O'Brien MP et al. (2023), SMC5 Plays Independent Roles in Congenital Hear...

XB-ART-60520

Int J Mol Sci 2023 Dec 28;251:. doi: 10.3390/ijms25010430.

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SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

O'Brien MP , Pryzhkova MV , Lake EMR , Mandino F , Shen X , Karnik R , Atkins A , Xu MJ , Ji W , Konstantino M , Brueckner M , Ment LR , Khokha MK , Jordan PW .

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Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

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Species referenced: Xenopus tropicalis
Genes referenced: actn1 mip pax6 smc5 trpa1
GO keywords: brain development [+]
gRNAs referenced: smc5 gRNA1 smc5 gRNA2 tpra1 gRNA1 tpra1 gRNA2 tpra1 gRNA3 tpra1 gRNA4

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Phenotypes: Xtr Wt + smc5 CRISPR (Fig. 1 E) [+]

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	Figure 1. SMC5 variant associated with impaired cardiac development in proband and frog knockdown model. (A) Graphic of hypoplastic left heart syndrome showing the small left ventricle (LV), small stenotic mitral valve (MV), aortic valve (AV), and hypoplastic aorta (Ao). (B) Fetal echocardiogram at 19 weeks gestation in an apical 4-chamber view demonstrates reduced left ventricular (LV) size compared to the right ventricle (RV) as well as the larger right atrium (RA) and small left atrium (LA). (C) A fetal echocardiogram at 23 weeks gestation in a view of the aortic arch demonstrates a small aortic arch (Ao) and larger ductus arteriosus (DA) with color Doppler imaging showing minimal blood flow (red) through the hypoplastic aortic arch and a much larger volume of blood flow (blue) across ductus arteriosus. Color scales estimate fluid velocity, with each color representing fluid flow toward (red) or away (blue) from the ultrasound probe located at the top of the image. (D) Postnatal echocardiogram at 28 days old in an apical 4-chamber view demonstrates a large right atrium (RA) and right ventricle (RV) with a thin hypoplastic left ventricle (LV). (E) Cross section of tadpole atria (A) and ventricle (V) of stage 45 live X. tropicalis embryos (n = 50). Scale bars indicating 50 m. p < 0.01, ** p < 0.0001 by t-test in (E).
	Figure 2. Smc5 is required for mouse cardiac development and function. (A) The outline of mESC cardiac differentiation and tamoxifen treatment. (BD) Smc5 cKO in cardiac progenitors causes a significant reduction in cell proliferation. Tamoxifen (TAM) was added on day 6, day 8, and day 10 of mESC differentiation. The cell outgrowth was evaluated on day 16 (B) and day 22 (C,D). Images of live cell cultures are shown for differentiation day 22 (D). Data are a cumulative of two independent experiments performed in replicas. Data was assessed using the MannWhitney t-test and significant comparisons are given as * p < 0.05, p < 0.01, and * p < 0.001, and other comparisons were deemed non-significant. (E,F). Smc5 cKO in cardiac progenitors causes a significant reduction in resulting cardiomyocytes based on the evaluation of sarcomere proteins -actinin (red) and cardiac troponin T (cTn) (green) expression. Tamoxifen (TAM) was added on day 6 and day 8 of mESC differentiation. Cells were evaluated on day 18. The ratio of tamoxifen-treated to untreated cells is shown (n 5 103 total cells). Examples of -actinin (red) (E) and cardiac troponin T (cTn) (green) (F) expression in mESC-derived cardiomyocytes are shown. Scale bar 25 m. Data was assessed using the MannWhitney t-test and significant comparisons were given as *** p < 0.001, and other comparisons were deemed non-significant. (G) Smc5 cKO in cardiac progenitors results in higher variability in beat rate (n = 616). (H) Examples of TAM-treated control (Cont) (Supplementary Movie S1), untreated (Unt) cKO (Supplementary Movie S2), and TAM-treated cKO cardiomyocytes with fast and slow beat rate are shown (Supplementary Movies S3S5) (scale, seconds). (I) Smc5 cKO in cardiac progenitors results in higher variability in beat rhythm (n = 712). (J) An example of TAM-treated cKO cardiomyocytes with short (red line) and long (blue line) intervals between contractions is shown (Supplementary Movie S6) (scale, seconds).
	Figure 3. smc5 knockout alters brain development in frogs and mice (A) Sagittal cross-section of stage 45 X. tropicalis brain in control and smc5 KO (CRISPR exon 1) embryos. Scale bars presenting 100 m. (B) Whole-mount in situ hybridization of pax6 in stage 28 X. tropicalis embryos, highlighting reduced expression in the smc5 KO (CRISPR exon1) brain, notochord, and developing eye. Images show pax6 distribution rather than scale. (C) Brain volume and brain volume normalized by weight in control and Smc5 cKO mice measured by MRI. (D) Brain volume normalized by weight compared between male (M) and female (F) mice, showing all, control, or Smc5 cKO mice. (E) Brain ventricular volume of control or Smc5 cKO mice is shown as raw volume, ventricular volume normalized by brain volume, or ventricular volume normalized by weight. Legend: left (L), right (R), dorsal (D), ventral (V), anterior (A) and posterior (P), * p < 0.05, **** p < 0.0001 by t-test in (A,CE) and by chi-square analysis in (B).
	Figure 4. Smc5 cKO reduces brain volume in most regions except medulla. (A) Percentage of subregion volume compared to total brain volume of control vs smc5 cKO mice. A significant increase in medulla size is noted with Smc5 cKO. (B) The volume of the medullary infracerebellar nucleus is significantly (p = 0.03) enlarged in Smc5 cKO animals as normalized to total brain volume. (C) The medulla is uniformly enlarged across all remaining medulla subregions as demonstrated by medulla volume normalized to total brain volume; however, we see no significant differences between groups. Nuc.: nucleus, * p < 0.05, n = 12 for all experiments.

