Intracellular Mg2+, which is implicated in arrhythmogenesis and transient cardiac ischemia, inhibits L-type Ca2+ calcium channel current (ICaL) of adult cardiomyocytes (CMs). We take the advantage of an in vitro model of CMs based on induced pluripotent stem cells to investigate the effects of intracellular Mg2+ on the phosphorylation or dephosphorylation processes of L-type Ca2+ channels (LTCCs) at early and late stages of cardiac cell differentiation. Using the whole-cell patch-clamp technique, we demonstrate that increasing intracellular Mg2+ concentration [Mg2+]i from 0.2 to 5 mM markedly reduced the peak of ICaL density, showing less effect on both the activation and inactivation properties in the late differentiation stage (LDS) of CMs more so than in the early differentiation stage (EDS). Increasing the [Mg2+]i from 0.2 to 2 mM in the presence of cAMP-dependent protein kinase A significantly decreased ICaL in LDS (70%) and in EDS (36%) CMs. In addition, the effect of forskolin was greatly attenuated in the presence of 2 mM [Mg2+]i in LDS but not in EDS CMs. The effect of forskolin was enhanced in the presence of ATP-γ-S in LDS CMs compared with EDS CMs. The exposure of both EDS and LDS CMs to 2 mM [Mg2+]i considerably reduced the effects of isobutylmethylxanthine (IBMX) and okadaic acid on ICaL. Our results provide evidence for differential regulation of LTCCs activities by cytosolic Mg2+ concentration in developing cardiac cells and confirm that Mg2+ acts under conditions that favor opening of the LTCCs caused by channel phosphorylation.
Get full access to this article
View all access options for this article.
References
1.
FabiatoA. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol, 245:C1–C14.
KobayashiT, YamadaY, FukaoM, TsutsuuraM and TohseN. (2007). Regulation of Cav1.2 current: interaction with intracellular molecules. J Pharmacol Sci, 103:347–353.
4.
XuX, MarxSO and ColecraftHM. (2010). Molecular mechanisms, and selective pharmacological rescue, of Rem-inhibited CaV1.2 channels in heart. Circ Res, 107:620–630.
5.
McDonaldTF, PelzerS, TrautweinW and PelzerDJ. (1994). Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev, 74:365–507.
6.
BersDM. (2008). Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol, 70:23–49.
7.
NguemoF, SasseP, FleischmannBK, KamanyiA, SchunkertH, HeschelerJ and ReppelM. (2009). Modulation of L-type Ca(2+) channel current density and inactivation by beta-adrenergic stimulation during murine cardiac embryogenesis. Basic Res Cardiol, 104:295–306.
8.
BrunetS, ScheuerT and CatterallWA. (2013). Increased intracellular magnesium attenuates beta-adrenergic stimulation of the cardiac Ca(V)1.2 channel. J Gen Physiol, 141:85–94.
9.
MaltsevVA, JiGJ, WobusAM, FleischmannBK and HeschelerJ. (1999). Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development. Circ Res, 84:136–145.
10.
PelzerS, LaC and PelzerDJ. (2001). Phosphorylation-dependent modulation of cardiac calcium current by intracellular free magnesium. Am J Physiol Heart Circ Physiol, 281:H1532–H1544.
11.
LansmanJB, HessP and TsienRW. (1986). Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore. J Gen Physiol, 88:321–347.
12.
YamaokaK and SeyamaI. (1998). Phosphorylation modulates L-type Ca channels in frog ventricular myocytes by changes in sensitivity to Mg2+ block. Pflugers Arch, 435:329–337.
13.
WangM and BerlinJR. (2007). Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes. Arch Biochem Biophys, 458:65–72.
14.
WhiteRE and HartzellHC. (1989). Magnesium ions in cardiac function. Regulator of ion channels and second messengers. Biochem Pharmacol, 38:859–867.
15.
WangM and BerlinJR. (2006). Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration. Am J Physiol Cell Physiol, 291:C83–C92.
