The Reversible Covalent Reaction of Levosimendan with Cardiac

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The Reversible Covalent Reaction of Levosimendan with Cardiac Troponin-C in vitro and in situ Brittney Ann Klein, Béla Reiz, Ian Michael Robertson, Malcolm Irving, Liang Li, Yin-Biao Sun, and Brian D. Sykes Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00109 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Title: The Reversible Covalent Reaction of Levosimendan with Cardiac Troponin-C in vitro and in situ Brittney A. Klein1, Béla Reiz2, Ian M. Robertson4, Malcolm Irving3, Liang Li2, Yin-Biao Sun3, and Brian D. Sykes1* 1

Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada 2 Department of Chemistry, Faculty of Science, University of Alberta, Edmonton, Alberta T6H 2H7, Canada 3 Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King’s College London, London SE1 1UL, UK 4 Pharmaceutical and Health Benefits Branch, Ministry of Health, Government of Alberta, Edmonton, Alberta, T5J 3Z5, Canada Abstract: The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target since cardiac troponin-C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analog is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; determine whether the presence of the cTnI switch peptide or changes in either [Ca2+] and pH modify the reaction kinetics; and determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that it would be the highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart. (243 words)

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Introduction Cardiovascular disease (CVD) continues to be the number one cause of death worldwide [1]. In 2013, CVD was determined to be the underlying cause of about 1 in every 3 deaths in the US [2]. Preventative measures such as diet and exercise to mitigate risk factors that contribute to heart failure have been offset by the increasing number of women affected, the rise of individuals with diabetes and obesity, and an aging population [3]. Heart failure occurs when the heart is unable to pump sufficient blood to satisfy the needs of the body. Impaired cardiac muscle contraction results in systolic heart failure, referred to as heart failure with reduced ejection fraction (HFrEF). The most common cause of HFrEF is ischemic cardiomyopathy [4]. Positive inotropes, which increase the contractility of heart muscle to enhance cardiac output, would theoretically be useful in the treatment of systolic heart failure. Two positive inotropes commonly used in acute decompensated heart failure are dobutamine and milrinone, both of which upregulate sympathetic β-adrenergic signaling pathways to increase heart rate and stroke volume [5]. Although beneficial in the short-term, long-term use of these inotropes can lead to arrhythmias and hypotension [6]. Compounds acting as calcium sensitizers, which could increase the contractile response of the heart to calcium without altering calcium homeostasis, would have potential in the treatment of systolic heart failure. The thin filament is an attractive target for Ca2+ sensitizers, given that it is the site of Ca2+ binding and the subsequent trigger for contraction. Contraction is regulated in the heart muscle by troponin (cTn). cTn is a complex composed of C, I, and T subunits (cTnC, cTnI and cTnT, respectively) localized to the thin filament of the sarcomere. cTnC contains two globular domains: the regulatory N-terminal domain (cNTnC) that acts as the Ca2+ sensor, and the structural C- terminal domain that anchors cTnC on the thin filament. During systole, when the cytosolic Ca2+ concentration increases, Ca2+ binds to cNTnC and increases the prevalence of the open conformation of cNTnC [7,8]. Following this conformational change, the switch region of cTnI (switch-cTnI) binds to cNTnC and drags the inhibitory and C-terminal regions of cTnI away from actin. This leads to an allosteric change in tropomyosin exposing the myosin binding sites on actin thereby promoting the formation of the force-producing cross-bridges [9,10]. However, cTnC is also a major contributor to Ca2+ buffering in the cell [11], so that a change in the intrinsic Ca2+ affinity of cTnC would alter Ca2+ homeostasis. There are several structures of drug-cTnC complexes [11], which have led to the proposal that an approach to increasing force at a constant Ca2+ level is the modulation of the affinity of cNTnC for the switch region of cTnI (stabilization leading to sensitization [13], and reduced affinity leading to desensitization[14]). Targeting of this cTnC – cTnI protein-protein interface has been difficult as the hydrophobic cleft and regulatory helical target is a common structural motif found in cTnC, skeletal TnC, calmodulin, and the regulatory and essential light chains of myosin. The highest affinity compound published to date


