Cavβ2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes

Abstract

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

Calcium-dependent inactivation controls cardiac L-type Ca2+ currents under β-adrenergic stimulation

“…6 C) revealed changes in the overshoot (Fig. 6 D) and dV/dt max (34.7 ± 9.8 and 74.3 ± 6.3 V/ms, respectively) when compared with RFP-overexpressing cardiomyocytes, likely as a result of increasing the number of channels at the plasma membrane (Moreno et al, 2015). Together, these results demonstrate that VDI is also instrumental in shaping the AP.…”

“…All studies were done in accordance and with the approval of the Institutional Bioethical Committee of Universidad de Chile. Cardiomyocytes were isolated enzymatically from neonatal Sprague-Dawley rats (P0-1), as previously described (Hermosilla et al, 2011; Moreno et al, 2015). Briefly, immediately after euthanasia, hearts were removed and minced in Hanks’ solution (in mM): 116 NaCl, 5.4 KCl, 0.8 NaH 2 PO 4 , 0.8 MgS0 4 , 5.6 glucose, 20 HEPES (pH 7.4, adjusted with NaOH).…”

Prolonged AT1R activation induces CaV1.2 channel internalization in rat cardiomyocytes

“…As shown, calcium transients from rat cardiomyocytes exposed to AngII displayed a faster transient decay as compared to control (extracted from single exponential fits to the Ca 2+ transient decay; Fig. 7D ) suggesting that AngII also modifies SERCA activity 28 . As expected from the reduced L-type calcium currents recorded, decreased calcium transient amplitudes are evident in rat cardiomyocytes treated with AngII (63.7 ± 1.3% reduction, Fig.…”

“…Ca V β 2b channel subunit (AF423193) was cloned as described in Moreno et al . 28 . The HA epitope inserted into the extracellular S5-H5 loop of domain II of Ca V 1.2 for the HA-tagged Ca V 1.2 construct is described previously 29 .…”

“…Cardiomyocytes were incubated with Fluo-4 (Molecular Probes, 1 µM) as described in ref. 28 and thoroughly washed with external solution (mM): 100 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 90 sorbitol, 5 glucose and 10 HEPES (pH 7.4 adjusted with Tris-Base). Fluo-4 loaded cardiomyocytes were excited at 480 nm and the fluorescence emission at 510 nm was collected and recorded at 12 Hz.…”