Simvastatin inhibits L-type Ca 2+ -channel activity through impairment of mitochondrial function

Plasma membrane ion channels and mitochondrial electron transport complexes (mETC) are recognised “off-targets” for certain drugs. Simvastatin is one such drug, a lipophilic statin used to treat hypercholesterolaemia, but which is also associated with adverse effects like myopathy and increased risk of glucose intolerance. Such myopathy is thought to arise through adverse actions of simvastatin on skeletal muscle mETC and mitochondrial respiration. In this study we investigated whether the glucose intolerance associated with simvastatin is also mediated via adverse effects on mETC in pancreatic beta-cells since mitochondrial respiration underlies insulin secretion from these cells, an effect in part mediated by promotion of Ca 2+ influx via opening of voltage-gated Ca 2+ channels (VGCCs). We used murine pancreatic beta-cells to investigate these ideas. Mitochondrial membrane potential, oxygen consumption and ATP-sensitive-K + -channel activity were monitored as markers of mETC activity, respiration and cellular ATP/ADP ratio respectively; Ca 2+ channel activity and Ca 2+ influx were also measured. In intact beta-cells, simvastatin inhibited oxidative respiration (IC 50 ~ 3 µM) and mETC (1< IC 50 < 10 µM), effects expected to impair VGCC opening. Consistent with this idea simvastatin > 0.1 µM reversed activation of VGCCs by glucose but had no significant effect in the sugar’s absence. The VGCC effects were mimicked by rotenone which also decreased respiration and ATP/ADP. This study demonstrates modulation of beta-cell VGCC activity by mitochondrial respiration and their sensitivity to mETC inhibitors. This reveals a novel outcome for the action of drugs like simvastatin for which mETC is an “off target”.


Introduction
Mitochondrial electron transport (mETC) complexes (Nadanaciva et al., 2007;Hargreaves et al., 2016;Wallace, 2008) and plasma membrane ion channels are well recognised "off targets" for drugs (Lynch et al., 2017;Real et al., 2018). For some drugs, both moieties may be "off targets"; a situation which confounds the interpretation of adverse drug effects both in the clinic and laboratory. An example of this occurs in insulin secreting beta cells with the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor, simvastatin, a lipophilic drug used to treat hypercholesteremia. Such pluripotent effects of simvastatin complicates the mechanistic understanding of how adverse effects arise in the clinic; for example the association between lipophilic statin use and increased risk of glucose intolerance and diabetes (Cederberg et al., 2015;Sattar et al., 2010).
Insulin secretion from the pancreatic beta-cell is promoted by glucose via oxidative metabolism (Maechler et al., 2010;Affourtit et al., 2018). The resultant increase in cytosolic Mitochondrial respiration The rate of oxygen consumption (OCR) was used to measure mitochondrial respiration insitu with Clark oxygen electrodes (Rank Brothers, Bottisham, UK) as previously described (Daunt et al., 2006). Known densities of MIN6 cells were incubated at 32 o C in Hanks solution which contained (in mM): 137 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 1.2 NaH2PO4, 4.2 NaHCO3 10 HEPES (pH 7.4 with NaOH). For OCR cells were first incubated in the absence of substrate, basal, followed by stimulation with10 mM glucose for 10 minutes after which a single concentration of drug was tested. The OCR was corrected for background consumption of O2 by the electrode by subtraction of the OCR measured in 6 mM NaN3, a treatment which blocks oxidative respiration at mETC complex IV. The effect of drugs is expressed as the change in OCR relative to that stimulated by glucose in the same suspension. Only data from glucose-sensitive suspensions was used (>10% increase in response to 10 mM glucose).

