Casein Kinase 2 Regulates the Intrinsic Activity of L-Type Calcium Currents in Cardiomyocytes

Figures

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  Figure 1. Functional characterization of LTCC currents in neonatal cardiomyocytes overexpressing CaM WT or variants. Representative LTCC current traces are recorded from neonatal rat cardiomyocytes under four conditions: with native/endogenous CaM (A), overexpressing CaM WT (B), CaM S81A (C) or CaM S81D (D). Following a 40 ms prepulse to −40 mV to inactivate fast Na+ and T-type Ca2+ currents, L-type Ca2+ currents were elicited by depolarizing voltage pulses ranging from −60 to +65 mV, starting from a holding potential of –80 mV (protocol shown in the inset above the current traces). The time scale is set to 100 ms, and the current density scale is 10 pA/pF throughout. The corresponding peak current densities were plotted against the applied voltages and fitted by a BoltzIV equation (E). The current density increased from −22 ± 6 pA/PF in control (native CaM) to −31 ± 6 pA/pF in overexpressing CaM WT (p < 0.01). The overexpression of phosphoresistant CaM S81A effectively nullified the upregulation of LTCC currents induced by CaM WT, leading to a significant reduction in peak current density to −9 ± 3 pA/pF (p < 0.01). Conversely, the overexpression of phosphomimetic CaM S81D led to a…

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  Figure 2. Impact of selective CK2 inhibitor TBB on LTCC currents in neonatal cardiomyocytes. Representative LTCC current traces were recorded from neonatal cardiomyocytes treated with 0.5 µM TBB under 4 conditions: with native/endogenous CaM (Aa), overexpressing CaM WT (Ba), CaM S81D (Ca) or CaM S81A (Da). In cells expressing native/endogenous CaM, TBB treatment induced a significant 80% reduction in peak current density, from −22 ± 6 pA/pF in control to −4 ± 2 pA/pF post-treatment (p < 0.01), as shown in (Ab). Similarly, TBB treatment significantly reduced the enhanced LTCC currents induced by overexpressing CaM WT (as shown in Figure 1B), resulting in an approximately 90% reduction in peak current density, from −31 ± 6 pA/pF under control conditions to −3.9 ± 1 pA/pF post-treatment (p < 0.01), as depicted in (Bb). Likewise, the LTCC current significantly increased by overexpressing the phosphomimetic CaM S81D (as shown in Figure 1D), which was markedly reduced following TBB treatment, achieving a peak current density of −3 ± 1 pA/pF (p < 0.01), as depicted in (Cb). For cells overexpressing the phosphoresistant CaM S81A (as shown in Figure 1C), TBB treatment further decreased the already…

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  Figure 3. Western blot analysis of cardiomyocyte protein samples. Lanes 1–3 contain control cardiomyocyte lysates, while lanes 4–9 contain total protein lysates from cardiomyocytes transfected with siRNA directed toward CK2. These wells contain proteins from transfected cardiomyocytes sourced from 5 different culture preparations. The transfections and protein extraction procedures were performed over the course of several months to ensure consistency and reproducibility. The membrane was incubated with primary Anti-Casein Kinase 2α (1: 1000) and secondary Anti-Rabbit (1: 10,000) antibodies; after, it was stripped and reprobed with Anti-GAPDH (1: 10,000) and secondary Anti-Rabbit (1: 10,000) antibodies to confirm protein loading. Protein bands were detected using enhanced chemiluminescence (ECL) and visualized by the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). Cardiomyocytes exposed to the siRNA showed a 89.6 ± 3.2% (mean ± SD) decrease in the band intensity of the CK2 protein when normalized to the expression of the housekeeping protein GAPDH.

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  Figure 4. Functional characterization of LTCC currents in neonatal cardiomyocytes post-Csnk2α1 gene silencing elucidated the pivotal role of CK2 in phosphorylating both native and heterologous expressed CaM. (Aa) Representative LTCC current trace recorded from cells expressing native/endogenous CaM. (Ab,Ac) Representative LTCC current traces from cells transfected with either the specific Csnk2α1 siRNA or universal negative control siRNA. In cells where the Csnk2α1 gene was silenced, the LTCC current density significantly decreased to −7 ± 3 pA/pF (p < 0.01), in contrast to cells transfected with the universal negative control siRNA, which showed no significant change. (Ba–Bc) LTCC current traces from cells overexpressing CaM WT under the same experimental conditions as those in (Aa–Ac). Subsequent to Csnk2α1 gene silencing, the LTCC current density decreased to only 20% of the control level, with a value of −6 ± 2 pA/pF (p < 0.01), whereas no significant change in current density was observed in cells transfected with the universal negative control siRNA. The corresponding current densities were plotted against the applied voltages and fitted using the BoltzIV equation as shown in (Ad,Bd).

