Discovery of novel small molecule inhibitors of cardiac hypertrophy using high throughput, high content imaging

Abstract

Chronic cardiac hypertrophy is maladaptive and contributes to the pathogenesis of heart failure. The objective of this study was to identify small molecule inhibitors of pathological cardiomyocyte hypertrophy. High content screening was performed with primary neonatal rat ventricular myocytes (NRVMs) cultured on 96-well plates and treated with a library of 3241 distinct small molecules. Non-toxic hit compounds that blocked hypertrophy in response to phenylephrine (PE) and phorbol myristate acetate (PMA) were identified based on their ability to reduce cell size and inhibit expression of atrial natriuretic factor (ANF), which is a biomarker of pathological cardiac hypertrophy. Many of the hit compounds are existing drugs that have not previously been evaluated for benefit in the setting of cardiovascular disease. One such compound, the anti-malarial drug artesunate, blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). These findings demonstrate that phenotypic screening with primary cardiomyocytes can be used to discover anti-hypertrophic lead compounds for heart failure drug discovery. Using annotated libraries of compounds with known selectivity profiles, this screening methodology also facilitates chemical biological dissection of signaling networks that control pathological growth of the heart.

Keywords: Cardiomyocyte; High content; High throughput; Hypertrophy; Screening; Small molecule.

Fig. 1

A novel high throughput assay of cardiomyocyte hypertrophy. (A) Neonatal rat ventricular cardiomyocytes (NRVMs) were seeded on 96-well plates and treated with candidate compounds in the presence of PE or PMA for 48 h. Cells were fixed, subjected to indirect immunofluorescence with antibodies against ANF and α-actinin, and stained with Hoechst dye prior to high content imaging. (B) Images were analyzed using Harmony software. Cells were identified based on nuclei segmentation (Hoechst), and subsequently individual cells were segmented and cell area was calculated based on area of α-actinin fluorescence (FITC). Non-myocytes were removed from analysis based on the absence of α-actinin signal. Finally, ANF expression was measured using a perinuclear mask (Cy3).

Fig. 2

Statistical assessment of the cardiomyocyte hypertrophy high throughput assay. The HDAC inhibitor TSA was used as a positive control to block NRVM hypertrophy and ANF expression in the presence of PE or PMA. Data shown are from two experimental plates: one with each hypertrophic agonist. Individual data points representing mean values per well (total value divided by cell count) are shown. (A and B: Plate one) Z-prime values for cell size, and ANF expression in the presence of PE were 0.63 and 0.72, respectively. (C and D: Plate two) Z-prime values for cell size and ANF expression in the presence of PMA were 0.72 and 0.76, respectively. These values indicate that the differences between the negative controls (PE + DMSO or PMA + DMSO) and the positive controls (PE + TSA or PMA + TSA) are sufficient, and consistent enough, to enable efficient high throughput screening for inhibitors of hypertrophy.

Fig. 3

Graphical representation of hit compounds that block cardiomyocyte hypertrophy. (A) Compounds from the Spectrum Library are depicted based on percent inhibition of ANF expression (X-axis) and cell area (Y-axis) relative to the positive control HDAC inhibitor, TSA; % inhibition by TSA was set to 100% for each plate. Reduction in the number of identified cells (nuclei) compared to the positive control TSA was used as an indicator of toxicity. Toxic compounds are indicated in red, and were removed from subsequent analysis. (B) Class I hits are those compounds that significantly reduce cell area and ANF expression. Class II hits are compounds that significantly reduce cell area, but increase ANF expression.

Fig. 4

The anti-malarial compound artesunate blocks hypertrophy of cultured NRVMs. (A) Chemical structure of artesunate. (B) Quantitative high content imaging demonstrates dose-dependent inhibition of PMA-mediated NRVM hypertrophy and ANF expression by artesunate, with sub-micromolar IC₅₀ values for each endpoint. (C) Representative images of NRVMs treated with PMA in the absence or presence of artesunate.

Fig. 5

Artesunate blocks cardiac hypertrophy in vivo. (A) Ten week-old mice were subjected to transverse aortic constriction (TAC) and treated with vehicle or artesunate for four weeks. (B–D) Echocardiography revealed that artesunate blunted TAC-induced systolic dysfunction, and reduced TAC-mediated thickening of the LV posterior wall and intraventricular septum. (E) LV weight-to-tibia length ratios were determined at necropsy. (F) Images of LV sections stained with fluorescein-conjugated peanut agglutinin were used to assess myocyte cross-sectional area. Scale bar = 10 mu;m. (G) Quantification of myocyte cross-sectional area (mu;m²); >100 myocytes were quantified per the indicated number of LVs. *P < 0.05 vs. sham; ^†P < 0.05 vs. TAC + vehicle.