Energy metabolism regulated by HDAC inhibitor attenuates cardiac injury in hemorrhagic rat model

A disturbance of energy metabolism reduces cardiac function in acute severe hemorrhagic patients. Alternatively, adequate energy supply reduces heart failure and increases survival. However, the approach to regulating energy metabolism conductive to vital organs is limited, and the underlying molecular mechanism remains unknown. This study assesses the ability of histone deacetylase inhibitors (HDACIs) to preserve cardiac energy metabolism during lethal hemorrhagic injury. In the lethally hemorrhagic rat and hypoxic myocardial cells, energy metabolism and heart function were well maintained following HDACI treatment, as evident by continuous ATP production with normal cardiac contraction. Valproic acid (VPA) regulated the energy metabolism of hemorrhagic heart by reducing lactate synthesis and protecting the mitochondrial ultrastructure and respiration, which were attributable to the inhibition of lactate dehydrogenase A activity and the increased myeloid cell leukemia-1 (mcl-1) gene expression, ultimately facilitating ATP production and consumption. MCL-1, the key target of VPA, mediated this cardioprotective effect under acute severe hemorrhage conditions. Our results suggest that HDACIs promote cardioprotection by improving energy metabolism during hemorrhagic injury and could therefore be an effective strategy to counteract this process in the clinical setting.


Cell damage and HDACIs rescue
In the hypoxia model, rat cardiomyoblasts H9c2 were cultured with 2 mM CoCl 2

Animal studies Animals
The protocol was approved by the Animal Ethics Committee of Beijing Institute of Transfusion Medicine. Male Wistar rats (7-8 weeks old, 255-265 g) were obtained from the Vital River Laboratory Animal Technology Company (Beijing, China) and given free access to a pellet diet and purified water ad libitum. Experiments were performed after an acclimatization period of 4 days.

Surgery
The rats were anesthetized with pentobarbital sodium, then two polyethylene catheters (0.1-mm diameter) filled with heparinized saline were annulated into the left femoral artery and vein to allow for continuous recording of arterial blood pressure, collection of blood samples, and controlled bleeding. MAP and ECG readings were continuously monitored with a polygraph (Biopac Systems, Inc., M150A). The rats were observed for 10 minutes after surgery to ensure stability of the MAP and ECG.
Body temperature was carefully maintained at 37 ± 0.5ºC using a heating pad (Softron, TMS-201). MAP was monitored at a minimum of 30 minutes after hemorrhage until the rats showed initial signs of recovery from anesthesia, and then the femoral catheters were removed. The skin over the incision sites was carefully closed by sutures and covered with antibiotic ointment. OCR was measured using the Seahorse XFe96 analyser. After the hemorrhage procedures were completed, the rats were kept under observation in heated cages at 37°C with free access to diet pellets and purified water. Exclusion criteria were as follows: MAP < 100 mmHg before hemorrhage, surgical bleeding > 0.5 ml during catheterization, and surgical time > 15 minutes.

Hemorrhage procedures
Hemorrhage was performed using adjustable pumps (Lange Corporation). For phase 1 (P1) hemorrhage, 40% volume of whole blood was rapidly withdrawn from the femoral artery over 10 minutes. For phase 2 (P2) hemorrhage, the femoral vein was allowed to bleed another 20% of total blood over the next 50 minutes to simulate the process of a chronic bleeding stage. Animals were randomly assigned to different groups; VPA was dissolved in 0.9% saline (250 µl) and administered through the right femoral vein. The first hemorrhage model included groups treated at the end of P1, the second hemorrhage model treated at the end of P2.

Western blotting and immunoprecipitation
For immunoblot analysis, rat tissues and cells were lysed in a lysis buffer (50 mM growth medium was replaced with XF assay medium. The cells were pre-incubated at 37°C for 1 hour before starting the analysis. Oligomycin was used at a final concentration of 2µM, FCCP at 0.5µM, Antimycin A and Rotenone at 0.5µM.

Mitochondrial microscopy imaging
MCL-1 over-expression and siRNA knockdown H9c2 cells were cultured in DMEM supplemented with 15% FBS and incubated overnight. The cell culture media was then replaced by hypoxic medium (obtained by DMEM media supplement with 2 mM CoCl2 solution) and cultured for additional 6h to induce hypoxic injury.
PCMV6-Entry and non-targeting siRNA transfected H9c2 cells were applied as control respectively. The hypoxic medium was replaced by the serum-free pre-warmed media containing Mitochondrion-Selective Probes (MitoTracker® Green 16 FM), incubation for 30 minutes under cell growth conditions. After staining is complete, replace the staining solution with fresh prewarmed media with or without VPA, and observe cells using a DeltaVision Microscopy Imaging Systems (Applied Precision) using 10 min frames during 12 hours.