Impact of Neurons on Patient-Derived Cardiomyocytes Using Organ-On-A-Chip and iPSC Biotechnologies.

In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establishes junctions at the sinus node and ventricular levels. Thus, an increase or decrease in neuronal activity acutely affects myocardial function and chronically affects its structure through remodeling processes. The neuro-cardiac junction (NCJ), which is the major structure of this system, is poorly understood and only a few cell models allow us to study it. Here, we present an innovant neuro-cardiac organ-on-chip model to study this structure to better understand the mechanisms involved in the establishment of NCJ. To create such a system, we used microfluidic devices composed of two separate cell culture compartments interconnected by asymmetric microchannels. Rat PC12 cells were differentiated to recapitulate the characteristics of sympathetic neurons, and cultivated with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). We confirmed the presence of a specialized structure between the two cell types that allows neuromodulation and observed that the neuronal stimulation impacts the excitation-contraction coupling properties including the intracellular calcium handling. Finally, we also co-cultivated human neurons (hiPSC-NRs) with human cardiomyocytes (hiPSC-CMs), both obtained from the same hiPSC line. Hence, we have developed a neuro-cardiac compartmentalized in vitro model system that allows us to recapitulate the structural and functional properties of the neuro-cardiac junction and that can also be used to better understand the interaction between the heart and brain in humans, as well as to evaluate the impact of drugs on a reconstructed human neuro-cardiac system.

Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics.

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases, including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of an active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics show that these new compounds, such as LJ2 or its specific isomers LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. In agreement with the established role of Casp2 in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~5,500,000 M-1 s-1), and the most selective inhibitor, LJ3a, has close to a 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that the spatial configuration of Cα at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit Casp2-mediated S1P cleavage and thus SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with ß-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

PIDDosome-SCAP crosstalk controls high-fructose-diet-dependent transition from simple steatosis to steatohepatitis.

Sterol deficiency triggers SCAP-mediated SREBP activation, whereas hypernutrition together with ER stress activates SREBP1/2 via caspase-2. Whether these pathways interact and how they are selectively activated by different dietary cues are unknown. Here, we reveal regulatory crosstalk between the two pathways that controls the transition from hepatosteatosis to steatohepatitis. Hepatic ER stress elicited by NASH-inducing diets activates IRE1 and induces expression of the PIDDosome subunits caspase-2, RAIDD, and PIDD1, along with INSIG2, an inhibitor of SCAP-dependent SREBP activation. PIDDosome assembly activates caspase-2 and sustains IRE1 activation. PIDDosome ablation or IRE1 inhibition blunt steatohepatitis and diminish INSIG2 expression. Conversely, while inhibiting simple steatosis, SCAP ablation amplifies IRE1 and PIDDosome activation and liver damage in NASH-diet-fed animals, effects linked to ER disruption and preventable by IRE1 inhibition. Thus, the PIDDosome and SCAP pathways antagonistically modulate nutrient-induced hepatic ER stress to control non-linear transition from simple steatosis to hepatitis, a key step in NASH pathogenesis.