Electrophysiologic Characteristics and Pharmacologic Response of Human Cardiomyocytes Isolated from a Patient with Hypertrophic Cardiomyopathy

Abstract

Background

Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiac disorder encountered in the clinic. Data relative to the electrophysiologic characteristics and pharmacologic responsiveness of human tissues and cells isolated from patients with hypertrophic cardiomyopathy are rare. As a consequence, cellular mechanisms underlying arrhythmogenicity are poorly understood.

Methods

Cardiomyocytes were enzymatically dissociated from a septal myectomy surgically removed from a patient with obstructive hypertrophic cardiomyopathy. Sharp microelectrodes and patch-clamp techniques were used to evaluate action potential and sodium channel current (I_Na) characteristics.

Results

Action potential morphology recorded was that typical of an M cell, but with a longer than normal duration (APD) and a relatively steep APD-rate relationship. APD at all rates was significantly reduced following exposure to ranolazine (10 μM). Whole cell patch clamp recording yielded robust peak I_Na and large late I_Na (1.1% of peak I_Na vs. 0.1–0.2% in healthy controls). A large window current was observed as well. Ranolazine (10 μM) shifted steady-state V_0.5 of inactivation by − 8 mV, reduced late I_Na by 82% and significantly diminished the window current.

Conclusion

Our results indicate the presence of cells with M cell characteristics in the septum of the human heart, as has previously been described in the canine heart. They also point to an ameliorative effect of ranolazine to reduce augmented late I_Na and thus to reduce the prolonged APD in the setting of HCM. These results suggest a potential therapeutic role for ranolazine in HCM.

Introduction

Hypertrophic cardiomyopathy (HCM), with a prevalence of 1:500, is the most common monogenic cardiac disorder encountered in the clinic.¹ HCM is also the most common cause of arrhythmic sudden cardiac death in young athletes.²

Its high prevalence notwithstanding, it is poorly understood from the standpoint of cellular mechanisms underlying arrhythmogenicity and diastolic dysfunction. Data relative to the electrophysiologic characteristics and pharmacologic responsiveness of human tissues and cells isolated from patients with hypertrophic cardiomyopathy are rare. HCM also lacks a disease-specific pharmacological treatment.³

Here we report action potential (AP) characteristics of myocytes enzymatically dissociated from a septal myectomy surgically removed from a patient with obstructive hypertrophic cardiomyopathy, together with characteristics of sodium channel current (I_Na) and its response to the late I_Na blocker ranolazine.

Methods

The patient, a 48-y/o male, presented with a systolic murmur and concentric left ventricular hypertrophy with a significant gradient of 39 mmHg. A septal myectomy and a mitral valve replacement, necessitated due to mitral stenosis, were performed. The procedure was done at the Mohawk Valley Heart Institute, Utica, NY, USA, with ethical committee approval for tissue retrieval. The sample was obtained from the left ventricular septal wall.

Cardiomyocytes were obtained by standard enzymatic dissociation methods.⁴ Transmembrane APs were recorded at 36.5°C from single cardiomyocytes using high resistance microelectrodes (DC resistance=40 to 50 MΩ) filled with 2.7 mol/L KCl. Macroscopic peak and late I_Na were recorded at room temperature from single cardiomyocytes using the whole cell patch-clamp technique. I–V relationship, steady-state activation and inactivation and window currents were determined as previously described.^5–7

Late I_Na was evaluated using a square depolarization pulse to −20 mV for 300 ms from at holding potential of −120 mV, applied once every 10 seconds. Late I_Na is presented as a fraction of peak I_Na. The effect of ranolazine to inhibit late I_Na was evaluated at a concentration of 10 μM. Recordings in the presence of the drug were performed >3 minutes after exposure to the drug as well as >5 minutes after washout.

Statistics

Data are expressed as mean±SEM. Statistical analysis was performed using Student paired t test or ANOVA, as appropriate. p<0.05 was considered to be statistically significant.

