pubmed.ncbi.nlm.nih.gov

Voltage-gated sodium channels are required for heart development in zebrafish - PubMed

  • ️Fri Jan 01 2010

Voltage-gated sodium channels are required for heart development in zebrafish

Sameer S Chopra et al. Circ Res. 2010.

Abstract

Rationale: Voltage-gated sodium channels initiate action potentials in excitable tissues. Mice in which Scn5A (the predominant sodium channel gene in heart) has been knocked out die early in development with cardiac malformations by mechanisms which have yet to be determined.

Objective: Here we addressed this question by investigating the role of cardiac sodium channels in zebrafish heart development.

Methods and results: Transcripts of the functionally-conserved Scn5a homologs scn5Laa and scn5Lab were detected in the gastrulating zebrafish embryo and subsequently in the embryonic myocardium. Antisense knockdown of either channel resulted in marked cardiac chamber dysmorphogenesis and perturbed looping. These abnormalities were associated with decreased expression of the myocardial precursor genes nkx2.5, gata4, and hand2 in anterior lateral mesoderm and significant deficits in the production of cardiomyocyte progenitors. These early defects did not appear to result from altered membrane electrophysiology, as prolonged pharmacological blockade of sodium current failed to phenocopy channel knockdown. Moreover, embryos grown in calcium channel blocker-containing medium had hearts that did not beat but developed normally.

Conclusions: These findings identify a novel and possibly nonelectrogenic role for cardiac sodium channels in heart development.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1. Function and developmental expression of zebrafish cardiac-type sodium channels

A, B. Whole cell sodium currents (A), and current-voltage relationships in CHO cells transfected with full-length scn5Laa and scn5Lab (B). C. Temporal expression of scn5Laa and scn5Lab in early embryos (0–12hpf) by RT-PCR. YPC = yoke plug closure stage, s = somites. D, E. By whole-mount in situ hybridization, sodium channel gene expression was detected diffusely in both heart chambers before displaying predominately ventricular expression by 104hpf. Transcripts were also detected in the brain and spinal cord. Developmental stages are as labeled. Arrow, heart. Arrowhead, spinal cord. A, atrium. V, ventricle.

FIGURE 2
FIGURE 2. Zebrafish scn5Laa and scn5Lab are each required for normal cardiac development

A. Embryos at 58–62hpf following treatment with active or control antisense morpholinos. All embryos were also treated with the p53 morpholino. Injection of p53 morpholino alone did not cause any identifiable phenotype (Online Figure VII). Note that head size is reduced in all sodium channel morphants and that many embryos injected with AB_MO1 display a yoke sac extension abnormality (arrowhead). All scale bars = 500µM. B. Cardiac developmental defects in morphant Tg(cmlc2:GFP) embryos. AA_MO1 and AB_MO2 caused the most severe cardiac phenotypes at the lowest doses. Knockdown of both scn5Laa and scn5Lab resulted in cardiac defects that were more severe than knockdown of either sodium channel alone, particularly with respect to ventricular morphogenesis. Arrows = ventricle. Arrowheads = atrium. Scale bar = 150µM. Treatments are as labeled: STD_MO = standard control morpholino, zerg_MO = cardiac potassium channel morpholino, AA_MIS1 = scn5Laa 5-mismatch control morpholino, AA_MO1 = scn5Laa translation-blocking morpholino (ATG initiation site), AA_MO2 = second scn5Laa translation-blocking morpholino (5’UTR), AB_MIS1 = scn5Lab 5-mismatch control morpholino, AB_MO1 = scn5Lab splice-blocking morpholino (E6I6), AB_MO2 = second scn5Lab splice-blocking morpholino (E25I25).

FIGURE 3
FIGURE 3. Sodium channel morphant embryos have reduced numbers of embryonic cardiomyocytes

A–H. Confocal reconstructions of hearts of control Tg(cmlc2:GFP) embryos (A, C) or clutchmates injected with the scn5Laa translation inhibitor morpholino (AA_MO1) (B, D) illustrate that sodium channels are required for normal numbers of embryonic cardiomyocytes. Arrows, ventricle and arrowheads, atrium at 58hpf (A, B) and 104hpf (C, D), respectively. Higher magnification of ventricular and atrial chambers presented in A’, B’ and A”, B”, respectively, illustrate similar chamber-specific cellular morphology in both control (A’, A”) and morphant (B’, B”) embryos. Scale bars = 50µm (A, B, C, D) and 20µm (A’, A”, B’, B”). E, F. Serial confocal sections through the ventricular chamber of control embryos revealed trabeculation by 104hpf (arrows in F). Scale bar = 20µm. G, H. At the same timepoint, scn5Laa morphant ventricles remained a single layer (shown are ventricles from two different morphant embryos). Scale bars = 20µm. I. Confocal reconstructions of the embryonic heart in Tg(cmlc2:DsRed2-nuc) embryos permitted quantification of deficits of embryonic cardiomyocytes in morphant embryos at 60–62hpf. Scn5Laa and scn5Lab morphant hearts have significantly fewer embryonic cardiomyocytes than those of mismatch control morpholino-injected clutchmates and embryos injected with a morpholino targeting the zerg cardiac potassium channel. Results are mean ± s.e.m. (N) in bar graph = number of hearts analyzed. *, P<0.01 versus AA_MIS1 and †, P<0.01 versus AB_MIS1, ANOVA. Embryos injected with both AA_MO1 and AB_MO2 morpholinos (scn5Laa, scn5Lab double knockdown) had fewer cardiomyocytes than embryos injected with either AA_MO1 or AB_MO2 alone (177±8 versus 228±14 and 226±12, respectively) but the results were not statistically significant following ANOVA of all 7 groups. Where indicated, p53 morpholino was co-injected with active morpholinos as a control for non-specific morpholino toxicity. Photos are representative wild type, scn5Laa control, and scn5Laa morphant embryo hearts at 78hpf. Arrows, ventricle. Arrowheads, atrium. Scale bars = 50µm.