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is of Tirasemtiv binding to the fast skeletal Tn complex [15]. An alternative target would be the myosin thick filaments which are responsible for force generation; examples include Omecamtiv Mecarbil [16] and EMD 57033 [17]. Developing drugs to target the heart presents difficulties beyond the litany of normal concerns such as specificity, toxicity and bioavailability. The most important concern is the determination of the optimal efficacy. In the case of a targeted drug to a disease like cancer, one wants to completely stop the cancer. However, for the heart, one wants to only make small changes and not stop the heart completely. Correlating the impact that small modifications in isolated sarcomeric proteins have on the heart has been challenging. Single mutations in the sarcomeric proteins observably cause large scale remodeling of the heart and lead to dilated cardiomyopathy (DCM) and/or hypertrophic cardiomyopathy (HCM) [18]. When proteins carrying these mutations are studied using biophysical methods, it is often difficult to discern significant changes in their physical properties as seen with L29Q in cTnC [19]. In a similar manner, a small change in TnI phosphorylation has been shown to alter myocardial function [20]. This exemplifies the significance small perturbations can have on the normal function of the heart. Designing a drug with specificity to only cardiac muscle has also proved difficult as the structures of the sarcomeric proteins in striated muscles are similar. These issues are undoubtedly reflected in the lack of existing drugs. The potential of covalent drugs has recently been reviewed, tracing the history from Aspirin and Penicillin to the most recent cancer drug, Ibrutinib [21-24]. These included targeted [25] and reversible covalent drugs [26]. The fact that C84 is unique to cTnC, raises the question of whether targeting of C84 with a reversible covalent drug would be a selective way to target the heart. One of the most widely studied Ca2+ sensitizers is levosimendan [27], which has been the focus of numerous large clinical trials for the treatment of various cardiovascular disorders, including heart failure [28]. While levosimendan binds to cTnC in vitro, the details of its in vivo action remain elusive. Besides Ca2+ sensitization, levosimendan has also been shown to have vasodilatory, anti-inflammatory, and anti-apoptotic effects [29,30]. Although levosimendan inhibits phosphodiesterase 3 (PDE3) at high concentrations, its positive inotropic effects are thought to be due to its interaction with cTnC and not to an increase in intracellular Ca2+ [31-34]. Studies have shown that C84 is essential for binding [35,36] and that levosimendan does not bind to the N-terminal domain of cTnC when C84 is mutated to a serine [37]. We have recently shown that when a covalent cTnC-levosimendan analog is exchanged into ventricular trabeculae, the cardiac muscle cells become constitutively active, even in the absence of Ca2+, demonstrating the potency of a levosimendan molecule bound to cTnC [38]. We have also established that levosimendan reacts specifically with C84 of cTnC to form a reversible covalent thioimidate bond, and that it only reacts with the Ca2+ bound conformation of cTnC [39]. We suggested that this


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results in a small fraction of levosimendan covalently bound at steady state and that this might be enough to enhance contractility in vivo. In the present study, we verify the in vitro mechanism of action for levosimendan so that rate constants can be derived. We determine whether the presence of the cTnI switch peptide or changes in either [Ca2+] or pH modify the reaction kinetics, and demonstrate that the reaction can occur in situ in porcine cardiac myofibrils. We show that the reaction is consistent with a reversible covalent mechanism, and quantify the rate and equilibrium constants associated with the various environments. Using these kinetic rate constants, we predict the concentration of reacted cTnC in vivo under the conditions studied. We calculate that the greatest amount of covalent levosimendan-cTnC complex would be formed under the acidotic conditions that are associated with late stage heart failure and discuss whether the predicted level would have appreciable therapeutic value. The results provide a rationale and basis for the development of reversible covalent drugs to target a failing heart.


