Late sodium current inhibitors to treat exercise‐induced obstruction in hypertrophic cardiomyopathy: an in vitro study in human myocardium

Abstract

Background and Purpose

In 30–40% of hypertrophic cardiomyopathy (HCM) patients, symptomatic left ventricular (LV) outflow gradients develop only during exercise due to catecholamine‐induced LV hypercontractility (inducible obstruction). Negative inotropic pharmacological options are limited to β‐blockers or disopyramide, with low efficacy and tolerability. We assessed the potential of late sodium current (I_NaL)‐inhibitors to treat inducible obstruction in HCM.

Experimental Approach

The electrophysiological and mechanical responses to β‐adrenoceptor stimulation were studied in human myocardium from HCM and control patients. Effects of I_NaL‐inhibitors (ranolazine and GS‐967) in HCM samples were investigated under conditions simulating rest and exercise.

Key Results

In cardiomyocytes and trabeculae from 18 surgical septal samples of patients with obstruction, the selective I_NaL‐inhibitor GS‐967 (0.5 μM) hastened twitch kinetics, decreased diastolic [Ca²⁺] and shortened action potentials, matching the effects of ranolazine (10μM). Mechanical responses to isoprenaline (inotropic and lusitropic) were comparable in HCM and control myocardium. However, isoprenaline prolonged action potentials in HCM myocardium, while it shortened them in controls. Unlike disopyramide, neither GS‐967 nor ranolazine reduced force at rest. However, in the presence of isoprenaline, they reduced Ca²⁺‐transient amplitude and twitch tension, while the acceleration of relaxation was maintained. I_NaL‐inhibitors were more effective than disopyramide in reducing contractility during exercise. Finally, I_NaL‐inhibitors abolished arrhythmias induced by isoprenaline.

Conclusions and Implications

Ranolazine and GS‐967 reduced septal myocardium tension during simulated exercise in vitro and therefore have the potential to ameliorate symptoms caused by inducible obstruction in HCM patients, with some advantages over disopyramide and β‐blockers.

Abbreviations

LV

left ventricle

LVOT

left ventricular outflow tract

HCM

hypertrophic cardiomyopathy

I_NaL

late sodium current

I_CaL

L‐type calcium current

EAD

early after‐depolarization

DAD

delayed after‐depolarization

APD

action potential duration

Introduction

Symptoms related to obstruction occurring at the left ventricular outflow tract (LVOT) are present in approximately 65% of hypertrophic cardiomyopathy (HCM) patients (Gersh et al., 2011; Authors/Task Force et al., 2014). Marked upper septal hypertrophy and LV hypercontractility lead to accelerated blood flow in the LVOT, exerting a drag force on the elongated anterior mitral valve leaflet that moves towards the septum, ultimately determining a significant pressure gradient (>30 mmHg) in the LVOT (Maron et al., 2006). While a significant gradient is present at rest in about half of patients with obstructive HCM, obstruction develops only during stress or exercise in the other 50% (‘dynamic’ or ‘inducible’ obstruction) (Maron et al., 2006; Pozios et al., 2015).

To date, pharmacological options to treat inducible obstruction are limited to http://www.guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId=4 antagonists (β‐blockers) or http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7167. High doses of non‐selective β‐blockers, such as 80–100 mg·day⁻¹ of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=554, are often required to reduce dynamic gradients (Nistri et al., 2012) but a large number of patients with exertional symptoms do not tolerate such aggressive β‐blocker treatments and are left with residual symptomatic gradients. Disopyramide is an non‐selective compound that primarily behaves as a http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=582 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=572 K⁺ channel blocker and is extensively used in HCM patients with rest obstruction to relieve symptoms associated with LVOT gradients or intraventricular gradients. The clinical efficacy of disopyramide on rest obstruction has been attributed to its negative inotropic effect. Through the reduction of LV contractility, disopyramide slows down the acceleration of flow in the LVOT during systole, thus delaying or abolishing mitral‐septal contact (Sherrid et al., 2005). However, the use of disopyramide for inducible obstruction is limited by its unpredictable efficacy on dynamic gradients, particularly those elicited by exercise (Maron et al., 2006), and by the important side effects of the drug (e.g. the anticholinergic effects). Thus, there is a strongly felt need to identify more effective and better tolerated agents for patients with obstructive HCM. However, the identification of new pharmacological options to reduce dynamic gradients is limited by the lack of studies on the cellular pathophysiology of exercise‐induced obstruction. In particular, the response to β‐adrenoceptor stimulation has never been studied in human myocardium from HCM patients.

We previously analysed the electromechanical profile of cardiomyocytes isolated from myectomy samples of patients with obstructive HCM (Coppini et al., 2013). When compared with control cells, HCM cardiomyocytes showed prolonged action potentials (APs), slower Ca²⁺ transients and elevated diastolic Ca²⁺, largely determined by a marked overexpression of the late sodium current (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=582). Such electro‐mechanical abnormalities were reversed in vitro by the I_NaL‐inhibitor http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7291, with beneficial effects on diastolic function and cellular arrhythmias (Coppini et al., 2013). Ranolazine is available in the clinic to treat stable angina and is being employed in HCM patients with angina symptoms (Ammirati et al., 2016). However, ranolazine is a non‐selective I_NaL‐inhibitor, with several potentially relevant pleiotropic effects, such as the inhibition of K⁺ and Ca²⁺ currents (Antzelevitch et al., 2004), the reduction of myofilament Ca²⁺ sensitivity (Lovelock et al., 2012) and the stabilization of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=125 (Parikh et al., 2012). Novel, highly selective I_NaL‐inhibitors (GS‐967 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8413) (Belardinelli et al., 2013; Rajamani et al., 2016) have been recently developed. GS‐967 (6‐[4‐(trifluoromethoxy)phenyl]‐3‐(trifluoromethyl)‐[1,2,4]triazolo[4,3‐a]pyridine) is a potent inhibitor of I_NaL (Belardinelli et al., 2013), with an IC₅₀ of 0.2 to 0.5 μM (IC₅₀ of ranolazine is 2–4 μM). Moreover, GS‐967 has no significant effects on either peak I_Na or hERG K⁺ current in the effective range of concentrations (Belardinelli et al., 2013). GS‐967 has been shown to suppress experimentally induced ventricular arrhythmias in rat and rabbit cardiomyocytes, via shortening of AP duration and reduction of intracellular Na⁺ and Ca²⁺ overload (Belardinelli et al., 2013; Sicouri et al., 2013; Pezhouman et al., 2014). This, in turn, abolished the occurrence of arrhythmogenic early and delayed after‐depolarizations (EADs and DADs) (Belardinelli et al., 2013; Sicouri et al., 2013; Pezhouman et al., 2014). Moreover, GS‐967, used in pigs, suppressed ischaemia‐induced and catecholamine‐triggered ventricular arrhythmias (Bonatti et al., 2014; Alves Bento et al., 2015), as well as atrial fibrillation triggered by autonomous stimulation (Carneiro et al., 2015). Notably, GS‐967 did not exert any effects on the mechanical and electrical function of the heart in healthy rats (Fernandes et al., 2014). This result is in line with the negligible effects of ranolazine in human myocardium from non‐hypertrophic, non‐failing patients (Coppini et al., 2013), due to the absence of I_NaL overexpression, which appears to be a specific, disease‐related target.

In the present study, we first used GS‐967 as a pharmacological tool to assess whether the previously observed effects of ranolazine in human HCM myocardium can be entirely attributed to I_NaL inhibition rather than its pleiotropic effects. We then compare the mechanical and electrophysiological responses of HCM myocardium to the β‐adrenoceptor agonist http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=536 with those observed in control cardiac samples, in order to characterize the features and possible abnormalities of β‐adrenoceptor signalling in the HCM heart. Finally, the main objective of this study was to investigate the in vitro effects of ranolazine and GS‐967 under β‐adrenoceptor stimulation, the latter used to simulate stress and exercise in the myocardium of patients with obstructive HCM. With this approach, we aimed to assess whether the in vitro pharmacological profile of I_NaL‐inhibitors supports their use to treat inducible obstruction in HCM patients as an alternative to disopyramide and β‐blockers or in combination with these commonly used compounds.

Methods

Details are available online (‘Expanded Methods’ section of the Online Data Supplement).

