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An Unbiased High-Throughput Screen to Identify Novel Effectors That Impact on Cardiomyocyte Aggregate Levels
Patrick M McLendon1, Gregory Davis1, James Gulick1
1From the Division of Molecular Cardiovascular Biology, Heart Institute, Cincinnati Children's Hospital Medical Center, OH (P.M.M., G.D., J.G., S.R.S., N.X., J.D.M., J.R.); Division of Biomedical Informatics, Cincinnati Children's Hospital, OH (N.S.); and UES, Inc, Dayton, OH (P.M.M.).
Insights
This study screened the entire genome to find genes affecting protein aggregate buildup in heart cells. It identified Janus kinase 1 (Jak1) as a potential therapeutic target for protein-related cardiomyopathies.
Area of Science:
- Cardiology
- Molecular Biology
- Genetics
Background:
- Postmitotic cells like cardiomyocytes are vulnerable to proteotoxic stress, leading to protein aggregate accumulation.
- Desmin-related and crystallin cardiomyopathies are characterized by large protein deposits in cardiomyocytes.
- Enhancing protein clearance pathways (proteasomal degradation, autophagy) benefits cardiomyocyte function under proteotoxic insults.
Purpose of the Study:
- To conduct an unbiased, genome-wide screen for RNA transcripts and protein products influencing cardiomyocyte aggregate accumulation.
- To identify novel therapeutic targets for desmin-related cardiomyopathies and other proteinopathies.
Main Methods:
- Utilized primary mouse cardiomyocytes expressing mutant αB-crystallin (CryAB) to model desmin-related cardiomyopathy.
- Performed a genome-wide screen using a short hairpin RNA (shRNA) lentivirus library to assess gene product effects on aggregate formation.
- Quantified changes in protein aggregate levels within cardiomyocytes.
Main Results:
- The screen identified numerous gene products across various cell signaling pathways that significantly reduced protein aggregate levels.
- Specific candidates were validated for their ability to modulate aggregate accumulation in cardiomyocytes.
Conclusions:
- The genome-wide screening approach is effective in identifying novel players in cellular proteotoxic processes.
- Janus kinase 1 (Jak1), a nonreceptor tyrosine kinase, was validated as a key mediator of aggregate reduction, highlighting its therapeutic potential.
Rationale:
Postmitotic cells, such as cardiomyocytes, seem to be particularly susceptible to proteotoxic stimuli, and large, proteinaceous deposits are characteristic of the desmin-related cardiomyopathies and crystallin cardiomyopathic diseases. Increased activity of protein clearance pathways in the cardiomyocyte, such as proteasomal degradation and autophagy, has proven to be beneficial in maintaining cellular and cardiac function in the face of multiple proteotoxic insults, holding open the possibility of targeting these processes for the development of effective therapeutics.
Objective:
Here, we undertake an unbiased, total genome screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes.
Methods And Results:
Primary mouse cardiomyocytes that accumulate aggregates as a result of a mutant CryAB (αB-crystallin) causative for human desmin-related cardiomyopathy were used for a total genome-wide screen to identify gene products that affected aggregate formation. We infected cardiomyocytes using a short hairpin RNA lentivirus library in which the mouse genome was represented. The screen identified multiple candidates in many cell signaling pathways that were able to mediate significant decreases in aggregate levels.
Conclusions:
Subsequent validation of one of these candidates, Jak1 (Janus kinase 1), a tyrosine kinase of the nonreceptor type, confirmed the usefulness of this approach in identifying previously unsuspected players in proteotoxic processes.
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