Inhibition of the late sodium current slows t-tubule disruption during the progression of hypertensive heart disease in the rat

Abstract

The treatment of heart failure (HF) is challenging and morbidity and mortality are high. The goal of this study was to determine if inhibition of the late Na(+) current with ranolazine during early hypertensive heart disease might slow or stop disease progression. Spontaneously hypertensive rats (aged 7 mo) were subjected to echocardiographic study and then fed either control chow (CON) or chow containing 0.5% ranolazine (RAN) for 3 mo. Animals were then restudied, and each heart was removed for measurements of t-tubule organization and Ca(2+) transients using confocal microscopy of the intact heart. RAN halted left ventricular hypertrophy as determined from both echocardiographic and cell dimension (length but not width) measurements. RAN reduced the number of myocytes with t-tubule disruption and the proportion of myocytes with defects in intracellular Ca(2+) cycling. RAN also prevented the slowing of the rate of restitution of Ca(2+) release and the increased vulnerability to rate-induced Ca(2+) alternans. Differences between CON- and RAN-treated animals were not a result of different expression levels of voltage-dependent Ca(2+) channel 1.2, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, ryanodine receptor type 2, Na(+)/Ca(2+) exchanger-1, or voltage-gated Na(+) channel 1.5. Furthermore, myocytes with defective Ca(2+) transients in CON rats showed improved Ca(2+) cycling immediately upon acute exposure to RAN. Increased late Na(+) current likely plays a role in the progression of cardiac hypertrophy, a key pathological step in the development of HF. Early, chronic inhibition of this current slows both hypertrophy and development of ultrastructural and physiological defects associated with the progression to HF.

Keywords: calcium handling; heart failure; late INa; ranolazine; t-tubule.

Fig. 1.

Measurements of t-tubule organization index (OI) in myocytes from hearts of control- and ranolazine-fed rats. A and B: example 2-dimensional images of a cardiac myocyte from a control (A) and a ranolazine-treated (B) rat. The graphs below each panel show the fast Fourier transform (frequency on the x-axis; power on the y-axis) for each cell indicated with a red arrow in the images. The frequency range for bands 1 and 2 (B1 and B2, respectively) is indicated for calculation of OI, as described in methods. C and D: frequency (y-axis) histograms showing examples of measurements of OI (x-axis) in cardiomyocytes from individual control- and ranolazine-fed rats. E and F: summary data for all myocytes (n = 1,003 and 1,230 for control and ranolazine, respectively) from all hearts (n = 8 rats in each group).

Fig. 2.

Summary of data for mean cardiomyocyte cell length and width in control- and ranolazine-fed spontaneously hypertensive rats (SHRs). A: 2-dimensional image of myocytes from a control heart. B: image of a ranolazine heart. C: summary of mean cell length. D: summary of variability (SD) of cell length. E: mean cell width. F: variability of cell width in hearts from control- (black bars) and ranolazine-fed (light gray bars) rats. N = 1,024 and 1,234 myocytes from 8 hearts in each group. *P < 0.05 vs. control; **P < 0.01 compared with control.

Fig. 3.

Calcium transients in myocytes of hearts from control- and ranolazine-fed rats. A: line scan image across multiple cells in an intact heart from a control rat. Horizontal black lines indicate cell boundaries. Average intensity profile for the entire site is shown above the image. Intensity profiles for selected myocytes are shown to the right of the image. B: line scan image from a ranolazine heart. F/F₀, fluorescence intensity ratio. C–F: summary data for Ca²⁺ transient rise time, time to peak, and durations at 50 and 90% recovery (TD₅₀ and TD₉₀, respectively) to baseline in myocytes from control- (red bars) and ranolazine-fed (green bars) rats. n = 72–146 myocytes in 3 hearts each. *P < 0.05 and **P < 0.01 compared with control.

Fig. 4.

