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.