Robust Overexpression of Dpr1 in a Rat Myocardial Infarction Model

We observed robust overexpression of Dpr1 protein and mRNA in the remote area of rat myocardial infarction tissue and in isolated cardiomyocytes, cultured under hypoxia conditions (Figure 1A and 1B, Figure S1A–S1D in the online-only Data Supplement), suggesting involvement of Dpr1 in early cardiac remodeling. In addition, Dpr1 accumulation in the cytoplasm and increased nuclear localization in hypoxia cells were determined (Figure 1C). This prompted us to elucidate the function of Dpr1 in the heart. Therefore, we genetically engineered transgenic mice (Dpr1-tg) with cardiac-specific overexpression of Dpr1 under the control of an α–myosin heavy chain promotor. Upregulation of Dpr1 in cardiac tissue was confirmed by immunoblotting (Figure S1E). Animals were characterized at 3 months of age.

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Figure 1.

Robust overexpression of Dapper-1 (Dpr1) in a rat myocardial infarction (MI) model. A, Western blot showing upregulation of Dpr1 in murine MI tissue obtained 1 week after surgery. B, Western blot showing increased Dpr1 protein content in cardiomyocytes after hypoxia. Coomassie staining was used as loading control. C, Fluorescence image of cardiomyocytes exposed to hypoxia for 24 hours. Dpr1 (red) accumulates in the cytoplasm and translocates into the nucleus (blue).

Dpr1 Induces Cardiac Hypertrophy and Impairs LV Function

Dpr1-tg mice exhibited a higher heart weight/tibia length ratio and increased left and right ventricular weight/tibia length ratio than wild-type (WT) animals (7.6±0.2 mg/mm versus 7.0±0.2 mg/mm, 5.6±0.1 mg/mm versus 5.2±0,1 mg/mm, 1.3±0.09 mg/mm versus 1.1±0.05 mg/mm, respectively; P<0.05; Figure 2A–2C). Cardiomyocyte cross-sectional area was increased in mice overexpressing Dpr1 (+21±2% versus WT animals; P<0.05; Figure 2D and 2E). In line with these findings, upregulation of prominent hypertrophy marker genes atrial natriuretic factor, brain natriuretic peptide, and β–myosin heavy chain was observed (Figure 2F). Baseline echocardiography revealed no significant difference between Dpr1-tg mice and WT animals in ejection fraction (Figure 2G), end-systolic, and end-diastolic diameter (data not shown). However, cardiac hypertrophy was associated with impairment of LV function. Pressure–volume loop analysis revealed a decrease in the maximal rise of LV pressure (dp/dt max; 10 878±2669 mm Hg/s in WT versus 8498±2044 mm Hg/s in Dpr1-tg mice; P<0.005; Figure 2H), indicating impaired contractility in Dpr1-tg mice. The reduction in dp/dt min levels (−8920±435 mm Hg/s in WT versus −7529±451 mm Hg/s in Dpr1-tg mice; P<0.01; Figure 2I) and the simultaneous increase in the time constant of isovolumic relaxation (10.24±0.6 m/s in WT mice versus 12.2±0.3 m/s in Dpr1-tg mice; P<0.01; Figure 2J) demonstrate signs of diastolic dysfunction compatible with the myocardial hypertrophy observed.

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Figure 2.

Dapper-1 (Dpr1) induces cardiac hypertrophy and impairs left ventricular function. A–C, Increase of heart weight (HW)/tibia length (TL) and left and right ventricular weight (LVW and RVW)/TL ratio in Dpr1-tg mice. CSA indicates cardiomyocyte surface area. D, Enlargement of individual cardiomyocyte in Dpr1-tg animals (n≥300 cardiomyocytes from 10 animals each group). E, Representative HE-stained left ventricle tissue sections of wild-type (WT) and Dpr-tg mouse. F, Enhanced mRNA expression of hypertrophy marker genes atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and β–myosin heavy chain (β-MHC). G, Ejection fraction of Dpr1-tg mice is unaltered. H, Reduced contractility of left ventricle indicates systolic dysfunction in Dpr1-tg animals. Attenuation of dp/dt min (I) and increased τ_g (J) indicates diastolic dysfunction. All data are presented as mean±SEM; *P<0.05 vs WT, n≥10 animals each group; n.s. = not significant.

