Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy

Abstract

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.

Fig. 1. Loss of Sun1 extends the longevity of LaminA (Lmna) mutant mice.

A The median postnatal lifespan of the Lmna^F/F/ZP3Cre mice in which a conditional (floxed) Lamin A (Lmna^F/F) is deleted in all tissues is 17.5 days. On a Sun1^–/– background, median longevity is increased to 32.5 days. B When Lmna^F/F is deleted specifically and constitutively in hearts by crossing the mice with the αMyHC-Cre line, the Lmna^F/FːαMyHC-Cre mice have a median lifespan of 26.5 days. On a Sun1^–/– background, these mice live for longer than 6 months. C 3–5-month-old Lmna^F/F mice were crossed with the tamoxifen (Tmx) inducible cardiomyocyte-specific Cre Tg(Myh6-Cre/Esr1) (abbreviated to mcm), after a single injection of Tmx the mice die within 3–4 weeks. On a Sun1^–/– background, these mice (Lmna^F/F:mcm) live for more than 1 year. D Lmna^N195K/N195K animals live for 78 days compared to Lmna^N195K/N195KSun1^–/– which had a median lifespan of 118 days. A–D (****P < 0.0001; **P = 0.0073 Log-rank (Mantel–Cox) test).

Fig. 2. Lifespan and cardiac phenotype of the Lmna^F/F:mcm mice.

A The median lifespan of the Lmna^F/F:mcm mice was 27 days after a single Tmx injection (****P < 0.0001; Log-rank (Mantel–Cox) test). B Lmna^F/F:mcm mice develop kyphosis (red arrow) by 21 days after injection. C PCR detected the floxed (deleted) Lmna gene (red arrowhead) only in whole heart tissue after Tmx induction and neither in other tissues or when Tmx was not injected. Assays were performed once with the number of animals as indicated in the graph. DNA marker in bp (D) LaminA/C levels were quantified by Western analysis of whole heart lysates 21 days after Tmx induction. A significant reduction in A-type Lamin protein was detected. Lamin C levels were not reduced as much in the Lmna^F/F:mcm hearts compared to Lmna^F/F:mcm controls (****P < 0.0001; unpaired two-tailed T-test, data presented as mean ± SD). Quantitative analysis was performed at 21 days post-Tmx induction (right panel). The presence of the LoxP sites in the WT-Lmna gene (Lmna^F/F) results in a reduction in Lmna transcript levels compared to Lmna^Wt/Wt levels. However, this had no overt effect on longevity or postnatal growth/viability. Markers in kDA (E, F) LaminA/C protein, detected by immunofluorescence, was reduced/absent in CM nuclei in both (E) isolated CMs (Scale bar 10 μm) and (F) heart sections (red arrows) with CM nuclei detected by PCM-1 staining, 21 days after Tmx induction. Nuclei are in blue. The images shown are representative of three independently performed experiments. Scale bar 10 μm. Source data are provided as a Source Data file.

Fig. 3. Cardiac function and histology in Lmna^F/F:mcm mice after Cre induction.

A Lmna^F/F:mcm mice show reduced cardiac contractile function at 21 days after Tmx injection. B Lmna^F/F:mcm hearts have reduced EF% and FS%, with increased LVID. Data were analyzed from the total number of animals (N) per genotype as indicated in the graph. (****P < 0.0001; **P = 0.0015; one-way ANOVA, mean ± SD). C Histological analysis revealed increased nucleated cell infiltration and intercellular spaces in Lmna^F/F:mcm hearts (I and ii). Significantly fewer viable (brick-like) CMs were isolated from Lmna^F/F:mcm hearts compared to Lmna^F/F:mcm controls (N = 3 repeats) (iii). The Lmna^F/F:mcm CMs contain large intracellular vacuoles (red arrowhead, iv). Scale bar 100 μm (i), and 50 μm (ii). D In Lmna^F/F:mcm hearts the left ventricular lumen was enlarged (i) with increased fibrosis (**P = 0.0007, blue staining) and apoptotic nuclei (*P = 0.0220; one-way ANOVA; mean ± SD) (ii–iv). Scale bar 500 μm (i), 50 μm (ii). All analyses were performed on hearts isolated 21 days after Tmx induction.

