Mitochondrial-Localized Versus Cytosolic Intracellular CO-Releasing Organic PhotoCORMs: Evaluation of CO Effects Using Bioenergetics

Abstract

While interactions between carbon monoxide (CO) and mitochondria have been previously studied, the methods used to deliver CO (gas or CO-releasing metal carbonyl compounds) lack subcellular targeting and/or controlled delivery. Thus, the effective concentration needed to produce changes in mitochondrial bioenergetics is yet to be fully defined. To evaluate the influence of mitochondrial-targeted versus intracellularly released CO on mitochondrial oxygen consumption rates, we developed and characterized flavonol-based CO donor compounds that differ at their site of release. These molecules are metal-free, visible light triggered CO donors (photoCORMs) that quantitatively release CO and are trackable in cells via confocal microscopy. Our studies indicate that at a concentration of 10 μM, the mitochondrial-localized and cytosolic CO-releasing compounds are similarly effective in terms of decreasing ATP production, maximal respiration, and the reserve capacity of A549 cells. This concentration is the lowest to impart changes in mitochondrial bioenergetics for any CO-releasing molecule (CORM) reported to date. The results reported herein demonstrate the feasibility of using a structurally tunable organic photoCORM framework for comparative intracellular studies of the biological effects of carbon monoxide.

Figure 1

Carbon monoxide releasing molecules (CORMs) previously used in mitochondria bioenergetics studies (top) and the framework of photoCORMs described in this work (bottom).

Figure 2

Absorption (a) and emission spectra (b,c) of 1–3 in media containing 10% FBS at 100 μM. Blue, green and red coloration of spectral sections in (c) indicate the confocal detection ranges.

Figure 3

Confocal microscopy images showing co-localization of 2 with MTR in A549 cells. Rows 1 and 3 show cells treated with vehicle control (0.4% DMSO). Rows 2 and 4 show cells treated with 2 at 100 μM for 4 h. Image panels depict the Hoechst nuclear stain ( ), the MitoTracker mitochondria stain ( ) the CO donor ( ) or a merge of the three fluorescence channels. Scale bar indicates 20 μm for rows 1 and 2. Scale bar indicates 10 μm for rows 3 and 4.

Figure 4

Confocal images of A549 cells co-stained with 2, MTR and Hoechst 33342. (a) Independent and co-localized pixels of 2 and MTR. (b) Overlaid intensity profile of regions of interest (ROIs) in the co-stained A549 cells as indicated by the white arrows.

Figure 5

Plots of percent cell viability in A549 cells for 1 (a), or 2 (b), their photo-induced reaction products, and 2-carboxyethyl-triphenylphosphonium bromide upon illumination in cells.

Figure 6

Cellular bioenergetics analysis in A549 cells. (a) OCR profiles of cells without light-triggered CO release. (b) OCR profiles of cells after 1 h illumination to trigger CO release in situ. (c) Bioenergetics data of illuminated cells (1 h post CO release) including basal respiration, proton leak, ATP production, maximal respiration, reserve capacity, and non-mitochondrial respiration in the presence of 1 (red), 2 (blue) and TPP tail (green) at the 0.1–10 μM concentration range. Abbreviations: FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; AA+Rot, antimycin A and rotenone. Values represent the means ± SEMs from three independent biological experiments. The values that are significantly different compared to control by one-way ANOVA test are indicated by asterisks as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Scheme 1

(1) Preparation of mitochondrial-targeting photoCORMs 2 and 3 via EDC/HOBt coupling between 1a and the TPP tail. (2) Visible light-induced CO release reactivity of 2 and 3 to generate 4 and 5, which are non-fluorescent molecules.