Cavß2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes.

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 CaVß2. To date, five distinct CaVß2 transcriptional start site (TSS) variants (CaVß2a-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 CaVß2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVß2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVß2 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 CaVß2a and smaller in CaVß2d. 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 CaVß2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVß2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

Single cell migration profiling on a microenvironmentally tunable hydrogel microstructure device that enables stem cell potency evaluation.

Cell migration is a key function in a myriad of physiological events and disease conditions. Efficient, quick and descriptive profiling of migration behaviour in response to different treatments or conditions is highly desirable in a series of applications, ranging from fundamental studies of the migration mechanism to drug discovery and cell therapy. This investigation applied the use of methacrylamide gelatin (GelMA) to microfabricate migration lanes based on GelMA hydrogel with encapsulated migration stimuli and structural stability under culture medium conditions, providing the possibility of tailoring the microenvironment during cell-based assays. The actual device provides 3D topography, cell localization and a few step protocol, allowing the quick evaluation and quantification of individual migrated distances of a cell sample by an ImageJ plugin for automated microscopy processing. The detailed profiling of migration behaviour given by the new device has demonstrated a broader assay sensitivity compared to other migration assays and higher versatility to study cell migration in different settings of applications. In this study, parametric information extracted from the migration profiling was successfully used to develop predictive models of immunosuppressive cell function that could be applied as a potency test for mesenchymal stem cells.

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

The cardiac L-type calcium channel is a multi-subunit complex that requires co-assembling of the pore-forming subunit CaV1.2 with auxiliary subunits CaVα2δ and CaVß. Its traffic has been shown to be controlled by these subunits and by the activation of various G-protein coupled receptors (GPCR). Here, we explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking. Bioluminescence Resonance Energy Transfer (BRET) assay between ß-arrestin and L-type channels in angiotensin II-stimulated cells was used to assess the functional consequence of AT1R activation, while immunofluorescence of adult rat cardiomyocytes revealed the effects of GPCR activation on CaV1.2 trafficking. Angiotensin II exposure results in ß-arrestin1 recruitment to the channel complex and an apparent loss of CaV1.2 immunostaining at the T-tubules. Accordingly, angiotensin II stimulation causes a decrease in L-type current, Ca2+ transients and myocyte contractility, together with a faster repolarization phase of action potentials. Our results demonstrate that prolonged AT1R activation induces ß-arrestin1 recruitment and the subsequent internalization of CaV1.2 channels with a half-dose of AngII on the order of 100 nM, suggesting that this effect depends on local renin-angiotensin system. This novel AT1R-dependent CaV1.2-trafficking modulation likely contributes to angiotensin II-mediated cardiac remodeling.