenol challenge with rapid pacing for 100 stimuli each. C) Quantification of VT duration in the application of isoproterenol in Pak2WT and Pak2ctg mice in vivo. D) Calculation of ventricular ectopic beats after TAC challenge for 5 weeks in WT and Pak2ctg mice in vivo with isoproterenol and caffeine challenge. E) Heat maps (indicating the spectral intensity difference of Ca2+ alternans) showing frequency‐dependent CaT alternans during progressive decrements of pacing cycle length between 100–70 ms. Corresponding CaT traces are also exhibited from the selected region within the boxes. F) Calculation of CaT alternans ratio for each group (n = 4‐7 hearts). Large alternans ratio (>20%) are counted for evaluation of the severity of CaT alternans. G) Typical AP traces during VT in a Pak2WT mouse heart after TAC for 7 weeks induced…

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  Figure 4. Pak2 deletion aggravates isoproterenol and TAC‐induced disruptions of cardiac intracellular calcium homeostasis. A) Calcium fluorescence was measured in isolated ventricular myocytes from Pak2f/f and Pak2cko mice after acute isoproterenol treatment. B) The pie diagrams of occurrence of premature Ca2+ transients (PCT) and Ca2+ oscillation (CO) from the indicated groups. C) The statistical histogram of severity of Ca2+ transient abnormality from the indicated groups. D) Calcium fluorescence was measured in isolated cardiomyocytes from Pak2f/f and Pak2cko mice after sham or TAC surgery for 5 weeks. E) The pie diagrams of occurrence of PCT and CO from the indicated groups. F) The statistical histogram of severity of Ca2+ transient abnormality from the indicated groups. G) The statistical histogram of τ of Ca2+ transient decays from the indicated groups. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 5. Pak2 deletion aggravates TAC‐induced cardiac oxidative stress. A) Heat map showing differentially expressed protein with proteomics in heart tissue from Pak2f/f and Pak2cko mice with sham or TAC surgery for 5 weeks. B) KEGG enrichment analysis of differentially expressed protein from Pak2f/f and Pak2cko mice at 5 weeks after TAC surgery. C) GO enrichment analysis of proteomics in heart tissue from different treatments. D) Heat map showing differentially expressed protein with phos‐proteome in heart tissue from different treatments. E) KEGG enrichment analysis of phos‐proteomics in heart tissue from different treatments. F) GO enrichment analysis of phos‐proteomics. G) Venn graph showing the significantly different proteins and phosphoprotein for Pak2cko/TAC versus Pak2f/f/TAC, and Pak2cko/TAC versus Pak2cko/sham respectively. H) Comparing the correlation between the proteomic and phosphoproteomic changes for TMT and PP proteomics results of each sample between Pak2cko/TAC and Pak2f/f/TAC. I) the relevant proteins enriched in the oxidative phosphorylation and tricarboxylic acid cycle pathways were analyzed for correlation between the phosphoproteomics and proteomics. K,M) The…

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  Figure 6. Pak2 deletion aggravates cardiac dysfunction by impairing mitochondrial function. A) Representative fluorescence images shows ROS levels of Pak2f/f and Pak2cko mice at 5 weeks after sham or TAC surgery. Scale bar 100 µm. B) Quantification of panel A. C) Representative transmission electron microscopy images of Pak2f/f and Pak2cko mice at 5 weeks after sham or TAC surgery. Scale bar 1 µm. D) ATP content in Pak2f/f and Pak2cko mice at 5 weeks after sham or TAC surgery. E) Pie diagrams showing the subcellular location of differentially expressed protein in Pak2f/f and Pak2cko mice at 5 weeks after TAC surgery in the comparison between the Pak2cko/TAC and Pak2f/f/TAC groups. F) Heat map showing differentially expressed mitochondrial respiratory electron transport chain protein in Pak2f/f and Pak2cko mice at 5 weeks after sham or TAC surgery. G) Representative immunoblotting images showing NDUFB6, NDUFS8, NDUFA8 protein from indicated groups. H) Quantification of G (n = 4 mice for each group). I) Statistical graph of relative activity of mitochondrial complex I. J) Representative immunoblotting images showing NOX4, NOX2, ox‐CaMKII, p‐CaMKII and t‐CaMKII protein from indicated groups.…

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  Figure 7. Cardiac‐specific Pak2 overexpression attenuates TAC or isoproterenol‐induced cardiac oxidative stress and abnormalities in mitochondrial structure. A,B) Representative fluorescence images (A) and statistical graph (B) shows ROS levels of Pak2WT and Pak2ctg mice at 7 weeks after sham or TAC surgery. Scale bar 100 µm. C) Representative transmission electron microscopy images of Pak2WT and Pak2ctg mice at 7 weeks after sham or TAC surgery. Scale bar 1 µm. D) Representative immunoblotting images showing NOX4, ox‐CaMKII, p‐CaMKII and t‐CaMKII protein in Pak2WT and Pak2ctg mice at 7 weeks after sham or TAC surgery. E) Quantification of D (n = 4 mice for each group). F,G) Representative fluorescence images (F) and statistical graph (G) shows ROS levels of Pak2WT and Pak2ctg mice at 7 weeks after sham or TAC surgery. Scale bar 100 µm. H) Representative immunoblotting images and statistical analysis showing p‐CaMKII and t‐CaMKII protein in Pak2WT and Pak2ctg mice after sham or acute isoproterenol treatment (n = 4 mice for each group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 8. Pak2 activator JB2019A attenuates TAC‐induced cardiac hypertrophy and susceptibility to ventricular arrhythmia. WT mice were subjected to sham or TAC surgery for 5 weeks. DMSO or JB2019A (80 mg kg−1/d, i.p.) was injected for 4 weeks from 1 weeks after sham or TAC surgery. A) Chemical structure of Pak2 activator JB2019A. Docking model of Alphafold2 predicted Pak2 structure and its activator JB2019A. Key residues involved in JB2019A binding are labeled. Hydrogen bonds and distances are highlighted in yellow; B) Representative immunoblotting images showing p‐Pak2 protein level from WT mice with DMSO or JB2019A treatment (n = 4 mice for each group). C) Representative in vivo cardiac electrophysiological studies of Pak2f/f and Pak2cko mice with different frequencies of burst stimulation. D) Representative in vivo electrophysiological recordings from WT mice at 5 weeks after sham or TAC surgery. Cardiac arrhythmia was induced with isoproterenol (2 mg kg−1) and caffeine (160 mg kg−1). Arrows indicate the ventricular ectopic beats (EB). Scale bar: 1 s or 0.2 s. E) statistical graph showing the VT occurrence of C. F) Statistical graph of ventricular ectopic beats in the four groups of…

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  Figure 9. Pak2 activator JB2019A attenuates TAC‐induced cardiac oxidative stress and abnormalities in mitochondrial structure. A) Representative fluorescence images shows ROS levels of WT mice 5 weeks after sham or TAC surgery with DMSO or JB2019A treatments. Scale bar 100 µm. B) Quantification of A. C) Representative transmission electron microscopy images of WT mice at 5 weeks sham or TAC surgery with DMSO or JB2019A treatments. Scale bar 1 µm. D,E) Representative immunoblotting images and statistical graph showing ox‐CaMKII and t‐CaMKII protein expressed 5 weeks after sham or TAC surgery with DMSO or JB2019A treatments (n = 4 mice for each group). F) Representative immunoblotting images showing Pak2 and NOX4 protein levels in the myocardium of control patients and patients with atrial fibrillation. G) Quantification of F. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.