Chronic heart failure slows late sodium current in human and canine ventricular myocytes: implications for repolarization variability.

BACKGROUND: Late Na(+) current (I(NaL)) in human and dog hearts has been implicated in abnormal repolarization associated with heart failure (HF). HF slows inactivation gating of late Na(+) channels, which could contribute to these abnormalities. AIMS: To test how altered gating affects I(NaL) time course, Na(+) influx, and action potential (AP) repolarization. METHODS: I(NaL) and AP were measured by patch clamp in left ventricular cardiomyocytes from normal and failing hearts of humans and dogs. Canine HF was induced by coronary microembolization. RESULTS: I(NaL) decay was slower and I(NaL) density was greater in failing hearts than in normal hearts at 24 degrees C (human hearts: tau=659+/-16 vs. 529+/-21 ms; n=16 and 4 hearts, respectively; mean+/-SEM; p<0.002; dog hearts: 561+/-13 vs. 420+/-17 ms; and 0.307+/-0.014 vs. 0.235+/-0.019 pA/pF; n=25 and 14 hearts, respectively; p<0.005) and at 37 degrees C this difference tended to increase. These I(NaL) changes resulted in much greater (53.6%) total Na(+) influx in failing cardiomyocytes. I(NaL) was sensitive to cadmium but not to cyanide and exhibited low sensitivity to saxitoxin (IC(50)=62 nM) or tetrodotoxin (IC(50)=1.2 muM), tested in dogs. A 50% I(NaL) inhibition by toxins or passing current opposite to I(NaL), decreased beat-to-beat AP variability and eliminated early afterdepolarizations in failing cardiomyocytes. CONCLUSIONS: Chronic HF leads to larger and slower I(NaL) generated mainly by the cardiac-type Na(+) channel isoform, contributing to larger Na(+) influx and AP duration variability. Interventions designed to reduce/normalize I(NaL) represent a potential cardioprotective mechanism in HF via reduction of related Na(+) and Ca(2+) overload and improvement of repolarization.

A multi-modal composition of the late Na+ current in human ventricular cardiomyocytes.

OBJECTIVE: We reported an ultraslow late Na+ current (INaL) in ventricular cardiomyocytes of human hearts. INaL has been implicated in regulation of action potential duration in normal hearts and repolarization abnormalities in failing hearts. We have also identified sodium channel (NaCh) gating modes including bursts (BM) and late scattered openings (LSM) that together comprise INaL; however, the contribution of these gating modes to Na+ current (INa) remains unknown. In the present study, the late NaCh activity was recorded, analyzed, and modeled for heterologously expressed NaCh, Nav1.5, and for the native NaCh of ventricular mid-myocardial cardiomyocytes from normal and failing hearts. METHODS AND RESULTS: We found that LSM gating was significantly slower in failing compared to normal myocytes and Nav1.5 (tau=474+/-10 vs. 299+/-9, and 229+/-12 ms, m+/-SEM; P<0.05, n=5-6). Total burst length of BM decreased with depolarization and was larger in failing compared to normal myocytes and Nav1.5. A complete INa decay was then numerically approximated as composed of NaCh populations operating in three gating modes described by separate Markov kinetic schemes: transient mode (TM), LSM, and BM. The populations of NaCh operating in each gating mode were estimated as 79.8% for TM, 20% for LSM, and 0.2% for BM, yielding an apparent four-exponential INa decay at -30 mV (maximum INa) (tau i approximately 0.4, 4, 50, and 500 ms). Whole-cell recordings confirmed the existence of all four predicted components. The model also predicted voltage and temperature dependence of INaL as well as INaL increase and slower decay in failing hearts and acceleration by amiodarone. CONCLUSIONS: The early phase of Na+ current decay (<40 ms) involves all three NaCh gating modes, the intermediate phase (from 40 to 300 ms) is produced by BM+LSM, although the contribution of BM decreases with depolarization, and ultra-late decay (>300 ms) is determined solely by LSM. The concept of multi-mode composition for INaL provides a new rationale for INaL modulation by factors such as voltage, temperature, pharmacological agents, and pathological conditions.

Reversal of chronic molecular and cellular abnormalities due to heart failure by passive mechanical ventricular containment.

