Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats

Figures

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  Figure 1. Characteristics of female Cacna1c+/− rats. (A) Heart weight to body weight ratio (HW/BW) and LV and RV weight normalized to tibia length (TL) did not differ between WT (+/+) and Cacna1c+/− (+/−) rats. Circles represent individual animals: N = 8 (WT); N = 8 (Cacna1c+/−); Student’s t-test, p-values as indicated. (B) Expression of L-type Ca2+ channels in Cacna1c+/− LV tissue. Western Blot analysis of expression of Cav1.2 (normalized to GAPDH) in Cacna1c+/− vs. WT (+/+) LV myocardium. Original immunoblot images show four LV samples from each genotype. Antibodies used are listed in Tables S1 and S2. First, the expression of Cav1.2 was normalized to the expression of GAPDH in each sample. Next, the Cav1.2/GAPDH ratios were averaged for the four WT samples on each Western Blot so that the mean value for WT amounts to 100%. Finally, the Cav1.2/GAPDH ratios of the four Cacna1c+/− samples were normalized to the averaged ratio of the four WT samples. Circles represent individual tissue samples. N = 8 for each genotype. Student’s t-test, p-values as indicated. In Cacna1c+/−, the expression of Cav1.2 was reduced. All original Western Blot images from this series are shown in Supplementary…

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  Figure 2. Characteristics of electrically stimulated CaTs, SR Ca2+ load, and fractional release in WT (+/+) and Cacna1c+/− (+/−) ventricular myocytes. Data from linescan imaging (A) and 2D confocal imaging (B) are shown. From left to right: diastolic Ca2+, systolic Ca2+, CaT amplitude, rise time, and tau of CaT decay. Circles represent individual myocytes: (A) n = 85, N = 8 (WT); n = 145, N = 14 (Cacna1c+/−); (B) n = 32, N = 3 (WT); n = 59, N = 6 (Cacna1c+/−); Mann–Whitney U-test, p-values as indicated. (C) Data from linescan imaging. Example trace of the caffeine protocol (left) used to estimate SR Ca2+ load and fractional release. Three electrically stimulated CaTs are followed by bolus application of caffeine (20 mM). Summarized data to the right: caffeine-induced CaT amplitude, fractional release, and tau of caffeine transient decay. Circles represent individual myocytes: n = 12, N = 4 (WT); n = 20, N = 3 (Cacna1c+/−); the number of cells is reduced for tau of decay (n = 11 WT; n = 16 Cacna1c+/−) because one of the following occurred: the recording ended before the decay was completed; the decay did not follow an exponential trend; the cell moved out of the field of view after caffeine…

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  Figure 3. Expression and phosphorylation of major Ca2+ handling proteins in WT and Cacna1c+/− LV myocardium. (A) Original Western Blot images of four Cacna1c+/− (+/−) and 4 WT (+/+) samples. Proteins used for normalization (GAPDH, actin) and derived from the same membrane are shown below the protein of interest. Note that some of the loading controls are identical (see GAPDH control for CSQ and pS2808 and actin control for PLB and pS16), as the respective proteins were derived from the same membrane, and in case of PLB/pS16 stripping was performed. (B) Summarized data normalized to the expression of WT. Expression of CSQ, NCX, and SERCA2a (top row), expression of RyR2 and phosphorylation of RyR2 at S2808 (pS2808) and S2814 (pS2814) (middle), and expression of PLB and phosphorylation of PLB at S16 (pS16) and T17 (pT17) (bottom row). In Cacna1c+/−, expression of NCX and SERCA2a is up-regulated, and phosphorylation of RyR2 at S2808 is elevated. Circles represent individual animals: N = 8 (WT); N = 8 (Cacna1c+/−); Student’s t-test, p-values as indicated. Further information and all original Western Blot images from this series are shown in Supplementary Material. *, p < 0.05; *** p < 0.001.

