Michiko Tashiro

Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes

The effects of changing cytosolic [Mg2+] ([Mg2+]i) on l‐type Ca2+ currents were investigated in rat cardiac ventricular myocytes voltage‐clamped with patch pipettes containing salt solutions with defined [Mg2+] and [Ca2+]. To control [Mg2+]i and cytosolic [Ca2+] ([Ca2+]i), the pipette solution included 30 mm citrate and 10 mm ATP along with 5 mm EGTA (slow Ca2+ buffer) or 15 mm EGTA plus 5 mm BAPTA (fast Ca2+ buffer). With pipette [Ca2+] ([Ca2+]p) set at 100 nm using a slow Ca2+ buffer and pipette [Mg2+] ([Mg2+]p) set at 0.2 mm, peak l‐type Ca2+ current density (ICa) was 17.0 ± 2.2 pA pF−1. Under the same conditions, but with [Mg2+]p set to 1.8 mm, ICa was 5.6 ± 1.0 pA pF−1, a 64 ± 2.8% decrease in amplitude. This decrease in ICa was accompanied by an acceleration and a –8 mV shift in the voltage dependence of current inactivation. The [Mg2+]p‐dependent decrease in ICa was not significantly different when myocytes were preincubated with 10 μm forskolin and 300 μm 3‐isobutyl‐1‐methylxanthine and voltage‐clamped with pipettes containing 50 μm okadaic acid, to maximize Ca2+ channel phosphorylation. However, when myocytes were voltage‐clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, ICa decreased only 25 ± 3.4% on changing [Mg2+]p from 0.2 to 1.8 mm. In the presence of 0.2 mm[Mg2+]p, changing channel phosphorylation conditions altered ICa over a 4‐fold range; however, with 1.8 mm[Mg2+]p, these same manoeuvres had a much smaller effect on ICa. These data suggest that [Mg2+]i can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca2+]p to 1, 100 or 300 nm also showed that the [Mg2+]p‐induced reduction of ICa was smaller at the lowest [Ca2+]p, irrespective of channel phosphorylation conditions. This interaction between [Ca2+]i and [Mg2+]i to modulate ICa was not significantly affected by ryanodine, fast Ca2+ buffers or inhibitors of calmodulin, calmodulin‐dependent kinase and calcineurin. Thus, physiologically relevant [Mg2+]i modulates ICa by counteracting the effects of Ca2+ channel phosphorylation and by an unknown [Ca2+]i‐dependent mechanism. The magnitude of these effects suggests that changes in [Mg2+]i could be critical in regulating l‐type channel gating.

Magnesium Homeostasis in Cardiac Myocytes of Mg-Deficient Rats

To study possible modulation of Mg2+ transport in low Mg2+ conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control) to Wistar rats for 1–6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg2+ concentration ([Mg2+]i) of ventricular myocytes was measured with the fluorescent indicator furaptra. Mg2+ transport rates, rates of Mg2+ influx and Mg2+ efflux, were estimated from the rates of change in [Mg2+]i during Mg loading/depletion and recovery procedures. In Mg-deficient rats, the serum total Mg concentration (0.29±0.026 mM) was significantly lower than in control rats (0.86±0.072 mM) after 4–6 weeks of Mg deficiency. However, neither total Mg concentration in ventricular tissues nor [Mg2+]i of ventricular myocytes was significantly different between Mg-deficient rats and control rats. The rates of Mg2+ influx and efflux were not significantly different in both groups. In addition, quantitative RT-PCR revealed that Mg deficiency did not substantially change mRNA expression levels of known Mg2+ channels/transporters (TRPM6, TRPM7, MagT1, SLC41A1 and ACDP2) in heart and kidney tissues. These results suggest that [Mg2+]i as well as the total Mg content of cardiac myocytes, was well maintained even under chronic hypomagnesemia without persistent modulation in function and expression of major Mg2+ channels/transporters in the heart.

Mg2+- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress

Transient receptor potential melastatin 7 (TRPM7) is a Ca(2+)- and Mg(2+)-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca(2+) overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H2O2) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H2O2 to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H2O2 under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H2O2 and NMM were dependent on free [Mg(2+)]i; the inhibition was stronger when cells were perfused with higher free [Mg(2+)]i solutions via pipette. In addition, TRPM7 current was not inhibited by H2O2 when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg(2+)]i, which is sufficient for TRPM7 inhibition by H2O2 in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H2O2 just like the wild-type TRPM7 in a [Mg(2+)]i- and [ATP]i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg(2+)]i elevation.