Patients

The study follows the principles of WMA Declaration of Helsinky for medical research involving human subjects. The experimental protocols were approved by the ethical committee of Careggi University‐Hospital of Florence (2006/0024713, renewed May 2009; 2013/0035305). Each enrolled patient gave written informed consent. We enrolled 22 HCM patients who were followed by the Cardiomyopathy Unit in Florence, consecutively referred to surgical septal myectomy, for relief of drug‐refractory symptoms related to LVOT obstruction. Among the 22 patients, 15 agreed to undergo mutational screening in sarcomeric genes. Clinical data are found in Table 1.

Table 1.

HCM patient characteristics

HCM Patients (n = 22)
Age at surgery	51 ± 8 years
Gender	Female 11/22 (50%)
NYHA Class II	13/22 (59%)
NYHA Class III	9/22 (41%)
Genotype	15/21 (7 MYBPC, 4 MYH, 4 SARCOMERE‐NEG.)

Arrhythmic risk
Syncope	7/22 (32%)
Non‐sustained ventricular tachycardia	11/22 (50%)
History of atrial fibrillation	10/22 (45%)
ICD implanted	3/22 (13%)

Echo features
Maximal septal thickness	27 ± 6 mm
Ejection fraction	66 ± 6%
LVOT gradient at rest >30 mmHg	22/22 (100%)
LVOT gradient at rest	81 ± 9 mmHg
LA end‐systolic volume	101 ± 37 mL
Severe diastolic dysfunction (pseudonormalized, restrictive)	10/22 (45%)

Pharmacological therapy
β‐blockers	22/22 (100%)
Disopyramide	11/22 (50%)
Amiodarone	4/22 (18%)
Diuretics/ACE‐inhibitors	11/22 (50%)

Clinical features of the HCM patients enrolled in the study. Clinical data refers to visits performed less than 1 month before the myectomy operation. Categorical data are expressed as proportion of patients; continuous values are expressed as mean ± SD. NYHA, New York Heart Association; MYPBC, myosin‐binding protein C; MYH, myosin heavy chain; ICD, implantable cardioverter defibrillator; LA, left atrium.

The control cohort comprised five patients aged <65 years undergoing heart surgery for aortic stenosis or regurgitation and who required a septal myectomy operation due to the presence of a bulging septum causing symptomatic obstruction. All control patients had septal thickness <14 mm and preserved left ventricular systolic function (ejection fraction >55%). Clinical data are found in Supporting Information Table S1.

Tissue processing and cell isolation

Surgical septal specimens from HCM and control patients were washed with standard cardioplegic solution and processed within 30 min from excision. Endocardial trabeculae suitable for mechanical measurements were dissected, and the remaining tissue was minced and subjected to enzymic dissociation to obtain viable single myocytes, as previously described (Coppini et al., 2014a).

Single cell studies

Perforated patch whole‐cell current‐clamp was used to measure membrane potential, as previously described (Coppini et al., 2013). [Ca²⁺] variations were simultaneously monitored using the Ca²⁺‐sensitive fluorescent dye Fluoforte (Enzo Life Sciences, Farmingdale, NY, USA), by measuring fluorescence at 515 ± 10 nm during excitation at 490 ± 8 nm. Cells were mechanically permeabilized at the end of the experiment, and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calculated from emitted fluorescence as previously described (Voigt et al., 2012), using 389 nmol·L⁻¹ as Fluoforte dissociation constant. Late I_NaL and L‐type Ca²⁺ current (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=529) were measured using whole‐cell voltage clamp as previously described (Coppini et al., 2013). I_CaL was also recorded under AP‐clamp conditions, using representative AP traces, recorded from HCM cardiomyocytes under the same conditions, as command voltage. The inward current recorded under AP‐clamp conditions was dihydropyridine‐sensitive (see Supporting Information Figure S6).

Intact trabeculae studies

Ventricular trabeculae were mounted between a force transducer and a motor for muscle length control, as previously described (Coppini et al., 2013); isometric force was recorded under different experimental conditions and stimulation protocols. Isometric force was recorded at 35 ± 2°C under various conditions including various pacing rates (0.1–2.5 Hz) and acute drug administration (see below). The occurrence of premature beats was evaluated during steady‐state stimulation (0.5 Hz) and during stimulation pauses.

Drug studies

For experiments on isolated cardiomyocytes and trabeculae, Ranolazine was used at the concentration of 10 μM, disopyramide at 5 μM and GS‐967 at 0.5 μM, unless otherwise specified. Each cell or trabecula was randomly assigned to be treated with one of the three test drugs (disopyramide, ranolazine or GS‐967). Test recordings in the presence of the drug were performed after >3 min from the beginning of drug exposure. Afterwards, the drug was washed out for >5 min, and measurements were repeated. Isoprenaline (10⁻⁷ M) was used to mimic β‐adrenoceptor stimulation during exercise in single cells and trabeculae.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Raw traces were analysed by a blinded operator, different from the one who performed the experiments. For each trace, we performed measurements in at least five subsequent events and averaged them to obtain a single value for a given cell/trabecula in a certain condition. Clinical data from patients are expressed as mean ± SD. Data from cells and muscles are expressed as mean ± SEM. Figures 1G and 2D, F show the effects of drugs in a normalized fashion: we calculated the drug‐induced variation of a given parameter from the baseline [(drug‐baseline)/baseline, expressed as percentage] in each cell/trabecula and used these values (percentages of variation) to calculate averages and perform statistical analyses. The number of cells/trabeculae and the number of different patient samples from which cells/trabeculae were isolated are indicated for each group/condition in the respective figure legends. For each group, we included data points from at least five patient samples, obtained from an even number of cells/trabeculae for each sample. Statistical analysis was performed as previously described using linear mixed models (Coppini et al., 2013), taking into account non‐Gaussian distribution, inequality of variances and within‐subject correlation. In brief, in order to reduce the risk of type I errors resulting from the closer interrelationship between cells/trabeculae isolated from the same patient sample, we used hierarchical statistics including two nested levels (patients and cells) (Sikkel et al., 2017); a third hierarchical level was added when drugs were tested in a cell/trabecula, in order to allow paired comparisons. This approach was implemented using linear mixed models in Stata 12.0 (StataCorp LLC, USA). For categorical data (e.g. occurrence of cellular arrhythmias), we used the Fisher exact test. P < 0.05 was considered statistically significant. Due to the lesser availability of control samples, the number of control cells/trabeculae included in the unpaired comparisons (Figure 2) is lower than that of HCM cells/trabeculae.

Figure 1.

GS‐967 shortens action potentials, hastens Ca transients and accelerates contraction in HCM myocardium. (A) Left: representative I_NaL traces from an HCM cardiomyocyte during depolarization to −20 mV in the absence (Basal) or presence of GS‐967 (0.5 μmol·L⁻¹). Right: Average integrals of I_NaL calculated between 50 and 750 ms from the onset of depolarization. Means ± SE from 14 HCM myocytes (five patients). (B) Representative superimposed APs at baseline and in the presence of GS‐967 (0.5 μM), elicited at 0.2 Hz. (C) APD at 90% of repolarization (APD90%) at baseline and in the presence of GS‐967 (0.5 μM). Means ± SE from 37 HCM cardiomyocytes from nine HCM patients. (D) Representative superimposed Ca transients at baseline and in the presence of GS‐967 (0.5 μM), elicited at 0.2 Hz (left) and 1 Hz (right). (E) Time from peak to 50% decay of Ca transients (50% decay) at baseline and in the presence of GS‐967 (0.5 μM), elicited at 0.2, 0.5 and 1 Hz. (F) Diastolic intracellular Ca‐concentration ([Ca²⁺]_i) at baseline and in the presence of GS‐967 (0.5 μM), during regular stimulation at 0.2, 0.5 and 1 Hz at steady state. (G) % variation of diastolic [Ca²⁺]_i and Ca‐transient amplitude (Systolic Ampl.) with the application of GS‐967 with respect to baseline in HCM cardiomyocytes. (E–G) Means ± SE from 26 cardiomyocytes from seven HCM patients. (H) Representative superimposed force twitches elicited at 0.5 Hz in HCM trabeculae in the absence and presence of GS‐967. (I) Time from stimulus to peak of force twitches elicited at different frequencies (0.1 to 3 Hz) at baseline, in the presence of GS‐967. Means ± SE from 10 trabeculae from seven patients. *P < 0.05, significant differences; linear mixed models corrected for paired comparisons.