Imaging studies
Changes in inner mitochondrial membrane potential (ΔΨmit) and cytosolic calcium concentration ([Ca 2+ ]i) were monitored with rhodamine-123 (Rh123) and the calcium fluophore FLUO-4 respectively as previously described (Daunt et al., 2006;Duchen et al., 1993). Cells were perifused in a modified Hanks solution at 32 o C. Regions of interest (ROI) were drawn around cells, corrected for background fluorescence by subtraction, and the average fluorescence intensity calculated. To sample the total cell population, ROIs were from both single cells and cell clusters. For each experimental group, samples were pooled from multiple visual fields from at least 4 different cell preparations. Image analysis was performed with custom scripts written in Labtalk (OriginLab Corporation, MA USA).
For ΔΨmit, images were captured at a frame rate of 1 Hz with a Photonics ISIS CCD camera, DT3155 frame grabber (Data Translation, UK) and Imaging workbench software (IW6 INDEC BioSystems, Santa Clara, USA). Only ROIs that responded 1 µM FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone), a mitochondrial protonophore which collapses ΔΨmit and results in a fluorescence increase, were chosen for further analysis (Duchen et al., 1993;Elmorsy et al., 2017). ROIs were normalised to the difference between the fluorescence measured in glucose (minimum fluorescence) and that in and that in the presence of 1 µM FCCP (maximum fluorescence) (Daunt et al., 2006). Measurement of L-type Ca 2+ channel activity Measurement of L-type Ca 2+ activity was performed with cell-attached patch-clamp, similar to that described (Smith et al., 1989). This method was used preferentially over whole-cell methods (Smith et al., 1989;Smith, 2009) to prevent K + channel contamination.
Patch pipettes were drawn from GC150TF capillary glass (Harvard Instruments), coated with Sylgard (Dow-Corning) and fire polished before use. Pipettes typically had resistances between 2-4 MΩ. The zero-current potential was adjusted with the pipette in the bath just before seal establishment. No corrections have been made for liquid junction potentials (<4 mV). Currents were low pass filtered at 2 kHz (-3db, 8 pole Bessel), digitized at 10 kHz using pClamp 8.3 (Axon Instruments, Foster City, USA).
To maximize channel detection, but minimize surface charge effects (Smith et al., 1993)  To record single Ca 2+ channel activity, Vm was held at -90 mV and Ca 2+ currents were elicited by pulses to -40 mV of 200 ms duration at 0.5 Hz. Leak and capacitance currents were removed by subtraction of the numerical average of records absent of activity. Channel activity, NPo, was recorded for 3-5 minutes, first in basal then after 10 minute incubation in 10 mM glucose, followed by the addition of drug or vehicle.
Single-channel data were analysed with custom scripts written in Labtalk using halfamplitude threshold techniques as described (Smith et al., 1993). Drug effects on NPo were quantified as a percentage of its respective control values. Ca 2+ currents are displayed conventionally as downward deflections.

Assessment of intracellular ATP: Cell-attached KATP ion channel activity as a biosensor
To monitor the acute intracellular ATP/ADP ratio, the activity of KATP channels in cellattached experiments was measured. This channel acts as a biosensor of the intracellular sub membrane ATP/ADP ratio and can be used to follow the energetic status of an intact cell in real time (Gribble et al., 2000). For this, KATP channel activity was measured in cellattached experiments with a pipette potential, Vp, of 0 mV. The pipette contained (in mmol/l): data were analysed with half-amplitude threshold techniques as implemented in Clampfit Ver. 10.6 (Axon Instruments, Foster City, USA). Channel activity, NPo, was measured continuously, in the absence then presence of 10 mM glucose followed by drug additions.
An increase in NPo reflects a decrease in ATP/ADP (Gribble et al., 2000); a phenomenon expected to occur with an impairment of glucose metabolism (Köhler et al., 1998;Kiranadi et al., 1991).

Drugs
Simvastatin and pravastatin were obtained from Tocris Bioscience, Bristol, UK. FCCP was obtained from Sigma-Aldrich, Poole, UK. Simvastatin was used in its lipophilic, lactone, form.
Simvastatin, rotenone and FCCP were dissolved in ethanol or DMSO; Pravastatin was dissolved in H2O. Drug additions were made from serially diluted stocks such that the vehicle was always applied at the same final concentration; for the electrophysiology this was 0.1% vol/vol and for the OCR this was 1% vol/vol.

