Pedigrees of the SRPK3/TTN myopathy families.

a, Segregation of the familial SRPK3 variants is shown. S indicates the SRPK3 variant and WT indicates the wild-type allele. Individuals presenting with skeletal muscle disease are indicated in black. Mild presentations are shown in gray (corresponding to YIII:4 and YIII:5, two female carriers with skewed X-inactivation, 80:20 and 65:35, in lymphocytes, respectively). b, Extended pedigree details of families M and Z. Individuals presenting with skeletal muscle disease are indicated in black. Cardiac involvement is indicated by gray/dotted symbols. Segregation of the familial SRPK3 (S) and TTN (T) variants is shown. S + T indicates cosegregating SRPK3/TTN variants; WT indicates both SRPK3 and TTN WT alleles. Individuals ZIV:1, ZIV:4, ZIV:6 and ZIV:7 carry the familial TTN variant (p.Arg16905*) previously reported in association with DCM (ref. ¹⁹) but are presymptomatic at ages 52, 44, 40 and 38 years old, respectively. Likewise, individual MIII:2 carries the familial TTN variant (p.Asp28805Metfs*6) but is also presymptomatic at age 46 years old. c, Cosegregation of the SRPK3 and TTN variants (S + T) with the myopathic phenotype (shown in black). All known genotypes are shown; WT, both SRPK3 and TTN WT alleles; empty symbols indicate that the sample was not available for testing (or failed testing). All affected individuals carry the SRPK3 and TTN variants (S + T), whereas their unaffected relatives carry one or the other, but never both. Two females carrying cosegregating SRPK3/TTN variants and showing a skewed X-inactivation pattern are mildly affected (YIII:4 and YIII:5), and those with random X-inactivation are unaffected (TI:2, UI:2, XII:2 and YII:2). A female carrying only the SRPK3 variant (but no TTN variant; ZII:5) and a complete X-inactivation pattern (3:97, in lymphocytes) is unaffected. Individual RI:2, with cosegregating SRPK3 and TTN variants whose fully inactivated chr X carries the SRPK3 deleterious variant, is also unaffected. Individuals RII:3 and SI:2 are noninformative for the CAG repeat analyzed in the X-inactivation assay.

Muscle pathology of the patients with SRPK3/TTN myopathy.

a–h, Examples of muscle histopathology (n = 23). a, Myopathic changes with increased internalized nuclei and fiber size variability (22/23) shown by hematoxylin and eosin (H&E) staining, as seen in patient XIII:1. b,c, Minicores and core-like structures (15/23) shown by NADH histochemistry, as seen in patients BII:1 and WIII:3. d,e, Type I fiber predominance and type I uniformity (19/23) shown by ATPase pH 4.6 and pH 9.2 staining, as seen in patients CII:2 and BII:1, respectively. f, More severe end of the disease spectrum, with vacuoles, necrosis, regeneration and fibrosis shown by H&E in patient YII:3. g,h, EM images confirmed the presence of core structures and revealed Z-line misalignment, accumulation of Z-band material and branching of myofibrils, as seen in patients XIII:1 and WIII:3. Representative images have been obtained as part of the diagnostic workup in accredited pathology laboratories. i, Lower limb MRI T1-weighted images from four patients with SRPK3/TTN myopathy (VII:1, YII:3, MIII:3 and ZIV:1). A pattern of fatty replacement involving the subscapularis muscle in the shoulder girdle was observed. In the pelvic girdle, the gluteus maximus was affected (arrows), but the gluteus minimus and medius muscles were spared even in the advanced stages of the disease. In the thigh, there was a predominant involvement of the hamstring muscles, while the sartorius and gracilis muscles were not involved in the advanced stages of the disease, with the adductor magnus muscle (arrowheads) almost completely spared. In the lower legs, there was predominant involvement of the medial gastrocnemius muscle (arrows) associated with the involvement of the soleus muscle. The peroneus and tibialis anterior muscles were also involved, but only in advanced stages.

Titin immunoanalysis of patients with SRPK3/TTN myopathy.

Muscle biopsy lysates of individuals DI:1, DII:1, LII:1, XII:3, XIII:1 and YII:3 were analyzed using different anti-titin antibodies. a, SRPK3/TTN patients LII:1, YII:3, DII:1, XII:3 and XIII:1 showed a normal pattern of C-terminal titin proteolytic fragments (13, 15 and 18 kDa in size), ruling out a C-terminal titinopathy. b,c, Antibodies against the N-terminal titin (Z1Z2 TTN-1, b) and distal I-band of titin (F146.9B9, c) showed that the full-length titin band is missing or highly reduced in the patients with SRPK3/TTN myopathy (XIII:1, XII:3 and YII:3), but it is present in an unaffected relative TTNtv carrier (DI:1, father of DII:1) and a disease control also carrying a heterozygous TTNtv. This could be attributed to changes in N-terminal protein sequence or structure, or otherwise, protein modifications preventing antibody recognition. d, Coomassie staining also showed the absence or reduction of the high molecular weight band representing the full-length titin protein, whereas the NEB and MyHC bands were normal. Western blots were repeated twice, from the same muscle lysates. Full-length blots are provided as source data. MW, molecular weight; MyHC, myosin heavy chain; NEB, nebulin.