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Materials and Methods Proteins Human cTnC (C35S) (MW 18386.5 Da) and cChimera_thm (MW 15936.7 Da) were prepared as previously described [38,40]. Wild-type human cTnC (UniProtKB/Swiss-Prot: P63316) differs from porcine cTnC (UniProtKB/Swiss-Prot: P63317) by a single amino acid, D115E. Endogenous porcine cTnC extracted from cardiomyofibrils is N-acetylated and has an expected mass of 18459.9 Da. [41]. Human cardiac switch peptide (cSp) (cTnI144-163) was purchased from G.L. Biochem Ltd. (MW 2199.7 Da). Myofibril Preparation Frozen hearts from young pigs aged 6-8 months were obtained from Pel-Freez Biologicals. Left ventricular muscle was excised from the porcine hearts and stored at -20 °C. Cardiomyofibrils were obtained from ~200 mg of porcine cardiac muscle in a series of homogenization and centrifugation steps. Initially, the muscle was partitioned into smaller pieces and rinsed with 900 µL of rigor buffer (50 mM imidazole, 100 mM KCl, 2mM MgCl2 and 1mM EGTA at pH 7), 100 µL of 10% (v/v) Triton-X100 and 10 µL of 100X protease inhibitor cocktail (PIC, CalBiochem 539131). The muscle was re-suspended in the above solution and homogenized on ice using a PowerGen 125 homogenizer at full speed for one minute. The homogenate was centrifuged at 9500 rpm for 3 minutes in a Beckman Coulter Microfuge 18. The supernatant was removed and the process repeated a total of 4 times, the last 2 in the absence of Trition-X100. The pellet obtained from the final homogenization step was gently re-suspended with 500 µL of rigor buffer and 5 µL PIC then centrifuged. The supernatant was removed, the rinsing process repeated and the cardiomyofibrils were stored overnight at 4 °C in 500 µL Ca2+ rigor buffer (rigor buffer with 2 mM CaCl2). In Situ Reaction of Levosimendan The protocol used is shown diagrammatically in Fig. S4. After the overnight incubation period, the myofibrils were rinsed twice with Ca2+ rigor buffer and centrifuged at 9500 rpm for 3 minutes. Maxchelator (http://www.stanford.edu/∼cpatton/maxc.html) was used to determine the volume of 1M CaCl2 needed to attain 2mM Ca2+ in solution. All stocks of ((4-[(4’R)-4-methyl6-oxo-1,4,5,6-tetrahydropyridazin3-yl] phenyl) hydrazo-no) propanedinitrile (levosimendan) (MW 280.28 Da) were prepared fresh to a concentration of 10 mM by dissolving in dimethyl sulfoxide (DMSO). Levosimendan is soluble up to 3 mM in phosphate buffer at pH 8 [42]. To investigate the reaction of levosimendan with cTnC in situ, the cardiomyofibrils were soaked with 10, 50 or 500 µM levosimendan in Ca2+ rigor buffer at room temperature. Every hour for 3 hours, the cardiomyofibrils were centrifuged and reinfused with 500 µL of levosimendan solution in an attempt to emulate the perfusion used in the intravenous clinical protocol [42].


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Extraction of cTnC from Cardiomyofibrils Upon completion of the last infusion of levosimendan solution, the cardiomyofibrils were spun down and re-suspended in 500 µL of extraction buffer (5mM EDTA, 40 mM Tris at pH 8.5). The cardiomyofibrils were subjected to extraction conditions for 45 minutes at room temperature before being centrifuged. The supernatant obtained from this centrifugation step was concentrated in a 3K Nanosep® centrifugal device (PALL) and a 50 µL aliquot of the retained volume was used for mass spectrometry experiments. In Vitro Levosimendan Reaction Studies To investigate if pH, [Ca2+] and/or the presence of cTnI influenced the rate a covalent bond formed between levosimendan and cysteine 84 (C84) of cTnC, the reaction was monitored at several time intervals using LC-MS. The conditions used to perform these experiments are outlined in Robertson et al. [39]. All experiments were conducted using 1 mg/mL solutions of recombinant human cTnC (C35S), cChimera_thm or 1:1 cTnC (C35S): cTnI144-163 complex and reacted with 500 µM levosimendan (in DMSO to a final concentration of 5% v/v). To study the effect of decreasing the [Ca2+] on the formation of the thioimidate bond in cTnC (C35S), 2 mM EGTA was added to the buffer and the volume of 1M CaCl2 needed to attain a pCa (-log10[Ca2+]) of 6 or 7 was calculated using Maxchelator. Changes in the rate of formation of the levosimendancTnC (C35S) complex at lower pH were also investigated by decreasing the buffer’s pH to 6.4. The fraction of levosimendan-protein complex at various time points was measured by mass spectrometry and used to determine the rate of formation of the thioimidate bond in each system. Liquid Chromatography- Mass Spectrometry To monitor the formation of the thioimidate bond in vitro, changes to the cTnC (C35S) were analyzed using reverse phase high performance liquid chromatography and detected by mass spectrometry (RP-HPLC-MS). These experiments were performed on an Agilent 1200 SL HPLC system using a 75 X 0.5 mm Poroshell 300SB-C8 column, which has a particle size of 5 µm (Agilent Technologies, USA) and an Opti-pak trap cartridge kit. A 5 µL aliquot of the sample was loaded onto the column at a rate of 0.15 mL/min using an initial buffer composition of 80% mobile phase A (0.1% formic acid (FA) in water) and 20% mobile phase B (0.1% FA in acetonitrile). After injection, the column was washed using 20% mobile phase B for 2 minutes to remove salts. To effectively elute proteins, a linear elution gradient was employed where the concentration of mobile phase B was increased to 98% over 12 minutes (See Supplementary Information Section). The column temperature was maintained at 60 °C throughout these experiments. Determination of the molecular weights of the proteins extracted from the reaction of levosimendan with porcine cardiomyofibrils were conducted on the same HPLC system as