Fig. 4. Sun1 loss in Lmna^F/F:mcm/Sun1^–/– hearts inhibits changes in nuclear morphology and organization caused by Lmna loss.

A Heart extracts from Lmna^F/F:mcm/Sun1^+/+ and Lmna^F/F:mcm/Sun1^−/− confirmed the absence of Sun1 in the Sun1^−/− hearts (red). GAPDH was used as a loading control (green). A representative blot is shown for the number of animals (N) per genotype is indicated below the image. Markers are in kDa (B) Immunofluorescence imaging of CMs isolated from 2-month-old Lmna^F/F:mcm/Sun1^+/+ and Lmna^F/F:mcm/Sun1^−/− mice. Absence of Sun1 results in the redistribution of Nesprin1 to the poles of the nuclei indicated by the yellow arrows. Note that the two nuclei in each cardiomyocyte are positioned closer to each other and are elongated. Images are representative for N = 3 per genotype shown. Hoechst staining is shown in blue, sarcomeric α-actinin is magenta, (scale bar 10 μm). C Immunostaining of CM nuclei in sections through the left heart ventricle 25 days after Cre induction (Tmx). CM nuclei with redistributed or absent Lamin A/C expression are indicated by yellow arrowheads (Ci, Cii). CM nuclei still expressing Lamin A/C are indicated by red arrowheads (Cii, Ciii and iv). Intensity staining and quantification of Lamin A/C levels in nuclei of cardiomyocytes shows a reduction after Cre-induction (***P = 0.0008; ****P < 0.0001; unpaired two-tailed T-test, mean ± SD) (lower panel). Data are analysed of nuclei from Lmna^FF:mcm/Sun1^+/+ Tmx (n = 400), Lmna^FF:mcm/Sun1^+/+ CTL (n = 384), Lmna^FF:mcm/Sun1^−/− Tmx (n = 328) and Lmna^FF:mcm/Sun1^−/− CTL (n = 399). Scale bar 10 μm. D Lmna^F/F:mcm/Sun1^+/+ CM nuclear morphologies are distorted with indentations (red arrowheads) at the nuclear periphery and DAPI intense foci localizing to the tips of the nuclei (yellow arrowheads). In the absence of Sun1, Lmna^F/F:mcm/Sun^−/− CMs show no nuclear indentations or chromatin redistribution. 70% of CM nuclei in Lmna^F/F:mcm/Sun1^+/+ hearts had NE ruptures/distortions or were misshapen compared to less than 1% of CM nuclei in Lmna^F/F:mcm/Sun^−/− Cre-induced mice (lower panel). Data are analysed were of nuclei from 2 animals per genotype Lmna^FF:mcm/Sun1^+/+ Tmx (n = 206), Lmna^FF:mcm/Sun1^+/+ CTL (n = 152), Lmna^FF:mcm/Sun1^−/− Tmx (n = 131) and Lmna^FF:mcm/Sun1^−/− CTL (n = 150), Lmna^Wt/Wt/Sun1^−/− Tmx (n = 144) (No statistical analysis was performed). Source data are provided as a Source Data file.

Fig. 5. Cardiac pathology of Lmna^F/F:mcm/Sun1^−/− hearts after Cre induction.

