Voltage-gated sodium channels in the mammalian heart

Abstract

Mammalian species express nine functional voltage-gated Na(+) channels. Three of them, the cardiac-specific isoform Nav1.5 and the neuronal isoforms Nav1.8 and Nav1.9, are relatively resistant to the neurotoxin tetrodotoxin (TTX; IC50 ≥ 1 μM). The other six isoforms are highly sensitive to TTX with IC50 values in the nanomolar range. These isoforms are expressed in the central nervous system (Nav1.1, Nav1.2, Nav1.3, Nav1.6), in the skeletal muscle (Nav1.4), and in the peripheral nervous system (Nav1.6, Nav1.7). The isoform Nav1.5, encoded by the SCN5A gene, is responsible for the upstroke of the action potential in the heart. Mutations in SCN5A are associated with a variety of life-threatening arrhythmias, like long QT syndrome type 3 (LQT3), Brugada syndrome (BrS) or cardiac conduction disease (CCD). Previous immunohistochemical and electrophysiological assays demonstrated the cardiac expression of neuronal and skeletal muscle Na(+) channels in the heart of various mammals, which led to far-reaching speculations on their function. However, when comparing the Na(+) channel mRNA patterns in the heart of various mammalian species, only minute quantities of transcripts for TTX-sensitive Na(+) channels were detectable in whole pig and human hearts, suggesting that these channels are not involved in cardiac excitation phenomena in higher mammals. This conclusion is strongly supported by the fact that mutations in TTX-sensitive Na(+) channels were associated with epilepsy or skeletal muscle diseases, rather than with a pathological cardiac phenotype. Moreover, previous data from TTX-intoxicated animals and from cases of human tetrodotoxication showed that low TTX dosages caused at most little alterations of both the cardiac output and the electrocardiogram. Recently, genome-wide association studies identified SCN10A, the gene encoding Nav1.8, as a determinant of cardiac conduction parameters, and mutations in SCN10A have been associated with BrS. These novel findings opened a fascinating new research area in the cardiac ion channel field, and the on-going debate on how SCN10A/Nav1.8 affects cardiac conduction is very exciting.

Figure 1.

Structure and function of voltage-gated Na⁺ channels. A) Proposed membrane topology of voltage-gated Na⁺ channels. Each of the four domains (DI-IV) is composed of six transmembrane helices. The fourth segment (S4; red) contains regularly arranged positive charges that are important elements of the voltage sensor. The intracellular loop between the third and fourth domain forms the inactivation gate composed of residues isoleucine, phenylalanine, and methionine (IFM motif). The proximal C terminus contains an EF-hand domain involved in the binding of Ca⁺⁺, and a downstream calmodulin-binding motif (IQ motif). B) Functional states of voltage-gated Na⁺ channels. C) Whole-cell recordings showing Na_v1.5 current family obtained in HEK293 cells. Currents were elicited by test potentials from − 80 mV to various test pulses in 5 or 10 mV increments at a pulsing frequency of 1.0 Hz (holding potential: − 120 mV). D) Single-channel recordings in cell-attached patches using transfected HEK293 cells. Na_v1.5 channel activity was measured by stepping from a holding potential of − 120 mV to a test potential of − 20 mV. The arrows indicate the beginning and the end of the test pulse (8 ms). The single-channel amplitude under these conditions was 1.26 pA. Modified from.

Figure 2.

Phylogenetic tree, tissue distribution, chromosomal localization, and TTX sensitivity of mammalian voltage-gated Na⁺ channels. The name of an individual channel consists of the chemical symbol of the permeating ion (Na), the principal physiological regulator (voltage), the gene subfamily, and the number of the specific channel isoform, assigned according to the approximate order in which each isoform was identified. Abbreviations: CNS – central nervous system, PNS – peripheral nervous system, TTX – tetrodotoxin. Adapted from.

Figure 3.

Subcellular localization of voltage-gated Na⁺ channels in mouse ventricular myocytes using a specific antibody against Na_v1.5. Intense fluorescence signals were obtained at the outer plasma membrane (PM) and at the intercalated disks (*). Staining along the Z lines suggests channel localization in the t-tubular membrane system, as shown at a higher magnification in the right image. Adapted from.