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  Figure 5. Functional characterization of LTCC currents post-Csnk2α1 gene silencing in neonatal cardiomyocytes overexpressing CaM variants S81A or S81D. (Aa–Ad) LTCC current traces from cells overexpressing CaM S81A under three distinct conditions. (Aa) is the baseline CaM 81A current, while (Ab,Ac) are CaM S81A currents from cells co-transfected with the Csnk2α1 siRNA or the universal negative control siRNA, respectively. The corresponding current–voltage are displayed in (Ad). Silencing the Csnk2α1 gene led to a significant reduction in the CaM S81A current density from −9 ± 3 pA/pF in the control to −5 ± 2 pA/pF (p < 0.01). Conversely, cells transfected with the universal negative control siRNA showed no significant change in current density. (Ba–Bd) illustrate LTCC current traces recorded from cells overexpressing CaM S81D under the same conditions described for (Aa–Ac). In the Csnk2α1 gene-silenced cells, the CaM S81D current density dropped to just 15% of the control level, reaching −5 ± 2 pA/pF (p < 0.01). In contrast, cells transfected with the universal negative control siRNA exhibited no notable change in current density.

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  Figure 6. CK2 phosphorylates CaM at multiple target sites. (Aa,Ab) Representative LTCC current traces from cells overexpressing the phosphoresistant CaM T79A/S81A/S101A with or without Csnk2α1 gene silencing. Overexpression of CaM T79A/S81A/S101A resulted in a significant nullification of the LTCC currents, leading to a dramatic reduction in peak current density to −6 ± 2 pA/pF. This reduction is notable when compared to cells expressing native CaM (p < 0.01) or cells overexpressing CaM WT (p < 0.01), as depicted in Figure 1A,B. (AC). Knocking out the Csnk2α1 gene did not induced any further reduction in the LTCC current, when compared to cells overexpressing single phosphoresistant CaM T79A or S81A, maintaining a current density of −6 ± 2 pA/pF. (Ba,Bb) LTCC current traces from cells overexpressing phosphomimetic CaM T79D/S81D/S101D with or without Csnk2α1 gene silencing. (Bc) The overexpression of phosphomimetic CaM T79D/S81D/S101D led to significantly larger LTCCs compared with the results obtained with native CaM or after transfection with CaM WT, T79D, or S81D, achieving a peak current density of −51 ± 5 pA/pF (p < 0.01). Csnk2α1 silencing failed to obliterate LTCCs under this…

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  Figure 7. Catecholamines (NE and ISO) promote the activity of LTCCs. (A,B) Representative LTCC current traces recorded from neonatal rat cardiomyocytes following treatment with 1 µM NE or 100 nM ISO. (C) The current–voltage relationships under these conditions with control (native CaM) and overexpressing CaM WT included for comparison. NE treatment significantly increased the current density from −22 ± 6 pA/pF (control) to −35 ± 5 pA/pF (p < 0.01). Similarly, ISO treatment resulted in a current density increase to −33 ± 3 pA/pF (p < 0.01).

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  Figure 8. Knocking out the Csnk2α1 gene does not abrogate the β-adrenergic stimulation of LTCC activity. (Aa). The augmentation of LTCC currents by overexpressing CaM WT, as demonstrated in Figure 1B, was potentiated by NE treatment, leading to a peak current density of −48 ± 8 pA/pF (p < 0.01). (Ab) indicates that Csnk2α1 gene silencing only partially inhibited the LTCC currents post-NE treatment, leaving a residual current density of −19 ± 4 pA/pF, although it remains significantly reduced compared to CaM WT (p < 0.01). The corresponding current–voltage relationships are detailed in (Ac), which also includes the I–V curves from cells overexpressing CaM WT alone or subjected to Csnk2α1 gene silencing for comparison. (Ba,Bb) LTCC current traces from cells overexpressing CaM S81D, with or without Csnk2α1 gene silencing, subjected to NE treatment. The upregulation of LTCC currents by overexpressing CaM S81D, as shown in Figure 1D, was further amplified by NE treatment, leading to a peak current density of −52 ± 8 pA/pF (p < 0.01). Csnk2α1 gene silencing partially inhibited these currents, resulting a residual current density of −15 ± 2 pA/pF (p < 0.01), which is significantly lower than in…