Calpain is an intracellular Ca2+ -activated protease that is involved in numerous Ca2+ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca2+ -dependent increase of the late sodium current (INaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell INaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which INaL was activated by the presence of a higher (1μM) intracellular [Ca2+] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1–2 h before INaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of INaL decay evaluated by the two-exponential fit (τ1 = 42±3.0 ms τ2 = 435±27 ms, n = 6, in MDL vs. τ1 = 52±2.1 ms τ2 = 605±26 control no vehicle, n = 11, and vs. τ1 = 52±2.8 ms τ2 = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA). MDL significantly reduced INaL density recorded at –30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage relationships demonstrated that the INaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca2+ accumulation in the pulse train.
Calpain inhibition reverses INaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca2+. Specifically it decreases INaL density and accelerates INaL kinetics resulting in improvement of myocyte electrical response and Ca2+ handling as predicted by our in silico simulations.
Citation: Undrovinas A, Maltsev VA, Sabbah HN (2013) Calpain Inhibition Reduces Amplitude and Accelerates Decay of the Late Sodium Current in Ventricular Myocytes from Dogs with Chronic Heart Failure. PLoS ONE8(4): e54436. https://doi.org/10.1371/journal.pone.0054436
Editor: Vladimir E. Bondarenko, Georgia State University, United States of America
Received: September 10, 2012; Accepted: December 12, 2012; Published: April 15, 2013
Copyright: © 2013 Undrovinas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by National Heart, Lung, and Blood Institute Grants HL-53819 and HL-074238, by a grant-in-aid from the American Heart Association (0350472Z; to AU), and, in part, by the Intramural Research Program of the National Institute on Aging (to VM; the numerical modeling part). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The role of the late sodium current (INaL) in electrophysiological remodeling and arrhythmias in chronic heart failure (HF) has been extensively studied during the last decade. It has been shown that INaL is augmented and its decay slowed in failing human and dog ventricular cardiomyocytes (VCMs)(see for review ). A remarkable contribution of INaL into HF mechanisms has been demonstrated in experiments where “correction” of INaL in failing VCMs resulted in: 1) rescue of normal repolarization, 2) decrease beat-to-beat action potential (AP) duration variability, and 3) improvement of Ca2+ handling and contractility . Accordingly, INaL has emerged as a novel target for cardioprotection to treat the failing heart ,  The new approaches may involve: 1) discovery new drugs that directly and specifically target INaL, 2) targeting intracellular signaling pathways (for example Ca2+-dependent signaling) that are altered in HF and may have modulatory effect on INaL, 3) modulation of altered Na+ channel (NaCh) microenvironment, such as different expression of auxiliary β-subunits and sub-sarcolemmal cytoskeleton that, in turn, may be responsible for the augmented slowed INaL in HF, 4) combination of two latter mechanisms. The new drug, ranolazine (RAN) that was developed as an antianginal agent, has been demonstrated to specifically inhibit INaL , . RAN reduced arrhythmias in the immediately post-MI patients in the recent MERILIN-TIMI trial  confirming the clinical relevance of INaL. Ca2+, calmodulin and CaMKII and this Ca2+ signaling pathway can significantly amplify INaL in HF affecting both contractile and electrical performance , . As to NaCh microenvironment, it has been shown that alterations in membrane phospholipids composition and/or in sub-sarcolemmal cytoskeleton, which consists of ankyrin, actin, spectrin (fodrin), can affect NaCh gating in heart in the way that the late openings may occur , , . Recently we have shown that silencing SCN1B but not SCN2B, the genes that are responsible for expression of the β1 and β2 NaCh subunits, could be a plausible mechanism to modulate INaL in HF with the aim to improve both contractility and rhythm .
Calpain is an intracellular Ca2+ -activated protease and an important mediator of the actions of the intracellular Ca2+ in heart. Cleavage by calpain is critical in a variety of calcium-regulated cellular processes such as muscle contraction, neuronal excitability, secretion, signal transduction, cell proliferation, differentiation, cell cycle progression, and apoptosis , . Deregulation of calpain caused by impaired Ca2+ homeostasis during cardiac pathologies such as atrial fibrillation, heart failure, hypertrophy, or ischemia reperfusion, is critically involved in the myocardial damage. One of the intracellular targets of calpain is fodrin, a dynamic structure that is altered under a variety of pathological conditions featuring poor Ca2+ handling (e.g. ischemia or heart failure , , , ). In the present study we tested the hypothesis that the membrane-permeant calpain inhibitor MDL-28170 (MDL) can prevent, in part, Ca2+-related INaL modulation in VCMs from dogs with chronic HF. We found that MDL reduces density of whole-cell INaL and makes INaL decay faster in the failing VCMs. Using the excitation – contraction coupling (ECC) numerical model  we also assessed physiological significance of the MDL effects. We show that these MDL-induced INaL alterations: 1) reduce AP duration, and 2) prevent diastolic intracellular Ca2+ accumulation during the excitation pulse train in silico.