Figure 4.

Cardiac currents and number of mutations (red) associated with cardiac arrhythmia. SCN5A is more frequently affected than all the other cardiac ion channel genes. It is likely that considerably more mutations have been recently identified by genetic screenings, and that even novel mutations may not be considered for publication in peer-reviewed journals or online data bases. Furthermore, it must be pointed out that most if not all ion channel subunits interact with other cardiac proteins, whose mutations can affect the cardiac action potential. More recently, several SCN10A mutations were identified in BrS patients. These mutations were not included, because the physiological significance of SCN10A/Na_v1.8 in the heart and its role in shaping the cardiac AP is still a matter of debate. Helpful databases were PubMed/Medline, http://www.fsm.it/cardmoc/ and http://www.qtsyndrome.ch/.

Figure 5.

Subcellular localization of Na_v1.1 and Na_v1.2 in mouse ventricular myocytes. Freshly dissociated cardiac cells were fixed, permeabilized and probed with the primary antibody against Na_v1.1 and Na_v1.2. Both brain-type Na⁺ channels were localized at the outer plasma membrane, at the intercalated disk regions and in t-tubular membranes. Scale bars represent 50 μm. Control – antibody against Na_v1.2 pre-absorbed against the respective antigen. Adapted from.

Figure 6.

Na⁺ channel transcript patterns in different adult mammalian hearts. Relative transcript levels were obtained by real-time RT-PCR. Relative transcript levels of TTX-sensitive channels decreased with increasing heart size (30% for mouse, 8% for rat, and 4% for pig/human). Furthermore, alternative splicing of Na_v1.5 RNA occurred also in a species-dependent manner. For further details see.

Figure 7.

Effects of TTX on the cardiovascular system. TTX blocks Na⁺ channels of the skeletal muscle, the diaphragm, afferent and efferent nerve fibers including the phrenic nerve and the sympathetic nerve system. This leads to muscular incoordination, respiratory dysfunction, a marked decrease in the total peripheral resistance (TPR) and a drop in blood pressure. The toxin also activates the medullary chemoreceptor trigger zone which initiates vomiting. Victims often remain conscious, but cannot speak, move or breathe, suggesting that the brain is largely protected by the blood-brain barrier. Death may occur in as little as 17 minutes after ingesting the toxin (human oral killing dose: 1 to 2 mg). Treatment may involve mechanical ventilation, normal saline infusion, gastric emptying procedures, application of activated charcoal, atropinization, and treatment with dopamine. Prognosis is good if the patient arrives at the emergency department conscious and prior to respiratory arrest. For details see.

Figure 8.

Correlation between TTX blood levels and the degree of intoxication. Each data point corresponds to one case of tetrodotoxication. Please note that only in a few cases, blood TTX levels were determined. The figure shows that TTX concentrations fairly correlated with the severity of the patients' symptoms. The clinical grading system for TTX poisoning was introduced by Fukuda and Tani. First-degree and second-degree cases are relatively mild cases; blood TTX levels were similar to or lower than the IC₅₀ of TTX-sensitive Na⁺ channels. The third degree is characterized by more severe disturbances, like ataxia, widespread paralysis, pronounced hyporeflexia, drop in blood pressure, fixed/dilated pupils, cyanosis, and respiratory failure (e.g., dyspnoea, decreased vital capacity or lower forced expiratory volume). Hypothermia also develops, when skeletal muscle contractions and conduction in nerve fibers are gradually blocked. Fourth-degree cases are severely intoxicated victims presenting with cessation of respiration, decreased arterial O₂, unconsciousness, bradycardia, and hypotension. TTX concentrations were between 40 and 164 nM. These concentrations would be indeed high enough to block the vast majority of TTX-sensitive Na⁺ channels in the body. Effects on the heart were rarely seen. Bradycardia was observed in few victims who had a pre-existing chronic disease (diabetes mellitus), and cardiac arrhythmia occurred secondary to hypoxia. Adapted from.