Binding of LncRNA-DACH1 to dystrophin impairs the membrane trafficking of Nav1.5 protein and increases ventricular arrhythmia susceptibility

  1. Zhenwei Pan
  2. Gen-Long Xue
  3. Jiming Yang
  4. Yang Zhang
  5. Ying Yang
  6. Ruixin Zhang
  7. Desheng Li
  8. Tao Tian
  9. Xiaofang Zhang
  10. Changzhu Li
  11. Xingda Li
  12. Jiqin Yang
  13. Kewei Shen
  14. Yang Guo
  15. Xuening Liu
  16. Guohui Yang
  17. Yanjie Lu
  18. Baofeng Yang

  • Curated by eLife

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    eLife assessment

    This study presents a valuable contribution to cardiac arrhythmia research by demonstrating long noncoding RNA Dachshund homolog 1 (lncDACH1) tunes sodium channel functional expression and affects cardiac action potential conduction and rhythms. Whereas the evidence for functional impact of lncDACH1 expression on cardiac sodium currents and rhythms is convincing, biochemical experiments addressing the mechanism of changes in sodium channel expression and subcellular localization are incomplete.

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Abstract

Dystrophin is a critical interacting protein of Nav1.5 that determines its membrane anchoring in cardiomyocytes. Long noncoding RNAs (lncRNAs) are involved in the regulation of cardiac ion channels, while their influence on sodium channel remains unexplored. Our preliminary data showed that lncRNA-Dachshund homolog 1 (lncDACH1) can bind to dystrophin, which drove us to investigate if lncDACH1 can regulate sodium channel by interfering with dystrophin. Western blot and immunofluorescent staining showed that cardiomyocyte-specific transgenic overexpression of lncDACH1(lncDACH1-TG) reduced the membrane distribution of dystrophin and Nav1.5 in cardiomyocytes. Meanwhile, peak I Na were reduced in the hearts of lncDACH1-TG mice than wild-type (WT) controls. The opposite data of western blot ,immunofluorescent staining and patch clamp were collected from lncDACH1 cardiomyocyte conditional knockout (lncDACH1-cKO) mice. Moreover, increased ventricular arrhythmia susceptibility was observed in lncDACH1-TG mice in vivo and ex vivo . The conservative fragment of lncDACH1 inhibited membrane distribution of dystrophin and Nav1.5, and promoted the inducibility of ventricular arrhythmia. Strikingly, activation of dystrophin transcription by dCas9-SAM system in lncDACH1-TG mice rescued the impaired membrane distribution of dystrophin and Nav1.5, and prevented the occurrence of ventricular arrhythmia. Furthermore, lncDACH1 was increased in transaortic constriction (TAC) induced failing hearts, which promoted the inducibility of ventricular arrhythmia. And the expression of lncDACH1 is regulated by hydroxyacyl-CoA dehydrogenase subunit beta (hadhb), which binds to lncDACH1 and decreases its stability. The human homologue of lncDACH1 inhibited the membrane distribution of Nav1.5 in human iPS-differentiated cardiomyocytes. The findings provide novel insights into the mechanism of Nav1.5 membrane targeting and the development of ventricular arrhythmias.

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  1. Version published to 10.21203/rs.3.rs-156367/v2 on Research Square
  2. Version published to 10.7554/elife.89690.1 on eLife
  3. eLife

    eLife assessment

    This study presents a valuable contribution to cardiac arrhythmia research by demonstrating long noncoding RNA Dachshund homolog 1 (lncDACH1) tunes sodium channel functional expression and affects cardiac action potential conduction and rhythms. Whereas the evidence for functional impact of lncDACH1 expression on cardiac sodium currents and rhythms is convincing, biochemical experiments addressing the mechanism of changes in sodium channel expression and subcellular localization are incomplete.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:

    In this study, the authors show that a long-non coding RNA lncDACH1 inhibits sodium currents in cardiomyocytes by binding to and altering the localization of dystrophin. The authors use a number of methodologies to demonstrate that lncDACH1 binds to dystrophin and disrupts its localization to the membrane, which in turn downregulates NaV1.5 currents. Knockdown of lncDACH1 upregulates NaV1.5 currents. Furthermore, in heart failure, lncDACH1 is shown to be upregulated which suggests that this mechanism may have pathophysiolgoical relevance.

