Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion

Fig. 1. Cardiac phenotype of KI/+ and KI/KI mice in comparison to WT mice. A – Characterisation of cardiac dimensions and function by echocardiography: Fractional shortening and end-diastolic dimensions are shown. For cohort characteristics and a wider set of echocardiographic parameters please refer to Table S1. B – Heart weight is increased in KI/KI mice, values are normalised to tibial length. ****p < 0.0001 versus WT. C – Invasive haemodynamic assessment of LV performance of the three genotypes: dP/dtmax, dP/dtmin and relaxation constant Tau are shown at baseline conditions, and under adrenergic stress (dobutamine infusion at 4 ng g−1 BW min−1 and 16 ng g−1 BW min−1). Where error bars are missing, they are smaller than the symbols. For cohort characteristics and a wider set of parameters refer to Table S2.

Fig. 2. Molecular phenotype of KI/+ and KI/KI hearts. A, B – Transcriptome profiling by RNAseq: A – KEGG Pathway analysis: The top five statistically (p < 0.001) enriched GO terms for KI/KI hearts when compared to WT hearts are shown. B – Heat map shows genes in the KEGG HCM gene set with the strongest up-regulation in the KI/KI hearts at the top and the genes showing the strongest down-regulation at the bottom. Colours range from dark red to dark blue representing respectively the highest and lowest expression of a gene. C – Assessment of transcriptional changes by qPCR for genes related to the fetal gene programme (top), hypertrophic signalling (middle) and fibrosis (bottom). All measurements are normalised to Gapdh; significant changes are observed in the hearts of KI/KI mice; *p < 0.05, ***p < 0.001, ****p < 0.0001 versus WT, n = 6 per group. D – Changes in hypertrophic signalling proteins assessed by Western blotting: Fhl1, Carp (Ankrd1) and Dscr1 (Rcan1) are induced in KI/KI hearts, in line with the activation of hypertrophic signalling pathways. Anti-hypertrophic Fhl2 is down-regulated in these hearts (for quantification see Fig. S5B). Gapdh serves as loading control. The position…

Fig. 3. MLP depletion in KI/KI hearts. A – Measurement of Csrp3 transcript by qPCR (left) and MLP protein (right) in the three groups, normalised to Gapdh transcript and protein, respectively. In the KI/KI mice, Csrp3 transcript is 2.5-fold up-regulated. The abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. MLP level is down-regulated to 50% in KI/+ mice and to 20% in KI/KI mice. ***p < 0.001, ****p < 0.0001 versus WT, n = 6 per group. B – Representative Western blot demonstrating reduced MLP levels in KI/+ and KI/KI mice. Gapdh serves as loading control. The position of marker proteins is indicated (molecular weight in kDa). N.B., both TaqMan probe target site and epitope of the anti-MLP antibody 79D2 are not overlapping with the site of the C58G mutation. C – Immuno-fluorescence on isolated adult cardiomyocytes from mice of the three genotype groups. Cells are stained for MLP (left), titin M-band epitope m8 (middle) and merged images are shown on the right (MLP red, titin m8 green). Scale bar represents 20 μm. Insert: Magnified area, scale bar represents 5 μm.

Fig. 4. The Csrp3 C58G allele cannot rescue the phenotype of Csrp3-/- knock out mice. A – Representative examples of M-mode echocardiography of Csrp3+/- mice with normal systolic function (top), Csrp3 KI/- mice (middle) and Csrp3-/- mice (bottom), the latter both with impaired systolic function. The calculated value of LV fractional shortening (FS) is given for each mouse. B – Cardiac morphology of the genotypes. Haematoxylin-eosin stained paraffin heart sections are shown.

Fig. 5. Protein depletion is a hallmark of HCM-associated MLP mutations. A – Recombinant HA-tagged MLP WT, HCM-associated MLP mutants (L44P, S44R/E54G, C58G), a DCM-associated MLP mutant (K69R) and empty parental vector were expressed in HEK cells, by transfection with a bicistronic vector system (see Section 1.6 of Material and Methods in Appendix A Supplementary data). Cell lysates were blotted for MLP (using either anti-HA antibody against the N-terminal tag, light grey, or anti-MLP antibody 79D2 against the C-terminus of the protein, dark grey) and hrGFP. In addition, CSRP3 and hrGFP transcripts were measured by qPCR in parallel transfected cells (black bars). The ratios of CSRP3 transcript or MLP protein to hrGFP were set to 100% for WT transfections and all other samples expressed relative to WT, n = 3 per group. Note that all HCM-associated mutants are affected by protein depletion, while the DCM-associated mutation is not affected. B – NRC were transduced with adenoviral particles coding for MLP WT, L44P, C58G and empty vector (see A) and cell lysates were blotted for MLP (using antibody 79D2) and hrGFP. The HA-tag on the recombinant MLP allows separation of this protein from…

Fig. 6. HCM-associated MLP mutant proteins are subjected to depletion by proteasomal pathways: A – NRC were transduced with adenoviral particles as in Fig. 5B and treated with proteasomal inhibitors MG-115 and MG-132. Cell lysates were blotted for HA, detecting the recombinant MLP WT, L44P and C58G. Pan-actin served as loading control and blotting for ubiquitin confirmed efficient inhibition of the proteasome by accumulation of ubiquitinated proteins. In control (ctr, DMSO-treated) cells, MLP L44P and C58G are destabilised, however they are restored to WT protein levels upon proteasomal inhibition. B – TUBE assay: Ubiquitinated proteins were pulled down from Csrp3 KI/KI heart lysate (treated in vivo with proteasomal inhibitor MG-262 1 μmol/kg BW for 20 h) using immobilised tandem ubiquitin-binding entities (TUBE). Agarose matrix without TUBE served as control (ctr). Lysate controls are shown (LC, 1% of input). Blotting for ubiquitin (ubi) indicates the enrichment of ubiquitinated proteins in the TUBE pulldown. Blotting for MLP shows specific pulldown of MLP with TUBE, indicating ubiquitination of the protein. Please note the higher molecular weight (>20 kDa) than normal endogenous MLP (<…

Fig. 7. Proteotoxic stress response in the KI/KI mice: A – Co-immunoprecipitation of MLP and Bag3 from KI/KI heart lysate (see Fig. 6B). MLP C58G was precipitated using anti-MLP 79D2 antibody (IP) or isotype antibody control (ctr). Lysate controls are shown as detection controls (LC, 1% of input); * indicates signal from antibody chains. Bag3 co-precipitates with MLP C58G, evidencing a complex formation between the endogenous proteins. The position of marker proteins is indicated (molecular weight in kDa). B – In the KI/KI mice with proteasomal overload, the protein quality control complex Bag3 - Hsp70 – Hsc70 is induced and small heat shock proteins Hsp27, HspB7 and αβ-crystallin (ABcryst) are upregulated. Please note, the blots for MLP and loading control Gapdh on the same membrane are already shown in Fig. 3B. The position of marker proteins is indicated (molecular weight in kDa). C – Left: Assessment of transcriptional changes by qPCR for genes related to protein quality control. All measurements are normalised to Gapdh (n = 6 per group); significant changes are observed in the hearts of KI/KI mice for all transcripts apart from Hsc70 and Hsp70. Right: Quantification of protein…