Gain-of-function mutation in ubiquitin ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues
Mathilde C.S.C. Vermeer, Maria C. Bolling, Jacqueline M. Bliley, Karla F. Arevalo Gomez, Mario G. Pavez-Giani, Duco Kramer, Pedro H. Romero-Herrera, B. Daan Westenbrink, Gilles F.H. Diercks, Maarten P. van den Berg, Adam W. Feinberg, Herman H.W. Silljé, Peter van der Meer
Mathilde C.S.C. Vermeer, Maria C. Bolling, Jacqueline M. Bliley, Karla F. Arevalo Gomez, Mario G. Pavez-Giani, Duco Kramer, Pedro H. Romero-Herrera, B. Daan Westenbrink, Gilles F.H. Diercks, Maarten P. van den Berg, Adam W. Feinberg, Herman H.W. Silljé, Peter van der Meer
View: Text | PDF
Research Article Cardiology

Gain-of-function mutation in ubiquitin ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues

Abstract

The start codon c.1A>G mutation in KLHL24, encoding ubiquitin ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation and ultimately causing epidermolysis bullosa simplex (EBS). The majority of patients with EBS are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homolog of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human-induced pluripotent stem cell–derived (hiPSC-derived) cardiomyocytes from 2 patients and 3 nonfamilial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients’ explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values, and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, which can be prevented by restoring desmin protein levels.

Authors

Mathilde C.S.C. Vermeer, Maria C. Bolling, Jacqueline M. Bliley, Karla F. Arevalo Gomez, Mario G. Pavez-Giani, Duco Kramer, Pedro H. Romero-Herrera, B. Daan Westenbrink, Gilles F.H. Diercks, Maarten P. van den Berg, Adam W. Feinberg, Herman H.W. Silljé, Peter van der Meer

×

Figure 2

Analysis of ex vivo heart and morphology of in vitro patient-derived dyn-EHTs.

Options: View larger image (or click on image) Download as PowerPoint
Analysis of ex vivo heart and morphology of in vitro patient-derived dyn...
(A) IFA labeling of desmin in paraffin sections of the explanted heart of patient II:3 with DCM (KLHL24-ΔN28) versus sections of the heart from a patient with idiopathic DCM (named as WT, KLHL24). Scale bars: 50 μm. Panel A is part of a larger panel depicted in Supplemental Figure 1. (B) Co-IFA of KLHL24 with desmin and desmoplakin in control hiPSC-CMs cultured in 2D. The arrows point to the colocalized areas. Scale bars: 10 μm. (C) Side view of contractile EHTs (day 14) right after pulling tissues out of the PDMS molds and after 14 days of dynamic loading dyn-EHTs (day 28). Pictures are representative of all control tissues (KLHL24), derived of 3 control individuals in the upper panels and both patients in the mid and lower panels (KLHL24-ΔN28). Strips are in white and the images are an overlay of shots taken at diastole (red) and systole (green). Scale bars: 1.5 mm. (D) Significant differences in morphological parameters between control- and patient-derived tissues imaged at day 14 (EHTs) and day 28 (dyn-EHTs). n = 11 (d14) and n = 20 (d28) for control-derived tissues; n = 13 (d28) and n = 23 (d14) patient-derived tissues; ****P < 0.0001 (tissue uniformity: unpaired t test, compared with control dyn-EHTs; diastolic length: 2-way ANOVA, post hoc Sidak’s multiple comparison test compared control dyn-EHTs at day 28). (E) IFA labeling of desmin (Y20) and cardiac Troponin T on paraffin sections of control- and patient-derived dyn-EHTs at day 28. Images are representative for n = 4 stainings of tissues/group. Scale bars: 200 um. (F) Western blots depicting affected proteins measured in the 3 control- and 2 patient-derived dyn-EHTs at day 28 (quantified graphs available in Supplemental Material). (G) Overview TEM pictures of control- and patient-derived dyn-EHTs at day 28, emphasizing the swollen-like mitochondria (M) and electron denser glycogen granules (G) observed throughout patient-derived dyn-EHTs. Scales bars: 75 nm.