FIGURE 4
FIGURE 4. Sodium channel knockdown perturbs the expression of cardiac precursor genes in anterior lateral mesoderm

A. Analysis of gene expression in the early heart-forming region of anterior lateral plate mesoderm at the 6-somite stage by in situ hybridization. Embryos injected with scn5Laa translation inhibitor morpholino (AA_MO1) had reduced expression of nkx2.5 gata4, and hand2 but no change in gata5 expression compared to embryos injected with 5-mismatch control morpholino (AA_MIS1). All embryos shown are from the same injected clutch. Scale bar = 200µm. B. Real-time quantitative RT-PCR performed using independent clutches of embryos also revealed significantly reduced nkx2.5 expression but no change in gata5 expression following injection of AA_MO1 compared to injection of AA_MIS1 control morpholino. Results are mean ± s.e.m for three independent experiments, each performed in triplicate. P<0.05 for nkx2.5, T-test. C. Quantification of numbers (n) of scn5Laa morphant embryos displaying absent, reduced or normal (comparable to wild type) expression levels of cardiac precursor genes as assessed by in situ hybridization. D. Knockdown of scn5Lab also resulted in markedly-reduced expression of nkx2.5 and gata4 at 6 somites. However, no significant change in the expression of hand2 was observed. E. Sodium channel morpholinos reduce nkx2.5 expression in a dose-dependent manner as shown by RT-PCR (AA_MO1) and in situ hybridization at 6 somites (AB_MO2).

FIGURE 5
FIGURE 5. Sodium channel knockdown results in reduced numbers of differentiating cardiomyocytes

A. By in situ hybridization, scn5Laa and scn5Lab morphant embryos at the 16 somite stage display markedly reduced expression of nkx2.5 and of the myocardial sarcomeric genes cmlc2 and vmhc compared to control embryos. Treatment groups are as labeled. The number of embryos displaying the phenotype/the total number of embryos assessed is indicated in the lower right corner of each panel. B. Deficiencies in cardiac differentiation are not due to developmental delay. Shown is an embryo injected with the AB_MO2 morpholino compared to an uninjected clutchmate. Despite dramatic differences in the size of the heart field and head, both embryos have equivalent numbers of somites. C. Confocal reconstructions of the developing heart cone in Tg(cmlc2:GFP) embryos at the 22-somite stage (embryo in top panel is shown for orientation). Scale bar = 50µm. Scn5Laa morphant heart cones (AA_MO1, n=4) have significantly fewer differentiating cardiomyocytes than the heart cones of wild type (WT, n=6) and control-injected (AA_MIS1, n=3) clutchmates. Results are mean ± s.e.m. *, P<0.01 versus wild-type and †, P<0.01 versus AA_MIS1-injected, ANOVA.

FIGURE 6
FIGURE 6. Voltage-gated sodium channels may regulate early cardiac development independent of membrane electrophysiology

A. Rearing of embryos in 10µM solution of the L-type calcium blocker nisoldipine inhibited heart-beating at all stages examined through 78hpf but did not perturb early chamber formation or the initiation of looping. B. Analysis of vehicle-treated and nisoldipine-treated Tg(cmlc2:DsRed2-nuc) embryos indicated that L-type calcium channel-blockade did not affect embryonic cardiomyocyte number through 78hpf. Results are mean ± s.e.m for vehicle-treated (n = 2) and nisoldipine-treated (n = 3) embryos, respectively. C. Prolonged exposure of developing embryos to pharmacological modulators of sodium channel function failed to disrupt either cardiac specification or differentiation. In situ hybridization for nkx2.5 at the 6–8 somite stage (top) and fluorescent microscopy of the heart tubes of Tg(cmlc2:GFP) embryos at 26–28hpf (bottom), after the indicated treatments (see Table 2). TTX = tetrodotoxin, ATX II = anemone toxin II. Scale bars = 200µm (top) and 100µm (bottom). D. In situ hybridization for nkx2.5 at the 6 somite stage after incubation from the 1 cell stage in sodium-free media, to inhibit inward sodium current (INa). Early heart development proceeded normally. Heart tubes also form normally (not shown). Scale bar = 500µm.

Similar articles

Cited by

References

    1. Yu FH, Catterall WA. Overview of the voltage-gated sodium channel family. Genome Biol. 2003;4:207. - PMC - PubMed
    1. Goldin AL. Resurgence of sodium channel research. Annu Rev Physiol. 2001;63:871–894. - PubMed
    1. Catterall WA, Goldin AL, Waxman SG. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev. 2005;57:397–409. - PubMed
    1. Roden DM, Balser JR, George AL, Jr, Anderson ME. Cardiac ion channels. Annu Rev Physiol. 2002;64:431–475. 431–75. - PubMed
    1. George AL., Jr Inherited disorders of voltage-gated sodium channels. J Clin Invest. 2005;115:1990–1999. - PMC - PubMed

MeSH terms

Substances