Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by β-adrenergic stimulation

Figures

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  Figure 1. Expression and localization of B56α in the heart.A, quantification of the B56α protein expression in hearts of WT and heterozygous (HZ) mice (left panel; ∗p < 0.05 versus WT; t test; N = hearts). A representative immunoblot of all genotypes is shown (right panel). Individual samples were used. Calsequestrin (CSQ) served as a reference protein. Molecular weight markers were included on the left side of the blots. B, photomicrographs showing detailed analysis of isolated cardiomyocytes from WT, HZ, and KO mouse hearts by confocal microscopy. Images were taken with the same microscope settings for all genotypes. Red fluorescence staining represents B56α. The control (Ctr.) was performed without the primary antibody raised against B56α. Note the loss of the B56α signal in KO cardiomyocytes. Detailed analysis (small insets) revealed both a diffuse and sarcomeric pattern in the distribution of B56α in WT and HZ suggesting a cytosolic and myofilamental localization.

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  Figure 2. Loss of B56α is accompanied by a lower PP2A activity.A, quantification of the protein expression of regulatory B and B′ subunits in hearts of WT and homozygous (KO) mice (left panel; ∗p < 0.05 versus WT; t test; N = hearts). Shown are representative Western blots (right panel). Calsequestrin (CSQ) served as a reference protein. One set of samples was available for detection of proteins that was probed with the CSQ antibody and then used for both Figures 2 and 4. Molecular weight markers were included on the left side of the blots. B, protein expression of structural (PP2A-Aα), catalytic (PP1-Cα & PP2A-Cα), and inhibitory (PP1-I1 and -I2) subunits in cardiac homogenates of WT and KO mice (left panel; ∗p < 0.05 versus WT; t test; N = hearts). Representative immunoblots are depicted at the right-hand side. CSQ served as a reference protein. The same CSQ loading control was used for all other blots. Molecular weight markers were included on the left side of the blots. C, PP activity was determined in heart samples of WT and KO mice under basal conditions and after acute administration with isoprenaline (ISO). PP1 and PP2A activities were obtained by incubation of heart samples with 3…

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  Figure 3. Improved basal left-ventricular pressure and relaxation in catheterized KO mice. Shown are the contractile data in anaesthetized left-ventricular catheterized WT and KO mice in the absence and presence of increasing doses of isoprenaline (ISO). A, heart rate in beats per minute. B, maximum left-ventricular pressure in mmHg. C, rate of contraction (dP/dtmax) in mmHg/s. D, rate of relaxation (dP/dtmin) in mmHg/s. E, cardiac output in RVU/min. F, ejection fraction in percentage (∗p < 0.05 versus WT: t test; &p < 0.05 versus WT: two-way ANOVA; #p < 0.05 versus WT: Holm–Sidak posthoc test; N = 7 WT mice; N = 8 KO mice).

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  Figure 4. Increased phosphorylation levels of Ca2+regulatory and myofilament proteins in KO hearts.A, representative immunoblots of cardiac Ca2+ regulatory and contractile proteins in WT and KO mice. Protein phosphorylation was measured in the absence (basal) and presence of isoprenaline (ISO). Calsequestrin (CSQ) served as a reference protein. One set of samples was available for detection of proteins that was probed with the CSQ antibody and then used for both Figures 2 and 4. Molecular weight markers were included on the left side of the blots. B, quantification of protein phosphorylation of sarcoplasmic reticulum (PLN, phospholamban; RyR2, ryanodine receptor type 2) and sarcolemmal proteins (Cav1.2, L-type Ca2+ channel) at basal (Bs) and β-adrenergic stimulated (ISO) conditions. Normalization of phosphorylation levels was performed to the nonphosphorylated individual protein. Subsequently, the protein was adjusted to WT levels both under basal and stimulated conditions. C, quantification of phosphorylated myofilament proteins (MyBP-C, myosin-binding protein C; MLC2, myosin light chain 2; TnI, troponin inhibitor) (∗p < 0.05 versus WT; t test; N = hearts).

