Title : Analysis of myocardial phase separation mechanisms driven by AI and thermodynamic laws and their clinical translation
Abstract:
Heart failure remains a major global cause of morbidity and mortality. A significant challengeincardiac regeneration is the inability to generate new cardiomyocytes (CMs) through cell division, whichmaybe linked to ribosome biogenesis dysfunction associated with liquid–liquid phase separation (LLPS). Thecircadian clock gene brain and muscle ARNT-like protein 1 (Bmal1) is known to confer cardiac protection, but its potential role in regulating RhoA—a key modulator of cell division—remains unclear. We aimedtoinvestigate whether Bmal1 promotes cardiac regeneration by orchestrating the circadian-dependent formation of a Bmal1/RhoA LLPS complex. We developed a novel iterative approach combining artificial intelligence (AI) with thermodynamiclaws to predict the LLPS potential of Bmal1 and RhoA. CMs-specific Bmal1 knockout (CKO) mice weregenerated. Using animal models, cellular assays, and molecular techniques—including RNApull-down, fluorescence recovery after photobleaching (FRAP), chromatin isolation by RNA purification (CHIRP), andRibo-Halo assays—we examined Bmal1/RhoA LLPS assembly and cardiac regeneration. We identifiedthat Bmal1 deficiency disrupted ribosomal biogenesis by downregulating the ribosomal protein RPS10, whichinturn impaired the binding of RPS10 to the 3′ untranslated region (3′UTR) of RhoA mRNAin the cytosol. This disruption prevented RhoA 3′UTR LLPS formation, leading to defective cell division and cardiacdysfunction. Strikingly, overexpression of the RhoA 3′UTR increased Bmal1 expression, restored ribosomebiogenesis, and rescued normal CM division. These findings define a novel Bmal1/RPS10/RhoA3′UTRaxisthat coordinates LLPS and drives cardiac regeneration. Moreover, we showed that targeted deliveryof Bmal1 via mesenchymal stem cell–derived extracellular vesicles (EVs) re-established Bmal1/RPS10/RhoA3′UTR LLPS and reinitiated cardiac regeneration in a circadian-dependent manner. In conclusion, the core circadian transcription factor Bmal1 regulates cardiac regenearationbydriving the circadian-dependent assembly of a Bmal1/RPS10/RhoA 3′UTR LLPS complex in the cytosol. Collectively, our findings reveal a promising therapeutic strategy whereby EV-mediated modulationof clock-controlled LLPS promotes cardiac regeneration.
