BMAL1 regulates mitochondrial fission and mitophagy through mitochondrial protein BNIP3 and is critical in the development of dilated cardiomyopathy

Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans. Electronic supplementary material The online version of this article (10.1007/s13238-020-00713-x) contains supplementary material, which is available to authorized users.


Western Blot
To obtain the total protein, RIPA buffer (50 mM Tris/pH 8.0, 150 mM NaCl, 2 mM EGTA, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) was used to obtain the whole cell lysate. Proteins were quantified by BCA Protein Assay Kit (TIANGEN, China). The equal amount of protein was run in Tris-glycine SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, USA). At room temperature, membranes were blocked for 1 h in 5% skim milk in Trisbuffered saline (20 mM Tris/pH 7.6,150 mM NaCl, 0.1% Tween-20) and then incubated in primary antibody diluted in 5% skim milk overnight at 4°C. Primary antibodies used were found in table 4. Membranes were gently washed three times with PBS-T buffer for 10 minutes at a time, then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature.
Membranes were washed again in PBS-T, the method is the same as above, then developed with Clarity Western ECL Substrate (Bio-Rad Laboratories, #170-5061) and imaged using an imaging system (Tanon, China). Densitometry was analyzed using ImageJ software (National Institutes of Health).

Transmission electron microscopy
The CMs were removed from the petri dish using a cell scraper, rinsed with PBS, pelleted at 800 rpm for 20 mins, and then fixed in 2.5% glutaraldehyde in PBS for 2 hours at 4°C. Pellets were wished 3 times with PBS, and then post-fixed with 1% osmium tetroxide in PBS for 2 hours on the rotator. The pellets were wished again before dehydration in a range of increasing ethanol concentrations (30%, 50%, 70%, 85%, 95%, 100%, 100%, 100%). After dehydration, pellets were infiltrated in a series of acetone with increasing concentrations (33%, 66%, 100%, 100%) and then polymerized in 60°C for 24 hours. The resin blocks were cut into 50-60 nm thick resin block, partially mounted on 300 mesh copper grids (Electron Microscopy Sciences).
The slices were stained positive with uranyl acetate and lead citrate and were observed by a PHILIPS CM-120 transmission electron microscope (PHILIPS).

Immunofluorescence and Alkaline phosphatase staining
The cells were rinsed with PBS and fixed with 4% paraformaldehyde for 5 minutes.
The cells were permeabilized with 0.05% Triton X-100 at room temperature for 15 minutes, and washed three times with PBS, and then incubated in 4% goat serum for 30 minutes. The cells were stained by primary antibodies overnight at 4°C.The next day, the cells were incubated with Alexa Fluor conjugated secondary antibodies at 37°C for 1 hour, and then counterstained with DAPI at room temperature for 5 minutes.
According to the manufacturer's instruction, Alkaline phosphatase Kit (Millipore) was used to identify the pluripotency. The stained cells were imaged by a Leica DMi8 microscope. The size of cell surface area was quantified with ImageJ software package (National Institutes of Health).