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described above, using a Phenomenex Aerius 3.6 µm, WIDEPORE XB-C8, 200 Å, 2.1 X 50 mm column with a guard column. A 50 µL sample was obtained from either concentrated extract or the supernatant from the first soaking with levosimendan. For analysis of these samples, a 5 or 10 µL aliquot was injected onto the column, respectively. The column was washed at a flow rate of 0.5 mL/min using 2% mobile phase B. A different linear gradient was used for elution of the proteins (Supporting Information). The column temperature was regulated to 40 °C. All mass spectra were acquired in positive mode of ionization using an Agilent 6220 Accurate-Mass Orthogonal Acceleration Time-of-Flight (oaTOF) HPLC/MS system (Santa Clara, CA, USA) equipped with a dual sprayer electrospray ionization source with the second sprayer providing a reference mass solution. Individual spectrums were mass corrected using peaks at m/z 121.0509 and 922.0098 from the reference solution and data analysis was performed on Agilient MassHunter Qualitative Analysis software package (version B.03.01 SP3). For further mass spectrometric conditions, see Supplementary Methods.


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Results 1. Verification of the Reaction Mechanism Robertson et al. [39] measured the rate of formation of a reversible covalent complex between recombinant human cTnC (C35S) and levosimendan at various levosimendan concentrations in vitro (Fig. S1). The starting point for all the in vitro experiments conducted in this study employed the same experimental conditions as previously described [39]. A repetition of the mass spectrometry experiment to monitor the formation of the levosimendan-cTnC (C35S) adduct at a levosimendan concentration of 500 µM was performed (Fig. 1A).

B

A

C

D

Figure 1. Formation of the levosimendan-troponin C adduct monitored by mass spectrometry. The fraction of covalent complex formed was calculated using peak intensities from deconvoluted mass spectra over a 5-hour time interval. This data was subsequently fit to Equation 1 to extract the kinetic rate constants which are presented in Table 1. A) Fraction of covalent complex derived from the peak intensities of cTnC (C35S) at 18387 Da and the levosimendan-cTnC (C35S) adduct at 18667 Da when 500 µM levosimendan was reacted with 1


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mg/mL cTnC (C35S) as described in Robertson et al. [39]. Not shown is a point equal to 0.75 taken at 24 hrs. B) Changes to the rate of formation of the covalent adduct in the presence of cSp (cTnI144-163) (blue) and cChimera_thm (red) using the peak intensities of cTnC (C35S) at 18387 Da, cChimera_thm at 15937 Da and the covalent adduct at 18667 Da and 16217 Da, respectively. C) Effect of increasing pCa on the formation of the levosimendan-cTnC(C35S) adduct: pCa 3.8 (black), pCa 6 (blue) and pCa 7 (red). D) Influence of lowering pH on the rate of formation of levosimendan-cTnC(C35S) adduct from pH 7 (black) to 6.4 (blue). The fraction of covalent complex formed (MW 18667 Da) was measured using the peak heights from the deconvoluted mass spectra after levosimendan was reacted with cTnC (C35S) for 1, 5, 15, 30, 60, 120 and 300 minutes. Before determining if the kinetics of this reaction were perturbed by altering the experimental conditions, we had to establish what the rate constants were for the conditions used in Figure 1A and if the data fit a reversible covalent mechanism (Eq.1). Equation 1 describes the mechanism wherein a protein (P) and a drug (D) first produce a Michaelis complex (P:D) which is then converted to a covalent complex (P-D). k-1 k-2 P + D D P:D D P-D

