Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis

Abstract

Regulation of cellular death is central to nearly all physiological routines and is dysregulated in virtually all diseases. Cell death occurs by two major processes, necrosis which culminates in a pervasive inflammatory response and apoptosis which is largely immunologically inert. As necrosis has long been considered an accidental, unregulated form of cellular death that occurred in response to a harsh environmental stimulus, it was largely ignored as a clinical target. However, recent elegant studies suggest that certain forms of necrosis can be reprogrammed. However, scant little is known about the molecules and pathways that orchestrate calcium-overload-induced necrosis, a main mediator of ischemia/reperfusion (IR)-induced cardiomyocyte cell death. To rectify this critical gap in our knowledge, we performed a novel genome-wide siRNA screen to identify modulators of calcium-induced necrosis in human muscle cells. Our screen identified multiple molecular circuitries that either enhance or inhibit this process, including lysosomal calcium channel TPCN1, mitophagy mediatorTOMM7, Ran-binding protein RanBP9, Histone deacetylase HDAC2, chemokine CCL11, and the Arp2/3 complex regulator glia maturation factor-γ (GMFG). Notably, a number of druggable enzymes were identified, including the proteasome β5 subunit (encoded by PSMB5 gene), which controls the proteasomal chymotrypsin-like peptidase activity. Such findings open up the possibility for the discovery of pharmacological interventions that could provide therapeutic benefits to patients affected by myriad disorders characterized by excessive (or too little) necrotic cell loss, including but not limited to IR injury in the heart and kidney, chronic neurodegenerative disorders, muscular dystrophies, sepsis, and cancers.

Figure 1

RNAi screen for regulators of necrosis. (A) Schematic diagram of the RNAi screen. (B) Scatter plot of log-transformed, nontargeting siRNA-normalized signed fold change of ATP levels in pooled siRNA-transfected cells. Data points are ordered from most negative (inhibitors) to most positive (enhancers). Dashed red or green lines are screen-specific cutoffs for inhibitors or enhancers, respectively. Dashed grey line indicates nontargeting control siRNAs. (C) Volcano plot of the t-test q-value (negative log₁₀) against the signed fold change (log₂) from the primary screen. The horizontal blue line indicates the statistical significance level (q = 0.05), and the vertical blue lines represent a signed fold change of ±2. Necrosis inhibitors (red dots, 1084) are defined as genes with a signed fold change less than −2 and q < 0.05; necrosis enhancers (green dots, 743) are defined as genes with a signed fold change greater than 2 and q < 0.05. (D) Pie chart showing the percentages of necrosis inhibitors and enhancers in the human genome based on data from the primary screen.

Figure 2

Enrichment of functional groups, biological processes and pathways in ionomycin-induced necrosis. (A) Candidate genes from the primary screen were grouped by molecular function using the PANTHER classification system. Top molecular functions related with necrosis were catalytic activity and binding. (B) Detailed protein categorization of the catalytic activity group in panel (A). Hydrolase and transferase activities were the primary catalytic activities involved in necrosis. (C) Candidate genes from the primary screen were grouped by biological process using the PANTHER classification system. More than half of all hits were associated with cellular and metabolic processes. (D) Detailed protein categorization of the cellular process group in panel (C). Cell communication and cell cycle were the predominant cellular processes identified. (E) Highly scored pathways in ionomycin-induced necrosis based on PANTHER pathway analysis.

Figure 3

Protein interaction networks in ionomycin-induced necrosis. Potential protein–protein interactions between top candidates from the secondary screen were analyzed using the STRING program. (A) Necrosis enhancers; (B) necrosis inhibitors. Specific groups of genes discussed in the text were indicated by arrows. The different colors for proteins were used only as a visual aid.

Figure 4

Tertiary RNAi screen data for regulators of cardiomyocyte necrosis as assessed by LDH. Bar graph of LDH log₂ (fold change) for significant (p < 0.05) targets which recapitulated the secondary screen results.

Figure 5

Proteasome inhibition suppresses ionomycin-induced necrosis in primary cardiomyocytes. (A) NRCMs were transfected with control (siControl) or PSMB5 siRNA (siPSMB5). Knockdown efficiency was assessed by Western blotting. (B) NRCMs transfected with siControl or siPSMB5 were incubated with ionomycin (1 μM) for 1 h (n = 3). Cell viability and LDH release were analyzed by MTT and LDH assays, respectively. **, p < 0.01 vs siControl. (C) NRCMs were pretreated with the proteasomal inhibitor bortezomib (BTZ, 1 μM) for 1 h prior to incubation with ionomycin (0.5 μM) for 1 h (n = 3). **, p < 0.01 vs Vehicle control.