Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila

Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.


Supplementary Figures and Legends
Median survival during acute exposure to 15 mM paraquat is increased about 50% in female H3S28A mutants (n H3S28S =77, n H3S28A =89; 7-10 days old). Each curve represents the average of at least 3 separate experiments. Fig. 3: Unchanged arrythmicity index in H3S28A mutants. The AI is defined as the standard deviation of the heart periods normalized to the median heart period. It increases with decreasing rhythmicity. All measurements were performed at room temperature (RT) and following thermal stimulation (37 °C) for at least five minutes (n H3S28S =22, n H3S28A =24; 2 days old males).

Suppl.
Suppl. Fig. 4: Heatmap of the 25 most up-and down-regulated genes. The color code represents the row z-score, where a value over 0 (bright to dark red) corresponds to an increased and a value below 0 (bright to dark blue) corresponds to a decreased expression compared to the arithmetic mean over all samples. The arrangement of the samples (columns) and genes (rows) corresponds to the hierarchical clusters (see dendrograms above an left) (n H3S28S,H3S28A = 3 samples à 15 heart tubes; 7 days old males).

OCT measurement of heart rate and fractional shortening
Cardiac function of two day old male flies was measured using a custom built OCT system (Clinical Sensoring and Monitoring, Technische Universitaet Dresden, 1 ). Animals were first anesthetized by exposure to Fly Nap® (Carolina Biological Supply Company) and then, with the dorsal side facing the OCT probe, immobilized on a plastic petri dish using double-sided adhesive tape. The conical chamber as the widest part of the heart tube was then imaged for five seconds in transverse direction.
After data acquisition at RT, the stage was heated to 37 °C for at least five minutes prior to the second recording after thermal stimulation. Two-dimensional B-mode images were analyzed using custom made, LabVIEW-based software in order to determine heart rate, fractional shortening and the arrhythmicity (AI) index. Heart rate was detected between the first and the last beat within the recording time, fractional shortening was calculated in the horizontal axis as ((EDD-ESD)/EDD)*100.
The arrhythmicity index was calculated as the heart period standard deviation normalized to the median heart period. The FD-OCT (fourier-domain) system had a center wavelength of 880 nm and a bandwidth of 130 nm at FWHM (full width at half maximum); an axial and transversal resolution of around 6 μm and 7 µm, respectively, in tissue; an A-scan rate of 12 kHz. For each measurement, around 512 frames (each covering an area of 0.38 × 3 mm², corresponding to 96 × 1024 pixels in the Y−Z direction) were obtained at 93 fps.

Differential gene expression analysis
Dissection of cardiac tubes: The cardiac tubes of seven day old male flies were dissected and exposed according to Vogler and Ocorr 2 . After exposure, cardiac tubes were grasped with forceps at the conical chamber and quickly transferred to an Eppendorf tube containing 350 µl of lysis buffer

Mitochondrial function:
Mitochondrial function analysis was performed as described before 7

and as detailed below
Isolation of mitochondria: 7-to 10-day-old male flies were anesthetized for 5 min at -20 °C. Twenty flies were collected and transferred to 1 ml of isolation buffer (pH 7.4) containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA with 0.5 % (w/v) bovine serum albumin (BSA). Flies were homogenized using a Teflon pestle. The homogenate was centrifuged at 300 × g for 5 min and the supernatant containing the mitochondria was collected. The supernatant was centrifuged at 6,000 × g for 10 min.
The pellet was washed with isolation buffer without BSA and centrifuged at 6,000 × g for 10 min. The pellet was re-suspended in isolation buffer and protein concentration was determined using the LOWRY assay (Biorad). All procedures were performed at 4 °C. Mitochondrial respiration was measured as described before 8,9 . In detail we used a Clark-type electrode (Strathkelvin) at 37 °C during magnetic stirring in respiration buffer containing 125 mM KCl, 10 mM MOPS, 2 mM MgCl 2 , 5 mM KH 2 PO 4 , 0.02 mM EGTA, with 5 mM glutamate and 5 mM malate as substrates for complex I.
The oxygen electrode was calibrated using a solubility coefficient of 217 nmol O 2 /ml at 37 °C. For the measurement of complex I respiration, suspended mitochondria (corresponding to a protein amount of 50 µg) were added to 500 µl of incubation buffer. After 2 min, 1 mM ADP was added and ADPstimulated respiration was measured for 3 min.
Hereafter, mitochondria were used to either measure maximal uncoupled oxygen uptake in the respiration chamber, or respiration buffer containing mitochondria was taken from the respiration chamber to measure ATP production or ROS production, respectively. Maximal uncoupled respiration was measured by adding 30 nM FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) and was analyzed for 30 sec. Respiration was calculated as nmol O 2 /min/mg protein using the Stratkelvin analysis software. For every sample, two measurements were performed, results were averaged and 13 normalized to maximal uncoupled respiration. Mitochondrial ATP production: After measurement of ADP-stimulated respiration, the incubation buffer containing mitochondria was taken from the respiration chamber and immediately supplemented with ATP assay mix (diluted 1:5). ATP production was determined immediately and compared with ATP standards using a 96-well white plate and a Cary Eclipse spectrophotometer (Varian) at 560 nm emission wavelength. Mitochondrial ROS production: ROS concentration in the extramitochondrial space was determined using the Amplex Red Hydrogen Peroxide Assay (Life Technologies). Amplex Red reacts in a 1:1 stoichiometry with peroxides under catalysis by horseradish peroxidase (HRP) and produces highly fluorescent resorufin. The incubation buffer containing mitochondria was removed from the respiration chamber and immediately supplemented with 50 µM Amplex UltraRed and 2 U/ml HRP. The supernatant was collected after 120 min of incubation in the dark. ROS concentration was determined and compared with H 2 O 2 standards using a 96-well black plate and a Cary Eclipse fluorescence spectrophotometer (Varian) at 540 nm emission and 580 nm extinction wavelengths.