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Published on: March 22, 2017
Identification of hypertrophy-modulating Cullin-RING ubiquitin ligases in primary cardiomyocytes
Maximillian Fischer1,2, Moritz Jakab1, Marc N Hirt3,4
1Institute of Pharmacology and Toxicology, Technische Universität München, Munich, Germany.
Insights
Researchers identified 13 novel Cullin-RING ubiquitin ligases (CRLs) that regulate cardiac hypertrophy in cardiomyocytes. F-box protein 25 (Fbxo25) was further studied, showing it promotes cardiac hypertrophy.
Area of Science:
- Cardiovascular Biology
- Molecular Cell Biology
- Biochemistry
Background:
- Cullin-RING ubiquitin ligases (CRLs) are crucial regulators of cellular processes, including cardiac hypertrophy.
- Identifying specific CRLs involved in cardiac hypertrophy is essential for understanding heart disease mechanisms.
Purpose of the Study:
- To identify novel hypertrophy-modulating CRLs in cardiomyocytes (CM).
- To characterize the role of specific CRLs, particularly CRLFbox25, in cardiac hypertrophy.
Main Methods:
- Functional genomic screening using siRNA-mediated depletion and automated microscopy in neonatal rat CM.
- Confirmation of screening hits via 3H-isoleucine incorporation assays.
- In vivo validation using transverse aortic constriction (TAC) model and Western blot analysis.
Main Results:
- Thirteen novel CRLs were identified as positive or negative regulators of CM hypertrophy.
- Depletion of Fbxo6, Fbxo45, and Fbxl14 decreased CM size; depletion of Fbxo9, Fbxo25, Fbxo30, Fbxo32, Fbxo33, Cullin1, Roc1, Ddb1, Fbxw4, and Fbxw5 increased CM size.
- Fbxo25 depletion increased CM size and protein synthesis, upregulating hypertrophy markers ANP and BNP; Fbxo25 protein levels increased 4.5-fold post-TAC.
Conclusions:
- Novel CRLs, including Fbxo25, are key regulators of cardiac hypertrophy.
- CRLFbox25 acts as a positive modulator of cardiac hypertrophy, suggesting it as a potential therapeutic target.
Abstract:
Cullin-RING ubiquitin ligases (CRL) regulate numerous biological processes in the heart and have been implicated in regulating cardiac hypertrophy. This study aimed to identify novel hypertrophy-modulating CRLs in cardiomyocytes (CM). A functional genomic approach using siRNA-mediated depletion and automated microscopy was employed to screen for cell size-modulating CRLs in neonatal rat CM. Screening hits were confirmed by 3H-isoleucine incorporation. Of 43 targets screened, siRNA-mediated depletion of Fbxo6, Fbxo45, and Fbxl14 resulted in decreased cell size, whereas depletion of Fbxo9, Fbxo25, Fbxo30, Fbxo32, Fbxo33, Cullin1, Roc1, Ddb1, Fbxw4, and Fbxw5 led to a markedly increased cell size under basal conditions. In CM stimulated with phenylephrine (PE), depletion of Fbxo6, Fbxo25, Fbxo33, Fbxo45, and Fbxw4 further augmented PE-induced hypertrophy. As a proof-of-concept, the CRLFbox25 was analysed by transverse aortic constriction (TAC) resulting in a 4.5-fold increase in Fbxo25 protein concentrations compared to control animals. In cell culture, siRNA-mediated depletion of Fbxo25 resulted in a ∼ 37% increase in CM cell size and ∼41% increase in 3H-isoleucine incorporation. Depleting Fbxo25 resulted in upregulation of Anp and Bnp. In summary, we identified 13 novel CRLs as positive or negative regulators of CM hypertrophy. Of these, CRLFbox25 was further characterized, as a potential modulator of cardiac hypertrophy.
