Characterization of Ca_V1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and Ca_V1.2 exon 33 expressions in human heart failure

Figures & data

Figure 1. Exon 33^+/− cardiomyocyte has similar Ca_V1.2 channel properties with WT cardiomyocyte. (A) The stimulus waveform was used to induce I_Ca of cardiomyocyte, briefly I_Ca was recorded under the different testing potentials, increased from −60 mV to 60 mV (10 mV increase each step) followed by 10 mV depolarizing-pulse to induce steady-state inactivation in cardiomyocytes when using the 1.8 mmol/L Ca²⁺ as charger carrier. (B) Exemplary current traces were recorded from WT (black), exon 33^+/− (blue) or exon 33^−/− (red) cardiomyocytes. (C) Current densities of Ca_V1.2 channels in WT (n = 11 cells, V_0.5 = 2.82±2.3 mV), exon 33^+/− (n = 20 cells, V_0.5 = 3.18±1.7 mV) and exon 33^−/− (n = 18 cells, V_0.5 = −7.28±3.3 mV) cardiomyocytes (*P<0.05 vs. WT, unpaired t test). (D) Normalized current-voltage relationships of Ca_V1.2 channels in WT, exon 33^+/− and exon 33^−/− cardiomyocytes. (E) Steady-state inactivation of Ca_V1.2 channels in WT (n = 16 cells, V_0.5,inact = −20.63±0.44 mV), exon 33^+/− (n = 14 cells, V_0.5,inact = −20.46±0.85 mV) and exon 33^−/− (n = 12 cells, V_0.5,inact = −31.94±0.64 mV) cardiomyocytes.

Figure 2. Exon 33^+/− cardiomyocyte did not show any signs of abnormal excitations. (A) AP waveforms recorded from WT (black), exon 33^+/− (blue) and exon 33^−/− (red) cardiomyocytes. (B) APD after 90% of repolarization (APD_90%) from WT (n = 11 cells), exon 33^+/− (n = 14 cells) and exon 33^−/− (n = 16 cells) (*P<0.05 vs. WT, unpaired t test). (C and D) Detection of EADs in cardiomyocytes of WT (n = 73 cells, 17 mice), exon 33^+/− (n = 24 cells, 5 mice) or exon 33^−/− mice (n = 73 cells, 16 mice) at a 0.5-Hz pacing rate. (P = 0.0139 one-way ANOVA; *P<0.05 vs. WT, Bonferroni post hoc test). (E and F) Detection of autonomous APs in cardiomyocytes from WT (n = 73 cells, 17 mice), exon 33^+/− (n = 24 cells, 5 mice) and exon 33^−/− (n = 73 cells, 16 mice) a 5-Hz pacing rate (P = 0.0052, one-way ANOVA; **P<0.01 vs. WT, Bonferroni post hoc test).

Figure 3. The mRNA expression of Rbfox1/2 and Ca_V1.2 α1_C in human hearts. (A) The mRNA levels of Rbfox1 were measured by real-time RT-PCR from the samples of human normal (NF, black, n = 8), dilated cardiomyopathy (DCM, red, n = 15) and ischemic cardiomyopathy (ICM, blue, n = 10) hearts. The relative expression was normalized by internal expression of human GAPDH and RPLPO mRNA (P = 0.0103, one-way ANOVA; *P<0.05 vs. WT, Bonferroni post hoc test). (B) The mRNA levels of Rbfox2 were also measured by real-time RT-PCR from NF (n = 8), DCM (n = 15) and ICM (n = 10) hearts. (C) Ca_V1.2 α1_C mRNA expression was measured by real-time RT-PCR from NF (n = 8), DCM (n = 15) and ICM (n = 10) hearts (**P<0.01, one-way ANOVA; **P<0.01 vs. WT, Bonferroni post hoc test). The correlations between alternative exon 33 expression and Rbfox1 (D) or Ca_V1.2 α1_C (E) mRNA level in human hearts were analyzed by linear regression, R² refers to the coefficient of determination.