Dock10 Regulates Cardiac Function under Neurohormonal Stress

Figures

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  Figure 1. Confirmation of Dock10 ablation in primary murine cardiac cells. We transduced neonatal mouse cardiomyocytes (NMCMs) and neonatal cardiofibroblasts (NMCFs) derived from mice with floxed Dock10 alleles with an adenovirus expressing Cre-recombinase or the GFP control. We prepared DNA samples from the cells 5 days post virus infection. We established two PCR reactions; primers for the null allele, which is produced upon the deletion of exon 4 of Dock10, and primers for the floxed allele. (A,B) Null- and floxed-allele PCR reactions in NMCMs and NMCFs, respectively. (C,D) Example of Western blot analyses confirming Dock10 protein knockout post-adenoviral transduction in NMCMs and NMCFs, respectively. We used Vinculin as a loading control. (E,F) Densitometric analysis of Dock10 expression following adenoviral transduction in NMCMs and NMCFs, respectively. n = 16–18 in each condition, obtained from 3 independent experiments. We performed the statistical analyses using the Mann–Whitney test. *** p ≤ 0.001.

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  Figure 2. Dock10 ablation in NMCMs inhibits p38 signaling following acute exposure to PE. We evaluated NMCMs transduced with adenovirus expressing Cre-recombinase or GFP following incubation with the α-adrenergic agonist PE (50 μM, 20 min). (A,B) Western blots for total and phosphorylated p38 and JNK. Ponceau staining of the same blots was performed as internal loading controls with representative images shown here. (C,D) Quantitation of the PE-induced phosphorylation of p38 and JNK relative to saline control treatment (control). Note the marked inhibition of PE-induced p38 phosphorylation in the Dock10 KO cells. We performed pairwise comparisons using the Mann–Whitney test. *** p ≤ 0.001.

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  Figure 3. Dock10 ablation in NMCF inhibits JNK signaling following acute exposure to PE. We evaluated NMCFs transduced with adenovirus expressing Cre-recombinase or GFP following incubation with the α-adrenergic agonist PE (50 μM, 20 min). (A,B) Western blots for total and phosphorylated p38 and JNK. Ponceau staining of the same blots was performed as internal loading controls with representative images shown here. (C,D) Quantitation of the PE-induced phosphorylation of p38 and JNK relative to saline control treatment (control). Note the marked inhibition of PE-induced JNK phosphorylation in the Dock10 KO cells without a notable effect on p38 signaling. We performed the statistical analysis using the Mann–Whitney test. * p ≤ 0.05.

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  Figure 4. Global Dock10 KO mice exhibit mild cardiac dysfunction. (A) q-PCR analyses for Dock 9, Dock10, and Dock11 in the ventricular tissue of Global Dock10 KO (KO) mice and littermate controls. Results were normalized to GAPDH and are presented as fold relative to WT gene expression. For used primers, see Table S1. We performed the statistical analysis using the Mann–Whitney test. (B–D) Twelve-week-old mice (n = 11–15) were studied by gravimetric analysis and echocardiography. HW/BW: heart-weight–body-weight ratio (mg/g); LVIDd and LVIDs: left ventricular interior diameter in diastole and systole, respectively (mm); FS: fractional shortening (%). We performed the statistical analysis using an unpaired t-test. (E) RT-qPCR analyses as in (A) for expression of the fetal genes Acta1, Acta2, Nppa, Nppb, and Myh7. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

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  Figure 5. Dock10 KO cardiomyocytes exhibit impaired contractile function and Ca2+ handling. We analyzed dissociated ventricular myocytes using an IonOptix HyperSwitch system. (A) Representative traces for sarcomere length during field stimulation (0.5 Hz); inset shows traces normalized to baseline sarcomere length. Parameters shown are resting sarcomere length, shortening (difference between resting and contracted length), fractional shortening (shortening/resting length), and maximal relaxation rate (n = 87, Global Dock10 KO, n = 95, W.T. control). (B) Representative traces for cytosolic Ca2+ as assayed using the ratio-metric dye Indo-1/AM, and emission measured at 405 nm (Ca2+ bound) and 480 nm (Ca2+ free), shown as relative terms (a ratio of F405/F480). Inset shows traces normalized for diastolic (resting) Ca2+. Parameters shown are diastolic (resting) Ca2+, transient amplitude, the rate of Ca2+ rise at the beginning of the transient and the rate of Ca2+ decline at the end of the transient (n = 53, Global Dock10 KO, n = 50, W.T. control). The analyzed cells were obtained from 3 mice per cohort. We performed the statistical analysis using the Mann–Whitney test. ** p ≤ 0.01, * p ≤ 0.05.

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  Figure 6. Global Dock10 KO mice exhibit attenuated cardiac JNK and p38 signals following acute exposure to PE. (A,B) Western blots for total and phosphorylated p38 and JNK from 2-month-old mice-heart lysates after exposure to PE (10 mg/kg) or saline (control). Ponceau staining of the same blots was performed as internal loading controls with representative images shown here. (C,D) Quantitation of the PE-induced phosphorylation of p38 and JNK relative control treatment. Note the marked inhibition of the PE-induced phosphorylation of both p38 and JNK in the global Dock10 KO hearts. We performed the statistical analysis using the Mann–Whitney test. *** p ≤ 0.001, ** p ≤ 0.01.

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  Figure 7. Dock10 KO mice exhibit exacerbated pathological hypertrophy and cardiac dysfunction following chronic exposure to Ang II. We subjected Dock10 KO and WT mice to chronic Ang II infusion at 2 mg/kg/day for 2 weeks. (A) Heart weight normalized by body weight (mg/g). (B) Fractional shortening by M-mode echocardiography. We performed the statistical analysis by one-way ANOVA with Tukey post hoc multiple comparisons. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

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  Figure 8. Dock10 KO mice exhibit exacerbated characteristics of pathological cardiac hypertrophy following chronic exposure to Ang II. (A) Masson trichrome-stained, paraffin-embedded sections for Ang II-infused and saline-infused, WT and Dock10 KO hearts. Black bar = 100 µm. (B) Quantitative analysis of the percentage of fibrosis in the ventricular tissue. We performed the analysis on 5 images, covering the entire LV base for each of 4 mice per experimental group. (C) RT-qPCR for collagen gene expression. Data are normalized to GAPDH and presented as fold relative to Ang II-treated WT mice (n = 5 per cohort). We performed the statistical analysis using the Mann–Whitney test. (D) Analysis of cardiomyocyte cell area. Note the markedly increased cross-section area of myocytes from Ang II-treated global Dock10 KO mice. We analyzed a total of 184–229 cells for each condition from n = 3–4 mice per cohort. We performed the statistical analysis by ordinary one-way ANOVA with Tukey post hoc multiple comparisons. *** p ≤ 0.001, * p ≤ 0.05.