Results

Action potential (AP) morphology recorded using sharp microelectrodes was that typical of an M cell (Figure 1A), although with a longer duration at baseline (450 ms at 1 Hz) than typically recorded in human M cells.^{8, 9} The prolonged AP duration (APD) is consistent with the prolonged QT/QTc interval recorded from this patient (476/455 ms) and HCM patients in general.¹⁰ APD-rate relationship is relatively steep (Figure 1B), consistent with what is reported in experimental models of HCM.¹¹ APD at all rates was significantly reduced following exposure to ranolazine (10 μM).

Figure 1. Rate-dependence of action potential duration (APD) of a cardiomyocyte isolated from a left ventricular septal myectomy from a patient with hypertrophic cardiomyopathy and the effect of ranolazine.

A: Action potential recorded using high resistance microelectrode at body temperature in the absence (black) and presence of 10 μM ranolazine and after 10 min of washout. B: Rate-dependence of APD in the absence and presence of 10 μM ranolazine and after 10 min of washout. Basic cycle length (BCL). n=1

Whole cell patch clamp recording yielded robust peak I_Na (Figures 2A and B) and a relatively large late I_Na as well as window current (Figures 2C and). Steady-state inactivation was shifted to more negative potentials (−8 mV) by ranolazine resulting in a marked reduction of window current (Figures 2D and E). Late Na⁺ channel current (1.1% of peak I_Na ) was larger than typically observed in normal myocardium (0.1–0.2%)^{12, 13} and was reduced by 82% by 10 uM ranolazine (Figure 2C). The ranolazine-induced reduction in late I_Na and window current likely contribute to the abbreviation of APD.

Figure 2. Characteristics of peak and late sodium channel current (I_Na) and the effect of ranolazine.

A: Macroscopic I_Na recorded under control condition and the presences of ranolazine (10 μM) using whole cell patch-clamp technique and the protocol shown in the inset. B: Current-voltage relationship of I_Na (n=3). C: Ranolazine reduced late I_Na (as % of peak) by 82% (p<0.05, n=3). D: Steady state activation and inactivation curves recorded under control conditions and after ranolazine (10 μM). Ranolazine shifts half-inactivation voltage (V_{0. 5}) by −8 mV (from −90.1 ± 0.9 to −98.2 ± 0.8; n=3; p<0.05). E: Region of overlap of activation and inactivation curves amplified showing window sodium current under control conditions (light shaded area) and in the presence of 10 μM ranolazine (dark shaded area). Hypertrophic Cardiomyopathy (HCM).

Discussion

Our findings indicate the presence of cells with M cells characteristics in the septum of the human heart, as has been described in the canine heart,¹⁴ and point to an ameliorative effect of ranolazine to reduce augmented late I_Na and thus to reduce the prolonged APD in the setting of HCM.¹⁵ Our finding suggest that augmented late I_Na is a major contributor to the prolonged APD of the septal cardiomyocytes isolated from the HCM patient and that inhibition of this current using ranolazine may be of therapeutic value.

Our findings are consistent with those of Coppini and coworkers¹⁵ showing that the phenotype of isolated cardiomyocytes derived from HCM patients differs significantly from that of controls. These authors reported that enhanced CaMKII activity slowed I_Ca inactivation and increased late I_Na thus contributing to APD prolongation and related arrhythmias. Their data also suggested that by altering the function of EC-coupling proteins, CaMKII might also contribute to the altered Ca²⁺-transient kinetics and elevation of diastolic [Ca²⁺]i, which are responsible for the development of delayed afterdepolarizations (DAD). As in our study, ranolazine, at therapeutic concentrations, was shown to be capable of partially reversing the HCM-related cellular abnormalities via inhibition of late I_Na, but with negligible effects in myocytes isolated from control hearts.

The effect of ranolazine to block late I_Na and to abbreviate APD in HCM cardiomyocytes is also expected to reduce diastolic [Ca²⁺]_i and thus to suppress DADs and ameliorate diastolic dysfunction.^{15, 16}