16.
WangM, TashiroM and BerlinJR. (2004). Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes. J Physiol, 555:383–396.
17.
YamaokaK, YukiT, KawaseK, MunemoriM and SeyamaI. (2002). Temperature-sensitive intracellular Mg2+ block of L-type Ca2+ channels in cardiac myocytes. Am J Physiol Heart Circ Physiol, 282:H1092–H1101.
18.
MoodyWJ and BosmaMM. (2005). Ion channel development, spontaneous activity, and activity-dependent development in nerve and muscle cells. Physiol Rev, 85:883–941.
19.
KorhonenT, RapilaR and TaviP. (2008). Mathematical model of mouse embryonic cardiomyocyte excitation-contraction coupling. J Gen Physiol, 132:407–419.
20.
JanowskiE, CleemannL, SasseP and MoradM. (2006). Diversity of Ca2+ signaling in developing cardiac cells. Ann N Y Acad Sci, 1080:154–164.
21.
HongTT, SmythJW, GaoD, ChuKY, VoganJM, FongTS, JensenBC, ColecraftHM and ShawRM. (2010). BIN1 localizes the L-type calcium channel to cardiac T-tubules. PLoS Biol, 8:e1000312
22.
MauritzC, SchwankeK, ReppelM, NeefS, KatsirntakiK, MaierLS, NguemoF, MenkeS, HausteinM, et al. (2008). Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation, 118:507–517.
23.
PfannkucheK, LiangH, HannesT, XiJ, FatimaA, NguemoF, MatzkiesM, WernigM, JaenischR, et al. (2009). Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility. Cell Physiol Biochem, 24:73–86.
MeissnerA, WernigM and JaenischR. (2007). Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol, 25:1177–1181.
26.
KolossovE, BostaniT, RoellW, BreitbachM, PillekampF, NygrenJM, SasseP, RubenchikO, FriesJW, et al. (2006). Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J Exp Med, 203:2315–2327.
27.
HamillOP, MartyA, NeherE, SakmannB, SigworthFJ. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch, 391:85–100.
28.
BersDM, PattonCW and NuccitelliR. (1994). A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol, 40:3–29.
29.
MaltsevVA, JiGJ, WobusAM, FleischmannBK and HeschelerJ. (1999). Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development. Circ Res, 84:136–145.
30.
KuzmenkinA, LiangH, XuG, PfannkucheK, EichhornH, FatimaA, LuoH, SaricT, WernigM, JaenischR and HeschelerJ. (2009). Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J, 23:4168–4180.
31.
ZhangF, SowersJR, RamJL, StandleyPR and PeulerJD. (1994). Effects of pioglitazone on calcium channels in vascular smooth muscle. Hypertension, 24:170–175.
32.
ZhangF, RamJL, StandleyPR and SowersJR. (1994). 17 beta-Estradiol attenuates voltage-dependent Ca2+ currents in A7r5 vascular smooth muscle cell line. Am J Physiol, 266:C975–C980.
33.
YangL, KatchmanA, MorrowJP, DoshiD and MarxSO. (2011). Cardiac L-type calcium channel (Cav1.2) associates with gamma subunits. FASEB J, 25:928–936.
34.
BrunetS, ScheuerT and CatterallWA. (2009). Cooperative regulation of Ca(v)1.2 channels by intracellular Mg(2+), the proximal C-terminal EF-hand, and the distal C-terminal domain. J Gen Physiol, 134:81–94.
35.
LiuW, YasuiK, AraiA, KamiyaK, ChengJ, KodamaI and ToyamaJ. (1999). beta-adrenergic modulation of L-type Ca2+-channel currents in early-stage embryonic mouse heart. Am J Physiol, 276:H608–H613.
36.
MatsudaT, KoyamaY and BabaA. (2005). Functional proteins involved in regulation of intracellular Ca2+ for drug development: Pharmacology of SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger. J Pharmacol Sci, 97:339–343.
