Voltage-Gated Sodium Channels Are Required for Heart Development in Zebrafish

Q: What is the clinical evidence from this study?

Population: Heart development (cardiogenesis) in zebrafish embryos. Intervention: Antisense morpholino knockdown of cardiac sodium channel genes (scn5Laa and scn5Lab) and pharmacological blockade/activation of sodium or calcium currents (TTX, ATX II, veratridine, nisoldipine) vs. 5-nucleotide mismatch control morpholinos, vehicle-treated embryos, and zerg potassium channel knockdown. Primary outcome: Cardiac chamber morphogenesis, looping, cardiomyocyte progenitor number, and cardiogenic gene expression (nkx2.5, gata4, hand2).

Q: What does this research mean for the field?

Voltage-gated sodium channels are required for early cardiomyocyte progenitor specification and cardiac chamber morphogenesis through a non-electrogenic mechanism. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.ESTABLISHES_NEW_DIRECTION.

Sameer Chopra; Dina Myers Stroud; Hiroshi Watanabe; Jeffrey S. Bennett; C. Geoffrey Burns; K. Sam Wells; Tao Yang; Tao Zhong; Dan M. Roden

doi:10.1161/circresaha.109.213132

March 26, 2010Circulation ResearchOpen Access

Voltage-Gated Sodium Channels Are Required for Heart Development in Zebrafish

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Key result

Sodium channel knockdown reduces cardiomyocytes by ~40% and impairs chamber morphogenesis via non-electrogenic signaling.

Why the study?

Does knockdown or pharmacological blockade of voltage-gated cardiac sodium channels (scn5Laa and scn5Lab) impair early heart development and cardiomyocyte progenitor specification in zebrafish embryos?

Key Points

To investigate the mechanism and role of cardiac voltage-gated sodium channels in vertebrate heart development using zebrafish models.
Targeted the conserved Scn5a homologs scn5Laa and scn5Lab in zebrafish embryos using translation- and splice-blocking morpholino antisense oligonucleotides, with and without concomitant p53 knockdown.
Assessed cardiac progenitor markers (nkx2.5, gata4, hand2) and chamber morphology in transgenic reporter lines Tg(cmlc2:GFP) and Tg(cmlc2:DsRed2-nuc).
Tested electrophysiological requirements by exposing embryos to pharmacological channel modulators (tetrodotoxin, ATX II, veratridine, 10µM nisoldipine) and sodium-free growth medium.
Antisense knockdown of scn5Laa or scn5Lab resulted in dysmorphic cardiac chambers, impaired looping, reduced anterior lateral plate mesoderm marker expression, and up to 40% fewer cardiomyocytes in dual morphants at 60–62hpf.
Embryos reared in 10µM nisoldipine failed to initiate heartbeats through 78hpf but preserved normal cardiac morphogenesis and cardiomyocyte numbers through 60hpf.
Direct sodium current blockade via tetrodotoxin (causing neuromuscular paralysis by ~28hpf) or sodium-free medium failed to phenocopy channel knockdown defects, indicating channel-dependent cardiogenesis is nonelectrogenic.

Structured PICO

Does knockdown or pharmacological blockade of voltage-gated cardiac sodium channels (scn5Laa and scn5Lab) impair early heart development and cardiomyocyte progenitor specification in zebrafish embryos?

P

Population

Zebrafish embryos (strains AB, TuAB, Tg(cmlc2:GFP), Tg(cmlc2:DsRed2-nuc)) evaluated from gastrulation through 104 hours post fertilization to study the role of cardiac sodium channels in heart development.

I

Intervention

Antisense morpholino oligonucleotide knockdown (translation- and splice-blocking) targeting cardiac voltage-gated sodium channel orthologs scn5Laa and scn5Lab (alone, together, or co-injected with p53 morpholino), as well as pharmacological channel manipulation (tetrodotoxin [TTX], ATX II, veratridine, nisoldipine 10 µM, or sodium-free media) delivered via bath exposure or microinjection.

C

Comparator

5-nucleotide mismatch control morpholinos, vehicle-treated control embryos, and cardiac potassium channel (zerg) morpholino knockdown.

O

Outcome

Cardiac chamber morphogenesis, cardiac looping, ventricular trabeculation, total cardiomyocyte number (at 22-somite stage and 60-62 hpf), and expression of early myocardial precursor transcription factors (nkx2.5, gata4, hand2) and sarcomeric genes (cmlc2, vmhc).surrogate

This preclinical study demonstrates that cardiac voltage-gated sodium channels are essential for early heart development and cardiomyocyte progenitor specification in zebrafish through a novel, non-electrogenic mechanism independent of membrane depolarization.

Limitations

Cannot exclude the possibility that intracellular sodium conductance on organelle membranes is required for normal development
Rescue of morphant phenotypes with full-length scn5Laa or scn5Lab mRNA was not possible due to inadequate translation of the 6kb transcripts
Cannot exclude the possibility that scn5Laa and scn5Lab subserve different functions not uncovered by the assays or that manipulating one gene altered the expression or function of the other
Cannot exclude the possibility that intracellular sodium conductance (e.g., channels acting on organelle membranes) is required for normal development
Rescue of morphant phenotypes with full-length scn5Laa or scn5Lab mRNA was not possible, likely due to inadequate translation of the 6kb transcripts
The two genes (scn5Laa and scn5Lab) may subserve different functions during heart development not uncovered by the assays, or manipulating one gene may have altered the expression or function of the other

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Implication

Raises caution for developmental effects of sodium channel modulation; leaves open whether non-electrogenic mechanisms apply in human cardiogenesis.

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.

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Cite This Study

Chopra et al. (2010) studied Heart development (cardiogenesis) in zebrafish embryos. Antisense morpholino knockdown of cardiac sodium channel genes (scn5Laa and scn5Lab) and pharmacological blockade/activation of sodium or calcium currents (TTX, ATX II, veratridine, nisoldipine) vs. 5-nucleotide mismatch control morpholinos, vehicle-treated embryos, and zerg potassium channel knockdown was evaluated on Cardiac chamber morphogenesis, looping, cardiomyocyte progenitor number, and cardiogenic gene expression (nkx2.5, gata4, hand2).

synapsesocial.com/papers/6a7d9211cf7e8b32dacb4b9a https://doi.org/10.1161/circresaha.109.213132

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Voltage-Gated Sodium Channels Are Required for Heart Development in Zebrafish

Key Points

Structured PICO

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Voltage-Gated Sodium Channels Are Required for Heart Development in Zebrafish

Key Points

Structured PICO

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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