RESUMO
Background: Doxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence. Objectives: The aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target. Methods: Biopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell-derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol. Results: Senescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function. Conclusions: Senescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.
RESUMO
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, ultimately causing epidermolysis bullosa simplex (EBS). The majority of these EBS-patients are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homologue 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 (hiPSC)-derived cardiomyocytes from two patients and three (non)familial 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, that can be prevented by restoring desmin protein levels.