Variability in Ca²⁺ cycling along the cell length in myocytes in intact hearts from control- and ranolazine-fed rats. A and B: line scan images of Ca²⁺ transients in individual myocytes recorded longitudinally from intact hearts of control and ranolazine treated rats, respectively, during pacing at a basic cycle length (BCL) of 700 ms. Average fluorescence intensity profiles are shown above each image. C–J: heterogeneity indexes (HIs; i.e., values of SD) of mean values of rise time, TD₅₀, TD₈₀, and TD₉₀ of Ca²⁺ transients recorded in cardiomyocytes from control- (red bars) and ranolazine-fed (green bars) rats during pacing at BCL = 700 (left) and 300 ms (right). * P < 0.05, ** P < 0.01 compared with control.

Fig. 5.

Calcium alternans and restitution of sarcoplasmic reticulum Ca²⁺ release in myocytes in hearts of control- and ranolazine-fed rats. A and B: line scan images across multiple cells in hearts of control- (A) and ranolazine-fed (B) rats during pacing at BCL = 300 ms. Cells are separated by horizontal black lines. C: relationship between the magnitude of the alternans ratio [AR = 1 − (small/large)] and BCL in hearts from control- (○) and ranolazine-fed (●) rats. ECL₅₀ is the estimated cycle length at which AR = 0.5. D: summary of ECL₅₀ data for all myocytes [n = 11 and 16 in 3 hearts each from control- (red bar) and ranolazine-fed (green bar) rats]. E: recovery of sarcoplasmic reticulum Ca²⁺ release as a function of recovery interval. R₅₀ is the cycle length at 50% recovery of Ca²⁺ transient magnitude (relative to magnitude recorded at a BCL of 700 ms). F: summary of R₅₀ data for all myocytes [n = 7 and 16 in 3 hearts each from control- (red bar) and ranolazine-fed (green bar) rats]. *P < 0.05 and **P < 0.01 compared with control.

Fig. 6.

Effects of acute ranolazine treatment on Ca²⁺ transient characteristics during slow (BCL = 700 ms) and rapid (BCL = 300 ms) pacing. A and B: line scan images showing Ca²⁺ transients in a control rat heart at BCL = 700 and 300 ms. Intensity profile is shown below. C and D: recordings from the same heart as in A and B after 10 min of exposure to ranolazine (10 μM). E–L: summary data for Ca²⁺ transient characteristics before (red bars) and after 10 min exposure to ranolazine (green bars). n = 9 myocytes in 3 hearts. *P < 0.05 and **P < 0.01 compared with control.

Fig. 7.

Expression of Ca²⁺ cycling proteins in left ventricular tissue from rats-fed control (C) or ranolazine-containing (R) chow for 3 mo. A: Western blot showing expression of voltage-dependent Ca²⁺ 1.2 (Cav1.2), Na⁺/Ca²⁺ exchanger (NCX), sarco(endo)plasmic reticulum Ca²⁺-ATPase 2a (SERCA2a), voltage-gated Na⁺ channel 1.5 (Nav1.5), and ryanodine receptor type 2 (RyR2), with GAPDH as control. B: relative protein expression normalized to GAPDH in hearts from control- (black bars) and ranolazine-fed (white bars) rats. Values indicate means ± SE; n = 4 for C and n = 5 for R.

Fig. 8.

Measurements of late sodium current (I_Na,L) in cardiac myocytes isolated from 7- and 10-mo-old SHRs. A: voltage-clamp protocol (top) along with representative recordings of total and late (I_Na,L) sodium current from a single myocyte (10-mo-old SHR) in the absence (control) and presence of sodium channel blockers ranolazine (RAN, 5 μM) and tetrodotoxin (TTX, 10 μM). Inset: expanded tracings of I_Na,L. Test potential was −20 mV. B: concentration-response relationships for inhibition by ranolazine of I_Na,L in 7- (●) and 10-mo-old (○) SHRs. n_H, Hill coefficient. C: summary of normalized I_Na,L magnitude in 3-mo-old Wistar rats (WRs, n = 9), 7-mo-old SHRs (n = 4) and 10-mo-old SHRs (n = 7). nH **P < 0.01.