k1 k2

Eqn. 1. Mechanism for the formation of a reversible covalent bond between a protein (P) and a drug (D). Forward rate constant, k1(M-1s-1), for the formation of Michaelis complex (P:D). Reverse rate constant, k-1(s-1) for the dissociation of P:D into starting materials. Forward rate constant, k2 (s-1), for the formation of covalently bound protein-drug complex (P-D). Reverse rate constant, k-1 2 (s ), for the dissociation of the covalently bound P-D complex into P:D. To extract the rate constants, the data from the in vitro experiment was fit to Equation 1 using simulations performed in Mathematica. Since it was impossible to uniquely determine all four rate constants, we first set k1 to a typical diffusion limited value of 1x107 M-1s-1 and constructed contour plots of the Root-Mean-Square Deviation of the fit versus k2, and k-2, for various values of the dissociation constant for the levosimendan:cTnC(C35S) Michaelis complex (Fig.S2). The reverse rate constant for the Michaelis complex, k-1, is directly related to KD1, therefore any variations described to KD1 can be associated with changes to k-1. When KD1 was set in the range of 0.1-1 mM, the value of k2 increased as KD1 was increased. In all subsequent RMSD plots, k-1 was set to 7500 s-1, corresponding to a KD1 of 750 µM. This value was chosen as previous studies [43] measured the dissociation constant for levosimendan bound to AcyscNTnC:Ca2+:cTnI144-163 to be approximately 0.7 mM. The data was fit with KD1 = 750 µM, k2 = 0.0016 s-1 , and k-2 = 0.00032 s-1 (Fig.1A) The resulting value for KD2 is 0.2, which predicts a


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dissociation constant for total bound drug [P:D + P-D] given by (KD1*KD2)/(1 + KD2) of roughly 125 µM. This dissociation constant is close to the value estimated in paramagnetic broadening experiments of 190 +/- 70 µM [39]. The data from Robertson et al. [39, and unpublished results] were fit with a KD1 = 750 µM, k2 = 0.0016 s-1, and k-2 = 0.0002 s-1 (Fig.S1). The resulting value for KD2 is 0.13, which predicts a dissociation constant for total bound drug of 90 µM, consistent with the dissociation constant reported in the present study. Kinetics of thioimidate reaction Predicted [P-D] k2 (s-1) k-2 (s-1) in vivo (%) Panel B cTnC C35S 0.0016 0.00032 0.9 cTnC C35S + cSp (cTnI144-163) 0.0006 0.00018 0.6 cChimera_thm 0.00085 0.00022 0.7 Panel C cTnC C35S pCa 3.8 0.0016 0.00032 cTnC C35S pCa 6.0 0.0007 0.00007 1.7 cTnC C35S pCa 7.0 0.00048 0.00006 1.4 Panel D cTnC C35S pH 7.0 0.0016 0.00032 cTnC C35S pH 6.4 0.0007 0.00004 2.9 Table 1. Kinetic rate Constants for the in vitro reaction of levosimendan with Troponin C and extent of reaction predicted in vivo using them. The rate constants k2 and k-2 were derived from fitting the data in Figure 1 to Equation 1 using a dissociation constant of 750 µM (k1 set to 1x107 M-1s-1 and k-1 set to 7500 s-1). The percent of the covalent complex formed in vivo after 24 hours was subsequently predicted for each set of rate constants using the in vivo concentrations that would be present in a clinical setting [42].


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2. Influence of cSp/ cChimera on Rate of Formation of Thioimidate Bond In Vitro cTnC:2 Ca2+ + Ca2+ cTnC:3 Ca2+ cTnC:3 Ca2+ cTnC:3 Ca2+:cSp or cChimera (Closed) (Closed) (Open) (Open) Diastole Systole Eqn. 2 Conformational changes induced in troponin C as a function of Ca2+ concentration and binding of cardiac switch peptide (cSp). cTnC:2 Ca2+ represents Ca2+ in sites III and IV and cTnC:3 Ca2+ represents Ca2+ in sites II, III and IV. Conformational changes to the regulatory N-terminal domain of cTnC are induced in the presence of calcium and cSp (Eqn. 2). When cTnI switch peptide is absent from the environment, the calcium-saturated form of cTnC adopts a predominantly closed conformation, restricting the area of the hydrophobic pocket that is exposed [44]. C84 is located on the edge of this hydrophobic pocket and has been identified as the residue that reacts specifically with levosimendan to form a reversible covalent bond [39]. The thioimidate bond forms as long as calcium is present [39], undeterred by the closed conformation that is favored. cSp stabilizes the open conformation of Ca2+-saturated cTnC and makes the hydrophobic region more accessible [45]. However, the presence of cSp can also impede the binding of compounds in the hydrophobic region [46] due to steric interactions caused by the competitive binding of cSp in the same area [39]. To investigate how the presence of cSp influenced the rate of formation of the covalent adduct, we studied how the cSp would influence the reaction rates of the covalent bond in two different scenarios. In one scenario, we reacted 500 µM levosimendan with a 1:1 mixture of 1 mg/mL cTnC (C35S):cSp (cTnI144-163). In the other scenario, 500 µM levosimendan was reacted with 1 mg/mL of cChimera_thm, where cTnI144-173 is secured to cNTnC by a flexible linker [40]. Using the deconvoluted mass spectra attained from RP-HPLC-MS, we compared the peak heights of the unreacted cTnC (C35S):cSp (cTnI144-163) at 18387 Da (expected MW 18386.5 Da) or unreacted cChimera_thm at 15936 Da (expected MW 15936.7 Da) to the peak heights of the covalent complexes formed at 18667 Da (expected MW 18666.7 Da) and 16217 Da (expected MW 16216.7 Da), respectively. We measured the fraction of covalent complex formed at numerous time points (Fig.1) and fit this data using rates extracted from RMSD plots (Fig. S3). The rates for the formation and dissociation of the thioimidate bond are slower in the presence of the cSp (Table 1). The slower rates of formation derived in both scenarios where the cSp is present indicate that there are likely steric interactions that hinder the binding of levosimendan to C84. The slower rates of dissociation provide evidence that the covalent thioimidate bond formed is stabilized, similar to the observed stabilization of the ternary intermediate formed between cTnC:cTnI with bepridil [46] and EMD 57033 [47].