Statistical Analysis
Statistical analysis was performed using either Graphpad PRISM version 7.03 (San Diego, California USA) or StatsDirect 3.1 (Cambridge, UK), data were tested with the D'Agostino & Pearson omnibus normality test and the appropriate statistical test used as given in the text.
The concentration-response relationship for the block of OCR by simvastatin was quantified by fitting the data with the equation: Where Y is the fractional OCR, relative to that measured under control conditions, h is the slope index, [S] is the free simvastatin concentration and IC50 the concentration that produces half-maximal inhibition. Since 0.1% BSA binds simvastatin to decrease its free concentration ~10 fold (Shi et al., 2017;Real et al., 2018), [S] was taken to be 1/10 th of the added concentration.
Data are quoted as either the mean  SEM or median with 5 to 95% confidence intervals (C.I.), where n is the number of separate determinations. Statistical significance is defined as P <0.05 and is flagged in graphics as *, ** (p<0.01) or *** (p<0.001).

Results
Simvastatin blocks oxidative respiration.
In 5  The effects of glucose and FCCP on Δψmit were similar to those previously described in both MIN6 and primary beta-cells (Duchen et al., 1993;Daunt et al., 2006;Elmorsy et al., 2017;Smith et al., 1999). In the presence of glucose, 10 µM simvastatin depolarised Δψmit  Fig 2B). At 1 µM, rotenone blocked OCR by ~70%, an amount greater than that of the highest concentration of simvastatin tested. (Fig 2B).

Simvastatin inhibits glucose stimulated Ca 2+ influx
To determine if the inhibitory effect of simvastatin on mitochondrial respiration was manifest on Ca 2+ influx, cytosolic Ca 2+ was measured in MIN6 beta-cells. In the absence of glucose, basal [Ca 2+ ]i was 78 nM (, 63 to 107, 95% C.I.; Fig 3A). After a delay of between 1 to 4 minutes, 10 mM glucose produced an initial decrease in [Ca 2+ ]I followed by a biphasic increase. The latter consisted of an initial peak in [Ca 2+ ]I which then decayed to an elevated level of 120 nM (100 to 156, 95% C.I.; Fig 3A); a value 1.5 fold (1.4 to 1.7, 95% C.I.) greater than basal (p<0.0001, Wilcoxon signed rank test). The latter was associated with transitory spikes indicative of underlying Ca 2+ -dependent action potential activity (Rorsman et al., 1992 Simvastatin reverses glucose-stimulation of L-type Ca 2+ channel activity Since L-type Ca 2+ channels in this cell type are regulated by mitochondrial respiration (Smith et al., 1989), and simvastatin inhibited mETC and oxidative respiration, the action of the drug on VGCCs in functionally intact cells was explicitly explored with the cell-attached patch-clamp technique (Smith et al., 1989). Figure 4 shows that membrane potential depolarization elicited single-channel activity in MIN6 beta-cells indicative of L-type voltagegated Ca 2+ channels that are found in pancreatic beta-cells (Smith et al., 1993;Schulla et al., 2003): voltage-dependent activation, a single channel current amplitude of ~ 1 pA at -40 mV and prolongation of open channel lifetimes by 0.1 µM BAY-K8644, a dihydropyridine Ltype Ca 2+ channel agonist (Smith et al., 1989(Smith et al., , 1993. Perifusion of glucose significantly increased channel activity (NPo) to 210 ± 9 % of basal (p = 0.013, Wilcoxon signed rank test relative to control, n = 14; Figs. 4B & D); an increase similar in magnitude to that previously reported for this channel in primary beta-cells (Smith et al., 1989). Figure 4C shows that subsequent addition of 1 µM simvastatin reversed the effect of glucose and decreased NPo back down to its basal value (Fig 4D; p <0.0001, Kruskal Wallis Dunn's multiple comparison test). In the absence of glucose 1 µM simvastatin failed to affect channel activity ( Fig. 4D; One sample t-test). Similar results were also seen with 10 µM simvastatin: a reversal of the stimulatory effect of 10 mM glucose on channel activity but with no effect in the absence of the sugar (Fig. 4D); these data are indicative of an indirect, glucose-dependent effect of simvastatin on this channel type. This latter idea is supported by the observation that the degree of inhibition in NPo produced by 1 µM simvastatin was linearly correlated (R 2 > 0.91; p<0.001, Pearson R) with the magnitude of increase in NPo stimulated by glucose (Fig. 4E). Figure 4D also shows that the ability of simvastatin to reverse the stimulatory effect of glucose on L-type Ca 2+ channel activity was mimicked by 1 µM rotenone, an established inhibitor of mETC and mitochondrial respiration.