A Left ventricular enlargement (LV) was apparent in the Cre-induced (TMX) Lmna^F/F:mcm/Sun1^+/+ (n = 3) hearts but not in the LV of the Cre-induced (TMX) Lmna^F/F:mcm/Sun1^−/− (n = 3) hearts. The Lmna^F/F:mcm/Sun1^+/+ induced (TMX) hearts had significantly increased fibrosis (***P = 0.0001; One-way ANOVA), compared to controls, with no significant fibrosis in the Lmna^F/F:mcm/Sun1^−/− hearts compared to controls (i–iii). The following genotypes served as controls: Lmna^FF:mcm/Sun^+/+ (CTL) (n = 3), Lmna^FF:mcmSun1^−/− (CTL) (n = 3), and Lmna^Wt/Wt:mcm/Sun1^−/− induced (TMX) (n = 4). bar 500 μm, 100 μm (i + ii). B Cardiac papillary muscle active force measurements were significantly reduced from the Cre-induced (TMX) Lmna^F/F:mcm/Sun1^+/+ mice (n = 5) compared to Lmna^F/F:mcm/Sun1^+/+ controls (CTL) (n = 5) (unpaired two-tailed T-test) and Cre-induced (TMX) Lmna^F/F:mcm/Sun1^−/− (n = 3) (**P = 0.0047, *P = 0.0113; unpaired two-tailed T-test). The following genotypes served as controls: Lmna^FF:mcm/Sun1^−/− (CTL) (n = 5), Lmna^FFSun1^−/− induced (TMX) (n = 4), and Lmna^Wt/Wt:mcm/Sun1^−/− induced (TMX) (n = 4). Quantification was performed based on five fibers per animals. A, B Data are presented as mean ± SD. C ECGs were performed up to 28 days after Tmx induction on 3–5-month-old mice. ECGs performed before and after Cre induction revealed a progressive worsening of cardiac contractility in Lmna^F/F:mcm/Sun1^+/+ Cre-induced mice (red line) compared to Lmna^F/F:mcm/Sun1^−/− mice. The loss of Sun1 preserved both EF, FS, and Global Longitudinal Strain (GLS) in Lmna^F/F:mcm/Sun1^−/− hearts compared to Lmna^F/F:mcm/Sun1^+/+ hearts. Echo images were recorded at 28 days after Tmx induction. Data were analysed from the total number of animals per genotype (N) as indicated in the graph. Data are presented as mean ± SD.

Fig. 6. Expression of the DNSUN1 miniprotein reduces Nesprin1 localization at the nuclear envelope in cardiomyocytes.

A The AAV9 human/murine SUN1 dominant-negative construct (AAV9-DNhSUN1). The dominant-negative SUN1 sequence encompasses parts of the luminal domain, including the SUN domain. To detect the transgene in tissues, either an HA-tag or a MYC-tag sequence was fused on the NH2 terminus of the murine and human DNSUN1, respectively. ITR = AAV2 inverted terminal repeat; cTnT = Chicken cardiac troponin promotor; intron = β-globin/IgG chimeric intron; Signal sequence = 1–25aa of human serum albumin (Uniprot P02768 signal peptide + propeptide); Tag = HA epitope tag used with the murine DNmSUN1; Myc epitope tag used with human DNhSUN1; SUN1DN = 1046–2404 nt of NM_001130965; KDEL = Golgi-to-ER retrieval sequence; RGB pA = Rabbit globin polyA tail. B The DNmSUN1 miniprotein competes with endogenous SUN1 for binding to the KASH-domain of the Nesprins (which in CMs is Nesprin1). It displaces Nesprin1 from the nuclear envelope, disrupting LINC complex-mediated attachment of the nucleus to the cytoskeleton. C Induced pluripotential stem cell derived CMs were transduced with the AAV AJ-D to express DNmSUN1. The DNmSUN1 miniprotein is not anchored in the INM and so competes with endogenous SUN protein complexes from binding to the KASH domains of Nesprin1 in CMs, so breaking the LINC complex as outlined in (B). It displaces the Nesprin1 from the NEs of IPS-derived cardiomyocytes (Cii and iii). CMs expressing the transduced DNmSUN1 are indicated by the red arrows and show loss of Nesprin1 from the nuclear envelope (NE) (red arrows). In contrast, in CMs not expressing DNmSUN1 or at lower levels, Nesprin1 still localizes to the NE (yellow and white arrows- panel iii). The image shown is representative of 3 independently performed experiments. D The recombined Lmna gene following Cre induction was confirmed by PCR of the heart tissues (upper panel). Robust expression of both AAV9-DNmSun1 and AAV9-GFP protein (Dosage: 5 × 10¹⁰ vg/g per mouse) was detected in extracts from whole hearts 99 days post AAV injection (lower panel). Western blot was repeated twice with N = 4 per AAV9 construct. Markers in kDA. E Isolated CMs from mice transduced with AAV9 delivering DNhSUN1 (DNhSUN1) or controls injected with PBS only (CTL). CMs were immunostained with anti-Nesprin1 and anti-hSUN1. The intensity or levels of Nesprin1 at the NE were reduced by the expressed DNhSUN1 revealing displacement of Nesprin1 protein (scale bar 10 μm). Nesprin1 signal intensity at the NE was quantified and showed a drop in the median levels of Nesprin1 intensity at the nuclear rim to 41.13% in CMs isolated from AAV9-DNhSUN1 transduced mice (****P < 0.0001, unpaired two-tailed T-test, mean ± SD). n = 35 over three independent experiments. Source data are provided as a Source Data file.