Materials and Methods
2.1. HF model and cardiomyocyte isolation
The study conforms to the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by the Animal Care and Use Committee (IACUC protocols 0816 and 0777) of the Henry Ford Health System. Chronic heart failure that is similar by vast array of functional and pathophysiological parameters  to that in humans was produced in 2 dogs by multiple sequential coronary artery microsphere embolizations as previously described . At the time of harvesting the heart (∼3 months after last embolization), left ventricular (LV) ejection fraction was approximately ∼25%. Ventricular cardiomyocytes (VCMs) were enzymatically isolated from the apical LV mid-myocardial slices as previously reported . The yield of viable rod-shaped, Ca2+-tolerant VCMs varied from 40 to 70%.
2.2. Patch clamp technique and data analysis
INaL was measured using a whole-cell patch-clamp technique . INaL was assessed by 2 s-long membrane depolarizations to various potentials from a holding potential of −130 mV applied with a stimulation frequency of 0.2 Hz. The bath solution contained (in mM):140 NaCl, 5.0 CsCl, 1.8 CaCl2, 2.0 MgCl2, 5 glucose, 0.002 nifedipine, and 5 HEPES-CsOH buffer (Ph 7.4). The pipette solution contained (in mM): 5 NaCl, 133 CsCl, 0.9 CaCl2 (free [Ca2+] = 1 μM) MgATP, 20 Tetraethylammonium chloride, 1.0 EGTA, and 5.0 HEPES-CsOH buffer. The free [Ca2+] of 1 μM was set in the pipette (and hence inside the cell) to exaggerate abnormal effect of Ca2+ on INaL in HF . Experiments were performed at room temperature (22–24°C). A stock solution of the cell-permeant MDL 28170 (MDL) was prepared in DMSO. MDL was then diluted in the bath solution to a final concentration of 0.2 μM and 2.6 mM DMSO , . Cells suspensions were exposed to MDL from 1–2 hours prior to patch-clamp experiments, and MDL was also added to the pipette solution , . All measurements were made in the presence of MDL in the bath solution and 8–25 min after the membrane rupture to complete cell dialysis with intracellular recording solutions , .
The time course of INaL decay has been approximated by a double exponential fit to INaL starting at 40 ms after the onset of depolarization to –30 mV as previously suggested : (1)where τ1 and τ2 are the time constants, I40 is INaL instant value 40 ms after membrane depolarization, k1 and k2 are the contributions of each exponents (k1 + k2 = 1), respectively. 5-15 experimental traces were averaged to improve the quality of analysis.
Original INaL recordings were also analyzed to assess the current density (pA/pF), i.e. INaL = (whole cell INaL)/Cm, where Cm is cell electric capacitance that was measured by a voltage ramp (16) in each cell. The INaL data points in the current-voltage relationships were measured as the averaged current density within 200–220 ms after depolarization onset (vertical bar in Fig. 1A). The steady-state activation (SSA) parameters were determined from the current-voltage relationships by fitting data points of the normalized current with the function :(2)Where Gmax is a normalized maximum Na+ conductance, Vr is a reversal potential; V½G, and kG are the midpoint and the slope of the respective Boltzmann function underlying the steady-state Na+ channel activation.
A. Representative raw traces were recorded at [Ca2+]i = 1 μM in failing cardiac myocytes before (control) and after exposure to 0.2 μM of MDL. Exponential fits (Eq.1, see Methods) are shown by solid lines, together with their parameters. Inset shows voltage-clamp protocol. Vertical bar indicate the time window (200–220 ms of depolarization) used to evaluate INaL density. B. Statistical data for the decay time constants τ1 and τ2. Bars represent average data mean ±SEM, n – number of cells. Statistical significant difference *P<0.05 were evaluated by the Student's t-test.
