Dysregulated Rbfox2 produces aberrant splicing of CaV1.2 calcium channel in diabetes-induced cardiac hypertrophy

Figures

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  Fig. 1. Diabetic hearts show impaired diastolic function and hypertrophic phenotype. (A) Diabetic rat models were established by using the combination of high-fat diet (HFD) and streptozotocin (STZ) (i.p. injection). Control rats received the same volume of vehicle (citrate buffer) and fed with standard diets. After 8 weeks, the cardiac functions were measured by echocardiography and hearts were collected for further investigations. (B) Representative M-mode traces and left ventricle (LV) Doppler images in control and HFD/STZ-treated rats. (C) E/A ratio (ratio of early left ventricular diastolic flow velocity and late left ventricular diastolic flow velocity) was analyzed and presented as a bar chart. n = 6 rats for each group. *P = 0.0121, unpaired t test. (D) Fractional shortening (FS) was also analyzed. n = 6 rats for each group. P = 0.8752, unpaired t test. ns indicates no significant differences. (E) Representative images of HE staining for cardiac tissue from control and HFD/STZ-treated rats. (F) Cardiomyocyte cell surface area (CSA) was analyzed and showed as a bar chart. n = 30 cells from 3 rats for each group. **P < 0.0001, unpaired t test. (G) Rat Nppa, Nppb and Myh7 mRNAs were…

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  Fig. 2. CaV1.2 alternative exon 9* is specifically increased in diabetic heart. (A) The membrane expression of CaV1.2 α1C was detected by Western blotting in heart tissues from control and HFD/STZ-treated rats, Na-K ATPase protein was detected as internal control. The relative band densities were analyzed and normalized to Na-K ATPase. n = 6 rats for each group. *P = 0.0166, unpaired t test. (B) Schematic diagram shows the locations of the PCR primers designed to amplify and detect rat CaV1.2 inclusive of or in the absence of alternative exons in cardiac tissues. Total RNA was extracted from hearts, and PCR products amplified from cDNA libraries were separated on 2.5% agarose gel. Actb mRNA was detected as internal control. Rat Cacna1c mRNAs with exon 8 or 8a were amplified by RT-PCR, followed by digestion with restriction endonuclease BamHI. The value for percent exon 8a inclusion were the lower 2 bands’ intensity divided by the sum of the intensities of upper and lower bands. n = 4 rats for each group. P = 0.4107, unpaired t test. (C) The value for percent exon 9* inclusion was the upper band intensity divided by the summed intensities of upper and lower bands. n = 6 rats for each…

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  Fig. 3. Expression of Rbfox2 is dysregulated in the hearts from diabetic rats. (A) The expression of Rbfox2 in the rat hearts was detected by Western blotting; β-actin was detected as loading control. (B) Relative expression of Rbfox2 was normalized to β-actin. n = 6 rats for each group. **P = 0.0003, unpaired t test. (C) Different from wild-type (WT) Rbfox2, dominant-negative (DN) Rbfox2 is generated by exclusion of exon 6, which encodes half of the RNA recognition motif (RRM). (D) Using specific primers, we determined the WT Rbfox2 mRNA levels of the hearts by real-time RT-PCR; rat Actb mRNA was measured as internal control. n = 7 rats for each group. *P = 0.0171, unpaired t test. (E) DN Rbfox2 mRNA was also checked by real-time RT-PCR. n = 7 rats for each group. *P = 0.0423, unpaired t test with Welch’s correction

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  Fig. 4. Rbfox2 specifically modulates CaV1.2 alternative exon 9* splicing in cardiomyocyte. (A) H9c2 cells were transfected with nontargeting (NT) or Rbfox2 siRNA for 48 h. The endogenous expression of Rbfox2 protein was detected by Western blotting, the β-actin was detected as internal control. PCR product of CaV1.2E9* channel was amplified from cDNA libraries and separated on 2.5% agarose gel. Actb mRNA was detected as internal control. (B) The relative expression of Rbfox2 was normalized to β-actin. (C) The value for percent CaV1.2E9* inclusion was the upper band intensity divided by the summed intensities of upper and lower bands, and presented as a bar chart. (D) H9c2 cells were transfected with an empty vector, WT Rbfox2, DN Rbfox2 or WT plus DN Rbfox2 expression plasmids, nontreated cells were set as negative control (NC). After 48 h incubation, the expression of Rbfox2 protein was detected by Western blotting, the β-actin was detected as internal control. PCR products were separated on 2–3% agarose gel, which was used to check the proportions of CaV1.2E9* channels. (E) Relative expression of Rbfox2 was normalized to β-actin. (F) The proportion of CaV1.2E9* channels were analyzed…

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  Fig. 5. Glycated serum (GS), not D-glucose, decreases Rbfox2 expression but increases CaV1.2E9* channels. (A) NRVMs were treated with mannitol or D-glucose, nontreated cells were set as control. After 48 h incubation, the endogenous expression of Rbfox2 protein was detected by Western blotting, the β-actin was detected as internal control. (B) Relative Rbfox2 expression was normalized with β-actin in differentially-treated cells. n = 4 independent experiments. P = 0.9854, one-way ANOVA followed by a Tukey’s post hoc test. (C) PCR products amplified from cDNA libraries of differentially-treated NRVM were separated on 2.5% agarose gel, and the values of proportion of CaV1.2E9* were analyzed. n = 4 independent experiments. P = 0.9109, one-way ANOVA followed by a Tukey’s post hoc test. (D) Serum levels of advanced glycation end-products (AGEs) were measured by an ELISA kit in control (n = 5) and HFD/STZ-treated rats (n = 6). **P = 0.0042, unpaired t test. (E) The levels of AGEs in the lysate of cardiac tissues from different rats’ models (n = 6 each group) were also measured by ELISA. **P = 0.0027, unpaired t test. (F) NRVMs were treated with 10% non-glycated (NG) or glycated serum (GS) for…

