Bromide does not affect survival and apoptosis of H9C2 cardiomyocytes
To investigate the effects of bromide on the cardiomyocytes, we firstly assessed the toxicity of NaBr. As shown in Fig. 1a, CCK-8 analysis demonstrated that NaBr was not toxic to H9C2 cardiomyocytes when the concentration was up to 600 μM. Besides, similar tendency was observed in isolated rat neonatal primary cardiomyocytes when treated with the same doses of NaBr (sFig. 1). Hence, the doses range from 50 μM to 400 μM were regarded as safe and were chosen for the subsequent experiments. In addition, bromide did not affect mRNA expression levels of apoptosis-associated factors Bcl-2, Bax and Caspase-3 (Fig. 1b). Consistently, the active form level of Caspase-3 (the cleaved type) was not altered by NaBr incubation (Fig. 1c and d). These results implied that cell apoptosis was not regulated in response to bromide stimulation.
Bromide dampens clock gene expression in H9C2 cardiomyocytes
As shown in Fig. 2a and b, treatment of NaBr at the doses of 200 μM and 400 μM robustly inhibited mRNA levels of key clock genes Bmal1, Cry1 and Rorα in a dose-dependent manner. In particular, 400 μM NaBr inhibited mRNA levels of Bmal1 by 41.5%, Cry1 by 59.5% and Rorα by 43.8% respectively. The protein expression of these genes showed similar trends in response to NaBr (Fig. 2a-c). Also, we detected other clock genes expression upon NaBr treatment in sFig 2. Of note, serum shock has been demonstrated to induce rhythmic clock gene expression in various cells. Here, in our system, serum shock also resulted in a robust oscillation of clock genes including Bmal1, Cry1 and Rorα. etc (Fig. 2d, h and l). However, the Per1 did not exhibit an obvious circadian oscillation in H9C2 cardiomyocytes, which is in consistent with previous findings (Fig. 2f). Notably, NaBr treatment did not alter the phase of oscillation patterns of clock genes, but dampened the amplitudes at most checked time-points, except for Clock, whose amplitudes was intensified by NaBr incubation (Fig. 2d-m and Table 2). All these findings suggested the detrimental role of bromide in dampening the circadian clock in H9C2 cardiomyocytes.
Bromide inhibits glycolytic gene expression in H9C2 cardiomyocytes
Given that circadian disruption in cardiomyocytes is closely correlated with the transition of fuel utilization from lipid oxidation to glycolysis (15), it is of great interest to identify the impact of bromide on the glycolysis. Unexpectedly, NaBr did not increase, however, dose-dependently decreased mRNA expression levels of Hk2 and Pkm2, which are hallmarks of glycolysis (Fig. 3a). Similar results were obtained in the protein levels of these genes (Fig. 3b and c). In contrast, PPARα, an important transcriptional factor that activates fatty acid β-oxidation in heart, was not altered upon NaBr incubation (Fig. 3a-c). Furthermore, serum shock also induced significant oscillation of Hk2, Pkm2 and Pparα mRNAs. However, bromide dampened the amplitudes of Pkm2 and Pparα, while leaving Hk2 unchanged in both its phase and amplitude, compared to NaCl-treated group (Fig. 3d-f and Table 3).
Bromide inhibits autophagy in H9C2 cardiomyocytes
Autophagy is a cellular process that delivers cytosolic components to lysosomes for degradation in response to metabolic stress, such as starvation, to provide a source of nutrients and metabolic fuel (16). As shown in Fig. 4a and b, NaBr dramatically reduced the formation of autophagic puncta evidenced by using adenovirus expressing GFP-RFP-LC3. Coincidence with these findings, NaBr significantly reduced the LC3 II/LC3 I ratio and mRNA expression levels of Ulk1, Gabarapl1 and Atg5, which are key factors in regulating the autophagosome formation (Fig. 4c). Consistently, the protein expression levels of ULK1 and ATG5 were inhibited by NaBr in a dose-dependent manner (Fig. 4d and e). In addition, serum shock successfully induced rhythmic oscillation of Ulk1, Gabarapl1 and Atg5 genes expression in H9C2 cells. While the amplitudes of Ulk1 and Atg5 were dampened, bromide modestly altered the Gabarapl1 expression oscillation pattern (Fig. 4f-h and Table 4).
Autophagy mediates the inhibitory effect of bromide on the circadian clock and glycolytic gene expression in H9C2 cardiomyocytes.
To investigate the role of autophagy in the regulation of metabolism and autophagy in H9C2 cardiomyocytes, we incubated cells with 100 nM rapamycin (inhibitor of mTOR activity, as an autophagy inducer). As shown in Fig. 5a and b, rapamycin restored the inhibitory effect of NaBr on the mRNA expression levels of clock genes (Bmal1, Cry1 and Rorα) and glycolytic genes (Hk2 and Pkm2). Also, protein levels of these genes showed similar tendency (Fig 5c-e). Additionally, the phosphorylation of mTOR protein was slightly inducd by NaBr treatment (increased to ~1.4 folds), which was then retarded by rapamycin incubation (Fig 5c and f), indicating that bromide may inhibit autophagy partially through activating mTOR pathway, and further dampening clock and glycolytic gene expression and their rhythmicity (Fig. 6). In addition, another autophagy inducer, QX77, also partially reversed the inhibitory effects of NaBr on the mRNA and protein expression levels of clock genes (Bmal1, Cry1 and Rorα) and glycolytic genes (Hk2 and Pkm2) (sFig 3). Given that the autophagy is vital for the maintaining homeostasis in the physiological state, we treated H9C2 cardiomyocytes with 1 μM H2O2 and 400 μM NaBr. As shown in sFig 4, NaBr-treated H9C2 cardiomyocytes are susceptible to H2O2 stimuli, indicating that the bromide senses the cardiomyocytes to external toxic signals.