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3. Influence of Ca2+ Concentration on Rate of Formation of Thioimidate Bond In Vitro To mimic the effect of a diastolic environment on the reaction between levosimendan and cTnC(C35S), the fraction of levosimendan-cTnC(C35S) formed at 18667 Da (expected MW 18666.7 Da) was measured at two different Ca2+ concentrations corresponding to pCa 6 and pCa 7 (Fig.1). The rate of formation and the rate of dissociation of the thioimidate bond at both pCa 6 and 7 were determined to be slower than the rates when levosimendan was incubated with cTnC(C35S) in a Ca2+ saturated environment (Table 1). Previous work showed the Ca2+ dependence of levosimendan binding to cTnC [48] and explicitly for the formation of the covalent thioimidate bond [39]. Here we show that the rate of formation of the covalent levosimendantroponin complex depends on Ca2+ concentration in the physiological range (Fig. 1, Table 1). Interestingly, at low Ca2+ concentrations the fraction of covalent complex formed after 5 hours was determined to be approximately the same within experimental error as the fraction of covalent complex formed in a Ca2+ saturated environment. The rate of dissociation of the covalent complex at low Ca2+ concentrations also slowed, indicating a stabilization of the thioimidate bond, as seen with the addition of the cSp. 4. Influence of pH on Rate of Formation of Thioimidate Bond In Vitro Patients with myocardial ischemia have impaired cardiac muscle contraction which impedes the distribution of oxygenated blood throughout the body. This decreased circulation results in acidosis, which can drop the intracellular pH from 7.4 to 6.5 or lower [49]. To simulate the effects of acidosis on the rate of formation of the thioimidate bond in vitro, we reduced the pH of our buffer to 6.4. The rate of formation of the covalent complex was determined by measuring the rate of formation of the thioimidate bond at 18667 Da (expected MW 18666.7 Da) from the deconvoluted mass spectra (Fig.1). The rates of formation and dissociation of the covalent adduct extracted from the RMSD plots (Fig. S3) were both slower when levosimendan was incubated with cTnC (C35S) in this slightly acidic environment (Table 1). The slower rate of formation of the thioimidate bond could be a result of a lower Ca2+ affinity at lower pH favoring the closed conformation of cTnC or the precipitation of levosimendan from solution as it goes from a negative charge state to a neutral charge state at lower pH [39]. The slower rate of dissociation of the levosimendan-cTnC(C35S) complex suggests that there is a stabilization of the thioimidate bond at pH 6.4. This may be the result of direct stabilization of the thioimidate bond, which is more stable when the imine nitrogen is protonated [50], or through electrostatic stabilization of the neutral thioimidate bond by negatively charged residues in the immediate vicinity [39]. The latter would explain the importance of preserving selected amino acids in this region and the deleterious effects of mutations to D87 and D88 on the binding of levosimendan to cTnC [51].