	Figure 6. Smc5 cKO disrupts somatomotor/somatosensory, cerebellar, and medial forebrain functional connectivity. Connectivity maps use each color in a semicircle to represent a different brain region, with the left and right semicircle representing each respective mouse brain hemisphere. While connections exist between all regions, lines shown between regions of interest (ROI, black arrow) denote a change in connectivity that distinguishes between control and Smc5 cKO mice. Red lines represent an increase in synchrony between ROIs (red text) and blue lines represent a decrease in synchrony between ROIs (blue text). (A) Combined connectivity changes across the brain, with all ROIs shown. (B–D) Connectivity patterns associated with each interrogated ROI (named by black text) are individually shown. (B) Somatosensory and somatomotor connectivity maps, with somatomotor ROI represented by innermost black arrows, and somatosensory ROI represented by outermost black arrows. (C) Cerebellar connectivity map and (D) Medial forebrain bundle system connectivity map. n = 12 for all experiments.
	Figure 7. SMC5 malfunction during embryonic development produces CHD and NDD through independent processes. The data presented indicates that neurodevelopmental defects can occur with and without concurrent CHD. Furthermore, SMC5 mutations can alter brain FC and cause developmental delays.
	Supplementary Figure S1. tpra1 knockout in Xenopus tropicalis is not associated with cardiac defects. CRISPR-Cas9 mediated knockout of tpra1 using sgRNAs targeting using 4 separate non-overlapping target sites (Supplemental Table S2) does not cause notable developmental cardiac defects. ns: nonsignificant, numbers in parentheses indicated n values.
	Supplementary Figure S2. CRISPR-Cas9 complexes targeting smc5 cause insertion/deletions in X. tropicalis genome (A-D) Bar graphs show frequency and size of insertion or deletion (indel) in smc5 induced by CRISPR-Cas9 activity. Pie graphs show type and frequency of indel caused by CRISPR-Cas9. Indel size in multiples of 3 correspond to in frame indels (brown), and samples without indels considered to have no edits. Indels of all other sizes considered out of frame edits (blue). DNA samples without edits not represented on bar graph. (A) CRISPR-exon 1 indel size and frequency, (B) CRISPR-exon 1 edit type and frequency, (C) CRISPR-exon 4 edit type and frequency, (D) CRISPR-exon 4 indel size and frequency. n=5 for all experiments.
	Supplementary Figure S3. Vessel volume was correlated with total brain volume Anatomical data from individual mice are registered to a common space. We use a reference space created from the nonlinear registration, and subsequent averaging, of n=162 mice. This reference space has been registered to an MRI compatible version of the Allen Atlas. This allows us to estimate the volume of different regions of interest (ROI) from the Allen Atlas for each mouse and to impose the Allen Atlas onto the fMRI data for functional connectivity measurements.
	Supplementary Figure S4. Connectivity matrix can be interrogated to assess mouse functional connectivity between and within groups. (A) Edge (region to region correlation) strengths averaged for control (salmon) and cKO (sky blue) mice. There are no group differences the distribution of edge strengths between groups, inter or intra-hemisphere. Kurtosis and skewness were found to show no groupwise differences when compared both inter- and intra-hemisphere. (B) Connectivity between each brain region is shown, compared interhemisphere and intrahemisphere. The connectivity patterns of control mice are shown in the top right half of the matrix, and those of cKO mice are shown in the bottom left half of the matrix. (C) To identify differences between control and cKO connectivity patterns, difference matrices are computed between all pairs of mice. Both within the control and cKO groups, as well as comparisons between groups, all show a weak, but significant, correlation between distinguishing connectivity profiles. (D) We average the difference matrices, threshold the edges, and binarize to generate two networks which distinguish control from cKO mice. The two networks consist of either edges that show higher connectivity strength in control than cKO mice (Positive network), or lower connectivity strength in control than cKO mice (Negative network). n=12 for all experiments.
	Supplementary Figure S5. Smc5 cKO disrupts functional connectivity in all brain regions interrogated. Connectivity maps and corresponding mouse brain representations with respective regions of interest (ROIs) described by black text atop of each section. Lines shown between the ROI and another region represent a significant change in connectivity compared to control mice. Red lines represent increased synchrony and blue lines represent decreased synchrony. List of brain regions above connectivity map summarize regions that show significant differences in connectivity. Volumetric representation of mouse brain from each visual angle, with topmost images showing dorsal (left) and ventral (right) aspects, middle images showing rostral (left) and caudal (right) aspects, and bottom set of images showing left lateral (left) and right lateral (right) aspects of the mouse brain. (A) Combined connectivity changes across the brain, with all ROIs shown. Additional connectivity maps with ROIs in somatosensory and somatomotor regions (B), cerebellum (C), Hippocampus (D), Orbital area (E), Medial forebrain bundle (F), Visual cortex (G), and Retrosplenial region (H). n=12 for all experiments.

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