Dpr1 Activates Canonical Wnt Signaling Pathway in the Murine Heart

Dpr1-tg mice had increased Dvl2 protein levels and mRNA transcripts, whereas expression of Dvl1 and Dvl3 isoforms remained unaltered (Figure 3A, Figure S2A and S2B). In addition, we observed enhanced Dvl2 phosphorylation (Figure 3A), indicating higher Dvl2 activity. Given the redundancy of Dvl proteins, we asked whether this was sufficient to activate canonical Wnt signaling and found increased total- and activated β-catenin amounts, but reduction of β-catenin (pS33/37, pT41; Figure 3B and Figure S2C). Phosphorylation of β-catenin primes this protein for degradation. Immunostaining of LV tissue slides revealed β-catenin accumulation in the cytoplasm and its translocation into the nucleus (Figure 3C). Steady-state levels of β-catenin mRNA transcripts suggest post-translational stabilization (Figure S2D). Stabilized β-catenin was accompanied by transcription of Wnt/β-catenin targets c-myc, cyclin D1, peroxisome proliferator-activated receptor-δ, and myoD, all indicating activation of canonical Wnt signaling (Figure 3D).

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Figure 3.

Dapper-1 (Dpr1) activates canonical Wnt signaling pathway in the murine heart. A, Western blot of left ventricular tissue using dishevelled (Dvl)1-, Dvl2-, and Dvl3-specific antibodies. Expression of Dvl2 protein and mRNA levels were increased in Dpr1-tg mice. B, Western blot showing increased protein levels of total-β-catenin, active-β-catenin, but decrease of β-catenin (pS33/37, pT41). C, Fluorescence image of tissue slide obtained from left ventricle showing translocation of β-catenin (red) into the nucleus in Dpr1-tg mice. ABC indicates active β-catenin. D, Increase of β-catenin target genes c-myc, peroxisome proliferator-activated receptor (PPAR)-δ, cyclinD1, and myoD. All data are presented as mean±SEM; *P<0.05 vs WT, n=7 animals each group for protein analysis; n≥10 animals each group for RNA analysis.

Dpr1 Is Both Necessary and Sufficient to Activate Canonical Wnt Signaling in Cardiomyocytes

We further mapped the molecular mechanism by which Dpr1 impacts canonical Wnt signaling in cultured cardiomyocytes by siRNA-mediated Dpr1 depletion. Consistent with our observations in Dpr1-tg mice, we detected decreased Dvl2 protein abundance but unaltered expression of Dvl1 and Dvl3 (Figure 4A and Figure S3A). Numbers of Dvl2 mRNA copies were not reduced, suggesting a mechanism on levels after transcription (Figure S3B). Inhibition of lysosome acidification with NH₄Cl stabilized Dvl2 proteins in Dpr1-depleted cells, indicating that downregulation of Dvl2 is a result of lysosomal degradation (Figure 4B and Figure S3C). Downstream of Dvl2, these cells displayed reduced total- and active β-catenin protein amounts, as well as inhibition of c-myc protein expression (Figure 4A and Figure S3B). Copy number of β-catenin mRNA was stable, suggesting that decreased protein content is a result of post-translational reduction (Figure S3E).

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Figure 4.

Dapper-1 (Dpr1) is necessary and sufficient to induce canonical Wnt signaling in cardiomyocytes. A, Representative Western blot analysis of Dpr1 knockdown in cultured cardiomyocytes. Expression of Wnt proteins dishevelled (Dvl)2, β-catenin, active β-catenin (ABC), and c-myc is reduced in Dpr1 knockdown cells. B, Representative Western blot demonstrating that lysosomal inhibition with NH₄Cl (2.5 mmol/L) blocks Dvl2 degradation in Dpr1-depleted cells. C, Western blot demonstrating that knockdown of Dpr1 inhibits Wnt3a-induced activation of β-catenin in cardiomyocytes. D, Immunostaining with β-catenin specific antibody shows Wnt3a-induced translocation of β-catenin into the nucleus. In Dpr1 knockdown cells, translocation of β-catenin into the nucleus is blocked. E, Increased TOPflash reporter activity after stimulation with Wnt3a-conditioned medium. Knockdown of Dpr1 inhibits Wnt3a-induced activation of the Wnt signaling pathway. F, Adenoviral overexpression of Dpr1 synergizes the stabilizing effect of Wnt3a on β-catenin. G, Overexpression of Dpr1 increases TOPflash reporter activity under basal conditions. Stimulation with Wnt3a-conditioned medium shows synergistic effect. All data are presented as mean±SEM of ≥3 individual experiments in duplicate; *P<0.05 vs unstimulated control; #P<0.05 vs Wnt3a-stimulated control; Ad indicates adenovirus; L-cell, supernatant from L-cell culture nonexpressing Wnt3a was used as control; and pDC316-Dpr1, vector encoding Dpr1.

