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

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 (INaL)‐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 INaL‐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 INaL‐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. INaL‐inhibitors were more effective than disopyramide in reducing contractility during exercise. Finally, INaL‐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.

Intracellular Ca 2+ was monitored during field stimulation at different pacing rates and under acute administration of drugs as detailed below. Action potentials (APs) were measured simultaneously using the perforated-patch configuration (amphotericin-B method). Specifically, for AP recordings, the pipette solution contained (in mM) 115 K methanesulfonate, 25 KCl, 10 HEPES, 3MgCl 2 and cells were superfused with Tyrode buffer (see above) containing 1.8mM CaCl 2 . APs were elicited with short depolarizing stimuli (<3ms) at different frequency of stimulation (0.2Hz, 0.5Hz and 1Hz, 1 minute at each frequency).
EADs and DADs: EAD and DAD events were considered when a spontaneous depolarization larger than 20mV was detected during the plateau of an AP or during the diastolic period, respectively, while the cell is stimulated with the 3-minute protocol described above. A cardiomyocyte was scored positive for EADs or DADs if it displayed > 2 events during 3'of stimulated activity.
Drug studies: test recordings in presence of 10 -7 M isoproterenol, 0.5 µM GS-967 or 10µM ranolazine were performed after >3 minutes from the beginning of drug exposure and repeated after >5minutes of washout. Each cell was randomly assigned to either GS-967 or ranolazine and no cell was exposed to both drugs.
Voltage-clamp studies: Late sodium current (I NaL ) was measured as described before 1 L-Type Ca 2+ -current was measures as previously described 1  10 HEPES; pH 7.3 (with CsOH). 100 or 500 ms depolarizing steps to 0mV from a holding potential of -90mV were applied to the cell at a rate of 0.5Hz: 20 subsequent episodes were averaged. The measurements were repeated in the presence of GS-967 or isoproterenol.
Action-potential clamp technique was used to record L-Type Ca 2+ current in the context of a physiological variation of membrane potential shaped like a real action potential recorded in the same conditions from a similar cardiomyocyte. We selected 3 representative AP traces, one for each condition (baseline, isoproterenol and iso+ranolazine), recorded during stimulation at 0.5Hz from an HCM cardiomyocyte, and used them as command voltages while recording I CaL under the same conditions (AP traces used as command voltages are shown in the respective figures). For Ca-current recordings under AP-clamp conditions, we used the same solutions used for I CaL recordings with standard square pulses (see above). 10 subsequent episodes were averaged. Afterwards, the integral of I CaL current across the duration of a single AP was calculated from the average traces in each condition.
Signal recording and processing: Potential and current signals were measured with a Multiclamp 700B amplifier, using a standard 10kHz longpass digital filter. Patch-clamp signals were simultaneously digitized through using Digidata 1440A. Acquisition and analysis was controlled by dedicated software (pClamp10.0). All products from Molecular Devices, Sunnyvale, California. Ca-fluorescence videos were recorded at >200 frames per second and the fluorescence of single cells was analysed using Metafluor software (molecular devices Muscles were allowed to stabilize for at least 30 min before recordings and muscle length was empirically adjust to "optimal length" as previously described. Briefly, slack muscles were progressively stretched while monitoring the increase in the development of active tension. Resting tension remained negligible during the initial stretch phase. To reach the optimal sarcomere length, muscles were stretched step-by-step (2% of muscle length per step) to a length where a small increase in length resulted in no further increases in active tension, while resting tension started to rise 6 . This "optimal length'' was selected to be comparable to the maximally attained length in vivo (2.15±0.05 µm sarcomere length) 7 .
Isometric force was recorded at 35±2°C under various conditions including various pacing rates (0.1-2.5 Hz) and acute drugs administration (below). The occurrence of premature beats was evaluated during steady state stimulation (0.5 Hz) ad during stimulation pauses. Specifically, one-minute stimulation pauses were inserted after the last contraction of a steady series at 3 Hz to promote the occurrence of the spontaneous contractions. At the end of each experiment, muscle section was measured for force normalization.
Drugs tests: The effects of drugs, Ranolazine 10µM, Disopyramide at 5 µM and GS-967 at 0.5 µM, unless otherwise specified. Test recordings in presence of the drug were performed after >3 minutes from the beginning of drug exposure. Afterwards, the drug was washed out for >5 minutes and measurements were repeated. Isoproterenol (10-7 M) was used to mimic β-adrenergic stimulation during exercise in single cells and trabeculae and the other three drugs were applied on top of Isoproterenol at the doses specified above. To faithfully compare different sets of measurements, sensitivity analysis was performed for each statistical comparison, in order to account for:

1-non Gaussian distribution
The data were tested for normality using the Skewness/Kurtosis test 8 .