The steady-state inactivation (SSI) was evaluated by a double-pulse protocol with 2 s-duration pre-pulses (Vp) ranging from –130 mV to −40 mV followed by a testing pulse to −30 mV. INa amplitudes were normalized to that measured at Vp = −130 mV and the data points were fitted to a Boltzmann function A(Vp):(3)
2.3. Numerical model simulation of calpain inhibition of effect in failing myocytes
We simulated effect of selective inhibition of calpain on AP shape and diastolic Ca2+ accumulation in silico using our previously reported modification of EC coupling model of failing canine ventricular myocyte (originally developed by Winslow et al. ). In short, our model has introduced a new detailed formulation of INaL lacking in the original model. This important model modification has allowed us to predict an important role of INaL to alter AP shape (increase AP plateau duration) and to contribute to diastolic Ca2+ accumulation in HF ventricular myocytes . In short, in our in silico examinations we use INaL data measured under voltage clamp at 24°C and then apply Q10 factors to calculate model parameters for our full INaL description at 37°C. The details of the model parameters calculations have been described in our previous publications . Specific parameter values of the present study are given in Table 1. The stair case phenomenon was simulated by assigning a relatively low [Ca2+] of 0.125 μM as an initial value in both network SR and junctional SR before application of stimulation pulse train (at 1 Hz or 1.5 Hz).
2.4. Statistical Analysis
Multiple comparisons between treatment groups were made using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test or by the non-pared Student's t-test if appropriate. Data are reported as mean±SEM. The significance of SSA or SSI changes were evaluated using F-test (StatMost, DataMost Corp., Salt Lake City, UT) for tabulated values predicted by the model (Eqs. 2, 3) at a confidence level of 0.95. Differences for both experimental data and model predictions were considered statistically significant for P<0.05.
3.1. Calpain inhibitor MDL makes INaL decay faster in VCM from failing dog hearts
First we compare INaL decay in VCMs exposed to MDL with that in control, in the absence of the drug, at the intracellular Ca2+ = 1 μM (this intracellular Ca2+ was used in all experiments presented in this study). As it is shown in Fig. 1A in VCM exposed to MDL the INaL decay becomes faster than in control cell. Shown are raw traces along with the two-exponential fit (solid lines). Statistical data are given in Fig 1. B. Note that decay time course on the INaL became significantly faster in MDL-treated cells as it is obvious from the reduction of the time constants τ1 (upper panel) and τ2 (lower panel).
3.2. In VCMs from failing dog hearts calpain inhibitor MDL decreases INaL density in wide range of the membrane potential without affecting the steady-state activation voltage-dependency
Treatment with MDL significantly reduced INaL density in VCMs compared with control and vehicle-treated cells (Fig. 2 A). The density was measured as an average current at the membrane potential of –30 mV within 200–220 ms after the depolarization onset (shown by the vertical bar in Fig. 1A). Fig. B shows an effect of MDL on INaL density in the wide range of the membrane potentials assessed in the IV relationship (dots). The solid lines represent theoretical fit to the Eq.2 (see Methods) with the aim to assess SSA parameters (Shown in the graph). The MDL does not affect the voltage-dependence of the SSA as it is evident of the mid-potential position and the slope of the curve, which we found to be unchanged. At the same time MDL reduced the maximum conductance, Gmax, for INaL, which is expected because of the density reduction (See Fig. 2 Legend).
A. Statistical data for the INaL density in control (no vehicle), with the vehicle (DMSO), and in the presence of MDL (0.2 μM). The INaL density was measured at 200–220 ms after depolarization to –30 mV from the holding potential of –120 mV. B. Data points represent current-voltage relationship in control (blue triangles, red squares), and in the presence of MDL (black circles). The solid lines show theoretical curves of the steady-state activation (SSA, Eq.2, Methods) fitted to data point. MDL caused significant reduction of the maximum INaL conductance Gmax from 5.5 (control) and 0.47(vehicle) to 0.27 pS/pF (MDL) (P<0.001, F-test). Other fit parameters (mid-point potential, V1/2, and the slope coefficient, kG, remained almost unchanged in augmented in these conditions (values are shown at the traces). In all these experiments depicted in the figure the intracellular [Ca2+]i = 1 μM.
3.3. In VCMs from failing dog hearts calpain inhibitor MDL decreases does not affect the SSI of INaL
Fig. 3A shows experimental points obtained by the two-pulse protocol along with the theoretical fit (solid lines, Eq.3 in Methods) for the SSI evaluation. There was no statistical difference (F-test) when the theoretical curves corresponding to a MDL, vehicle or control (no vehicle) were compared. Fig. 3B shows statistics for the SSI parameters, mid-point potential and slope coefficient KA. There was no significant difference for these parameters pointing to the absence of the effect on SSI by the MDL.
A. Data points and theoretical fit (solid lines, Eq. 3, Methods) for the steady-state inactivation in control (blue triangles, red squares), and in the presence of MDL(black circles). B, C- statistical analysis of SSI parameters; midpoint potential (V1/2), and slope (KA). There was no statistical difference between these groups. Data points were pooled from 8 cells. The intracellular [Ca2+] = 1 μM in these experiments.
