Cavβ2 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 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 Ca2+ 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 Ca2+ 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 Ca2+ influx and Ca2+ 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.
KeywordsCalcium transients Auxiliary subunits Cardiomyocytes L-type calcium current
We are thankful to Rocio K. Finol-Urdaneta, Andrés Stutzin, and Luis Michea for constructive discussion to the manuscript. This work was supported by research grants from Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt) 1120240 to DV, and Fondecyt 1121078 and Millennium Institute on Immunology and Immunotherapy P09-016-F to FS.
D.V. and T.H. designed the project. C.M., T.H., L.T-D., D.M., M.E., P.D., D.S., and D.V. performed the experiments. D.V., T.H., and F.S. analyzed the data. D.V. wrote the manuscript.
Conflict of interest
The authors declare no conflict of interests.
- 6.Cingolani E, Ramirez Correa GA, Kizana E, Murata M, Cho HC, Marban E (2007) Gene therapy to inhibit the calcium channel beta subunit: physiological consequences and pathophysiological effects in models of cardiac hypertrophy. Circ Res 101:166–175. doi: 10.1161/CIRCRESAHA.107.155721 CrossRefPubMedGoogle Scholar
- 7.Colecraft HM, Alseikhan B, Takahashi SX, Chaudhuri D, Mittman S, Yegnasubramanian V, Alvania RS, Johns DC, Marban E, Yue DT (2002) Novel functional properties of Ca(2+) channel beta subunits revealed by their expression in adult rat heart cells. J Physiol 541:435–452PubMedCentralCrossRefPubMedGoogle Scholar
- 8.Chen X, Nakayama H, Zhang X, Ai X, Harris DM, Tang M, Zhang H, Szeto C, Stockbower K, Berretta RM, Eckhart AD, Koch WJ, Molkentin JD, Houser SR (2011) Calcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy. J Mol Cell Cardiol 50:460–470. doi: 10.1016/j.yjmcc.2010.11.012 PubMedCentralCrossRefPubMedGoogle Scholar
- 10.Chen X, Zhang X, Kubo H, Harris DM, Mills GD, Moyer J, Berretta R, Potts ST, Marsh JD, Houser SR (2005) Ca2+ influx-induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular myocytes. Circ Res 97:1009–1017. doi: 10.1161/01.RES.0000189270.72915.D1 CrossRefPubMedGoogle Scholar
- 16.Fatkin D, McConnell BK, Mudd JO, Semsarian C, Moskowitz IG, Schoen FJ, Giewat M, Seidman CE, Seidman JG (2000) An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy. J Clin Invest 106:1351–1359. doi: 10.1172/JCI11093 PubMedCentralCrossRefPubMedGoogle Scholar
- 17.Foell JD, Balijepalli RC, Delisle BP, Yunker AM, Robia SL, Walker JW, McEnery MW, January CT, Kamp TJ (2004) Molecular heterogeneity of calcium channel beta-subunits in canine and human heart: evidence for differential subcellular localization. Physiol Genomics 17:183–200. doi: 10.1152/physiolgenomics.00207.2003 CrossRefPubMedGoogle Scholar
- 18.Goonasekera SA, Hammer K, Auger-Messier M, Bodi I, Chen X, Zhang H, Reiken S, Elrod JW, Correll RN, York AJ, Sargent MA, Hofmann F, Moosmang S, Marks AR, Houser SR, Bers DM, Molkentin JD (2012) Decreased cardiac L-type Ca(2)(+) channel activity induces hypertrophy and heart failure in mice. J Clin Invest 122:280–290. doi: 10.1172/JCI58227 PubMedCentralCrossRefPubMedGoogle Scholar
- 22.Herzig S, Khan IF, Grundemann D, Matthes J, Ludwig A, Michels G, Hoppe UC, Chaudhuri D, Schwartz A, Yue DT, Hullin R (2007) Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. FASEB J Off Publ Fed Am Soc Exp Biol 21:1527–1538. doi: 10.1096/fj.06-7377com Google Scholar
- 24.Hullin R, Matthes J, von Vietinghoff S, Bodi I, Rubio M, D'Souza K, Friedrich Khan I, Rottlander D, Hoppe UC, Mohacsi P, Schmitteckert E, Gilsbach R, Bunemann M, Hein L, Schwartz A, Herzig S (2007) Increased expression of the auxiliary beta(2)-subunit of ventricular L-type Ca(2) + channels leads to single-channel activity characteristic of heart failure. PLoS One 2, e292. doi: 10.1371/journal.pone.0000292 PubMedCentralCrossRefPubMedGoogle Scholar
- 25.Link S, Meissner M, Held B, Beck A, Weissgerber P, Freichel M, Flockerzi V (2009) Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart. J Biol Chem 284:30129–30137. doi: 10.1074/jbc.M109.045583 PubMedCentralCrossRefPubMedGoogle Scholar
- 26.Miranda-Laferte E, Ewers D, Guzman RE, Jordan N, Schmidt S, Hidalgo P (2014) The N-terminal domain tethers the voltage-gated calcium channel beta2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions. J Biol Chem 289:10387–10398. doi: 10.1074/jbc.M113.507244 PubMedCentralCrossRefPubMedGoogle Scholar
- 30.Semsarian C, Ahmad I, Giewat M, Georgakopoulos D, Schmitt JP, McConnell BK, Reiken S, Mende U, Marks AR, Kass DA, Seidman CE, Seidman JG (2002) The L-type calcium channel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J Clin Invest 109:1013–1020. doi: 10.1172/JCI14677 PubMedCentralCrossRefPubMedGoogle Scholar
- 38.Voigt N, Li N, Wang Q, Wang W, Trafford AW, Abu-Taha I, Sun Q, Wieland T, Ravens U, Nattel S, Wehrens XH, Dobrev D (2012) Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na + -Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation. Circulation 125:2059–2070. doi: 10.1161/CIRCULATIONAHA.111.067306 CrossRefPubMedGoogle Scholar