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Arrhythmogenic calmodulin mutations disrupt intracellular cardiomyocyte Ca2+ regulation by distinct mechanisms.

Yin G, Hassan F, Haroun AR, Murphy LL, Crotti L, Schwartz PJ, George AL, Satin J - J Am Heart Assoc (2014)

Bottom Line: LQTS-CaM mutants do not consistently affect L-type Na current in heterologous cells or native cardiomyocytes, suggesting that the Na channel does not contribute to LQTS pathogenesis in the context of CaM mutations.LQTS-CaM mutants led to loss of Ca2+-transient entrainment with the rank order from greatest to least effect: CaM-D130G~CaM-D96V>CaM-F142L.CaM mutations associated with LQTS may not affect L-type Na+ current but may evoke defective Ca2+-dependent inactivation of L-type Ca2+ current.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Kentucky College of Medicine, Lexington, KY (G.Y., F.H., A.R.H., J.S.).

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Long QT syndrome (LQTS) CaM mutation effects on persistent Na current are limited to a single splice variant of NaV1.5 and a single CaM mutation. A, CaM‐D130G evoked increased persistent sodium current with fetal NaV1.5. Averaged tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 200‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. Summary data are provided in Table 1. B, CaM‐D130G has no significant effect on persistent Na current in fetal ventricular cardiomyocytes. Tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 300‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. CaM indicates calmodulin; WT, wild type.
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fig01: Long QT syndrome (LQTS) CaM mutation effects on persistent Na current are limited to a single splice variant of NaV1.5 and a single CaM mutation. A, CaM‐D130G evoked increased persistent sodium current with fetal NaV1.5. Averaged tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 200‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. Summary data are provided in Table 1. B, CaM‐D130G has no significant effect on persistent Na current in fetal ventricular cardiomyocytes. Tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 300‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. CaM indicates calmodulin; WT, wild type.

Mentions: Because CaM is known to regulate cardiac Na channel inactivation and a specific defect in Na channel inactivation (increased persistent Na current) occurs in LQTS,19 we investigated the effects of LQTS‐CaM mutants on heterologously expressed NaV1.5 as well as on native Na current. We performed heterologous coexpression of WT or mutant CaM with human NaV1.5 in tsA201 cells and then measured electrophysiological properties of the expressed channels. When recording Na current with a nominally Ca2+‐free intracellular solution, we did not observe differences in the level of persistent Na current (expressed as a percentage of peak current) for any of the CaM mutations compared with WT CaM. Consequently, we repeated these experiments with elevated intracellular calcium (~1 μmol/L free Ca2+) to provide a saturating concentration of Ca2+ to promote binding of CaM to NaV1.5.2,17 Furthermore, because of the early age of onset of LQTS in the reported probands, we also tested the effects of CaM mutations on a fetal and neonatal‐expressed NaV1.5 splice variant.14 Cells coexpressing CaM‐D130G with fetal NaV1.5 exhibited 7.5‐fold larger persistent Na current (1.5±0.4%) compared with cells coexpressing the fetal splice variant with WT CaM (0.2±0.1%; P<0.05) when recordings were made with high intracellular calcium (Figure 1A). This greater level of persistent current was not observed when CaM‐D130G was coexpressed with the canonical (adult expressed) splice isoform of NaV1.5 or with low intracellular calcium (Table 1). In contrast, cells coexpressing fetal or canonical NaV1.5 splice isoforms with either CaM‐D96V or CaM‐F142L did not exhibit abnormal levels of persistent Na current under high calcium conditions (Table 1). Other Na channel properties including conductance‐voltage relationship, voltage dependence of steady‐state inactivation, inactivation kinetics, and recovery from inactivation were largely unaffected by CaM mutants (Table 2). Furthermore, expression of CaM‐D130G in mouse FVM did not evoke detectable differences in the level of persistent Na current compared with cells transfected with WT CaM (Figure 1B). Given the inconsistent effect of CaM mutants on heterologous and native Na current, we concluded that NaV1.5 dysfunction was not the major cause of LQTS in the setting of CaM mutations.


Arrhythmogenic calmodulin mutations disrupt intracellular cardiomyocyte Ca2+ regulation by distinct mechanisms.