Passive mechanical containment of failing left ventricle (LV) with the Acorn Cardiac Support Device (CSD) was shown to prevent progressive LV dilation in dogs with heart failure (HF) and increase ejection fraction. To examine possible mechanisms for improved LV function with the CSD, we examined the effect of CSD therapy on the expression of cardiac stretch response proteins, myocyte hypertrophy, sarcoplasmic reticulum Ca2+-ATPase activity and uptake, and mRNA gene expression for myosin heavy chain (MHC) isoforms. HF was produced in 12 dogs by intracoronary microembolization. Six dogs were implanted with the CSD and 6 served as concurrent controls. LV tissue from 6 normal dogs was used for comparison. Compared with normal dogs, untreated HF dogs showed reduced cardiomyocyte contraction and relaxation, upregulation of stretch response proteins (p21ras, c-fos, and p38 alpha/beta mitogen-activated protein kinase), increased myocyte hypertrophy, reduced SERCA2a activity with unchanged affinity for calcium, reduced proportion of mRNA gene expression for alpha-MHC, and increased proportion of beta-MHC. Therapy with the CSD was associated with improved cardiomyocyte contraction and relaxation, downregulation of stretch response proteins, attenuation of cardiomyocyte hypertrophy, increased affinity of the pump for calcium, and restoration of alpha- and beta-MHC isoforms ratio. The results suggest that preventing LV dilation and stretch with the CSD promotes downregulation of stretch response proteins, attenuates myocyte hypertrophy and improves SR calcium cycling. These data offer possible mechanisms for improvement of LV function after CSD therapy.

Ranolazine improves abnormal repolarization and contraction in left ventricular myocytes of dogs with heart failure by inhibiting late sodium current.

BACKGROUND: Ventricular repolarization and contractile function are frequently abnormal in ventricular myocytes from human failing hearts as well as canine hearts with experimentally induced heart failure (HF). These abnormalities have been attributed to dysfunction involving various steps of the excitation-contraction coupling process, leading to impaired intracellular sodium and calcium homeostasis. We previously reported that the slow inactivating component of the Na(+) current (late I(Na)) is augmented in myocytes from failing hearts, and this appears to play a significant role in abnormal ventricular myocytes repolarization and function. We tested the effect of ranolazine, a novel drug being developed to treat angina, on (1) action potential duration (APD), (2) peak transient and late I(Na) (I(NaT) and I(NaL), respectively), (3) early afterdepolarizations (EADs), and (4) twitch contraction (TC), including after contractions and contracture. METHODS: Myocytes were isolated from the left ventricle of normal dogs and of dogs with chronic HF caused by multiple sequential intracoronary micro-embolizations. I(NaT) and I(NaL) were recorded using conventional whole-cell patch-clamp techniques. APs were recorded using the beta-escin perforated patch-clamp configuration at frequencies of 0.25 and 0.5 Hz. TCs were recorded using an edge movement detector at stimulation frequencies ranging from 0.5 to 2.0 Hz. RESULTS: Ranolazine significantly (P<0.05) and reversibly shortened the APD of myocytes stimulated at either 0.5 or 0.25 Hz in a concentration-dependent manner. At a stimulation frequency of 0.5 Hz, 5, 10, and 20 microM ranolazine shortened the APD(90) (APD measured at 90% repolarization) from 516+/-51 to 304+/-22, 212+/-34 and 160+/-11 ms, respectively, and markedly decreased beat-to-beat variability of APD(90), EADs, and dispersion of APDs. Ranolazine preferentially blocked I(NaL) relative to I(NaT) in a state-dependent manner, with a approximately 38-fold greater potency against I(NaL) to produce tonic block (IC(50)=6.5 microM) than I(NaT) (IC(50)=294 microM). When we evaluated inactivated state blockade of I(NaL) from the steady-state inactivation mid-potential shift using a theoretical model, ranolazine was found to bind more tightly to the inactivated state than the resting state of the sodium channel underlying I(NaL), with apparent dissociation constants K(dr)=7.47 microM and K(di)=1.71 microM, respectively. TCs of myocytes stimulated at 0.5 Hz were characterized by an initial spike followed by a dome-like after contraction, which was observed in 75% of myocytes from failing hearts and coincided with the long AP plateau and EADs. Ranolazine at 5 and 10 microM reversibly shortened the duration of TCs and abolished the after contraction. When the rate of myocyte stimulation was increased from 1.0 to 2.0 Hz, there was a progressive increase in diastolic "tension," that is, contracture. Ranolazine at 5 and 10 microM reversibly prevented this frequency-dependent contracture.