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  Figure 5. Depressed contraction and Ca2+cycling but improved β-adrenergic response in KO cardiomyocytes. Myocellular contraction and Ca2+ transient parameters were determined in electrically stimulated (0.5 Hz) cardiomyocytes of WT and KO mice under basal conditions and after stimulation with 1 μM isoprenaline (ISO). A, quantification of maximum sarcomere length (SL) shortening (ΔSL). B, quantification of the time to 50% relengthening. C, peak amplitude of Ca2+ transients. D, shown are the summarized data of the time to 50% decay of the Indo-1 signal (∗p < 0.05 versus WT: t test; n = cardiomyocytes (stated in the columns) of N = 6 WT hearts and N = 6 KO hearts). E, basal and ISO-stimulated Indo-1 ratio and SL were correlated. The slow (mx) and fast (my) slopes (see Table 2) of this relation can be used as indicators of the myofilament Ca2+ sensitivity (Basal: n = 109 cardiomyocytes of 11 WT hearts and n = 123 cardiomyocytes of N = 12 KO hearts; ISO: n = 60 cardiomyocytes of N = 6 WT hearts and n = 60 cardiomyocytes of N = 6 KO hearts).

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  Figure 6. Higher Ca2+spark frequencies after application of isoprenaline in KO cardiomyocytes. Intracellular Ca2+ signaling was investigated in more detail by measurement of SR Ca2+ load and Ca2+ spark characteristics. A, shown are the maximum amplitude (left panel) and decay kinetics (right panel) of caffeine-induced Ca2+ transients in WT and KO cardiomyocytes (∗p < 0.05 versus WT; t test; n = cardiomyocytes (stated in the columns) of N = 6 WT hearts and N = 8 KO hearts). B, illustration of characteristic X-T scans of WT and KO cardiomyocytes after 1 Hz prestimulation and application of 1 μM isoprenaline detecting Ca2+ sparks. C, quantification of data for Ca2+ sparks amplitude (left panel) and frequency (CaSpF, right panel) in cells of both genotypes (∗p < 0.05 versus WT: t test; n = cardiomyocytes (stated in the columns) of N = 6 WT hearts and N = 6 KO hearts). SR, sarcoplasmic reticulum.

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  Figure 7. Reduced ICaLcurrent densities are normalized by β-adrenergic stimulation in KO cardiomyocytes. The effects of ISO on LTCC current parameters were determined in ventricular myocytes. A, current-voltage relations of L-type Ca2+ currents (ICaL) were measured in KO and corresponding WT cardiomyocytes in the absence (Basal) and presence of 1 μM ISO (∗p < 0.05 versus WT: 2-way RM ANOVA; n = cardiomyocytes/N = mice, see Table 3). B, bar graphs show the summarized maximum (upper panel) and normalized (lower panel) ICaL current densities in the 4 groups (∗p < 0.05 versus WT: t test; n = cardiomyocytes (stated in the column) of N = mice, see Table 3). C, steady-state activation of ICaL (VDA, voltage-dependent activation) in basal and ISO-treated WT and KO cardiomyocytes show the voltage dependence of normalized conductance (G) to Gmax. Symbols represent the measured values fitted with Boltzmann function represented. D, shown is the steady-state inactivation of ICaL (VDI, voltage-dependent inactivation) in all 4 groups. E, shown are representative traces of steady-state ICaL, normalized to peak, measured in the absence (left panel) and presence (right panel) of ISO in WT (gray) and KO…

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  Figure 8. Augmented contractile response to increasing stimulation frequencies in KO cardiomyocytes. Graphical representation of Ca2+ transients and myocellular contraction under the influence of increasing stimulation frequencies in WT and KO cardiomyocytes. Data, collected at 0.5 Hz, were set to 100% for both genotypes. The following parameters were determined: A, the peak amplitude of Ca2+ transients. B, the maximum sarcomere length shortening. C, the time to 50% decay of Ca2+ transients. D, the time to 50% relengthening (&p < 0.05 versus WT: two-way ANOVA; #p < 0.05 versus WT: Holm–Sidak post hoc test; n = 49 cardiomyocytes of N = 5 WT hearts and n = 63 cardiomyocytes of N = 6 KO hearts).