Figure 2.

Mechanical and electrical response to β‐adrenoceptor stimulation of HCM myocardium. (A) Representative superimposed force twitches elicited at 0.5 Hz in control (left) and HCM (right) trabeculae in the absence and presence of isoprenaline (Iso; 0.1μM). (B) Isometric tension during steady‐state stimulation at 0.5 in the absence and presence of isoprenaline in control and HCM trabeculae. (C) Duration of force twitches (from stimulus to 90% of relaxation) elicited at 0.5 Hz in the absence and presence of isoprenaline (Iso; 0.1μM). (D) Percentages of change in the parameters of twitch kinetics (0.5 Hz) upon exposure to isoprenaline in control and HCM trabeculae: time from stimulus to peak contraction (peak time) and time from peak to 50% of relaxation (RT50%). (B–D) Means ± SE from six trabeculae from five control patients and 30 trabeculae from 20 HCM patients. (E) Representative superimposed APs elicited at 0.5 Hz in control (left) and HCM (right) cardiomyocytes, in the absence and presence of isoprenaline. (F) Percentages of change in the parameters of AP kinetics upon exposure to isoprenaline in control and HCM cardiomyocytes: time from stimulus to 50% repolarization (APD50%) and time from peak to 90% of repolarization (APD90%). (F) Means ± SE from 13 cardiomyocytes from five control patients and 31 cardiomyocytes from nine HCM patients. #P < 0.05, significantly different as indicated; linear mixed models, unpaired comparisons; *P < 0.05, significantly different as indicated; linear mixed models corrected for paired comparisons.

Materials

The compounds used in these studies were supplied as follows: disopyramide by Sigma‐Aldrich (St. Louis, Missouri, USA), ranolazine by Menarini International (Luxembourg, Luxembourg) and GS‐967 from Gilead Sciences (Foster City, California, USA).

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a,b,c,d).

Results

GS‐967 shortens action potentials and accelerates the kinetics of Ca²⁺ transients and twitches in HCM cardiomyocytes

GS‐967 was tested at 0.5 μM in isolated cardiomyocytes from HCM patients during patch‐clamp and Ca²⁺‐fluorescence measurements (Figure 1A). GS‐967 consistently reduced I_NaL (Figure 1A). Moreover, GS‐967 shortened the duration of APs at all frequencies studied by about 20% (Figure 1B, C). Moreover, GS‐967 reduced the rate of arrhythmogenic EADs occurring spontaneously during regular stimulation for 3 min. The number of cells showing EADs during 3 min of stimulation was halved by GS‐967 (Supporting Information Figure S2). Notably, GS‐967 had no effects on the upstroke time nor on the amplitude of the APs (Supporting Information Table S3). The kinetics of intracellular Ca²⁺ transients was hastened by GS‐967 at all frequencies investigated (Figure 1D, E): both the rise and decay times of Ca²⁺ transients were shortened by GS‐967 application (Supporting Information Table S3). GS‐967 reduced diastolic [Ca²⁺], while the amplitude of Ca²⁺ transients was unaffected (Figure 1F, G). Accordingly, GS‐967 reduced the occurrence of delayed after‐depolarizations (Supporting Information Figure S2). GS‐967 was then tested in intact trabeculae from HCM patients, while recording force under isometric conditions (Figure 1H) during stimulation at different frequencies (0.1 to 3 Hz). GS‐967 shortened twitch duration mainly by reducing time to peak contraction (Figure 1I). GS‐967 (0.5 μM) had no effects on APs and Ca²⁺ transients recorded from cardiomyocytes isolated from control patients (Supporting Information Figure S3).

Stimulation of β‐adrenoceptors exerts comparable mechanical effects in HCM and control myocardium but causes a paradoxical prolongation of action potentials in HCM cardiomyocytes

The effects of β‐adrenoceptor stimulation with isoprenaline (0.1μM) on isometric twitches were tested in trabeculae from control and HCM septal samples (Figure 2A–D). This β‐adrenoceptor agonist exerted qualitatively and quantitatively similar effects on twitch amplitude and kinetics in control and HCM trabeculae. In particular, isoprenaline increased peak force by about three‐fold in both control and HCM trabeculae (Figure 2B). Twitch kinetics was slower in HCM versus control trabeculae both at baseline and during β‐adrenoceptor stimulation (Figure 2C). Indeed, isoprenaline accelerated the kinetics of relaxation by a similar amount in HCM and control trabeculae, although the acceleration of twitch force development was less pronounced in HCM trabeculae (Figure 2D). All in all, the kinetics of contraction and relaxation, despite being accelerated by isoprenaline, was still slower in HCM than in control trabeculae, even during maximal β‐adrenoceptor stimulation.

In contrast, the effects of isoprenaline on APs were profoundly different in HCM with respect to control cardiomyocytes (Figure 2E). While isoprenaline shortened the duration of APs in control cardiomyocytes, this β‐adrenoceptor agonist prolonged repolarization in all cardiomyocytes from HCM patients (Figure 2F).

Ranolazine and GS‐967 decrease isometric force following β‐adrenoceptor stimulation

We tested the effects of disopyramide (5 μM), ranolazine(10 μM), GS‐967 (0.5 μM) and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=564 (0.1 μM) on isometric twitch amplitude and kinetics in trabeculae from HCM patient samples, in the absence and presence of β‐adrenoceptor stimulation with isoprenaline(0.1 μM), used to simulate exercise or stress conditions. Under baseline conditions, disopyramide reduced twitch amplitude by approximately 50%, while force was unaffected by ranolazine, GS‐967 and propranolol (Figure 3A, C). Activation of β‐adrenoceptors with isoprenaline led to the expected increase of twitch amplitude and hastening of twitch kinetics (Figure 3A, B, D). Interestingly, disopyramide, ranolazine, GS‐967 and propranolol reduced twitch amplitude when added to isoprenaline (Figure 3A–C). The reduction of twitch amplitude under isoprenaline was quantitatively less pronounced with disopyramide (16 ± 7%) compared with ranolazine (56 ± 23%, P < 0.05) or GS‐967 (32 ± 21%, P < 0.05; Figure 3A, C). The reduction of twitch force in the presence of ranolazine or GS‐967 was comparable with that obtained by blocking β‐adrenoceptors with propranolol (Figure 3C). Nonetheless, the acceleration of twitch kinetics by isoprenaline was not antagonized by the application of disopyramide, ranolazine or GS‐967 (Figure 3A, B, D). Indeed, after the addition of these drugs on top of isoprenaline, both time to peak and 50% relaxation time remained as fast as in the presence of isoprenaline alone (Figure 3D). However, propranolol completely antagonized the lusitropic effect of isoprenaline (Figure 3D).

Figure 3.

GS‐967 and ranolazine reduce contractile force only during β‐adrenoceptor stimulation in HCM myocardium. (A) Representative superimposed force twitches elicited at 1 Hz in HCM trabeculae in the absence and presence of disopyramide (5 μM), at basal conditions (left) and in the presence of isoprenaline (Iso; 0.1μM). (B) Representative superimposed force twitches elicited at 1 Hz in HCM trabeculae in the absence and presence of GS‐967 (0.5 μM), at basal conditions (left) and in the presence of isoprenaline. (C) Amplitude of force twitches elicited at 1 Hz at baseline, in the presence of disopyramide, ranolazine, GS‐967 or propranolol, at basal conditions (left) or in the presence of isoprenaline(right). (D) Duration of force twitches (from stimulus to 90% repolarization) elicited at 1 Hz at baseline (black), in the presence of disopyramide, ranolazine , GS‐967 or propranolol, at basal conditions (left) or in the presence of isoprenaline(right). (C, D) Means ± SE from 10 trabeculae from seven HCM patients. *P < 0.05, significantly different as indicated; linear mixed models corrected for paired comparisons.