Fig. 7. Lmna^F/F:mcm mice transduced with AAV9-DNhSUN1 show improved cardiac function and extended longevity.

A Transduction of AAV9-DNhSUN1 extends the lifespan of the Lmna^F/F:mcm mice after tamoxifen induction. Lmna^F/F:mcm + AAV9-DNhSUN1 transduced mice with the standard dose (2 × 10¹⁰ vg g⁻¹) live for a median of 66 days post-Tmx induction, whereas the Lmna^F/F:mcm + AAV9-GFP transduced mice have a shorter lifespan (36.5 days) (P < 0.0001; Log-rank (Mantel–Cox) test). All animals were induced with Tmx. B Transduction of a double dose (4 × 10¹⁰ VG/g) of AAV9 DNhSUN1 increases the lifespan of Lmna^F/F:mcm animals. Male Lmna^F/F:mcm injected with a double dose of DNhSUN1 live for an average of 205 days and females to 309 days post-Tmx induction, whereas the Lmna^F/F:mcm injected with the standard dose DNhSUN1 have a shorter median lifespan (males 66 days, females 104 days). C Experimental procedure for AAV transduction. Tmx induction by IP injection at postnatal day 14, followed on day 15 in initial experiments by intrathoracic injection of AAV9-DNmSUN1 or AAV9-GFP as control (AAV). In subsequent experiments with the DNhSUN1 delivery was by retro-orbital injection. The expected lifespan of Lmna^F/F:mcm injected with AAV9-GFP was 33 days after Tmx induction. The endpoint of this study is the date of death of Lmna^F/F:mcm animals injected with AAV9-DNhSun1 (DOD). D The DNhSUN1 protein is detected by western blot analysis at 3 weeks after injection and after 1 year following injection at similar levels in whole heart lysates with an antibody specific to the C-terminus of human SUN1. A representative blot is shown for the number of animals (N = 2) per condition. Western blots were performed in triplicate. Markers in kDA (E + F) At 21 days after Tmx induction, extensive fibrosis (blue staining) was detected in Lmna^F/F:mcm mice injected with AAV9-GFP (N = 5) in comparison to Lmna^F/F:mcm animals treated with AAV9-DNhSUN1 (N = 5). The following genotypes served as controls: Lmna^F/F + AAV9-DNhSUN1 (N = 5), Lmna^F/F + AAV9-GFP (N = 4). Data are presented as mean ± SD. (scale bars 500 μm, 40 μm). G ECG analysis of mice injected with a standard AAV-DNhSUN1 dose shows improved cardiac function at day 28 after Tmx induction. H Cardiac function after transduction with AAV9-DNhSUN1 is improved in Lmna^F/F:mcm animals. ECG analysis revealed an significant improvement of Fractional Shortening (FS), Global Longitudinal Strain (GLS) and Ejection Fraction in Lmna^F/F:mcm + AAV9-DNhSun1 animals compared to Lmna^F/F:mcm + AAV9-GFP control animals at day 28 (FS ***p = 0.0009; GLS ***p = 0.0003; EF ****p < 0.0001; One-way ANOVA with Tukey correction). Data were analyzed from the total number of animals (N) per genotype as indicated in the graph. Data are presented as mean ± SD. Source data are provided as a Source Data file.

Fig. 8. Breaking the LINC by disrupting Sun1 protects cardiomyocytes from contraction-induced stress.

A CM nuclei expressing normal Lmna, withstand mechanical stress and tension forces transmitted via the LINC complex from the sarcoplasmic cytoskeleton, predominantly the MT system to the NE. B The loss of or introduction of a mutation within the Lmna gene results in loss/or incorrect assembly of the nuclear lamina, resulting in a compromised and weakened Lamina/NE. The weakened nuclei are damaged due to the tension/stress forces exerted via the MT network and LINC complex from the contractile sarcomeres of the cardiomyocytes. C, D In the absence of SUN1 or by disrupting its binding to the KASH domains by expression of DNSUN1, the now untethered LINC complexes exert less tensional force on the CM nuclei, enabling the survival of the Lmna mutant CMs.