Over-expression of a cardiac-specific human dopamine D5 receptor mutation in mice causes a dilated cardiomyopathy through ROS over-generation by NADPH oxidase activation and Nrf2 degradation

Fig. 1. D5R functional deficiency produces dilated cardiomyopathy. A: Representative echocardiograms from cardiac-specific hD5WT-TG and hD5F173L-TG mice. B: Cardiac sections stained with H&E (top) and Masson's trichrome stain (bottom) from 3-month-old mice. Scale bar: 5 µm. C–G: Echocardiographic analysis of cardiac volume and function (n = 12 mice/group); *P < 0.05 vs. D5WT-TG. EF = ejection fraction, FS = fractional shortening, HW = heart weight.

Fig. 2. D5R functional deficiency increases myocardial oxidative stress. Plasma membrane proteins were extracted from hearts of 3-month-old cardiac-specific human D5WT-TG (hD5WT) and hD5F173L-TG mice (hD5F173) and 4-month-old cardiac-specific hD5F173 mice treated with saline (hD5F173 + Sal) or apocynin (hD5F173 + APO, 1 mmol/kg/day) for 4 weeks, starting at 3 months of age (n = 12 mice/group, regardless of age). A and F: NADPH oxidase activity was quantified by lucigenin chemiluminescence assay. *P < 0.05 vs. hD5WT mice; #P < 0.05 vs. hD5F173L + Sal. B and F: ROS production was quantified by H2DCF-DA. *P < 0.05 vs. hD5WT mice; #P < 0.05 vs. hD5F173L + Sal. C–E: p40phox and p47phox expression was semiquantified by western blot. *P < 0.05 vs. hD5WT-TG mice; #P < 0.05 vs. hD5F173L + Sal. F: NADPH oxidase activity, ROS production, and p40phox and p47phox protein expression were quantified in H9c2-D5WT and H9c2-D5F173L cells. *P < 0.05 vs. H9c2-D5WT cells (n = 8/group).

Fig. 3. Mitochondrial ROS and assessment in cardiac-specific hD5F173-TG mice. Temporal analysis of ROS levels in (A) D5RWT-TG and D5RF173L-TG mice (3 months) or in (B) H9c2-hD5F173L and H9c2-hD5WT cells. ROS levels were measured at the indicated time points by incubating with H2DCF-DA (total intracellular ROS) or MitoSox fluorescent probes (mitochondrial ROS). *p < 0.05 vs. D5RWT-TG mice (n = 7), **p < 0.05 vs. H9c2-hD5WT cells (n = 5). C: Fold changes of sub-mitochondrial particles gene expression in D5RF173L-TG mice relative to D5RWT-TG mice (n = 7). Fold changes of Complex I, II and IV enzyme activities for (D) in vivo study (n = 7) and (E) in vitro study (n = 5).

Fig. 4. Apocynin prevents the cardiac enlargement and improves cardiac function in cardiac cardiac-specific hD5F173-TG mice (hD5F173L). A: Representative echocardiograms of 4-month-old cardiac-specific hD5F173L-TG mice treated with saline (hD5F173L + Saline) or apocynin (APO, 1 mmol/kg/day) (hD5F173L + APO) for 4 weeks, starting at 3 months of age (n = 10 mice/group). B: Heart size and weight. C–F: Echocardiographic analysis of cardiac volume and function. (n = 10 mice/group); *P < 0.05 vs. hD5F173 + Saline. EF = ejection fraction, FS = fractional shortening.

Fig. 5. D5R functional deficiency impairs nuclear Nrf2 translocation and downstream pathway. A: Nuclear proteins were extracted from the hearts of 3-month-old cardiac-specific human D5WT-TG (hD5WT) and hD5F173L-TG (hD5F173) mice and 4-month-old cardiac-specific hD5F173 mice treated with saline (hD5F173 + Sal) or apocynin (hD5F173 + APO, 1 mmol/kg/day) for 4 weeks, starting at 3 months of age (n = 12 mice/group). Nrf2 and P84 (nucleus marker) were semiquantified by western blot. B: and C: NQO1 and HO1 expression was semiquantified by western blot. *P < 0.05 vs. hD5WT mice (3-month-old). D: Nrf2 location in H9c2-D5WT and H9c2-D5F173L cells was determined by immunofluorescence. Green: Nrf2 Blue: DAPI. E: Protein expressions of Nrf2, NQO1, and HO1 were quantified by western blotting in H9c2 cells. *P < 0.05 vs. H9c2-D5WT cells (n = 8 /group).