Yin G, Hassan F, Haroun AR, Murphy LL, Crotti L, Schwartz PJ, George AL, Satin J - J Am Heart Assoc (2014)

Long QT syndrome (LQTS) CaM mutation effects on persistent Na current are limited to a single splice variant of NaV1.5 and a single CaM mutation. A, CaM‐D130G evoked increased persistent sodium current with fetal NaV1.5. Averaged tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 200‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. Summary data are provided in Table 1. B, CaM‐D130G has no significant effect on persistent Na current in fetal ventricular cardiomyocytes. Tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 300‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. CaM indicates calmodulin; WT, wild type.
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fig01: Long QT syndrome (LQTS) CaM mutation effects on persistent Na current are limited to a single splice variant of NaV1.5 and a single CaM mutation. A, CaM‐D130G evoked increased persistent sodium current with fetal NaV1.5. Averaged tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 200‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. Summary data are provided in Table 1. B, CaM‐D130G has no significant effect on persistent Na current in fetal ventricular cardiomyocytes. Tetrodotoxin‐sensitive currents were normalized to the peak current measured at −30 mV during a 300‐ms depolarization recorded under high intracellular Ca2+ concentration (see Methods). The inset represents the same data plotted on an expanded vertical scale. CaM indicates calmodulin; WT, wild type.
Mentions: Because CaM is known to regulate cardiac Na channel inactivation and a specific defect in Na channel inactivation (increased persistent Na current) occurs in LQTS,19 we investigated the effects of LQTS‐CaM mutants on heterologously expressed NaV1.5 as well as on native Na current. We performed heterologous coexpression of WT or mutant CaM with human NaV1.5 in tsA201 cells and then measured electrophysiological properties of the expressed channels. When recording Na current with a nominally Ca2+‐free intracellular solution, we did not observe differences in the level of persistent Na current (expressed as a percentage of peak current) for any of the CaM mutations compared with WT CaM. Consequently, we repeated these experiments with elevated intracellular calcium (~1 μmol/L free Ca2+) to provide a saturating concentration of Ca2+ to promote binding of CaM to NaV1.5.2,17 Furthermore, because of the early age of onset of LQTS in the reported probands, we also tested the effects of CaM mutations on a fetal and neonatal‐expressed NaV1.5 splice variant.14 Cells coexpressing CaM‐D130G with fetal NaV1.5 exhibited 7.5‐fold larger persistent Na current (1.5±0.4%) compared with cells coexpressing the fetal splice variant with WT CaM (0.2±0.1%; P<0.05) when recordings were made with high intracellular calcium (Figure 1A). This greater level of persistent current was not observed when CaM‐D130G was coexpressed with the canonical (adult expressed) splice isoform of NaV1.5 or with low intracellular calcium (Table 1). In contrast, cells coexpressing fetal or canonical NaV1.5 splice isoforms with either CaM‐D96V or CaM‐F142L did not exhibit abnormal levels of persistent Na current under high calcium conditions (Table 1). Other Na channel properties including conductance‐voltage relationship, voltage dependence of steady‐state inactivation, inactivation kinetics, and recovery from inactivation were largely unaffected by CaM mutants (Table 2). Furthermore, expression of CaM‐D130G in mouse FVM did not evoke detectable differences in the level of persistent Na current compared with cells transfected with WT CaM (Figure 1B). Given the inconsistent effect of CaM mutants on heterologous and native Na current, we concluded that NaV1.5 dysfunction was not the major cause of LQTS in the setting of CaM mutations.

Bottom Line: LQTS-CaM mutants do not consistently affect L-type Na current in heterologous cells or native cardiomyocytes, suggesting that the Na channel does not contribute to LQTS pathogenesis in the context of CaM mutations.LQTS-CaM mutants led to loss of Ca2+-transient entrainment with the rank order from greatest to least effect: CaM-D130G~CaM-D96V>CaM-F142L.CaM mutations associated with LQTS may not affect L-type Na+ current but may evoke defective Ca2+-dependent inactivation of L-type Ca2+ current.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Kentucky College of Medicine, Lexington, KY (G.Y., F.H., A.R.H., J.S.).

Show MeSH
Related in: MedlinePlus