Cellular mechanisms underlying the effects of I_NaL‐inhibitors in the presence of isoprenaline

Ranolazine and GS‐967 were similarly tested in isolated HCM cardiomyocytes (Figure 4, Table 2). In line with the effects observed on twitch amplitude in trabeculae, ranolazine and GS‐967 did not affect Ca²⁺‐transient amplitude at baseline (Figure 4A). Ca²⁺‐transient amplitude increased in response to isoprenaline by 14 ± 3%. The application of either ranolazine or GS‐967 in the presence of isoprenaline significantly reduced Ca²⁺‐transient amplitude (Figure 4B). Interestingly, while isoprenaline did not affect intracellular diastolic [Ca²⁺], ranolazine and GS‐967, applied in the presence of isoprenaline, significantly reduced diastolic [Ca²⁺] (Figure 4C). Interestingly, isoprenaline accelerated Ca²⁺‐transient decay but prolonged Ca²⁺‐transient rise time in HCM cardiomyocytes (Figure 4A, Table 2). Accordingly, isoprenaline slowed time‐to‐peak shortening in HCM cardiomyocytes but accelerated relaxation (Supporting Information Figure S1). Ranolazine and GS‐967 shortened Ca²⁺‐transient rise when applied with β‐adrenoceptor stimulation with isoprenaline but did not affect Ca²⁺‐transient decay kinetics (Figure 4A, Table 2). In line with that, ranolazine and GS‐967 reversed the prolongation of APs induced by isoprenaline (Figure 4A, D). However, ranolazine (added to isoprenaline) did not affect the duration of APs in control myocytes (Supporting Information Figure S4A, B). In line with that, GS‐967 did not reduce twitch force during β‐adrenoceptor stimulation in control trabeculae (Supporting Information Figure S4C, D). We excluded that the reduction of Ca²⁺ transients and force by I_NaL‐inhibitors under β‐stimulation depends on a partial block of β‐adrenoceptors by these compounds (Supporting Information Figure S5). The binding studies showed that ranolazine has a very mild interaction with http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=28 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=29 receptors at the concentration used in this work (10 μM), while GS‐967 does not bind β‐receptors, even at very high concentrations. In further support of this hypothesis, we found that ranolazine exerted a negative inotropic effect also when the positive inotropic effect of β‐adrenoceptor stimulation was mimicked by http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5190, thereby bypassing the β‐receptors (Supporting Information Figure S4E, F). Finally, we investigated whether inhibition of L‐Type Ca²⁺ current by I_NaL‐inhibitors contributed to the observed effects (Figure 5). We found that neither ranolazine nor GS‐967 affected the peak density of I_CaL in HCM cardiomyocytes (Figure 5A, B). Interestingly, we noticed that isoprenaline not only increased the amplitude of I_CaL but also markedly slows down the current's inactivation in HCM cells (Figure 5C, D). Using the AP‐clamp technique, we found that the prolongation of AP by isoprenaline increased the total inward flow of Ca²⁺ during the plateau of the AP (Figure 5E, F): this is mainly determined by the direct effects of β‐adrenoceptor stimulation on I_CaL (i.e. the increased I_CaL amplitude and prolonged inactivation) rather than being a consequence of AP prolongation (Supporting Information Figure S6). Ranolazine, added to isoprenaline, markedly reduced the total inward flow of Ca²⁺ in HCM myocytes (Figure 5E, F), as a consequence of the marked shortening of APs caused by I_NaL inhibition during β‐adrenoceptor stimulation (Supporting Information Figure S6).

Figure 4.

GS‐967 and ranolazine shorten action and Ca transients during β‐adrenoceptor stimulus. (A) Representative simultaneously recorded APs (above) and Ca transients (below) at baseline (left), in the presence of isoprenaline (Iso; 0.1μM; centre) and in the presence of ranolazine (10 μM) added to isoprenaline (right), elicited at 0.5 Hz in an HCM cardiomyocyte. (B) Ca‐transient amplitude (0.5 Hz) at basal conditions, in the presence of isoprenaline, in the presence of ranolazine (10 μM) added to isoprenaline (Iso + Ran) or with GS‐967 (0.5 μM) added to isoprenaline (Iso + GS). (C) Diastolic intracellular Ca‐concentration ([Ca²⁺]_i) at basal conditions, in the presence of isoprenaline, in the presence of (Iso + Ran) or with (Iso + GS), during regular stimulation at 0.2, 0.5 and 1 Hz (steady state). (D) APD at 90% repolarization (0.5 Hz) at baseline, with isoprenaline alone, with (Iso + Ran) or with (Iso + GS). (B–D) Means ± SE from 21 cardiomyocytes from seven HCM patients. *P < 0.05, significantly different as indicated; linear mixed models, corrected for paired data.

Table 2.

Effects of ranolazine (Ran) and GS‐967 on APs and intracellular Ca²⁺ in the presence of isoprenaline (Iso)

Kinetics 0.5 Hz (ms)	Ca2+ transients				Action potentials
	TTP	50%	90%	TOT. T.	APD50	APD90
Baselinea	163 ± 41	568 ± 54	1010 ± 71	1178 ± 78	699 ± 47	792 ± 50
Iso (0.1μM)a	315 ± 58	445 ± 58	904 ± 61	1219 ± 79	854 ± 59	981 ± 68
Iso + Ranb	207 ± 29	435 ± 41	903 ± 53	1132 ± 61	706 ± 34	796 ± 40
Iso + GSc	209 ± 28	432 ± 38	894 ± 56	1128 ± 60	708 ± 32	799 ± 38
P (Baseline vs. Iso)	<0.05	<0.05	<0.05	n.s.	<0.05	<0.05
P (Iso vs. Iso + Ran)	<0.05	n.s.	n.s.	n.s.	<0.05	<0.05
P (Iso vs. Iso + GS)	<0.05	n.s.	n.s.	n.s.	<0.05	<0.05

Data are expressed as means ± SEM. P values were calculated using linear mixed models corrected for paired comparisons. TTP, time from stimulus to peak; 50%, time from peak to 50% decay of Ca transients; 90%, time from peak to 90% decay of Ca transients; TOT. T., Total Ca²⁺‐transient duration (from stimulus to 95% decay); APD20, APD at 20% of repolarization; APD50, APD at 50% of repolarization; APD90, APD at 90% of repolarization.

^aData from 25 HCM cardiomyocytes isolated from seven HCM patient samples;

^b13 cells, 6 pts.

^c12 cells, 5 pts.

Figure 5.

Ranolazine and GS967 reduced total Ca²⁺ entry through I_CaL during β‐adrenoceptor stimulation. (A) Representative superimposed I_CaL traces at baseline and in the presence of GS‐967 (0.5 μM) from an HCM cardiomyocyte (the pre‐pulse at −40 mV is omitted from the traces). (B) I_CaL density at baseline, in the presence of GS‐967 (0.5 μM) or ranolazine (Ran; 10 μM). Means ± SE from 15 HCM cardiomyocytes from five patients. (C) Representative superimposed I_CaL traces at baseline and in the presence of isoprenaline (Iso). (D) I_CaL inactivation time‐constant (left) and density (right) at baseline and with isoprenaline in HCM cells (13 myocytes, five patients). (E) Representative I_CaL traces recorded during AP‐clamp at baseline, with isoprenaline and with (Iso + Ran); command voltage is shown below. (F) Integral of Ca‐current during AP‐clamp. Means ± SE from 14 cells (five patients). *P < 0.05, significantly different as indicated; linear mixed models, corrected for paired data.

I_NaL‐inhibitors abolish catecholamine‐induced arrhythmia in HCM myocardium

We then evaluated the effects of ranolazine and GS‐967 on cellular arrhythmias evoked by β‐adrenoceptor stimulation (Figure 6A, B). Isoprenaline markedly increase the occurrence of both early and delayed after‐depolarizations in HCM cardiomyocytes (Figure 6). Interestingly, both ranolazine (10 μM) and GS‐967 (0.5 μM) reduced the occurrence of EADs and DADs.

Figure 6.

GS‐967 and ranolazine reduce the occurrence of EAD during β‐adrenoceptor stimulation. (A) Representative action traces at baseline, in the presence of isoprenaline (Iso; 0.1μM) and in the presence of GS‐967 (0.5 μM) added to isoprenaline, elicited at 1 Hz pacing rate. Ranolazine (Ran) suppresses EADs (marked by black arrows) that occur under isoprenaline. (B) Representative AP traces at baseline, with isoprenaline alone or with (Iso + Ran), 0.5 Hz pacing rate. Ranolazine suppresses DADs (marked by black arrows) that occur under isoprenaline. (C, D) Percentage of HCM cardiomyocytes showing at least two EADs (EADs, in C) or two DADs (in D) during 3 min of continuous stimulation, at baseline, in the presence of isoprenaline alone, with (Iso+Ran) and with (Iso+GS‐967). Means ± SE from 31 HCM cardiomyocytes from nine HCM patients. *P < 0.05, significantly different as indicated; linear mixed models corrected for paired comparisons.