Metabolic Inhibition Strongly Inhibits Na+-Dependent Mg2+ Efflux in Rat Ventricular Myocytes

We measured intracellular Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using the fluorescent indicator furaptra (25 degrees C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+]i: the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Delta[Mg2+]i/Deltat after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 microM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg2+]i from approximately 0.9 mM to approximately 2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg2+) and cell shortening to approximately 50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg2+]i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for > or = 90 min. The initial Delta[Mg2+]i/Deltat was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Delta[Mg2+]i/Deltat (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na+ concentration ([Na+]i) estimated with a Na+ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Delta[Mg2+]i/Deltat measurements, which is well below the [Na+]i level for half inhibition of the Mg2+ efflux (approximately 40 mM). Normalization of intracellular pH using 10 microM nigericin, a H+ ionophore, did not reverse the inhibition of the Mg2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (approximately 0.4 mM estimated indirectly in the this study), rather than elevation of [Na+]i or intracellular acidosis, inhibits the Mg2+ efflux, suggesting the absolute necessity of ATP for the Na+/Mg2+ exchange.

Magnesium Influx in Primary Cultured Ventricular Myocytes of Adult Rats

We have characterized physiological pathway of Mg2+ influx in acutely isolated ventricular myocytes of rats [Biophys J 107:2049-2058, 2014]. In this study, we measured the rate of Mg2+ influx in primary cultured ventricular myocytes dissociated from adult rat hearts. Acutely isolated cells were plated onto laminin-coated glass bottom dishes, and maintained in DMEM supplemented with 10% FBS and 5% horse serum at 37°C and 5%CO2 for 72 hours. On the first day of culture, acutely isolated cells were in rod shape with sharp edges and clear cross-striations. The edges of cells became rounder gradually over time in culture; some cells were in round shape on the third day. We used the cells that kept rod shape and clear intracellular structure for experiments. Cytosolic free Mg2+ concentration ([Mg2+]i) was measured with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. The basal level of [Mg2+]i in primary cultured cell was 0.85±0.068 mM (n=7), which was not significantly different from that of acutely isolated cells (0.92±0.039 mM, n=10). [Mg2+]i decreased by incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution for 20 min, and recovered by perfusion with Ca2+-free Tyrodes solution containing 1 mM Mg2+. The initial rate of Mg2+ influx was estimated from the time course of the [Mg2+]i recovery as described previously. The Mg2+ influx rate obtained from primary cultured cell was, on average, 0.28±0.11 μM/s with initial [Mg2+]i at 0.38±0.03 mM. This value was similar to that obtained from acutely isolated cells (0.27±0.043 μM/s at 0.35±0.016 mM [Mg2+]i). Since it is suggested that function of cellular Mg2+ regulation system is not altered by primary culture for 72 hours, the primary cultured ventricular myocytes may be used to study physiological roles of Mg2+ channel/transporter proteins.

Effects of TRPM7 Inhibitors on Physiological Mg2+ Influx in Rat Ventricular Myocytes

We measured free Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using a fluorescent indicator furaptra (mag-fura-2). [Mg2+]i decreased from ∼0.9 mM to 0.2-0.5 mM by incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution, and recovered by perfusion with Ca2+-free Tyrodes solution containing 1 mM Mg2+. The time course of the [Mg2+]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as an initial Mg2+ influx rate. In order to characterize physiological Mg2+ influx pathways, we used known TRPM7 inhibitors, 2-Aminoethoxydiphenyl borate (2-APB) and NS8593. The initial rate of Mg2+ influx was decreased to 43±10 % (n=6) by 100 μM 2-APB, and to 12±8.6 % (n=5) by 10 μM NS8593. These compounds inhibited the Mg2+ influx with half inhibitory concentrations (IC50) of 17 μM (2-APB) and 2.0 μM (NS8593). 2-APB and NS8593 also inhibited Ni2+ influx when estimated by quenching of furaptra fluorescence with IC50 values of, respectively, 20 μM and 4.4 μM; these values are comparable to those for Mg2+ influx. Under the whole-cell patch-clamp configuration, removal of intracellular and extracellular divalent cations induced large inward and outward currents, IMIC, carried by monovalent cations likely via TRPM7 channels. The IMIC measured at −120 mV was diminished to 48±3.6 % (n=7) by 100 μM 2-APB, and to 50±12 % (n=4) by 10 μM NS8593. These results support our previous conclusion [Biophys J 102:664a, 2012] that TRPM7/MIC channels serve as a major physiological pathway of Mg2+ influx in rat ventricular myocytes.

Cellular Basis of Magnesium Transport

About 40% of magnesium that is contained in food and drinking water is absorbed from the gastrointestinal tract. On the other hand, the kidneys are the most important for control of body magnesium balance through its urinary excretion, which is primarily regulated by tubular re-absorption. The amount of magnesium excretion in the urine is regulated by hormones and other factors, and can vary widely.