Gain-of-function mutation in ubiquitin ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues
Mathilde C.S.C. Vermeer, … , Herman H.W. Silljé, Peter van der Meer
Mathilde C.S.C. Vermeer, … , Herman H.W. Silljé, Peter van der Meer
Published July 22, 2021
Citation Information: J Clin Invest. 2021;131(17):e140615. https://doi.org/10.1172/JCI140615.
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 5

Analysis of patient-derived dyn-EHTs with RNAi of KLHL24 or desmin overexpression.

Options: View larger image (or click on image) Download as PowerPoint
Analysis of patient-derived dyn-EHTs with RNAi of KLHL24 or desmin overe...
(A) Representative side view pictures of patient-derived shNT, shKLHL24, and oeDES dyn-EHTs at day 28. Strips are in white and the images are overlays of shots at tissue diastole (red) and systole (green). Scale bars: 1.5 mm. (B) Desmin protein levels in shKLHL24 versus shNT and patient versus oeDES-derived patient dyn-EHTs, measured on blot (quantified graphs in the supplement). (C) Morphological and functional parameters of patient, shNT, shKLHL24, and oeDES compared with control-derived dyn-EHTs (n = ±15), shKLHL24 (n = 15), and oeDES (n = 10) patient tissues at day 28 (dyn-EHTs), relative to control (n = 20). #P < 0.05 (1-way ANOVA, post hoc Sidak’s multiple comparison test of patient shKLHL24 versus shNT dyn-EHTs); ##P < 0.01 (patient shKLHL24 versus shNT); ###P < 0.001 (patient shKLHL24 versus shNT); ####P < 0.0001 (patient shKLHL24 versus shNT); ^P < 0.05 (patient oeDES versus shNT); ^^P < 0.01 (patient oeDES versus shNT); ^^^^P < 0.0001 (patient oeDES versus shNT); **P < 0.01 (patient shKLHL24 versus control); ****P < 0.0001 (patient shKLHL24 versus control). (D) Peak contractile stress values of above dyn-EHTs. *P < 0.05 (1-way ANOVA, post hoc Sidak’s multiple comparison test of patient shKLHL24 versus control); #P < 0.05 (patient shKLHL24 versus shNT); ^^P < 0.01(patient oeDES versus shNT). (E) Representative maps of regional diastolic stress values derived from FEM in patient, shNT, shKLHL24, and oeDES compared with control-derived dyn-EHTs. White dots represent the tissue attachment point with the PDMS strip. Scale bar: 10 mm. (F) Western blots containing OXPHOS complex proteins (I–V) from patient-derived shNT compared with shKLHL24 dyn-EHTs and patient-derived oeDES compared with control-derived dyn-EHTs. mtHSP70 poses here as a specific loading control. Quantifications are in the supplement.