Fig. 6. Nrf2 ubiquitination in hD5F173Lmice. A: Total heart lysates from cardiac-specific hD5WT- and hD5F173L-TG mice (3- and 4-month-old) were immunoprecipitated with anti-Nrf2 antibody and immunoblotted with anti-ubiquitin antibody. B: Total heart lysates from cardiac-specific D5WT- and hD5F173L-TG mice (3- and 4-month-old) were immunoprecipitated with anti-ubiquitin antibody and immunoblotted with anti-Nrf2 antibody. C: Data are expressed as mean ± S.E. n = 4mice/group.*P < 0.05 vs. others, one-way ANOVA, Holm-Sidak test, NC = negative control, PC = positive control.

Fig. 7. D5R deficiency induces cardiomyocyte dysfunction A–C: Total proteins were extracted from hearts of 3-month-old cardiac-specific human D5WT-TG (hD5WT) and hD5F173L-TG (hD5F173) and 4-month-old cardiac-specific hD5F173 mice treated with saline (hD5F173L + Sal) or apocynin (hD5F173L + APO) for 4 weeks, starting at 3 months of age (n = 12 mice/group). PLN and SERCA2 expression was semiquantified by western blot. D–F: BAL2 and BAX expression was semiquantified by western blot. *P < 0.05 vs. hD5WT mice; #P < 0.05 vs. hD5F173L + Sal.

Fig. 8. D5R deficiency induces cardiomyocyte injury that is aggravated by Nrf2 inhibition. A: and B: H9c2-D5WT and H9c2-D5F173L cells were treated with ML385 (1 μM), an inhibitor of Nrf2, for 12 h. Cytosol BAX (B) and BAL2 (C) expressions are increased by ML385 treatment. Red: BAX; Blue: DAPI; and Green: BAL2.

Fig. 9. D5R functional deficiency upregulates p-JNK and p-ERK1/2. Proteins were extracted from hearts of 3-month-old cardiac-specific hD5WT-TG (hD5WT) and hD5F173L-TG (hD5F173) and 4-month-old cardiac-specific hD5F173 mice treated with saline (hD5F173L + Sal) or apocynin (hD5F173L + APO) for 4 weeks, starting at 3 months of age (n = 12 mice/group). Protein expression was semiquantified by western blot using anti-p-JNK and anti-p-ERK1/2. A: and B: p-JNK, A: and C: p-ERK1/2, (D) p-P38 (n = 10 mice/group). The data were normalized with total ERK1/2, total JNK, and total P38; *P < 0.05 vs. hD5WT mice; #P < 0.05 vs. hD5F173L + Sal.

Fig. 10. JNK pathway is downstream of Nrf2. H9c2-D5WT and H9c2-D5F173L cells were treated with ML385 (1 μM), an inhibitor of Nrf2, or SP600125 (10 μM), an inhibitor of JNK, for 12 h. JNK inhibition does not affect Nrf2 expression (A and C). However, Nrf2 inhibition (A, B, D) increases further the D5R functional deficiency-induced increase in JNK in H9c2-D5F173L cells. *P < 0.05 vs. untreated H9c2-D5WT cells. #P < 0.05 vs. untreated H9c2-D5F173L cells. n = 8/group.

Fig. 11. D5R mutation induces dilated cardiomyopathy that is related to oxidative stress. D5R deficiency causes an increase in NADPH oxidase activity (I), and prevents the translocation of Nrf2 into the nucleus (II), resulting in a failure of the transcription of a number of antioxidative genes via masculoaponeurotic fibrosarcoma (MAF) and antioxidant response elements (ARE). Antioxidant proteins include NAD(P)H quinone 1 dehydrogenase (NQO1) and heme oxygenase 1 (HO1). Oxidative stress accelerates the signaling of the ERK1/2 and JNK pathway, impairing the balance between apoptosis (BCL2 associated X, apoptosis regulator [BAX]) and anti-apoptosis (B aggressive lymphoma protein 2 (BAL2, PARP14 gene). Phospholamban (PLN) is also stimulated which inhibits cardiac muscle sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, encoded by ATP2A2), which in turn causes cardiac remodeling and finally, dilated cardiomyopathy. D5R deficiency also allows Kelch-like ECH Associated Protein 1 (Keap1) to target Nrf2 in the cytoplasm for ubiquitination and degradation in the proteasome.

Table S1. Primer sequence for real-time PCR. The mRNA expression levels of mitochondrial genes were examined by Real-time PCR using the primer pairs with the sequences below. m: mouse; h: human.