3.4. ECC model predictions of physiological importance
We tested if these experimentally measured INaL changes caused by the calpain inhibitor are physiologically significant in HF myocytes. Using INaL characteristics measured in the present study, we carefully calculated all INaL parameters (Table 1) required for numerical modeling of INaL effects as we previously established for VCMs of dog failing heart . Specifically, INaL,200ms is the density of INaL measured at 200 ms after membarne depolarization onset; τBM and τLSM are τ1 and τ2, respectively in Equation 1; kBM and kLSM are k1 and k2, respectively in Equation 1; parameters k G, V 1/2G, and k A, V1/2A are defined in Equations 2 and 3, respectively. We corrected INaL decay constants and maximum amplitude density for 37°C using Q10 factors (2.2 and 1.5, respectively) as we described in our previous studies ,  as follows: τ37 = τ24 ⋅2.2(24–37)/10 and Imax_37 = Imax_24 ⋅1.5(37–24)/10. Finally, Imax = Imax_BM + Imax_LSM and GNaL_max are the maximal total INaL peak current density and conductance, respectively. Fig. 4 upper panel shows results of the in silico test of how MDL-induced INaL changes affect AP shape and duration at a physiological temperature of 37°C at a pacing rate of 1 Hz. Note that simulated APs are shorter with lower plateau in VCMs treated by MDL. Lower panel of Fig. 4 shows the prediction of INaL dynamics (profile) during the AP in control and after MDL treatment. Our simulations show that the amplitude and duration of INaL become substantially smaller in MDL-treated cells vs. control (untreated) cells. Fig. 5 upper panel shows in silico simulations of the intracellular [Ca2+] dynamics in VCMs of failing heart. In response to the pulse train stimulation with the rate of 1.5 Hz, the diastolic Ca2+ level gradually increases in control conditions. The MDL-induced changes in INaL amplitude and decay kinetics almost completely eliminate this diastolic Ca2+ accumulation pattern. Lower panel of Fig. 5 shows simultaneous AP simulations for this condition. Note shorter AP with the lower plateau similarly to that shown in Fig. 4.
All simulations are based on the respective changes of INaL parameters measured experimentally under voltage clamp (see Methods for the modeling details and Table 1). Pacing rate 1.0 Hz.
Shown are model predictions for AP shape (lower panel) at 1 Hz pacing rate (steady-state) and cytosolic [Ca2+] (upper panel) in a train of 10 consecutive excitation pulses applied with a rate of 1.5 Hz. Note a substantial diastolic [Ca2+]i accumulation at the end of the pulse train in control but not in the presence of MDL. At the same time AP duration significantly decreased by MDL during the pulse train (lower panel).
For the first time we demonstrate at the single cell level that INaL alterations in amplitude and decay kinetics associated with chronic HF can be rescued by calpain inhibition. Our in silico simulations also demonstrate that the calpain modulation of INaL is physiologically important in HF myocytes, specifically, calpain inhibition greatly improves the myocyte function by reducing the action potential duration, and intracellular diastolic Ca2+ accumulation in the pulse train.
The calpain family is a group of cysteine proteases unique in their dependency on calcium to attain functionally active forms . Calpain is involved in a wide range of Ca2+-regulated cellular processes such as signal trasduction, secretion, cell proliferation, differentiation and apoptosis . Calpain deregulation resulting from the impaired Ca2+ handling is one of the important mechanisms for the pathological processes such as apoptosis and necrosis, reperfusion-induced heart stunning, ischemia and hypoxia, hyperthrophy and heart failure, and atrial fibrillation , . Therefore calpain inhibition is considered a therapeutic strategy targeting multiple disease states .
Our findings thus suggest a novel cellular and molecular mechanism to modulate NaCh that could be targeted to prevent pathophysiological consequences related to the augmented INaL in HF. There are some indications of the involvement of calpain into ion channel gating regulation, namely L-type Ca2+ channels , . In this context the calpain inhibition may serve to improve Ca2+ handling in failing heart and may be considered as a novel approach to modulate INaL current its related arrhythmias, and improve contractility , . Below we discuss possible cellular and molecular mechanisms of calpain effect on INaL.
4.1. Calpain and fodrin cytoskeleton
Fodrin-based cytoskeleton, an important element of the NaCh microenvironment in heart, is a dynamic structure that is altered under a variety of pathological conditions (e.g. ischemia or heart failure , , ). The role of the fodrin-based cytoskeleton in INaL modulation has been confirmed in our previous studies . It has also been shown that fodrin breakdown that occurs in some disease states featuring poor Ca2+ handling can be mediated by calpain , . Therefore prevention of Ca2+- induced fodrin cytoskeleton degradation will likely improve Ca2+ handling in HF.