Rotenone but not Simvastatin reactivate glucose-blocked KATP channel activity
To investigate if Δψmit depolarization was associated with a decrease in intracellular ATP levels, the level of sub-membrane ATP was monitored indirectly by measurement of KATP channel activity in cell-attached patches (Gribble et al., 2000;Köhler et al., 1998;Kiranadi et al., 1991). Figure 5 demonstrates the decrease in KATP channel activity associated with the rise in intracellular ATP/ADP that occurs with oxidative metabolism of glucose (Köhler et al., 1998;Kiranadi et al., 1991). Subsequent addition of 1 µM rotenone led to an increase in channel activity (Figs. 5F 5G), whereas 1 µM FCCP produced an even larger increase ( Fig. 5g) to effectively reverse the inhibitory effect of glucose on KATP channel activity.
Addition of 10 µM of simvastatin failed to mimic the ability of rotenone to reactivate KATP channels blocked by glucose, but instead produced further inhibition (Figs. 5c, 5G); an effect previously shown to be due to a direct block of the channel protein itself by this drug (Real et al., 2018). Antimycin at 1 µM, similarly activated KATP like FCCP. Channel identity was confirmed by its abolition with 200 µM tolbutamide; a specific sulphonylurea inhibitor of KATP channels.

Discussion
In the pancreatic beta-cell, the lipophilic lactone form of the statin, simvastatin at concentrations greater than 1 µM depolarised Δψmit, reversed the glucose activation of Ltype Ca 2+ channels and inhibited glucose-stimulated Ca 2+ influx. In contrast, 10 µM of the hydrophilic statin pravastatin neither affected Δψmit or Ca 2+ channel activity. Since all the effects of simvastatin were mimicked by rotenone, which also decreased the intracellular ATP/ADP ratio, supports the idea that this statin mediates its toxic effect on VGCCs via inhibition of oxidative respiration and a decrease in intracellular ATP/ADP; not by a direct action on the ion channel itself Effect of simvastatin on mitochondrial membrane potential and respiration We show that in pancreatic β-cells acutely applied simvastatin depolarizes Δψmit . In most cells tested 10 µM, simvastatin collapsed Δψmit, whereas at 3 µM it produced more variable affects, whilst at 1 µM it was without detectable effect. These results suggest that the IC50 for the lipophilic statin effect on mitochondrial function lies between 1 to 10 µM; a range that encompasses the IC50 of 3 µM for OCR; a potency similar to that seen in skeletal muscle: IC50 ~2 µM (Sirvent, Mercier, et al., 2005). Although the IC50 value we report is extracellular in origin this is probably representative of the value experienced by the mETC since our MIN6 cell line do not possess p-glycoprotein that would affect cellular levels of lipophilic drugs (Daunt et al., 2006), moreover simvastatin has been reported to block p-glycoprotein (Wang et al., 2001). Based on data from respiration studies on intact mitochondria, it is argued that Complex I is the primary target for simvastatin (Sirvent, Bordenave, et al., 2005); an idea support by the similarly of the effects of rotenone, a selective complex I inhibitor, to simvastatin in the present study. The inability of hydrophilic pravastatin to affect mitochondrial function is consistent with that previously reported for murine beta-cells (Zhou Effect of simvastatin on L-type Ca 2+ channel activity. L-type VGCCs are positively modulated by Mg 2+ nucleotide complexes (O'Rourke et al., 1992;Ohya and Sperelakis, 1989), such that changes in the cytosolic ATP/ADP ratio are expected to affect the activity of these ion channels. Indeed, both increases and decreases in the activity of L-type VGCCs in response to metabolic stimulation and mitochondrial inhibition respectively has already been described for both pancreatic beta-cells (Smith et al., 1989) and smooth muscle (McHugh and Beech, 1996). Our observations that glucose stimulated L-type VGCCs activity and that both simvastatin and rotenone reversed this effect, are consistent with this idea, where a depolarization in Δψmit , like that produced by simvastatin and rotenone, is known to decrease the cytosolic ATP/ADP ratio (Duchen et al., 1993) and inhibit this channel. Indeed, we