Finally, we evaluated the frequency of premature spontaneous beats and sustained triggered activity occurring in HCM trabeculae during stimulation pauses in the presence of isoprenaline (Figure 7A, B). When added to isoprenaline, ranolazine and GS‐967 markedly reduced the occurrence of arrhythmic events (Figure 7C), in keeping with the reduction of EADs and DADs observed in isolated HCM cardiomyocytes.

Figure 7.

GS‐967 and ranolazine reduce the occurrence of spontaneous contractions during β‐adrenoceptor stimulus. (A) Representative force traces at baseline, in the presence of isoprenaline (Iso; 0.1μM) and in the presence of (Iso+Ran), elicited at 0.5 Hz pacing rate. Ranolazine (Ran) suppresses the spontaneous activity that occur with isoprenaline. (B) Representative force traces at baseline, in the presence of isoprenaline alone and in the presence of (Iso+Ran). After 10 s of burst pacing at 3 Hz, the stimulation was abruptly interrupted to induce spontaneous activity. (C) Summary data from isoprenaline alone, ((Iso + Ran) and (Iso + GS). Means ± SE from 15 HCM trabeculae from 12 HCM patients. *P < 0.05, significantly different as indicated; linear mixed models, corrected for paired data.

Discussion

Selective I_NaL inhibition: a disease‐specific pharmacological option for HCM treatment

I_NaL is three times larger in the myocardium of HCM patients compared to that of non‐failing non‐hypertrophic subjects, leading to a prolonged APD that we observed consistently in myocardial samples from over 30 HCM myectomy patients (Coppini et al., 2013). In our view, these results highlight I_NaL inhibition as a disease‐specific pharmacological strategy to treat HCM. We previously confirmed this hypothesis by acutely applying ranolazine on myocardial samples from myectomy HCM patients, where we observed marked beneficial effects on diastolic function and arrhythmogeneicity (Coppini et al., 2013). In this work, a novel, more selective I_NaL‐inhibitor, GS‐967, was tested in intact cardiomyocytes and trabeculae from 18 HCM patients, in order to confirm that the beneficial effects previously observed with ranolazine (Coppini et al., 2013) were indeed consequence of I_NaL inhibition and were not mediated by the other pleiotropic effects of the drug [such as inhibition of K⁺ and Ca²⁺ currents (Antzelevitch et al., 2004), reduction of myofilament Ca²⁺ sensitivity (Lovelock et al., 2012), stabilization of ryanodine receptors (Parikh et al., 2012) or a mild β‐blocker effect (Flenner et al., 2016)]. Here, we found that all the effects of GS‐967 observed in HCM myocardium are qualitatively and quantitatively similar to those previously obtained with ranolazine, but they are achieved at a 20 times lower concentration (0.5 μM GS‐967 vs. 10 μM ranolazine), owing to the increased potency and selectivity for I_NaL. As an example, 0.5 μM GS‐967 reduced I_NaL in HCM myocytes by 67% (Figure 1), while ranolazine inhibited 72% of I_NaL at 10 μM (Coppini et al., 2013). Moreover, GS‐967, just like ranolazine, shortened AP duration, reduced the risk of arrhythmogenic EADs (Figure 1), lowered diastolic [Ca²⁺], suppressed the risk of DADs, accelerated Ca²⁺‐transient kinetics and hastened twitch relaxation in HCM myocardium (Figure 1). All in all, our results indicate that GS‐967 may exert the same possible beneficial effects as ranolazine on arrhythmias and diastolic function, the main determinants of symptoms and outcome in HCM patients (Coppini et al., 2014b; Olivotto et al., 2015).

These results also have important mechanistic implications. Because GS‐967 does not affect any other relevant target besides I_NaL at this concentration (Belardinelli et al., 2013), our results confirm that the reduction of arrhythmogenicity and the amelioration of diastolic function, previously observed with ranolazine (Coppini et al., 2013), are a direct consequence of I_NaL inhibition. Of note, we observed no major effects of GS‐967 in cardiomyocytes from control patients (Supporting Information Figure S3), as previously observed with ranolazine (Coppini et al., 2013), suggesting that the efficacy of I_NaL‐inhibitors is specific to disease conditions where a pathological increase of I_NaL is observed.

In addition to the acute effects on myocardial function, I_NaL inhibition may lead to additional benefits during long‐term treatment. Work from our group showed that lifelong I_NaL inhibition in a mouse model of HCM prevents the development of hypertrophic phenotype and LV dysfunction (Coppini et al., 2017) and suggested that I_NaL inhibition with ranolazine or more selective agents may be an effective disease‐modifying strategy in patients with HCM.

I_NaL‐inhibitors: novel options to treat inducible obstruction?

The principal aim of this work is to investigate the potential of I_NaL‐inhibitors in HCM, beyond the two already demonstrated effects, that is, reduction of the arrhythmic risk and the amelioration of symptoms related to diastolic dysfunction. Here, we tested these compounds as negative inotropic agents to decrease exercise‐induced LV septal hypercontractility, which is considered a direct cause of obstruction‐related symptoms in patients with obstructive HCM.

The negative inotropic agents that are currently used to treat obstructive HCM are limited, particularly for the cases of inducible obstruction. Despite its widespread use, disopyramide is far from being the perfect drug to control obstructive symptoms. Although it has been successfully employed for more than 40 years to treat rest obstruction, this drug has a very limited efficacy on exercise‐induced LVOT gradients (Maron et al., 2006). In addition, undesired anticholinergic side‐effects (mouth dryness, constipation, drowsiness) are relatively common and may require discontinuation of the drug unless they may be controlled by pyridostigmine (Sherrid, 2016). β‐blockers, the only available therapeutic option to treat inducible obstruction, are often insufficient to control symptoms, and the high dosages required to significantly affect dynamic LVOT gradients may be poorly tolerated (Nistri et al., 2012).

Our results show that the selective I_NaL‐inhibitors reduced myocardial force only during intense β‐adrenoceptor stimulation, mimicking stress or exercise, while leaving baseline (‘rest’) contractility unaffected. Interestingly, the lusitropic effect of β‐adrenoceptor stimulation (i.e. acceleration of relaxation) is maintained, while only the positive inotropic effect is lost. This is at variance with β‐blockers which, by acting at receptor level, antagonize the full spectrum of β‐adrenoceptor‐mediated effects, including the acceleration of relaxation (Figure 3) and the increase in heart rate, both essential for tolerating intense exercise activity. Interestingly, this effect appears to be specific of HCM myocardium, as the reduction of contractile force by I_NaL‐inhibitors during β‐adrenoceptor stimulation is not observed in myocardial samples from control patients (Supporting Information Figure S4). The cellular mechanisms underlying the unexpected negative inotropic action of I_NaL‐inhibitors under β‐adrenoceptor stimulation in HCM myocardium have been extensively investigated.

Mechanisms underlying the negative inotropic action of I_NaL‐inhibitors under β‐adrenoceptor stimulation in HCM myocardium

• 1 Altered electrical response to β‐adrenoceptor stimulation in HCM myocardium

By directly comparing the response of HCM myocardium β‐adrenoceptor stimulation with that of control cardiac muscle (Figure 2), we here provided the first characterization of the features and abnormalities of β‐adrenoceptor signalling in human hearts from HCM patients. In particular, we observed that the mechanical response to isoprenaline in terms of increased force amplitude and relaxation velocity was comparable in HCM and control trabeculae, but contractile kinetics remained slower in HCM, than in control trabeculae, even during maximal β‐adrenoceptor stimulation. These results indicate that the subcellular mechanisms responsible for the lusitropic effects of the β‐adrenoceptor cascade are essentially preserved in HCM myocardium: HCM myofilaments retain the ability to decrease Ca²⁺ sensitivity upon phosphorylation of troponin‐I by http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=284 (Sequeira et al., 2013), and PKA‐mediated phosphorylation of phospholamban occurs normally in human HCM myocardium (Coppini et al., 2013; Helms et al., 2016).