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Reaction of Levosimendan with cTnC In Situ in Cardiac Myofibrils To determine if the thioimidate bond forms between levosimendan and cTnC in situ, cardiomyofibrils were prepared from porcine left-ventricular cardiac muscle and subjected to low salt extraction for 45 minutes. The deconvoluted mass spectrum (Fig.2) from the concentrated supernatant immediately following extraction shows a series of peaks within the mass range of 18000 Da to 22000 Da. The peaks were identified as components of the thin and thick filaments [41,52,53]. Endogenous N-acetylated-cTnC (18459.9 Da) can be detected in the cardiomyofibrils, along with Na - tri methylated ventricular regulatory light chain (RLCv, 18803.2 Da), monophosphorylated RLCv (18881.4 Da), and the Na - trimethylated essential light chain (ELCv, 21753.3 Da). The expected amount of cTnC in wet tissue is 48 pmol/mg and the amount of RLC and ELC is 147-159 pmol/mg [54]. 500000

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Figure 2. Deconvoluted mass spectrum of endogenous thin and thick filament proteins from porcine cardiomyofibrils. Myofibrils were immersed in extraction buffer for 45 minutes. The components of the sarcomere that were extracted from left to right correspond to N-acetylated cTnC (18459.9 Da), Na - trimethylated RLCv (18801.4 Da), pRLCv (18881.4 Da) and Na - trimethylated ELCv (21753.3 Da). The peak at MW 20935 Da remains unidentified. These peaks were identified from Gregorich et al. [41,53]. Cardiomyofibrils from a separate preparation were reacted with 500 µM levosimendan 2+ in Ca -rigor solution at room temperature before undergoing extraction. The deconvoluted mass


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spectrum demonstrates that levosimendan reacted with cTnC (18459 Da) to form a covalent thioimidate bond (18739 Da) whilst cTnC is complexed with other proteins in the muscle fiber (Fig.3C). The percentage of levosimendan-cTnC adduct formed corresponds to 35% of the native cTnC that was extracted from the cardiomyofibrils in this sample (Fig.3C). The total amount of cTnC (unreacted native cTnC and levosimendan-cTnC adduct) extracted from the cardiomyofibrils soaked with levosimendan is less than the amount of cTnC extracted from cardiomyofibrils with no exposure to levosimendan, while RLCv is similar to the control (Fig.3A). This suggests either a loss of cTnC in steps preceding the extraction or that cTnC is not as readily extracted from the cardiomyofibrils after being immersed in a levosimendan solution. The experiment was repeated at a levosimendan concentration of 50 µM. Less native cTnC and no reacted complex were observed in the extraction solution (Fig.3B). The supernatants obtained from the first wash of the cardiomyofibrils with levosimendan were analyzed using mass spectrometry. The deconvoluted mass spectra detected small amounts of endogenous cTnC, ELCv and RLCv in the washes at both levosimendan concentrations (unpublished results). The in situ experiments demonstrate that a covalent complex between levosimendan and cTnC is observed when cTnC is anchored within the myofibril. One of the limitations of the mass spectroscopy method used in this work is that it does not indicate where the covalent bond forms, as the endogenous cTnC contains both C35 and C84. However, no species with two covalent adducts is observed, supporting the targeted interaction with C84 observed in vitro [39].


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Counts vs. Deconvoluted Mass (amu) Figure 3. Deconvoluted mass spectrum of endogenous thin and thick filament proteins extracted from porcine cardiomyofibrils. The components extracted from left to right are cTnC (18458 Da), levosimendan-cTnC complex (18738 Da), Na-trimethylated RLCv (18801 Da), pRLCv (18884 Da) and ELCv (21767 Da). A) Figure 2 scaled to provide a control. B) Extraction of cardiomyofibrils after soaking with 50 µM levosimendan. C) Extraction of cardiomyofibrils after soaking with 500 µM levosimendan. Insert shows the formation of the covalent levosimendan-native cTnC adduct within the myofibrils (18739 Da).