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  Fig. 6. GS application induces hyperpolarized window currents of CaV1.2 channel in NRVMs. (A) NRVMs were treated with 10% NG or GS for 48 h, then the membrane protein was extracted. CaV1.2 α1C subunit was detected, and Na-K ATPase was detected as internal control. (B) Relative expression of CaV1.2 α1C subunit was normalized with Na-K ATPase. n = 3 independent experiments. P = 0.7251, unpaired t test. ns indicates no significant differences. (C) CaV1.2 channel currents were recorded from NRVMs with NG, GS or GS + Gö6983 treatment in 10 mmol/L Ba2+ bath solution. (D) I-V relationship curves of CaV1.2 channel were recorded under different testing potentials in NRVMs. (E) Current densities of CaV1.2 channel in NRVMs treated with NG, GS or GS + Gö6983 were presented. (F) Plots of steady-state activation (SSA) curve of CaV1.2 channel were derived from I-V currents in differentially-treated NRVMs. (G) Plots of the steady-state inactivation (SSI) curve were recorded and analyzed in NRVMs. (H) Window currents were superimposed from SSI (f∞) and SSA (d∞) curves of NG, GS or GS + Gö6983-treated NRVMs

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  Fig. 7. GS application induces cardiomyocyte hypertrophy. (A) NRVMs treated with 10% NG or GS, real-time [Ca2+]i was measured by Ca2+ fluorescence indicator Fluo-4 AM, and the fluorescent intensity was monitored by time series scanning mode under a confocal microscope. Δ[Ca2+]i fluorescence intensities were measured by dividing the changes in the fluorescent signal by the average resting fluorescence. (B) Plots of time course of fluorescent intensity after application with NG (n = 11 cells) or GS (n = 10 cells) in NRVMs from 3 independent experiments, Δ[Ca2+]i was presented as ΔF/F0. *P < 0.05, two-way ANOVA followed by Sidak’s multiple comparisons test. (C) Representative images of NRVMs treated with NG or GS showed immunofluorescence staining by using anti-α-actinin antibody to determine cell surface area (CSA). (D) Analyzed CSA was shown as scatter plots in NG (n = 22 cells) or GS-treated NRVMs (n = 23 cells) from 3 independent experiments. **P < 0.0001, unpaired t test with Welch’s correction. (E) Rat Nppa, Nppb and Myh7 mRNAs were determined by real-time RT-PCR in differentially-treated NRVMs, rat Actb mRNA was detected as internal control. n = 4 independent experiments. *P < 0.05,…

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  Fig. 8. Knockdown of Rbfox2 hyperpolarizes window currents of CaV1.2 channel in NRVMs. (A) The protein expression of Rbfox2 and β-actin were detected in whole-cell lysate of isolated NRVMs by using Western blotting after transfecting with NT or Rbfox2 siRNAs. The membrane protein was also extracted, and membrane expression of CaV1.2 α1C was checked, Na-K ATPase was detected as a membrane loading control. (B) Relative expression level of Rbfox2 was normalized with β-actin in differentially transfected cells, and presented as a bar chart. n = 4 independent experiments. **P < 0.001, one-way ANOVA followed by a Tukey’s post hoc test. (C) Relative CaV1.2 α1C membrane expression was normalized with Na-K ATPase in differentially-transfected cells. n = 4 independent experiments. P = 0.6679, one-way ANOVA followed by a Tukey’s post hoc test. (D) Raw traces of CaV1.2 whole-cell calcium current recorded from NRVMs treated with NT or Rbfox2 siRNA in 10 mmol/L Ba2+ external solution. (E) I-V relationship of calcium channel current recorded under the different testing potential, increased from − 50 to 50 mV in NRVMs transfected with NT or Rbfox2 siRNA. (F) CaV1.2 channel current density in NRVMs was…

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  Fig. 9. Knockdown of Rbfox2 induces cardiomyocyte hypertrophy. (A) NRVMs treated with NT or Rbfox2 siRNAs, real-time [Ca2+]i was measured by Ca2+ fluorescence indicator Fluo-4 AM, Δ[Ca2+]i fluorescence intensities were measured by dividing the changes in the fluorescent signal by the average resting fluorescence. (B) Plots of time course of fluorescent intensity after application with NT (n = 20 cells) or Rbfox2 siRNAs (n = 13 cells) in NRVMs from 3 independent experiments, Δ[Ca2+]i was presented as ΔF/F0. *P < 0.05, two-way ANOVA followed by Sidak’s multiple comparisons test. (C) Representative images of NRVMs transfected with NT or Rbfox2 siRNA, immunofluorescence staining by using anti-a-actinin antibody was applied to determine cell surface area (CSA). (D) Analyzed CSA was shown as a bar chart in NT (n = 18 cells) or Rbfox2 siRNA (n = 18 cells) transfected NRVMs from 3 independent experiments. **P = 0.0005, unpaired t test. (E) Rat Nppa, Nppb and Myh7 mRNAs were determined by real-time RT-PCR in differentially-treated NRVMs, rat Actb mRNA was detected as internal control. n = 3–4 independent experiments. *P < 0.05, **P < 0.01, unpaired t test. (F) Illustration of main findings in…