Post-transcriptional alterations in the expression of cardiac Na+ channel subunits in chronic heart failure.

Clinical and experimental evidence has recently accumulated about the importance of alterations of Na(+) channel (NaCh) function and slow myocardial conduction for arrhythmias in infarcted and failing hearts (i.e., heart failure, HF). The present study evaluated the molecular mechanisms of local alterations in the expression of NaCh subunits which underlie Na(+) current (I(Na)) density decrease in HF. HF was induced in five dogs by sequential coronary microembolization and developed approximately 3 months after the last embolization (left ventricle (LV), ejection fraction = 27 +/- 7%). Five normal dogs served as a control group. Ventricular cardiomyocytes were isolated enzymatically from LV mid-myocardium and I(Na) was measured by whole-cell patch-clamp. The mRNA encoding the cardiac-specific NaCh alpha-subunit Na(v)1.5, and one of its auxiliary subunits beta 1 (NaCh beta 1), were analyzed by competitive reverse transcription-polymerase chain reaction. Protein levels of Na(v)1.5, NaCh beta 1 and NaCh beta 2 were evaluated by western blotting. The maximum density of I(Na)/C(m) was decreased in HF (n = 5) compared to control hearts (33.2 +/- 4.4 vs. 50.0 +/- 4.9 pA/pF, mean +/- S.E.M., n = 5, P < 0.05). The steady-state inactivation and activation of I(Na) remained unchanged in HF compared to control hearts. The levels of mRNA encoding Na(v)1.5, and NaCh beta 1 were unaltered in FH. However, Na(v)1.5 protein expression was reduced about 30% in HF, while NaCh beta 1 and NaCh beta 2 protein were unchanged. We conclude that experimental HF in dogs results in post-transcriptional changes in cardiac NaCh alpha-subunit expression.

Gating of the late Na+ channel in normal and failing human myocardium.

We previously reported an ultraslow inactivating late Na+ current (INaL) in left ventricular cardiomyocytes (VC) isolated from normal (NVC) and failing (FVC) human hearts. This current could play a role in heart failure-induced repolarization abnormalities. To identify properties of NaCh contributing to INaL, we examined early and late openings in cell-attached patches of HEK293 cells expressing human cardiac NaCh alpha-subunit (alpha-HEK) and in VC of one normal and three failing human hearts. Two types of the late NaCh openings underlay INaL in all three preparations: scattered late (SLO) and bursts (BO). Amplitude analysis revealed that slope conductance for both SLO and BO was the same compared to the main level of early openings (EO) in both VC (21 vs 22.7pS, NVC; 22.7 vs 22.6pS, FVC) and alpha-HEK (23.2 vs 23pS), respectively. Analysis of SLO latencies revealed voltage-independent ultraslow inactivation in all preparations with tendency to be slower in FVC compared to NCV. EO and SLO render one open voltage-independent state (tau approximately 0.4ms) for NVC and FVC. One open (voltage-dependent) and two closed states (one voltage-dependent and another voltage-independent) were found in BO of both specimens. Burst duration tend to be longer in FVC ( approximately 50ms) than in NVC ( approximately 30ms). In FVC we found both modes SLO and BO at membrane potential of -10mV that is attribute for take-off voltages (from -18 to -2mV) for early afterdepolarizations (EAD's) in FVC. In conclusions, we found a novel gating mode SLO that manifest slow (hundreds of ms), voltage-independent inactivation in both NVC and FVC. We were unable to reliably demonstrate any differences in the properties of the late NaCh in failing vs a normal human heart. Accordingly, the late current appears to be generated by a single population of channels in normal and failing human ventricular myocardium. Both SLO and BO could be implicated in EADs in HF.