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Discussion In this study, we have demonstrated that the kinetics of the reaction of levosimendan to form a thioimidate bond with cardiac TnC in vivo is consistent with a reversible covalent reaction [25] wherein C84 is preferentially reacted over C35 which is located outside the hydrophobic cleft of cTnC. This allows us to extract the rate constants that characterize the reaction in order to predict the extent of the reaction in vivo. Further, we have shown that under all of the in vitro conditions used, including in the presence of the cTnI switch peptide and changes in the [Ca2+] and pH, none of the rate constants are greatly affected. We have also demonstrated that the reaction can occur to a similar extent when the cTnC is in situ in cardiac myofibrils. The most significant change observed is at low pH, consistent with the increased stability of a thioimidate bond upon protonation [50]. These changes could be clinically significant in vivo (see below). This mechanism provides an explanation of the earlier puzzling NMR spectroscopy results of Sorsa et al. [55] who observed very slow changes in their NMR spectra of cTnC in the presence of levosimendan, and the fact that the spectroscopic changes observed during their levosimendan titration did not go to completion but reached a plateau; both consistent with a reversible covalent mechanism. Using their cTnC and levosimendan concentrations and our derived rate constants we predict 64% reaction which is very close to their results. This mechanism also explains why the apparent levosimendan binding constants we have observed were different for the Cys-null and wild type cTnC [39,43]. The remaining question to be then addressed is to what extent does this reaction occur in vivo? Clinically, administration of levosimendan 8 or 24 µg/kg IV in patients undergoing elective cardiac surgery significantly increased cardiac output, heart rate and stroke volume without significantly increasing myocardial oxygen consumption [56]. For a 75-kg person, the total amount is 0.6-1.8 mg and the concentration in blood is about 0.12-0.36mg/L (0.43-1.3 μM). Sometimes a higher dose is used, e.g. a 10-minute loading dose (6 or 12 µg/kg) followed by a 24hour infusion (0.1 or 0.2 µg/kg/min). This ends up with about 8 or 16 μM levosimendan in the blood. The bioavailability of levosimendan has been measured to be 85% [57]. Using a levosimendan concentration of 1.3 µM, and cTnC concentration in the tissue of 50 µM [54], we can predict the amount of cTnC which would form a covalent bond with levosimendan using the rate constants from Table 1. The kinetic simulations are the same as those used to fit the in vitro data with the exception that the levosimendan concentration is held constant in the in vivo simulation to emulate the IV perfusion in the clinical protocol. The amount of covalent complex formed after 24 hours (also from the clinical protocol) are shown in Table 1. The predicted amount raises from approximately 0.6% using the rate constants from the pH 7 experiments to 3% at low pCa, low pH that would occur in ischemic acidosis. This could have significant therapeutic implications, implying the drug becomes more effective when the heart is ischemic.


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The predicted concentration of covalent complex is directly proportional to the concentration of levosimendan under the conditions studied. One could then ask whether this small amount of covalent adduct would be sufficient to be clinically effective? For consideration: first, mutant proteins from familial hypertrophic cardiomyopathy diseases (such as L29Q in cTnC) show only minor changes in their in vitro biophysical and physiological properties, and yet result in large remodeling of the heart [19,58]. Similarly, small changes in TnI phosphorylation have been shown to alter left ventricular and myocardial function in mice [20]; second, the force-pCa curve for cardiac muscle is very cooperative, and much of the cooperativity is from the thin filament [59]; third, the heart works in low end of pCa curve (see for example the dashed line in Figure 3 of [13]); and fourth, only 1030% of myosin molecules are involved in force generation at any given time and an individual myosin head may go through only one power stroke in the course of systolic contraction [58]. We therefore propose that a small percentage of levosimendan-covalently bound TnC could make a significant contribution to the action of levosimendan in the clinical setting. Finally, the goal of developing a cardiac modulator which does not alter the effective pCa of the cTnC (and therefore Ca2+ levels because cTnC is a Ca2+ buffer in the cell [11]) may be thermodynamically improbable given that all of the binding equilibria are coupled. Under these circumstances, reaction of only a small percentage of the cTnC with a very effective covalent drug would get around this issue because the change in buffering capacity would be small. Whether or not the clinically effectiveness of levosimendan is a result in part of this reversible covalent interaction, the results presented in this paper suggests that this mechanism could be an effective direction for the design of drugs to specifically target the heart. The approach targets cardiac TnC, which reduces off-target reactions, and it is covalent to be effective, tunable to achieve appropriate efficacy, most effective when needed ( i.e., during acidosis), and reversible to go back to normal when finished. Acknowledgements This research has been supported by grants from The Heart and Stroke Foundation of Canada (G-14-0005884, BDS) and The British Heart Foundation (FS/15/1/31071, YBS). The authors thank Dr. Thomas Kampourakis for helpful discussions, and Dr. Mark Holt and Professor Leo Spyracopoulos for help with Mathematica. Supporting Information Includes LC-MS experimental conditions, fitting of previous data to a reversible covalent mechanism, contour plots used to extract rate constants, and experimental protocol for cardiac myofibril extraction experiments.


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The Reversible Covalent Reaction of Levosimendan with Cardiac

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