ROS Regulate Cardiac Function via a Distinct Paracrine Mechanism

SUMMARY Reactive oxygen species (ROS) can act cell autonomously and in a paracrine manner by diffusing into nearby cells. Here, we reveal a ROS-mediated paracrine signaling mechanism that does not require entry of ROS into target cells. We found that under physiological conditions, nonmyocytic pericardial cells (PCs) of the Drosophila heart contain elevated levels of ROS compared to the neighboring cardiomyocytes (CMs). We show that ROS in PCs act in a paracrine manner to regulate normal cardiac function, not by diffusing into the CMs to exert their function, but by eliciting a downstream D-MKK3-D-p38 MAPK signaling cascade in PCs that acts on the CMs to regulate their function. We find that ROS-D-p38 signaling in PCs during development is also important for establishing normal adult cardiac function. Our results provide evidence for a previously unrecognized role of ROS in mediating PC/CM interactions that significantly modulates heart function.


Temperature Shift Assays
The overexpression of UAS transgene was induced only during the embryonic and larval phases (from $0 hr after egg laying [AEL] to white pupae formation) or only during the pupal and adult phases (from white pupae formation to 7-day-old adulthood). To induce transgene overexpression only during the embryonic and larval phases, fertilized eggs were collected at room temperature (RT) on standard food vials, after which vials were transferred to 29 C. Larvae were maintained at 29 C until the onset of puparium formation (white pupae). Upon white pupae formation, vials were transferred to 17 C for culture until the eclosion of adult flies. Adult flies were continued to be raised at 17 C for about 7 days before being analyzed for their cardiac function (at RT). To induce UAS-transgene overexpression only during the pupal and adult phases, the same procedures were carried out except that the temperatures for fly rearing were reversed.

ROS Detection
ROS detection with DHE dye (Molecular Probes, Invitrogen) was performed using a published method (Owusu-Ansah and Banerjee, 2009), with minor modifications. In brief, adult fly hearts were dissected and cleaned in freshly prepared PBS and removed from the cuticle. Hearts were incubated with 30 mM DHE (freshly reconstituted in anhydrous DMSO and diluted in PBS) for 7-10 min at RT in the dark, washed three times with PBS for 5 min each in the dark, and then fixed for 5 min with 7% paraformaldehyde (PFA). Hearts were mounted in ProLong Gold antifade reagent (Invitrogen) and examined under a laser confocal microscope (Zeiss). The endogenous redox state in PCs and CMs was monitored using a genetically encoded fluorescent redox reporter, cyto-roGFP2-Orp1 (Albrecht et al., 2011).Orp1-mediated oxidation induces a conformational change in the linked roGFP2 variant that decreases its fluorescence. Detection of ROS (H 2 O 2 ) with cyto-roGFP2-Orp1 in the hearts was performed as previously described (Albrecht et al., 2011). In brief, adult fly hearts were dissected and incubated for 10 min at RT in freshly prepared PBS containing 20 mM N-ethylmaleimide. Hearts were rinsed once with PBS, fixed with 4% PFA for 15 min at RT, and then washed twice with PBS for 10 min. Hearts were mounted overnight in ProLong Gold antifade reagent and examined under a laser confocal microscope (Zeiss) with excitation at 488 and 405 nm.

Immunostaining
Third-instar wandering-stage larvae and adult female flies (7-10 days old) were collected and dissected in PBS. Hearts were fixed in a solution comprising picric acid/glacial acetic acid/formaldehyde in a ratio of 15:1:5 for 15 min at RT. After washing in PBS plus 0.1% Triton X-100 (PBT), the fixed hearts were incubated overnight at 4 C with primary antibodies diluted in PBT. Hearts were then washed with PBT and incubated for 2 hr at RT with the appropriate fluorescence-conjugated secondary antibodies (Jackson ImmunoResearch) diluted in PBT. Hearts were then washed again with PBT and mounted in ProLong Gold antifade reagent. Samples were examined under an epifluorescence-equipped (Olympus) or laser confocal (Zeiss) microscope.

Fly Heartbeat Analysis
Cardiac contractility measurements on semi-intact preparations of fly hearts were performed as described previously by Fink et al. (2009). Highspeed 30 s movies were recorded at a rate of >150 frames per second using a Hamamatsu CCD camera on a Nikon 80i upright microscope with a 103 dipping immersion lens (see Fink et al. [2009] for further details). The images were processed using SimplePCI software (Compix). M-modes and quantitative data were generated using a MATLAB-based image analysis program (Fink et al., 2009). To generate the M-mode figures, a single pixel-wide column was selected from the most posterior portion of the adult heart at the abdominal A3 segment that encompassed both edges of the heart tube. The corresponding columns were cut from all movie frames and aligned horizontally according to time. HPs or heartbeat lengths were defined as the time between the ends of two consecutive diastolic intervals. The AI was defined as the SD of all recorded HPs for an individual fly, normalized to the median HP to compensate for variability between flies (Ocorr et al., 2007). Diastolic and systolic diameters represent the relaxed and contracted state of the heart tube, respectively. Measurements were made in the exact same location in abdominal segment A3.

SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2014.02.029.

ROS regulate cardiac function in
(G,H) Representative confocal images of fixed heart preparations stained for alphaactinin (red) and phosphorylated histone H3 (green), which marks the nuclei of PC only.
Images are from wild-type hearts (G) and D-p38a -/b -/hearts (H) at 1 week of age.