4.2. Interplay between Ca2+, CAM/CaMKII cascade and calpain
It has been shown that INaL depends on the [Ca2+]i signaling cascade in the way that increased [Ca2+]i binds to EF-hand motif on NaCh C-terminal domain , ,  or via activating CaM/CaMKII cascade resulting in the augmented and slowed INaL , . This is very important mechanism of INaL regulation in HF because Ca2+ homeostasis is impaired in this disease stage. Inhibition of calpain results in reduced density of INaL despite of the presence of high [Ca2+]i that works in the opposite direction , . At the same time SSI and SSA parameters remain unchanged pointing to the fact that all channels are available for INaL and that the parameters of SSI and SSA depend on [Ca2+]i, rather than on calpain-dependent proteins. Indeed we have shown that the direct binding of Ca2+ to NaCh  (likely to E–F hand domain of NaCh C-terminus) is responsible for shifts of the half membrane potential of SSI voltage dependence towards depolarizing potentials . Therefore, reduction of INaL density produced by MDL likely results from reduced probability of NaCh transitions into different modes (burst and late scattered modes) that are involved in INaL formation . The faster INaL decay in the presence of calpain inhibition (Fig. 1) also indicates that gating of these modes is also affected by calpain.
4.3. Interplay between calpain and NaCh β-subunits
It has been shown that besides the main pore-forming α subunit of NaCh , the β2-subunit of NaCh is attached to the subsarcolemmal cytoskeleton . Therefore prevention of cleavage of fodrin by calpain may stabilize the cytoskeleton and enhance the β2-subunit dependent modulation of INaL that we have recently reported . We have shown that reduction of β2 expression by the siRNA increased INaL density and delayed its decay in VCMs from dogs with HF, i.e. very similar to that caused by the increased [Ca2+]i  (via activation of calpain) and opposite to that caused by the calpain inhibition by MDL shown herein.
4.4. Physiological relevance of Ca2+-calpain signaling to modulate INaL and to improve contractility and rhythm of failing heart
It has been established that INaL plays an important role in both electrical and contractile (via Ca2+ handling) deficiencies caused by chronic HF , . Then an important question is whether the magnitude of the effect of calpain inhibition on INaL reported in the present study is physiologically relevant. To address this question, we have carefully measured and analyzed specific characteristics of INaL in control and in the presence of MDL (Table 1) and then integrated them into our recently published ECC numerical model for ventricular cardiomyocytes of the failing dog , . As it is evident from Figs. 4 and 5, MDL substantially reduces effects of INaL on AP duration, which is known to increase in HF . The resultant decrease of INaL during AP plateau is observed as INaL becomes scaled (decreased) by about a factor of two during most of the plateau. This substantial “scaling” contributes not only to in AP duration shortening but also in to that AP plateau becomes substantially lower. This insight does not directly follow from the voltage clamp data and even not from AP simulations, because of a complex interplay of many Na+- and Ca2+- dependent mechanisms in ventricular cells reproduced by the dynamic ECC model.
Since shorter AP plateau and a smaller inward current during AP plateau are associated with less incidence of EADs , , one expected beneficial effect of calpain inhibition is also to reduce the probability of the EADs , a major mechanism for the triggered arrhytmia. Recently we have demonstrated that the augmented and slowed INaL in HF contributes to the diastolic [Ca2+]i accumulation in VCM of failing hearts during the pulse train . Reduction of INaL by the MDL significantly reduces this accumulation [Ca2+]i as it is predicted in silico (Fig. 5). Previously it has been demonstrated that delayed afterpotentials are linked to the diastolic Ca2+ accumulation associated with INaL, , . Therefore this predicted effect of MDL to prevent the diastolic [Ca2+]i accumulation indicates, in turn, that calpain inhibition can reduce probability of occurrence of the DADs.
Based on our present results with the specific calpain inhibitor MDL in ventricular cardiomyocytes isolated from failing dog hearts, we conclude that Ca2+-dependent calpain activation is able to strongly modulate INaL density and kinetics in failing myocardium. We illustrate in silico that the range of this modulation is physiologically relevant and remarkable as the calpain inhibition substantially improves (shortens) AP duration and prevents diastolic Ca2+ accumulation. Therefore, this Ca2+-dependent signaling cascade may serve as a plausible target to regulate INaL and its related electrical and contractile deficiencies in failing heart.
Conceived and designed the experiments: AU. Performed the experiments: AU. Analyzed the data: AU VM. Contributed reagents/materials/analysis tools: HNS. Wrote the paper: AU VM HNS.
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