Source data

ttn.1 heterozygosity induces a severe phenotype in homozygous srpk3-null mutant zebrafish larvae.

a–h, Lateral view of Alexa Fluor phalloidin filamentous actin (green) and α-actinin Z-band marker (red) staining in skeletal fast muscle fibers in WT (a,e), srpk3^+/−; ttn.1^+/− (b,f), srpk3^−/−; ttn.1^+/+ (c,g) and srpk3^−/−; ttn.1^+/− larvae (d,h) at 5 dpf. Compared to WT (a,e) or double heterozygotes (srpk3^+/−; ttn.1^+/−; b,f), homozygous srpk3-null alone only causes very mild muscle fiber defects (c,g), while ttn.1 heterozygosity in homozygous srpk3^−/− larvae severely affects muscle fiber integrity (d,h). i–t, Isolated myofiber immunostaining and electron microscopy (EM) in skeletal fast muscle fibers in WT (i,m,q), srpk3^+/+; ttn.1^+/− (j,n,r), srpk3^−/−; ttn.1^+/+ (k,o,s) and srpk3^−/−; ttn.1^+/− (l,p,t) larvae at 5 dpf. Isolated myofiber immunostaining showed that titin expression is largely reduced in the double mutant (srpk3^−/−; ttn.1^+/−; l,p) but not in the single heterozygous ttn.1 mutant (srpk3^+/+; ttn.1^+/−; j,n) or the srpk3-null (srpk3^−/−; ttn.1^+/+; k,o). EM showed that srpk3-null zebrafish (srpk3^−/−; ttn.1^+/+; s) had well-defined sarcomeres, with mildly disorganized myofibrils. The double-mutant fish (srpk3^−/−; ttn.1^+/−; t) displayed pronounced disruption of the sarcomere structure. White scale bars are 25 µm. Black scale bar is 500 nm. Representative images from >15 pooled fish per genotype.

Transcriptome analysis of mutant zebrafish larvae.

a, Number of DE genes between WT and double mutant (top: srpk3^−/−; ttn.1^+/−; n = 794), srpk3-null (middle: srpk3^−/−; ttn.1^+/+; n = 572) and heterozygous ttn.1 (bottom: srpk3^+/+; ttn.1^+/−; n = 128) zebrafish. Upregulated genes are in blue and downregulated genes are in red. b, GO term enrichment analysis. GO term enrichment was done using the topGO package using a one-sided Fisher’s exact test without adjustment for multiple testing. The top enriched GO terms (P < 0.001) for the three comparisons in a ordered by −log₁₀(P). The bars are colored according to the GO domain. Blue indicates BP; orange indicates CC; green indicates MF. c, ClueGO network diagram showing the overlap in enriched GO terms between double mutant (srpk3^−/−; ttn.1^+/−) and srpk3-null (srpk3^−/−; ttn.1^+/+). Nodes represent individual enriched GO terms; edges connect nodes that share annotated genes from the DE genes. Nodes are colored according to the contribution to the enrichment from DE genes from each comparison. Blue indicates >60% DE genes from the srpk3^−/−; ttn.1^+/− comparison; red indicates >60% DE genes from srpk3^−/−; ttn.1^+/+; purple indicates 40–60% from each comparison. BP, biological process; CC, cellular component; MF, molecular function.

SRPK3 phosphorylates RBM20 in vitro.

a, The RBM20_517–664-V5 reporter was transfected into 293T cells with or without GFP-SRPK3. GFP-SRPK3/RBM20_517–664-V5 co-expression resulted in RBM20_517–664-V5 hyperphosphorylation (lanes 4 and 5), as indicated by a mobility shift that was abolished by incubation with lambda phosphatase (P, lane 6). U indicates untreated samples; N indicates control samples incubated without phosphatase. In the absence of the SRPK3 construct, a less pronounced but still noticeable mobility shift can be observed (lanes 1 and 2), consistent with RBM20 phosphorylation by endogenous kinases such as SRPK1, CLK1 or AKT2. Assay was performed in quadruplicate. b, mRNA counts of the zebrafish RBM20 ortholog (BX649294.1 ENSDARG00000092881) are increased in srpk3-null zebrafish (srpk3^−/−; ttn.1^+/+ and srpk3^−/−; ttn.1^+/−), likely as a feedback loop due to the srpk3 deficiency. The box blots represent the first and third quartiles (25% and 75% percentile) with the center line at the median value. The whiskers extend from the hinge to the furthest value not beyond 1.5 times the interquartile range from the hinge. Differential expression was done using a two-sided Wald test with Benjamini–Hochberg adjustment for multiple testing⁶³. For srpk3^−/−; ttn.1^+/+ versus srpk3^+/+; ttn.1^+/+, *P = 0.0379. n = 6 for each condition. Full-length blots are provided as source data.

Source data