    Strengths:

    1. This study presents a novel mechanism of Na channel regulation which may be pathophysiologically important.

    2. The experiments are comprehensive and systematically evaluate the physiological importance of lncDACH1.

    Weaknesses:

    1. What is indicated by the cytoplasmic level of NaV1.5, a transmembrane protein? The methods do not provide details regarding how this was determined. Do you authors means NaV1.5 retained in various intracellular organelles?

    2. What is the negative control in Fig. 2b, Fig. 4b, Fig. 6e, Fig. 7c? The maximum current amplitude in these seem quite different. -40 pA/pF in some, -30 pA/pF in others and this value seems to be different than in CMs from WT mice (<-20 pA/pF). Is there an explanation for what causes this variability between experiments and/or increase with transfection of the negative control? This is important since the effect of lncDACH1 is less than 50% reduction and these could fall in the range depending on the amplitude of the negative control.

    3. NaV1.5 staining in Fig. 1E is difficult to visualize and to separate from lncDACH1. Is it possible to pseudocolor differently so that all three channels can be visualized/distinguished more robustly?

    4. The authors use shRNA to knockdown lncDACH1 levels. It would be helpful to have a scrambled ShRNA control.

    5. Is there any measurement on the baseline levels of LncDACH1 in wild-type mice? It seems quite low and yet is a substantial increase in NaV1.5 currents upon knocking down LncDACH1. By comparison, the level of LncDACH1 seems to be massively upregulated in TAC models. Have the authors measured NaV1.5 currents in these cells? Furthermore, does LncDACH1 knockdown evoke a larger increase in NaV1.5 currents?

    6. What do error bars denote in all bar graphs, and also in the current voltage relationships?

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript by Xue et al. describes the effects of a long noncoding RNA, lncDACH1, on the localization of Nav channel expression, the magnitude of INa, and arrhythmia susceptibility in the mouse heart. Because lncDACH1 was previously reported to bind and disrupt membrane expression of dystrophin, which in turn is required for proper Nav1.5 localization, much of the findings are inferred through the lens of dystrophin alterations.

    The results report that cardiomyocyte-specific transgenic overexpression of lncDACH1 reduces INa in isolated cardiomyocytes; measurements in whole heart show a corresponding reduction in conduction velocity and enhanced susceptibility to arrhythmia. The effect on INa was confirmed in isolated WT mouse cardiomyocytes infected with a lncDACH1 adenoviral construct. Importantly, reducing lncDACH1 expression via either a cardiomyocyte-specific knockout or using shRNA had the opposite effect: INa was increased in isolated cells, as was conduction velocity in heart. Experiments were also conducted with a fragment of lnDACH1 identified by its conservation with other mammalian species. Overexpression of this fragment resulted in reduced INa and greater proarrhythmic behavior. Alteration of expression was confirmed by qPCR.

    The mechanism by which lnDACH1 exerts its effects on INa was explored by measuring protein levels from cell fractions and immunofluorescence localization in cells. In general, overexpression was reported to reduce Nav1.5 and dystrophin levels and knockout or knockdown increased them.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:

    In this manuscript, the authors report the first evidence of Nav1.5 regulation by a long noncoding RNA, LncRNA-DACH1, and suggest its implication in the reduction in sodium current observed in heart failure. Since no direct interaction is observed between Nav1.5 and the LncRNA, they propose that the regulation is via dystrophin and targeting of Nav1.5 to the plasma membrane.

    Strengths:

    1. First evidence of Nav1.5 regulation by a long noncoding RNA.
    2. Implication of LncRNA-DACH1 in heart failure and mechanisms of arrhythmias.
    3. Demonstration of LncRNA-DACH1 binding to dystrophin.
    4. Potential rescuing of dystrophin and Nav1.5 strategy.

    Weaknesses:

    1. Main concern is that the authors do not provide evidence of how LncRNA-DACH1 regulates Nav1.5 protein level. The decrease in total Nav1.5 protein by about 50% seems to be the main consequence of the LncRNA on Nav1.5, but no mechanistic information is provided as to how this occurs.
    2. The fact that the total Nav1.5 protein is reduced by 50% which is similar to the reduction in the membrane reduction questions the main conclusion of the authors implicating dystrophin in the reduced Nav1.5 targeting. The reduction in membrane Nav1.5 could simply be due to the reduction in total protein.

  7. Version published to 10.21203/rs.3.rs-156367/v1 on Research Square