KLHL24 mutations in EBS: from skin blisters to heart damage

Epidermolysis bullosa simplex (EBS) is best known as a blistering skin disease. Yet, when caused by mutations in the KLHL24 protein, it can also lead to heart failure. Researchers from the lab of reNEW PI Karine Raymond used both patient material and stem cell-based heart models to uncover how these mutations affect cardiac tissue. Their work not only validated stem cell-based models as a powerful tool for future studies but also provided new insights into how KLHL24 mutations contribute to heart failure. The findings were published December 5th in Cardiovascular Research.

EBS: beyond the skin
Epidermolysis bullosa simplex (EBS) is a rare disease that causes severe skin blistering and affects about 1 in 150,000 people worldwide. In the vast majority of cases, the condition is caused by defects in structural proteins of the skin – called keratins. However, in rarer cases, other proteins are involved.

Raymond: “Our main focus is on EBS caused by a defect in the KLHL24 protein. While patients with this defect often show relatively mild skin symptoms, we report on two patients who recently developed life-threatening heart failure. Although the consequences of this defect in KLHL24 in the skin have been studied before, its role in the heart remains poorly understood. In this study, we set out to explore that role further by investigating the defect in cardiac tissue.”

Seeing the whole picture
Through their Chilean collaborator, Assistant Professor Ignacia Fuentes, the researchers obtained cardiac tissue from the two patients. Analyzing these tissues allowed them to gain new insights into how the KLHL24 defect affects cardiac tissue.

Dr. Veronika Ramovs, first author of the paper, explains: “KLHL24 is a protein that determines which proteins in a cell will be targeted for breakdown and removal. It is known that the defect in EBS patients leads to increased breakdown of its targets. Thus far, others have identified one of these targets – desmin – in cardiac tissue. To shed more light on additional targets, we performed proteomic studies on the patient material and compared this to healthy heart tissue from age-matched healthy donors. Through this approach, we were now able to see the whole picture and identify many more proteins that showed increased breakdown in affected patients.”

Stem cells as a model
Because EBS heart tissue with KLHL24 mutations is scarce and difficult to obtain, the researchers then explored if they could employ a simple, alternative model to study the disease: a 2D layer of stem cell-derived heart muscle cells.

“We performed similar proteomic studies on these simple models and were struck by how well they mirrored the situation in heart tissue from patients,” says Ramovs. “We thus combined lessons learned from this model with data from patient material.”

Novel insights
Using this combined approach, the team identified several novel targets of excessive protein breakdown in EBS patients with KLHL24 mutations. Of these, synemin was the most striking.

Ramovs: “Synemin and desmin are closely related proteins that work together as structural units of heart muscle cells. This makes it challenging to determine if KLHL24 directly targets synemin or if its breakdown is a consequence of desmin loss. It was only through combining our stem cell-based model with human sample analysis that we could conclude synemin is independently targeted.”

Strikingly, the researchers identified several effects of KLHL24 mutations beyond increased breakdown of structural proteins. These included reduced PKA signaling activity, increased mitophagy, and sarcomere shortening in heart muscle cells.

Way forward
The study has direct implications for monitoring EBS patients. “Only ~100 EBS patients with mutations in KLHL24 have been identified to date and since their skin symptoms are typically mild, they are commonly monitored less regularly. Our study flags the importance of looking beyond these milder skin symptoms and monitoring different organs closely,” says Ramovs.

At the same time, the study validated the use of simple stem cell-based model systems for future research. Raymond concludes: “This opens possibilities to gain deeper insight in the effect of KLHL24 mutations on the heart. More importantly, the fact we have molecular read-outs in this system means they can be readily applied for high-throughput drug screening and development of innovative gene therapies. We hope that this will ultimately lead to novel treatment options for patients with this devastating disease.”