On the contrary, the electrophysiological response to isoprenaline in isolated HCM cardiomyocytes was profoundly abnormal (Figure 2E, F). Among the many different effects on sarcolemmal and SR targets, isoprenaline enhances both L‐Type Ca²⁺ current and the slow delayed rectifier K⁺ current (I_Ks), with the latter prevailing in healthy human cells (Terrenoire et al., 2005), thus leading to a net reduction of the APD (Taggart et al., 2003) in control myocardium (Figure 2E, F). In HCM cardiomyocytes, hypertrophic remodelling leads to the unbalanced changes in the expression of Ca²⁺ and K⁺ currents (Coppini et al., 2013), that is, the expression of all K⁺ channels (including those responsible for I_Ks) is greatly reduced in the presence of a normal (or even slightly increased) density of I_CaL, as compared with control myocardium. In HCM myocytes, in response to β‐adrenoceptor stimulation, the potentiation of I_CaL prevails over the increase of K⁺ currents, thus leading to a net increase of depolarizing currents during the plateau of the AP, causing the observed ‘paradoxical’ prolongation of APD (Figure 2E, F). Moreover, in HCM cardiomyocytes, β‐adrenoceptor stimulation not only increases peak I_CaL amplitude but also markedly slows down I_CaL inactivation (Figure 5). The marked slowing of I_CaL inactivation is likely to contribute to the prolongation of APs by β‐adrenoceptor agonists. Additionally, it was previously shown that β‐adrenoceptor stimulation of ventricular cardiomyocytes with isoprenaline leads to I_NaL enhancement (Dybkova et al., 2014) through activation of CaMKII. A further increase of I_NaL following β‐adrenoceptor stimulation in HCM myocytes may have contributed to the paradoxical prolongation of APs observed in these cells. This paradoxical response might be relevant for the pathophysiology of exercise or stress‐induced arrhythmias in HCM patients, as AP prolongation increased the risk of arrhythmogenic early‐afterdepolarizations and triggered activity in HCM myocardium (Figures 5 and 6).

• 2 Altered calcium response to β‐adrenoceptor stimulation in HCM myocardium

In HCM cardiomyocytes, unlike control myocardium, the β‐adrenoceptor‐dependent increase of Ca²⁺ release and force may greatly rely on the increase in the duration of Ca²⁺ entry via L‐Type Ca²⁺ channels caused by the slower I_CaL inactivation. In addition to the enhancement of I_CaL, increased Ca²⁺ entry via the reverse‐mode action of the http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=180#945) contributes to augment Ca²⁺ transients upon β‐adrenoceptor stimulation (Perchenet et al., 2000). The latter effect is likely to be a consequence of the elevation of intracellular [Na⁺] in response to the enhancement of I_NaL and the prolongation of AP plateau that follows β‐adrenoceptor stimulation (Dybkova et al., 2014; Coppini et al., 2013). Indeed, we found that the rise time of Ca²⁺ transients is lengthened in response to isoprenaline in HCM cardiomyocytes (Figure 4, Table 2). The prolongation of Ca²⁺‐transient rise time may have direct mechanical consequences: indeed, we observed that the acceleration of twitches' rising phase (time to peak) upon β‐adrenoceptor stimulation was significantly less pronounced in HCM versus control trabeculae (Figure 2D). The idea that the increase of force in response to β‐adrenoceptor stimulation mainly depends on the increase of sarcolemmal Ca²⁺ entry is in apparent contrast with the commonly accepted idea that the positive inotropic effects of β‐adrenoceptor stimulation derive from the increase of sarcoplasmic reticulum Ca²⁺ content (Desantiago et al., 2008), due to enhancement of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=159 function by PKA‐mediated phospholamban phosphorylation. The observed acceleration of Ca‐transient decay by isoprenaline in HCM cardiomyocytes (Figure 4 and Table 2) suggests that the β‐adrenoceptor‐induced increase of SERCA function is preserved in HCM myocardium. The increase of SR Ca²⁺ content following β‐stimulation may be limited by the increased diastolic leakage from hyper‐phosphorylated ryanodine receptors (due to increased calmodulin‐kinase activity, see Coppini et al., 2013 and Ferrantini et al., 2016), thus rendering inotropic responses more dependent on the increase of Ca²⁺ entry through the sarcolemma. Another possible contributor to this phenomenon is the reduced density of T‐tubules (Orchard and Brette, 2008). We performed a preliminary investigation of the density of T‐tubules in cells isolated from the 10 HCM patient samples included in the study (Supporting Information Figure S7): in all the cells observed, T‐tubule density was extremely low much lower than the expected for healthy myocardium (Lyon et al., 2009). The functional consequences of β‐adrenoceptor activation may be radically different in disease myocytes with a markedly reduced density of T‐tubules. In the near‐absence of T‐tubules and with a large redistribution of L‐Type Ca²⁺ channels to the surface sarcolemma (Coppini et al., 2013), modulation of myocardial inotropism becomes largely dependent on the amplitude of Ca²⁺ trigger (Ferrantini et al., 2014), that is, the density and duration of I_CaL plus the rate of NCX‐mediated Ca²⁺ entry (reverse mode). Due to the reduced density of T‐tubules in HCM cardiomyocytes, the prolongation of APs is likely to be essential for the positive inotropic effect of β‐adrenoceptor activation. All in all, the abnormal Ca²⁺ response to β‐adrenoceptor agonists may contribute to impair relaxation during exercise, leading to exercise intolerance and exertional symptoms in HCM patients, regardless of obstruction.

• 3 Effects of I_NaL‐inhibitors on APs during β‐adrenoceptor stimulation

The abnormal response of HCM myocardium to stimulation of β‐adrenoceptors in terms of AP duration and Ca²⁺ handling underlies the unexpected negative inotropic action of I_NaL‐inhibitors. We found that both ranolazine and GS‐967, when applied with isoprenaline, cause shortening of AP and Ca²⁺‐transient duration and reduce Ca²⁺‐transient amplitude (Figure 4). On the contrary, AP duration does not change in response to ranolazine in control cardiomyocytes in the presence of isoprenaline (Supporting Information Figure S4). Interestingly, AP shortening by I_NaL‐inhibitors occurs also in the absence of β‐adrenoceptor stimulation (see Figure 1 for GS‐967 and Coppini et al., 2013, for ranolazine). However, when we compare the degree of APD shortening by I_NaL‐inhibitors at baseline (in a total of >50 cells combined) with that obtained with isoprenaline (in a total of >30 cells), the effect is significantly more pronounced in the presence of the β‐adrenoceptor agonist. At 0.5 Hz pacing rate, reduction of APD was −27.1 ± 3.7% in the presence of isoprenaline , while it was −17.2 ± 2.4% at baseline (P < 0.05, linear mixed models). When APD is prolonged and repolarizing K⁺ currents are reduced, the duration of AP plateau is more dependent on I_NaL (Wu et al., 2011; Shattock et al., 2017): therefore, the reduction of APD following a proportionally comparable inhibition of I_NaL is larger when APD is longer and I_NaL is further increased (i.e. in the presence of isoprenaline). The prolongation of APD in response to isoprenaline leads to an increase in the frequency of EADs in HCM cardiomyocytes. Interestingly, when I_NaL‐inhibitors are added with β‐adrenoceptor agonists, the shortening of APD brings the rate of EADs back to baseline, suggesting a possible efficacy of I_NaL‐inhibitors in preventing stress‐induced arrhythmias in HCM.

• 4 Effects of I_NaL‐inhibitors on Ca²⁺ transients and diastolic [Ca²⁺] under β‐adrenoceptor stimulation

I_NaL‐inhibitors, via shortening of APD, antagonize the increase of the duration of I_CaL during the plateau of APs induced by isoprenaline in HCM cardiomyocytes, as demonstrated here by AP‐clamp experiments (Figure 5 and Supporting Information Figure S6), ultimately leading to a marked reduction of total Ca²⁺ entry via I_CaL (−65 ± 12% with respect to Iso). This effect is a direct consequence of the shortening of APs and does not depend by any direct interactions of I_NaL‐inhibitors with Ca²⁺ channels (Figure 5). Moreover, in the presence of β‐adrenoceptor stimulation, I_NaL inhibition with ranolazine may reduce intracellular [Na⁺] by a greater extent as compared with the ‘rest’ condition. The marked reduction in intracellular [Na⁺] due to I_NaL inhibition (Coppini et al., 2013; Kornyeyev et al., 2015) during β‐adrenoceptor stimulation may lead to a pronounced decrease in reverse‐mode NCX activity, which in turn may contribute to diminish Ca²⁺ entry during AP plateau.

