A Seven Amino Acid Long Region in the Cardiac Cav1.2, Encompassing Ser1487, Is Essential for Mice Embryonic Development and “Fight or Flight” Responses

Figures

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  Figure 1. Probability of survival and THW/TL measurements in S1487A, S1487E, and 7aa del mice. Expression of the S1487A mutation resulted in viable pups (A) and no changes in THW/TL (B) in neonatal mice. Mice homozygote for the S1487E mutation were embryonic lethal (C) and developed an increased THW/TL ratio (D). The homozygote 7aa del mutation was also embryonic lethal (E). Analysis of THW/TL ratio in 52-week-old surviving mice showed increased THW/TL ratio in S1487E+/− mice compared to all other genotypes (F). THW/TL comparisons in neonates were performed using one-way ANOVA and in adults using Welch’s ANOVA with Dunnet’s post hoc test for multiple comparisons. The number of samples in each group is labelled within the graphs. (G) Representative neonatal hearts excised from the genotypes as shown. Scale bar = 4 mm. (* = vs wt, # = vs S1487A+/+, and + = vs 7aa del+/−). * p < 0.05, **** p < 0.0001 (THW/TL: total heart weight to tibia length ratio). Symbols denote group comparisons and number of symbols denotes level of significance.

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  Figure 2. Structural analysis of the effects of the 7aa deletion and S1487E mutations. (A) Cryo-EM structure of human Cav1.2 (pdb: 8EOG) showing the various subunits. The positions of the EF-hand (beige) interacting with the III-IV linker (pink) are shown. The inset shows a close-up of the region, with the 7 amino acids (residues 1483–1489) affected by the deletion in black. Select residues within this linker and surrounding areas are labelled for reference (B) Alphafold model of mouse Cav1.2. The interactions are similar to those observed in the experimental structure in panel A. Two residues in the IVS6 segment (Y1481 and L1482) are highlighted in green (C) Alphafold model of mouse Cav1.2 lacking the 7 residues affected by the 7aa deletion. This shows that several helical residues in IVS6 need to unwind to provide a new linker, as otherwise the EF-hand domain would clash with the transmembrane region. (D) Alphafold model of mouse Cav1.2 containing S1487E mutation (blue).

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  Figure 3. Mutations in S1487 and deletion of the 7aa region alter echocardiography parameters. (A) Representative echocardiograms. Scale bar = 25 mm/0.125 s. (B–I) Echocardiography parameters confirm the hypertrophic phenotype associated with S1487E+/− model (panel (A) bottom left) and slightly dilated 7aa del+/− hearts (panel (A) bottom right). Data reported as mean ± SEM. Multiple comparisons were performed using one-way ANOVA with Kruskal–Wallis test and Dunn’s post hoc test. Number of mice for each group is labelled within the graphs. Abbreviations: IVSd: interventricular septum thickness during diastole; IVSs: interventricular septum thickness during systole; LVIDd: left ventricular internal diameter during diastole; LVIDs: left ventricular internal diameter during systole; LVPWd: left ventricular posterior wall thickness during diastole; LVPWs: left ventricular posterior wall thickness during systole; EF%: ejection fraction; FS%: fractional shortening (* = vs wt, # = vs S1487A+/+, and + = vs 7aa del+/−); * p < 0.05, ** p < 0.01, **** p < 0.0001. Symbols denote group comparisons and number of symbols denotes level of significance.

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  Figure 4. NFAT3, GATA4, and GSK3 pathways are responsible for hypertrophy observed in S1487E+/− mutant model. (A) Representative Western blots and (B) quantitative analysis as mean ± SEM optical density on densitometer shown for each signaling pathway. Statistical analysis using one-way ANOVA with Dunnett’s post hoc test. * p < 0.05, ** p < 0.01.

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  Figure 5. S1487A+/+ mice are less responsive to acute β-adrenergic receptor stimulation. (A) Representative echocardiograms. Scale bar = 25 mm/0.125 s. (B–I) Echocardiography results depicting the fold change in each parameter in response to acute β-adrenergic receptor stimulation through treatment with ISO. Multiple comparisons were performed using one-way ANOVA with Kruskal–Wallis test and Dunn’s post hoc test. Numbers of mice for each group are labelled within the graphs. Abbreviations: IVSd: interventricular septum thickness during diastole; IVSs: interventricular septum thickness during systole; LVIDd: left ventricular internal diameter during diastole; LVIDs: left ventricular internal diameter during systole; LVPWd: left ventricular posterior wall thickness during diastole; LVPWs: left ventricular posterior wall thickness during systole; EF%: ejection fraction; FS%: fractional shortening. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. S1487A+/+ mice are less responsive to chronic β-adrenergic receptor stimulation. (A) Representative echocardiograms. Scale bar = 25 mm/0.125 s. (B–I) Echocardiography results depicting the fold change in each parameter in response to chronic β-adrenergic receptor stimulation following treatment with ISO (A–H). Multiple comparisons were performed using one-way ANOVA with Kruskal–Wallis test and Dunn’s post hoc test. Number of mice for each group are labelled within the graphs. Abbreviations: IVSd: interventricular septum thickness during diastole; IVSs: interventricular septum thickness during systole; LVIDd: left ventricular internal diameter during diastole; LVIDs: left ventricular internal diameter during systole; LVPWd: left ventricular posterior wall thickness during diastole; LVPWs: left ventricular posterior wall thickness during systole; EF%: ejection fraction; FS%: fractional shortening. * p < 0.05, *** p < 0.001, **** p < 0.0001.

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  Figure 7. β-adrenergic receptor activation alters Cav1.2 kinetics and calcium transients in wt but not S1487A+/+, and S1487E+/− cardiac myocytes. (A) Representative ICaL traces and I/V curves (inset) before and after ISO and proof of Cav1.2 current by Nisoldipine. (B) Fold change from basal in ICaL and biophysical parameters that occurred following exposure to ISO. Mean ± SEM; comparisons were performed using one-way ANOVA with Kruskal–Wallis test with Dunn’s post hoc test. n = no. of cardiomyocytes (C) Analysis of Fluo-4 fluorescence in myocytes from wt, S1487A+/+, and S1487E+/− hearts’ average calcium transients elicited with Fluo-4 in wt, S1487A+/+, and S1487E+/− mutant cardiomyocytes (N = no. of mice, n = no. of cardiomyocytes) and relative increase in F/FO (presented as mean ± SEM) (inset); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 8. PKA phosphorylation at S1487 is necessary for increased IBa through the Cav1.2 channel. (A) Representative IBa elicited through step depolarization to 10 mV from the holding potential of −80 mV recorded before/after forskolin addition in wt-Cav1.2 (no Rad) (left), wt-Cav1.2+Rad (black/gray) (middle), and S1487A-Cav1.2+Rad (dark red/red) (right) in heterologous expressed channels in HEK cells. (B) (left) Time courses of IBa fold changes before and after 5 μM forskolin addition. Forskolin was added at time 0 s. Two-way ANOVA mixed effect followed by Šídák’s multiple comparisons test. (right) Corresponding forskolin-induced IBa fold changes. Unpaired two-way Mann–Whitney t-test. (C) I/V curves and corresponding fractional activations. (D) All parameters represented in wt-Cav1.2+Rad (left) and S1487A-Cav1.2+Rad (right) and all recorded before and after 5 μM forskolin addition. Data are represented as mean ± SEM. Two-way ANOVA followed by Sidák’s multiple comparisons test and paired 2-tailed t-test; * p < 0.05, ** p < 0.01, **** p < 0.0001. No. of cells studied is shown in brackets.