37.
GusevK and NiggliE. (2008). Modulation of the local SR Ca2+ release by intracellular Mg2+ in cardiac myocytes. J Gen Physiol, 132:721–730.
38.
YangXY, YangTT, SchubertW, FactorSM and ChowCW. (2007). Dosage-dependent transcriptional regulation by the calcineurin/NFAT signaling in developing myocardium transition. Dev Biol, 303:825–837.
39.
HartzellHC and WhiteRE. (1989). Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes. J Gen Physiol, 94:745–767.
40.
KampTJ and HellJW. (2000). Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res, 87:1095–1102.
41.
AnRH, DaviesMP, DoevendansPA, KubalakSW, BangaloreR, ChienKR and KassRS. (1996). Developmental changes in beta-adrenergic modulation of L-type Ca2+ channels in embryonic mouse heart. Circ Res, 78:371–378.
42.
KojimaM, SadaH and SperelakisN. (1990). Developmental changes in beta-adrenergic and cholinergic interactions on calcium-dependent slow action potentials in rat ventricular muscles. Br J Pharmacol, 99:327–333.
43.
Abi-GergesN, JiGJ, LuZJ, FischmeisterR, HeschelerJ and FleischmannBK. (2000). Functional expression and regulation of the hyperpolarization activated non-selective cation current in embryonic stem cell-derived cardiomyocytes. J Physiol Lond, 523:377–389.
44.
BuddeT, MeuthS and PapeHC. (2002). Calcium-dependent inactivation of neuronal calcium channels. Nat Rev Neurosci, 3:873–883.
45.
BrunetS, ScheuerT, KlevitR and CatterallWA. (2005). Modulation of CaV1.2 channels by Mg2+ acting at an EF-hand motif in the COOH-terminal domain. J Gen Physiol, 126:311–323.
46.
WalshKB and ParksGE. (2002). Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels. Cardiovasc Res, 55:64–75.
47.
HeschelerJ, MieskesG, RueggJC, TakaiA and TrautweinW. (1988). Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes. Pflugers Arch, 412:248–252.
48.
HirayamaY and HartzellHC. (1997). Effects of protein phosphatase and kinase inhibitors on Ca2+ and Cl- currents in guinea pig ventricular myocytes. Mol Pharmacol, 52:725–734.
49.
SchroderF, HandrockR, BeuckelmannDJ, HirtS, HullinR, PriebeL, SchwingerRH, WeilJ and HerzigS. (1998). Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation, 98:969–976.
50.
duBellWH and RogersTB. (2004). Protein phosphatase 1 and an opposing protein kinase regulate steady-state L-type Ca2+ current in mouse cardiac myocytes. J Physiol, 556:79–93.
51.
StotzSC, JarvisSE and ZamponiGW. (2004). Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels. J Physiol, 554:263–273.
52.
FuY, WestenbroekRE, YuFH, ClarkJP3rd, MarshallMR, ScheuerT and CatterallWA. (2011). Deletion of the distal C terminus of CaV1.2 channels leads to loss of beta-adrenergic regulation and heart failure in vivo. J Biol Chem, 286:12617–12626.
53.
GaoTY, CuadraAE, MaH, BunemannM, GerhardsteinBL, ChengT, Ten EickR and HoseyMM. (2001). C-terminal fragments of the alpha(1C) (Ca(v)1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha(1C) subunits. J Biol Chem, 276:21089–21097.
54.
BretteF and OrchardC. (2003). T-tubule function in mammalian cardiac myocytes. Circ Res, 92:1182–1192.
55.
KeY, LeiM, CollinsTP, RakovicS, MattickPA, YamasakiM, BrodieMS, TerrarDA and SolaroRJ. (2007). Regulation of L-type calcium channel and delayed rectifier potassium channel activity by p21-activated kinase-1 in guinea pig sinoatrial node pacemaker cells. Circ Res, 100:1317–1327.
