High-Throughput Cardiac Hypertrophy Phenotyping Supports Lead Optimization of GRK5 Inhibitors

Figures

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  1. Comparison of high-throughput and high-content assay platforms for hypertrophic phenotype assessment in mixed cardiac cells versus purified CMs. (A) Neonatal rat hearts are either purified via Percoll gradient centrifugation to isolate CMs, or used as a mixed cardiac cell population retaining all cardiac cell types. Cells are seeded into 384-well plates and stimulated with pro-hypertrophic agents, followed by treatment with test compounds after 24 h. After 96 h, cells are fixed and stained for α-actinin and nuclei. Imaging (4 fields/well) and automated high-content analysis with a customized CellProfiler pipeline enables quantitative evaluation of CM hypertrophy and cell viability. (B) Mixed cardiac cells from 5-day-old-rats (P5) results in higher cell yield compared to P1-derived cells. (C) Semiautomated image analysis enables robust CM quantifications in both P1- and P5-derived heart cell preparations. (D) Mixed cardiac cells maintain a high proportion of CMs without fibroblast overgrowth, preserving a balanced, more physiological environment. Data represents mean ± SEM (n ≥ 5).

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  2. Systematic evaluation of cardiac hypertrophy stimulants defines distinct levels of phenotype induction (Hypertrophy Score) and morphometric signatures. (A) Representative immunofluorescence images of mixed cardiac cells treated with 20 μM PE and 50 ng/mL LIF or DMSO control. (B) Phenotypic profile of mixed cardiac cells following PE-LIF stimulation compared to DMSO control (n = 3, shaded area indicates SEM). (C) Hypertrophic stimulants were tested at three concentrations (left to right): phenylephrine (PE, 10/20/50 μM), endothelin-1 (ET-1, 50/100/200 nM), angiotensin II (Ang. II, 50/100/200 nM), norepinephrine (NE, 1/2/5 μM), leukemia inhibitory factor (LIF, 20/50/100 ng/mL), cortisol (1/2/5 μM), TGFβ-1 (50/100/200 ng/mL), PE-LIF (PE, 20 μM + LIF 50 ng/mL), PE-timolol (PE, 20 μM + TML, 2 μM). To facilitate direct comparison across conditions, key phenotypic parameters were integrated into a single metric, i.e., the Hypertrophy Score, which enhances the dynamic range of the assay (n = 3). (D) Cytokine-induced hypertrophy was more pronounced in purified CM cultures compared to mixed cardiac cells, highlighting model-specific differences in responsiveness (n = 3, shaded area…

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  3. GRK5 is a tractable target in the new hypertrophy assay. (A) Role of GRK5 in the context of cardiac hypertrophy. The canonical pathway regulates GPCR signaling via phosphorylation of activated receptors, whereas the noncanonical pathway involves GRK5 translocation to the nucleus in a Ca2 +/calmodulin-dependent manner where it regulates hypertrophic gene expression via phosphorylation of HDAC5 (created with biorender.com). (B–D) GRK5 siRNA knockdown in mixed cardiac cells (B) efficiently reduces protein levels as shown in representative immunoblots (C) and after semiquantitative blot analysis (D, n = 2), resulting in significant prevention of hypertrophy induction by PE-LIF (20 μM and 50 ng/mL) versus DMSO treatment (E, n = 3) (Mean ± SEM, unpaired t test; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

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  4. Phenotypic profiling of a sunitinib-derived GRK5 inhibitor series for in cellulo SAR as antihypertrophic agents. (A) Chemical structure of sunitinib-derived GRK5 inhibitors with reversible-covalent α-ketoamide warhead (3), α-hydroxyamide enantiomers (4) and a series of amides with distinct heterocyclic substituents (5). (B) Cardiotoxicity and antihypertrophic effects (Hypertrophy Score) were evaluated in mixed cardiac cells after 96 h stimulation with PE-LIF (20 μM, 50 ng/mL) and treatment with inhibitors (n = 3, mean ± SEM). Biochemical IC50 values are stated for each inhibitor.

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  5. Integrated evaluation of antihypertrophic GRK5 inhibitor efficacies. (A, B) Side-by-side comparison of inhibitor efficacies at 8 μM (Hypertrophy Score) underline superior cellular activity of 4a (A). Antihypertrophic efficacy was further validated by a significant reduction of hypertrophic gene expression (Nppa and Nppb) in purified CMs as determined by qPCR (B); data is shown as mean ± SEM (n = 4), one-way ANOVA: *** p ≤ 0.001, **** p ≤ 0.0001). (C) Correlation plot of biochemical GRK5 inhibition potency (pIC50) and antihypertrophic efficacy in the cellular assay (Hypertrophy Score, I8 μM). The dashed red line connects diastereomer pairs 4a/b to visualize an optimal relationship of on-target potency and cellular efficacy, independent from physicochemical differences. Note: 5d is marked in gray as an outlier due to likely cytotoxicity. (D) Relationship between lipophilicity (cLogP) and cellular efficacy (Hypertrophy Score, I8 μM); cLogP values were calculated using Schrödinger’s Maestro 14.3.129.