too demonstrate that rotenone can decrease the cytosolic ATP/ADP ratio as illustrated by its ability to activate KATP channels blocked by glucose like that observed for other chemical disruptors of mitochondrial function such as antimycin, FCCP and others (Köhler et al., 1998;Kiranadi et al., 1991). We presume that simvastatin also decreases the cytosolic ATP/ADP given it ability to inhibit OCR and depolarize Δψmit just like rotenone. However these effects of simvastatin were not associated with activation of the KATP channel since they were masked by the potent ability of simvastatin to directly interact with the ion channel protein and inhibit it as we have previously demonstrated (Real et al., 2018).
Our data are consistent with whole-cell voltage-clamp studies on pancreatic beta-cells where simvastatin blocked L-type Ca 2+ currents with an IC50 of 2 µM (Yada et al., 1999), a value similar to what we found for oxidative respiration. Although, other lipophilic compounds, such as Triton X-100 (Narang et al., 2013) and barbiturates (Kozlowski and Ashford, 1991) can also block L-type Ca 2+ channels they appear to do so via a direct, anaesthetic-like, mechanism. The fact that the inhibition of VGCC activity was glucosedependent and was mimicked by rotenone suggest that this block is mechanistically indirect and occurs via an inhibition of oxidative respiration and decreased metabolic regulation of this channel type. This idea is supported by the fact that simvastatin failed to affect the Ltype Ca 2+ channel in the absence of glucose and only abolished the stimulatory effect of glucose. Indeed, one technical advantage of the cell-attached patch clamp method over other configurations is that the cell is maintained in an intact functional state. This means that mitochondrial metabolism and signalling pathways continue to modulate channel activity within the patch.
We also demonstrate that the use of micromolar concentrations of simvastatin to study mitochondrial actions preclude any parallel investigation of changes in KATP-channel activity that may result from an alteration in cytosolic ATP due to the fact that this drug acts to directly block and silence this particular ion channel species as we reported previously (Real et al., 2018).
Effect of simvastatin on Ca 2+ influx.
The reduction in the [Ca 2+ ]I variance we observed with 10 µM simvastatin is consistent with a decline in the Ca 2+ dependent electrical activity that would result from a decrease in L-type Ca 2+ channel activity (Rorsman et al., 1992). The observation that 1 µM simvastatin was effective in the patch-clamp studies but failed to affect intracellular Ca 2+ levels may relate to the dissimilarity in temperatures employed: 22°C and 32°C respectively. Since oxidative respiration in these cells has a Q10 of 5 (Ohta et al., 1990;Escolar et al., 1990) a greater concentration of simvastatin may be required to block the higher metabolic flux of glucosemetabolism at 32°C. This reason may also contribute to the explanation of why, in a previous study (Real et al., 2018), we failed to see an effect of 1 µM simvastatin on glucose stimulated insulin secretion at 32°C.

Physiological Implications
The concentrations of simvastatin that we found to acutely affect mitochondrial function and VGCC activity (1-10 µM) are similar to those used in other in-vitro studies (Ishikawa et al., 2006;Zhou et al., 2014;Sirvent, Mercier, et al., 2005), but are in excess of those normally channels. In fact, the actual impact on insulin secretion will be the result of a convolution of these two process combined with other ATP-dependent process that will also be compromised such as exocytosis (Eliasson et al., 1996).
In conclusion our data highlights the fact that the cellular toxicity of an agent, which can acutely target and impair mitochondrial function, may in part result from decreased L-type Ca 2+ channel activity, Ca 2+ influx and compromised cell functions that are Ca 2+ dependent.

Funding Information
Dr Hani Almukhtar and Jala Alahmed were supported by the Islamic Development Bank.

Conflict of interest
No authors have a conflict of interest that might bias their work and have nothing to declare