Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity

  1. Katja Birker
  2. Shuchao Ge
  3. Natalie J Kirkland
  4. Jeanne L Theis
  5. James Marchant
  6. Zachary C Fogarty
  7. Maria A Missinato
  8. Sreehari Kalvakuri
  9. Paul Grossfeld
  10. Adam J Engler
  11. Karen Ocorr
  12. Timothy J Nelson
  13. Alexandre R Colas
  14. Timothy M Olson
  15. Georg Vogler
  16. Rolf Bodmer

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Abstract

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex’s role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6 . Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 ( goliath , E3 ubiquitin ligase), or SPTBN1 ( β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

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  1. Version published to 10.7554/elife.83385 on eLife
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)): Major comments:

    1. The authors mentioned that the heart dysfunction observed upon CHCHD3/6 KD may be mediated via defects in ATP synthase. Then, how does CHCHD3/6 KD affect ATP synthase? Additionally, OPA1 also affects ATP synthase, why does OPA1 KD just reduce fractional shortening (S.T.2) without reducing F-actin staining?
    • Since MICOS is in involved ETC assembly/sorting in cristae, ATPase subunits may not be assembled correctly and thus causing defects mitochondrial morphology and function, which is further supported by reduced mito-GFP staining (see Fig. 4B&H) (see discussion, lines 438-41 in red; see also lines 434-438 and 441-44 that are unchanged). F-actin staining with OPA1 KD: it may not be a strong enough KD to cause reduced F-actin staining, since only strong CHCHD3/6 KD shows reduced F-actin.
    • To address this issue and ask whether OPA1 interacts with CHCHD3/6 in affecting sarcomeric protein levels, we plan to do an interaction experiment with and OPA1 KD with CHD3/6-C1 KD at 21oC (as in Fig. 6B) and probe for reduced FS; and as well at 25oC and probe for reduced F-actin staining (as with beta-Spectrin KD in Fig. 6D and with Sam50 in Fig. 5D).
    1. It has been reported that CHCHD3 KD in HeLa cells causes fragmented mitochondria, so how does CHCHD3/6 KD caused mitochondrial aggregation? What is the mechanism?
    • Thank you for pointing that out. It is actually more appropriate to call this phenotype “fission-fusion defects”. See lines 279-86, 416.
    • To address this issue further, we propose to do co-KD or co-overexpression (OE) of Drp1 with KD of CHCHD3/6 (as above). Specifically, we will use the strong CHCHD3/6-RNAiA to in conjunction with Drp-1 OE (in the presence of mito-GFP) and see if this can rescue the mitochondrial morphology defects, thus concluding that CHCHD3/6 KD is likely to causes aggregation that is normalized with Drp-1 OE. If this is not the case, but a parallel experiment with Drp-1 KD can rescue, this would support the conclusion that CHCHD3/6 causes increased fragmentation that is counteracted with Drp-1 KD. A complementary experiment would be to use a CHCHD3/6 sensitizer (as in Fig. 5&6). These experiments are intended to address the question whether CHCHD3/6 causes primarily fusion or fission defects.
    1. The ultrastructure of mitochondria (especially aggregated mitochondria) in control and CHCHD3/6 KD heart of drosophila should be analyzed by TEM.
    • That’s difficult, since cardiac tissue is so thin. It is not clear of fission vs fusion defects can easily be distinguished. Instead, we propose to do genetic interaction experiments (see above 2.).

    Reviewer #1 (Significance (Required)): The manuscript partially illustrate the relationship between MICOS complex with Hypoplastic left heart syndrome (HLHS), which is interesting to the reader.

    • We thank the reviewer for his/her appreciation of our study

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): This study performed whole genome sequencing (WGS) on a large cohort of hypoplastic left heart syndrome (HLHS) patients and their families to identify candidate. Nine candidate genes with rare, predicted damaging homozygous variants were identified. Of the candidate HLHS gene homologs tested, cardiac-specific knockdown (KD) of the mitochondrial contact site and cristae organization system (MICOS) complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These heart defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC complex assembly. Analysis of 183 genomes of HLHS patient-parent trios revealed five additional HLHS probands with rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, the authors tested 60 additional prioritized candidate genes in these cases for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (E3 ubiquitin ligase), or SPTBN1 (scaffolding protein) caused synergistic heart defects, suggesting the potential involvement of a diverse set of pathways in HLHS. General Comments: The authors performed an elegant series of experiments that implicate variants of dCHCHD3/6 in HLHS patients as contributing to mitochondrial and sarcomeric defects and contractile function defects. Demonstrating in Drosphilia the functional and biochemical implications of knocking out dCHCHD3/6 provides some potentially important insights into the functional and biochemical implications of dCHCHD3/6 variants in HLHS patients. The data is also complemented by hiPSC-CM studies in which knockdown of CHCHD6 and CHCHD3 showed similar alterations in ATP synthase and mitochondrial morphology. The authors nicely show that knock down of the subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects in the Drosphilia. What is not clear is how these changes mirror the phenotype of HLHS in humans. It would helpful to speculate to a greater extent as to how these changes would manifest as a decreased left ventricular development in HLHS.