In line with the reduction of Ca²⁺ entry via I_CaL and reverse‐mode NCX, time to peak Ca²⁺ transients (markedly prolonged by isoprenaline) were brought back to baseline levels by the addition of I_NaL‐inhibitors (Figure 4 and Table 2). The reduction of isoprenaline‐induced AP prolongation by I_NaL inhibition combined with the decrease of [Na⁺] may prevent the increase of Ca²⁺ entry through I_CaL and reverse‐mode NCX contractility by β‐adrenoceptor stimulation in HCM myocardium. The marked reduction of Ca²⁺ entry appears to be large enough to counteract the isoprenaline‐induced increase of Ca²⁺‐transient amplitude and force in HCM myocardium, while I_NaL‐inhibitors are unable to significantly reduce Ca²⁺ transient and twitch amplitude in the absence of isoprenaline (Figures 1, 3 and 4). In parallel with the decrease of reverse‐mode NCX, the marked reduction of [Na⁺] by I_NaL inhibition also leads to increased forward activity of the exchanger (Coppini et al., 2013), thus increasing the rate of Ca²⁺ efflux through the sarcolemma during the diastolic period. This may have directly contributed to the marked reduction of diastolic [Ca²⁺] observed when ranolazine or GS‐967 are added to isoprenaline (Figure 4A–C). The increased sarcolemmal efflux combined with the decreased Ca²⁺ influx through I_CaL is likely to cause a reduction of SR Ca²⁺ content (Trafford et al., 2001; Eisner et al., 2013) in response to I_NaL inhibition during isoprenaline. A decrease of SR Ca²⁺ load may reduce diastolic leakage, contributing to the decrease of diastolic cytosolic [Ca²⁺] (Ferrantini et al., 2016). In line with that, the frequency of diastolic Ca²⁺ waves leading to DADs (markedly increased by isoprenaline) is reduced by I_NaL‐inhibitors in HCM cardiomyocytes during β‐adrenoceptor stimulation.

The weak β‐blocker action of ranolazine is not responsible for its negative inotropic effect in the presence of isoprenaline

Ranolazine was previously shown to behave as a weak β‐adrenoceptor blocker (Letienne et al., 2001; Flenner et al., 2016), albeit at higher concentrations than that used in our experiments (20 μM or higher). We here confirmed that ranolazine mildly binds to β₁ and β₂ adrenoceptors at the concentration used in our experiments (10 μM). In contrast, GS‐967 does not interact with β‐adrenoceptors, even at high concentrations (Supporting Information Figure S5). The mild β‐blocking effect of ranolazine is unlikely to play a major role in the observed response of HCM muscle to ranolazine in the presence of isoprenaline, for the following reasons: (i) the acceleration of relaxation by isoprenaline was not antagonized by 10 μM ranolazine , only the increase in force amplitude is reduced. (ii) The same negative inotropic effect as ranolazine was observed with 0.5 μM GS‐967 (Figure 3E), which does not have any β‐blocking capabilities (Supporting Information Figure S5), in line with previous observations in different healthy and diseased animal models (Belardinelli et al., 2013; Fernandes et al., 2014; Alves Bento et al., 2015). (iii) Finally, when we mimicked the inotropic and lusitropic response to β‐adrenoceptor stimulation with forskolin in HCM trabeculae (thereby bypassing β‐receptors), ranolazine, added in the presence of forskolin, exerted the same negative inotropic effect that was observed in the presence of β‐adrenoceptor stimulation with isoprenaline (Supporting Information Figure S4). These observations confirm that the interaction of I_NaL‐inhibitors with the physiological consequences of β‐adrenoceptor stimulation occurs downstream of the β‐adrenoceptor and of adenylyl cyclase activation and is a sole consequence of the inhibition of I_NaL.

Clinical implications and conclusions

In HCM myocardium, disopyramide exerts a potent negative inotropic effect at rest but is less effective under β‐adrenoceptor stimulation (Figure 3). This provides a clear explanation of why disopyramide effectively reduces resting gradients but is inadequate to control symptoms in patients with inducible obstruction. Conversely, I_NaL‐inhibitors (ranolazine and GS‐967) reduce myocardial force only during β‐adrenoceptor stimulation, used here to simulate exercise and stress. Because of this effect, ranolazine and selective I_NaL‐inhibitors (Justo et al., 2016; Rajamani et al., 2016) may be effective in reducing septal tension during exercise and therefore have a theoretical potential to ameliorate symptoms caused by inducible obstruction in HCM. Furthermore, by limiting Ca²⁺ overload during stress, I_NaL‐inhibitors may also play a protective role against stress‐induced arrhythmias (Lyon et al., 2009; Maron et al., 2006). Intriguingly, both ranolazine and GS‐967 abolished the increase in spontaneous contractions and triggered activity induced by isoprenaline (Figures 5 and 6). By acting downstream of the receptor, I_NaL‐inhibitors only counteract the positive inotropic effect of β‐adrenoceptor stimulation on septal tension and do not reduce the lusitropic and the chronotropic effects of noradrenaline, which are essential to tolerate exercise. High doses of non‐selective β‐blockers, such as nadolol, are often required to reduce dynamic gradients in patients with exertional symptoms due to inducible obstruction (Nistri et al., 2012): a large number of patients do not tolerate such aggressive β‐blocker treatments and are left with residual symptomatic gradients. In other patients, residual dynamic gradients persist even at the maximal target dose of β‐blockers (e.g. 80–100 mg·day⁻¹ of nadolol; see Nistri et al., 2012). Based on our results, we can envision the use of I_NaL‐inhibitors in addition to β‐blockers in patients with residual obstruction‐related symptoms at the maximal tolerated dose of β‐blockers, with the purpose of abolishing the remaining inducible gradients.

In conclusion, the novel selective I_NaL‐inhibitor GS‐967 improved diastolic function and reduced the arrhythmogenic potential of HCM myocardium in vitro, matching the effects of ranolazine at 20 times lower concentrations. I_NaL‐inhibitors (ranolazine and GS‐967) appeared to reduce septal contractility at peak exercise without affecting resting performance. These data suggest improved efficacy of I_NaL‐inhibitors over disopyramide, and a synergistic action with β‐adrenoceptor antagonists, in HCM patients with exercise‐induced obstruction.

Acknowledgements

This work was supported by Fondazione Telethon (GGP13162), by the European Commission (STREP Project 241577 ‘BIG HEART’, 7th European Framework Program), by the Italian Ministry of Health (RF 2010‐2313451, RF‐2013‐02356787 and GR‐2011‐02350583) and by Regione Toscana (FAS‐Salute 2014, project ToRSADE).

Author contributions

C.F. devised the project, performed intact trabeculae experiments, analysed the data and wrote the manuscript. J.M.P. contributed to intact trabeculae experiments and to analyse the data. L.M. collected and processed cardiac samples and contributed to single cell experiments. F.G. and B.T. contributed to intact trabeculae experiments. A.R. collected clinical data from patients. L.B. contributed to devise the project and critically reviewed the manuscript. R.M. performed β‐receptor binding experiments. C.P. contributed to single cell experiments. C.T. and E.C. contributed to data analysis and interpretation and edited the manuscript. I.O. recruited HCM patients and reviewed the manuscript. C.P. and A.M. contributed to conceive the project and critically edited the manuscript. R.C. devised the experiments, performed single cell experiments, analysed and interpreted the results and wrote the manuscript.

Conflict of interest

L.B. was employed by Gilead Sciences, Inc., till 2016.

Declaration of transparency and scientific rigour

This http://onlinelibrary.wiley.com/doi/10.1111/bph.13405/abstract acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1 Control Patients Characteristics. Clinical features of the control patients enrolled in the study. Clinical data refers to visits performed less than 1 month before the operation. Categorical data is expressed as proportion of patients; continuous values are expressed as mean ± SD. NYHA = New York Heart Association; LVOT = Left‐Ventricular Outflow Tract; LA = Left Atrium.