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  Figure 4. Effects of isoprenaline (ISO) on electrically stimulated CaTs, SR Ca2+ load, and fractional release in WT and Cacna1c+/− ventricular myocytes. Representative CaTs with their respective linescan images (edited as described in Supplementary Methods) of a WT (A) and Cacna1c+/− (B) ventricular myocyte before and during exposure to 100 nM ISO. ISO increased and accelerated CaTs in both cells. (C–F) Comparison of CaT characteristics between WT (+/+) and Cacna1c+/− (+/−) ventricular myocytes before (left panels) and during ISO exposure (right panels): (C) diastolic Ca2+, (D) systolic Ca2+, (E) CaT amplitude, (F) tau of CaT decay. In the presence of ISO, systolic Ca2+ and CaT amplitude were significantly lower in Cacna1c+/−. Circles represent individual cardiomyocytes: n = 22, N = 5 (WT); n = 34, N = 8 (Cacna1c+/−); Mann–Whitney U-test, p-values as indicated. (G) The amplitude of caffeine-induced CaTs (a measure for SR Ca2+ load), fractional SR Ca2+ release, and tau of decay of the caffeine-induced CaT in the presence of ISO. Fractional release in the presence of ISO is smaller in Cacna1c+/− myocytes, while SR Ca2+ load and tau of decay are comparable between genotypes. Circles represent…

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  Figure 5. Concentration dependence of isoprenaline (ISO) effect on CaT amplitude in WT and Cacna1c+/− ventricular myocytes. (A) Example CaT traces from one WT (left) and one Cacna1c+/− (right) ventricular myocyte treated with increasing concentrations of ISO (as indicated). (B) Resulting concentration-response curves for WT (+/+, squares) and Cacna1c+/− (+/−, circles) ventricular myocytes. Values for CaT amplitude were rundown-corrected and normalized (0% = CaT amplitude before ISO; 100% = CaT amplitude at 100 nM ISO) as detailed in Supplementary Methods. Number of cells/animals (WT/Cacna1c+/−): 0 nM: n = 52/55, N = 10/10, 1 nM: n = 27/17, N = 5/4, 3 nM: n = 17/26, N = 4/6, 10 nM: n = 22/24, N = 5/6, 30 nM: n = 21/31, N = 5/6, 100 nM: n = 52/55, N = 10/10. In Cacna1c+/− myocytes, the concentration-response curve was shifted to the right. EC50 values: 4.1 nM (WT, +/+) and 13.0 nM (Cacna1c+/−, +/−).

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  Figure 6. Phosphorylation of RyR2 in WT (+/+) and Cacna1c+/− (+/−) LV myocardium treated with 100 nM isoprenaline (ISO). Untreated LV myocardium served as control (Ctr). Phosphorylation of RyR2 at S2808 and at S2814. Original immunoblot images (A) and summarized data (B,C) is shown. Phosphorylation increases of RyR2 in ISO-treated hearts were stronger at (B) S2808 and (C) S2814 in WT compared to Cacna1c+/−. One representative Western Blot is presented for each protein/phosphorylation site and genotype with four Ctrl and four ISO-treated samples. GAPDH was used for normalization. For immunoblot images in (A), i.e., RyR2 and pS2808, GAPDH is the same for both proteins in each genotype because stripping was performed. Data is normalized to the average value of Ctr (=100%). Circles represent individual animals: N = 8 for each genotype (WT, Cacna1c+/−) and treatment (Ctrl vs. ISO); one-way ANOVA followed by Tukey’s post hoc test, p-values as indicated. All original Western Blot images from this series are shown in Supplementary Material. **, p < 0.01; ***, p < 0.001.

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  Figure 7. Sarcolemmal Ca2+ influx in WT (+/+) and Cacna1c+/− (+/−) ventricular myocytes before and during exposure to 100 nM isoprenaline (ISO). (A) Representative CaTs with their respective linescan images (edited as described in Supplementary Methods) of thapsigargin-treated myocytes of WT (left) and Cacna1c+/− (right) before and during exposure to 100 nM ISO. (B) CaT of Cacna1c+/− and WT myocytes did not differ after treatment with thapsigargin. SR depletion and inhibition of SERCA are evident from the small CaT amplitude and slow tau of decay. (C) ISO leads to a significantly greater increase in CaT amplitude and systolic Ca in WT myocytes. Circles represent individual cardiomyocytes: (B): n = 49/47, N = 5/5 (WT/Cacna1c+/−); tau of decay: n = 44/42 (WT/Cacna1c+/−). (C): n = 49/47, N = 5/5 (WT/Cacna1c+/−); tau of decay: n = 47/35 (WT/Cacna1c+/−). Mann–Whitney U test, p-values as indicated. Cell counts for tau of decay are reduced because, in some cases, the decline did not follow an exponential trend. *, p < 0.05; **, p < 0.01; ***, p < 0.001.