    • This is indeed a very important question we comment on in the discussion (see text revision, lines 415-22 in red; see also lines 402-415 and 423-33 that are unchanged). We want to stress that we focus on genetic interactions in heart development, not on convergent endpoint phenotypes between flies and humans. However, our studies do support the idea that mitochondrial defects could contribute to HLHS. We show that MICOS deficiency causes mitochondrial defects manifest in diminished ATP production in addition to diminished sarcomeric actin and myosin causing diminished contractility. Impaired contractility during development has previously been proposed to contribute to defective human cardiac growth (no flow – no growth, Goldberg and Rychik, 2016; Grossfeld et al., 2019), thereby compounding the potentially polygenic effects from damaging gene variants.
    • Why there would a preferential effect on the left ventricle is another interesting question. We speculate that some of the patient-specific variants are in genes preferentially affecting the left ventricle thus preferentially affecting its growth, thus affecting its contractility, then again compounded by impaired blood flow feeding back to diminishing growth. Specific Comments:

    Line 139: Figure 1A does not show echos from the siblings.

    • We apologize that the “(Figure 1A)” was in wrong position (after echocardiograms), causing confusion. We moved it to the previous sentence (line 138). In case the reviewers require that echocardiograms are shown as supplemental data, we can provide these.

    Line155: This table is listed as "Table 1" not Supplemental Table 1.

    • We apologize for mislabeling. This table is now listed as Supplementary Table 1.

    Reviewer #2 (Significance (Required)): This is a highly significant study. The main audience would be pediatric cardiologists and geneticists.

    • We thank the reviewer for his/her appreciation of our study
  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    This study performed whole genome sequencing (WGS) on a large cohort of hypoplastic left heart syndrome (HLHS) patients and their families to identify candidate. Nine candidate genes with rare, predicted damaging homozygous variants were identified. Of the candidate HLHS gene homologs tested, cardiac-specific knockdown (KD) of the mitochondrial contact site and cristae organization system (MICOS) complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These heart defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC complex assembly. Analysis of 183 genomes of HLHS patient-parent trios revealed five additional HLHS probands with rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, the authors tested 60 additional prioritized candidate genes in these cases for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (E3 ubiquitin ligase), or SPTBN1 (scaffolding protein) caused synergistic heart defects, suggesting the potential involvement of a diverse set of pathways in HLHS.

    General Comments:

    The authors performed an elegant series of experiments that implicate variants of dCHCHD3/6 in HLHS patients as contributing to mitochondrial and sarcomeric defects and contractile function defects. Demonstrating in Drosphilia the functional and biochemical implications of knocking out dCHCHD3/6 provides some potentially important insights into the functional and biochemical implications of dCHCHD3/6 variants in HLHS patients. The data is also complemented by hiPSC-CM studies in which knockdown of CHCHD6 and CHCHD3 showed similar alterations in ATP synthase and mitochondrial morphology.

    The authors nicely show that knock down of the subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects in the Drosphilia. What is not clear is how these changes mirror the phenotype of HLHS in humans. It would helpful to speculate to a greater extent as to how these changes would manifest as a decreased left ventricular development in HLHS.

    Specific Comments:

    Line 139: Figure 1A does not show echos from the siblings.

    Line155: This table is listed as "Table 1" not Supplemental Table 1.

    Significance

    This is a highly significant study. The main audience would be pediatric cardiologists and geneticists.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript titled "mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity", Katja Birker et al. reveal that CHCHD3/6 cardiac-specific KD caused reduced contractility and decreased sarcomeric F-Actin and Myosin staining in fly due to impaired ATP synthase. The findings shown in this manuscript are interesting. However, the additional experiments are needed to confirm the conclusion before publication.

    Major comments:

    1. The authors mentioned that the heart dysfunction observed upon CHCHD3/6 KD may be mediated via defects in ATP synthase. Then, how does CHCHD3/6 KD affect ATP synthase? Additionally, OPA1 also affects ATP synthase, why does OPA1 KD just reduce fractional shortening (S.T.2) without reducing F-actin staining?
    2. It has been reported that CHCHD3 KD in HeLa cells causes fragmented mitochondria, so how does CHCHD3/6 KD caused mitochondrial aggregation? What is the mechanism?
    3. The ultrastructure of mitochondria (especially aggregated mitochondria) in control and CHCHD3/6 KD heart of drosophila should be analyzed by TEM.

    Significance

    The manuscript partially illustrate the relationship between MICOS complex with Hypoplastic left heart syndrome (HLHS), which is intertesing to the reader.

  5. Version published to 10.1101/2022.06.13.22276366v1 on medRxiv