Table S2 Effects of GS‐967 on action potentials and intracellular Ca2+ in HCM cardiomyocytes. Data are expressed as means ± standard error of mean. Action potentials: data from 37 HCM cardiomyocytes isolated from 9 HCM patient samples. Ca‐transients = data from 26 myocytes from 7 HCM patients. P values were calculated using linear mixed models corrected for paired comparisons. T. to Peak = time to peak; 50% decay = time from peak to 50% decay of Catransients; 90% decay = time from peak to 90% decay of Ca‐transients; MDP = mean diastolic potential; Ampl. = amplitude of action potentials; Upstr. = maximal upstroke speed; APD20 = action potential duration at 20% of repolarization; APD50 = action potential duration at 50% of repolarization.

Figure S1 Human HCM cardiomyocytes and trabeculae: representative images and cell shortening. (A) Representative videomicroscopy images of a HCM cardiomyocyte suspension right after isolation and reintroduction of calcium. Of note, a large amount of debris as well as dead cells are visible at this stage. White bar is 15 μm. (B) Representative videomicroscopy images of HCM trabeculae mounted between the tip of a force transducer and a length controlling motor. Wire is used to tie the trabecula's end to the attachments at both sides. White bar is 1 mm. (C) Representative images of a contracting HCM myocyte at end diastole (above) and at peak shortening (below). White bar is 15 μm. A video is also provided as online supplement. (D) Left: representative superimposed sarcomere shortening traces from a HCM myocyte, recorded in the absence (black trace) and presence (dark green) of isoproterenol 10–7 M. Top right: average sarcomere shortening during stimulation at 0.5 Hz in HCM myocytes. Bottom right: kinetics of sarcomere shortening in HCM cardiomyocytes; TTP = time from stimulus to peak, RT50 = time from peak to 50% relaxation. Means ± SEM from 14 myocytes from 3 HCM patients.

Figure S2 GS‐967 suppresses cellular arrhythmias in HCM cardiomyocytes. (A) Representative superimposed trains of action potentials elicited at 0.5 Hz at baseline (black traces) and in the presence of GS‐967 0.5 μM (red traces). Early after‐depolarizations (EADs) are marked by arrows. (B) Representative superimposed trains of action potentials elicited at 0.5 Hz. Delayed afterdepolarizations (DADs) are marked by arrows (C) Fraction of HCM cardiomyocytes showing at least 2 early after‐depolarizations (EADs) during 3 min of continuous stimulation, at baseline (black) and in the presence of GS‐967 0.5 μM (red). (D) Fraction of HCM cardiomyocytes showing at least 2 delayed after‐depolarizations (DADs) during 3 minutes of pacing, at baseline (black) and in the presence of GS‐967 0.5 μM (red). (C‐D) Means ± standard error from 37 HCM cardiomyocytes from 9 HCM patients. * = P < 0.05, linear mixed models corrected for paired comparisons.

Figure S3 Effects of GS‐967 in control cardiomyocytes. (A) Representative superimposed action potentials at baseline (grey trace) and in the presence of GS‐967 0.5 μM (orange trace), elicited at 0.2 Hz (left) and 1 Hz (rught) from a control cardiomyocyte. (B) Action potential duration at 90% of repolarization (APD90%) at baseline (grey) and in the presence of GS‐967 0.5 μM (orange) at different frequencies of stimulation. Means ± standard error from 10 control cardiomyocytes from 3 patients. (C) Representative superimposed Ca‐transients at baseline (grey traces) and in the presence of GS‐967 0.5 μM (orange traces), elicited at 0.5 Hz (left). (D) Ca‐transient amplitude (Δ[Ca2+]i) at baseline (grey) and with the application of GS‐967 (orange) in control cardiomyocytes. (E) Time to peak (TP), Time from peak to 50% decay of Ca‐transients (T50%) and time from peak to 90% decay (T90%) at baseline (grey) and in the presence of GS‐967 0.5 μM (orange), elicited at 0.5 Hz. (D‐E) Means ± standard error from 9 cardiomyocytes from 3 patients.

Figure S4 Late Na current blockers under β‐stimulation in control samples and effects of forskolin (A) Representative action potentials at baseline (top, grey), in the presence of Isoproterenol 10–7 M (Iso, centre, green traces) and in the presence of Ran 10 μM added on top of Iso (right, purple traces), elicited at 1 Hz in a CTR cardiomyocyte. Means ± standard error from 8 cardiomyocytes from 3 HCM patients. (B) AP duration at 90% repolarization. Means± SEM from 7 control myocytes (2 pts.) (C) Representative twitches recorded at 0.5 Hz at basal conditions (grey), in the presence of GS (green), in the presence of ran+GS (purple). (D)Twitch (0.5 Hz) amplitude (top) and duration (bottom, from stimulus to 90% relax.) at baseline (grey), with Iso (green), with Iso + GS (plum). Means ± standard error from 3 trabeculae from 2 control patients. (E) Superimposed twitches at 0.5 Hz at baseline (black), with 1 μM forskolin (ochra) and with GS‐967 added on top of forskolin (pink) in a HCM trabecula. (F) Twitch amplitude and duration in response to forskolin and forskolin+ GS‐967 Means ± standard error from 4 trabeculae from 2 HCM patients.

Figure S5 Binding studies with β1 and β2‐adrenergic receptors. (A) Attenuation of 3H‐CGP12177 bound to HEK cell membranes expressing β1‐adrenergic radioactivity by different concentrations of ranolazine, GS‐967 and propranolol. Curves of ranolazine and propranolol are fitted with hill equations. (B) Attenuation of 3H‐CGP12177 bound to HEK cell membranes expressing β2‐ adrenergic radioactivity by different concentrations of ranolazine, GS‐967 and propranolol. Curves of ranolazine and propranolol are fitted with hill equations. (C) Normalized comparison of the binding curves of ranolazine with membranbes expressing β1 or β2‐adrenergic receptors. Calculated IC₅₀ values are indicated. All experiments were conducted in triplicate and the average value is shown with the calculated standard error.

Figure S6 Additional AP‐Clamp results. (A) Representative L‐type Ca‐current traces recorded during AP‐clamp at baseline (black), with Iso‐AP as Command Voltage but in the absence of Iso in the solution (light green) and after the addition of Iso to the bathing solution (green); command voltage APtraces are shown in figure 5E (main text). (B) Integral of L‐Type Ca‐current recorded during AP‐clamp in the conditions of panel A. Notably, the application of a prolonged AP (Iso‐AP) as a command voltage does not significantly increase the total amount of Ca‐entry; the actual addition of isoproterenol to the solution is necessary. (C) Representative Ca‐current traces recorded during AP‐clamp with Iso (green), with Iso + Ran‐ AP as Command Voltage but in the absence of Ran in the solution (light pink) and after the addition of Ran (on top of Iso) to the bathing solution (magenta). (D) Integral of L‐Type Ca‐current recorded in the conditions of panel C. Notably, the application of a shortened AP (Iso + Ran‐AP) as a command voltage significantly reduces the total amount of Ca‐entry; the actual addition of ranolazine to the solution does not significantly increase the reduction of Ca‐entry. (B‐D) Means ± standard error from 14 cells (5 patients). (E‐F) Ca‐current traces recorded during AP‐clamp in the absence and presence of lacidipine (10–5 M): representative traces (E) and average values (F) from 7 cells (2 patients).

Figure S7 The density of T‐tubules is markedly low in HCM cardiomyocytes. Representative confocal images of single cardiomyocytes isolated from the same HCM samples used for the functional experiment. Each cell derives from a different patient sample (ID of the patient is indicated next to the cell in each respective image). Cells were stained with Di‐3‐ANEPPDHQ (Thermo‐Fisher) and imaged with a Leica Confocal microscope using the 488 nm laser line. Sections were taken at mid cell. While the outer sarcolemma is well stained in all myocytes, T‐tubules are barely visible in most of them and some cells are completely devoid of T‐tubules. White bars equal 10 μm.

Ferrantini, C. , Pioner, J. M. , Mazzoni, L. , Gentile, F. , Tosi, B. , Rossi, A. , Belardinelli, L. , Tesi, C. , Palandri, C. , Matucci, R. , Cerbai, E. , Olivotto, I. , Poggesi, C. , Mugelli, A. , and Coppini, R. (2018) Late sodium current inhibitors to treat exercise‐induced obstruction in hypertrophic cardiomyopathy: an in vitro study in human myocardium. British Journal of Pharmacology, 175: 2635–2652. doi: 10.1111/bph.14223.