2-heteroschedasticity (inequality of variances)
We used the F test for equality of variances in two-group comparison studies and the Bartlett's test for variance homogeneity in the multiple comparison design.
Non-parametric test based on rank transformation (Wilcoxon's sum of rank) was used to check robustness of results under violation of condition 1-or 2-.

3-within-subject correlation
Most of the average data derives from multiple myocytes or trabecuale from different patients. We estimated within-subject correlation for each variable with One-way ANOVA. In order to account for the correlation among different cells/muscles from the same patient, we used linear mixed models 9 to compare couples of data groups, both paired and unpaired. Correction for heteroschedasticity was applied to linear mixed models in unpaired comparisons whenever the variances of the two groups were unequal (as calculated by F-test).
All the results of the new statistical analysis are available on request. The Probability (P) values that are shown in the manuscript and in the online supplement were calculated with linear mixed models according to the aforementioned procedure, both when comparing repeated measurements on the same samples (e.g. effect of ranolazine) and when comparing unpaired datasets. P-values <0.05 were considered statistically significant.
To quantify EADs/ DADs and spontaneous contractions occurrence, including their confidence intervals, the binomial proportion confidence interval was calculated as approximating the binomial distribution with a normal distribution (central limit theorem). The confidence interval was calculated as: where p � is the fraction of successes in a Bernoulli trial process estimated from the statistical sample and N is the sample size. In our case p � is the fraction of myocytes scored positive for EAD or DAD events as described above.

6
The central limit theorem was not applied to a binomial distribution where the fraction p � ≈ 0. In these cases the Wilson score interval was applied: The statistical significance of differences in DAD or EAD occurrence was assessed using the Fisher exact test.
Statistical analysis was performed using Stata 12 software (StataCorp LP, College Station, Texas, USA).
An example of the application of this statistical analysis is reported in the section "Detailed Statistics" fo this Online Data Supplement.

LEGEND:
The For each comparison, the table displays the results of single sta�s�cal tests in different columns: A) The results of Skewness/Kurtosis test for normality are shown: P>χ 2 corresponds to the probability of the given distribu�on to be normal. A distribu�on is considered normal when P>0.05. B) Variance ra�o test is used to test for heteroschedas�city (unequality of variances). The probability of variance ra�o to be equal to 1 is shown: variances are considered unequal when this probability is <0.05. C) Two-sample Wilcoxon rank-sum (Mann-Whitney) test is performed to check the robustness of differences between the two compared groups in case heteroschedas�city or non-normality are present, using a non-parametric test. The results of the test are given: when Prob > |z| is >0.05 the difference between the two groups is considered to be robust. D) Mul�-level one-way analysis-of-variance (ANOVA) is used to check the correla�on of data values within subjects. In brief, the variance among the cells/trabeculae from each single subject is compared with the overall variance. The shown values are the calculated intra-class correla�on and its 95% confidence intervals. When the es�mated correla�on exceeds 0.10 it is considered to have a significant effect on the results, requiring mul�-level analysis for comparison(see below). E) In order to account for the correla�on within subjects, linear mixed models were used to compare couples of data groups. The results provided here are: the es�mate of random effects (and their standard error), which is correlated with the amount of intra-class correla�on, z value , probability (Prob) > |z| and the 95% confidence intervals of the average difference between the two compared groups. When Prob > |z| is <0.05, the difference between the two groups is considered sta�s�cally significant. When Variance ra�o test (in B) reported unequal variances in unpaired comparisons, linear mixed models were adjusted for heteroschedas�city (via independent calcula�on of residuals in the two groups). Linear mixed models were also used to compare paired measurements (effect of drugs), by adding an addi�onal level to data hierarchy (the two repeated measurements within the same cell/trabecula, in the absence and presence of the drug). F) The average difference (with standard error) between the two groups is given here. Note that for both unpaired comparisons the difference is es�mated using linear mixed models, for paired comparisons it is calculated directly (from the output of paired t-test analysis). G) Finally, the results of standard Student's t-test are given in the last columns. t-test for unequal variances was used when heteroschedas�city was detected (see column B), here labeled with #. Paired t-test was used to test the effects of drugs in paired comparisons, here labeled with *. Calculated averages, standard error and P values are reported. Of note, the variable F.Ampl. (mN/mm2), highlighted in yellow, had a very high intra-class correla�on: the results of the standard t-test indicated a significant difference, while the results of linear mixed models did not.
Here we present an example of data analysis performed using the aforemen�oned approach with Stata 12 so�ware. We tested the effects of GS-967 (0.5 µmol/L) on diastolic [Ca 2+ ] i at 1Hz s�mulus rate on HCM myocytes.