Wnt3a-conditioned medium specifically activates β-catenin–dependent Wnt signaling. Cellular distribution of Dpr1 on Wnt3a stimulation changed from the cytoplasm to the nucleus and underlines its role in canonical Wnt signaling (Figure S3F). To determine whether Dpr1 depletion was sufficient to inhibit transmission of canonical Wnt signals, we first assayed β-catenin activation after Wnt3a treatment and found it inhibited (Figure 4C and Figure S3G). Furthermore, nuclear translocation of β-catenin was completely blocked (Figure 4D). Downstream, β-catenin interacts with transcription factors of the TCF/LEF family. The TOPflash luciferase reporter activity assay is an established readout of β-catenin/TCF/LEF interaction. TOPflash activity was reduced in Dpr1-depleted cardiomyocytes, and Wnt3a stimulation failed to enhance TOPflash reporter activation (Figure 4E); Conversely, Dpr1 overexpression enhances both β-catenin and TCF/LEF reporter activity under basal conditions and synergized with the effect of Wnt3a (Figure 5F and 5G, Figure S3H).

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Figure 5.

Dapper-1 (Dpr1)-induced activation of canonical Wnt signaling is dishevelled (Dvl)2-dependent. A and B, Representative Western blot and bar graph showing Dvl2 overexpression in cardiomyocytes cultured in hypoxia conditions. C, Dpr1 was coprecipitated with Dvl2 in protein lysate of cultured cardiomyocytes. D, Representative immunoblot confirming Dvl2-specific siRNA knockdown reveals unaltered levels of Dpr1 and active β-catenin (ABC). E, ABC-specific immunoblot from Dvl2-depleted and Wnt3a-stimulated cardiomyocytes. F, Wnt3a-induced activation of β-catenin is inhibited in Dvl2-depleted cells. G, TOPflash reporter assay demonstrates that there is no interaction between β-catenin and T-cell factor transcription factors after knockdown of Dvl2. H, Dpr1-induced TOPflash activity is inhibited in Dvl2 knockdown cardiomyocytes. All data are presented as mean±SEM of 3 individual experiments in duplicate; *P<0.05 vs unstimulated control; #P<0.05 vs Wnt3a-stimulated control. Ab indicates antibody; CoIP, coimmunoprecipitation; and L-cell, supernatant from L-cell culture nonexpressing Wnt3a was used as control.

Dpr1-Induced Activation of Canonical Wnt Signaling Is Dvl2 Dependent

Correlation between the protein abundances of Dpr1 and Dvl2 was observed in Dpr1-tg mice, in Dpr1-depleted cardiomyocytes, and furthermore in cardiomyocytes, cultured in hypoxia conditions (Figure 1B, Figure 5A and 5B). We next determined whether there is a causal association between inhibition of the canonical Wnt pathway and the downregulation of Dvl2 observed in Dpr1 knockdown cells. Dpr1 was coprecipitated with Dvl2 in cultured cardiomyocytes, indicating a physical interaction (Figure 5C). Reciprocal downregulation of Dpr1 in Dvl2-depleted cardiomyocytes was not observed (Figure 5D), suggesting Dpr1 regulates Wnt upstream of Dvl2. However, Wnt3a-induced activation of canonical Wnt signaling also failed in these cells. Neither β-catenin activation (Figure 5E and 5F) nor TCF/LEF reporter activity (Figure 5G) was increased after Wnt3a stimulation. In addition, Dpr1-induced TOPflash reporter activation was completely inhibited in cells lacking Dvl2 (Figure 5H), demonstrating that Dvl2 is a required cofactor for transmitting canonical Wnt signals downstream of Dpr1.

Wnt3a- and PE-Induced Hypertrophy Is Inhibited in Dpr1-Depleted Cardiomyocytes

Myocyte surface area increased in response to Wnt3a stimulation (Figure S4A) as did protein synthesis (Figure S4B), indicating that stimulation of the canonical Wnt pathway alone is sufficient to induce a hypertrophic response. In a complementary approach, we found that Dpr1 depletion and concomitant inhibition of the canonical Wnt pathway rescued Wnt3a-induced cardiomyocyte hypertrophy. The cardiomyocyte surface area was smaller in Dpr1 knockdown cells (Figure S4A), and Wnt3a-induced protein synthesis was completely blocked, indicating that Dpr1 is required for Wnt3a-induced hypertrophy (Figure S4B). In addition, Dpr1 depletion moderately inhibited PE-induced cardiac myocyte hypertrophy (Figure S4C). Even more apparent was the fact that PE treatment strongly enhanced protein synthesis in control cardiomyocytes, whereas Dpr1-depleted cells completely blocked PE-induced protein synthesis, indicating a crucial role of Dpr1 in PE-mediated cardiomyocyte hypertrophy (Figure S4D).