Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to β-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.

Some of us recentlyr eportedt he synthesis and structuralc haracterisation of am ixed-valence diruthenium adduct of lysozyme, ([HEWL/Ru 2 (OAc) 2 ], hereafter) originating from its direct reactionw ith the lantern-like complex diruthenium tetraacetate chloride,u nder physiological-likec onditions. [1] Interestingly,c ombined electrospray ionisation mass spectrometry (ESI-MS) and X-ray diffraction studies revealed that the binding of the [Ru 2 (m-O 2 CCH 3 ) 2 -(H 2 O) 2 ] 3 + fragment occurs selectively in two different sites, at the level of two exposed aspartate groups,a cting as bidentate ligands for the ruthenium atoms ( Figure 1). We were intriguedb yt he possible use of [HEWL/Ru 2 (OAc) 2 ] in catalysis and speculated that the persistence of the intact diruthenium motif would guarantee preservation of the interesting and peculiar catalytic properties exhibitedb y Ru 2 (OAc) 4 Cl. This latter complex showed significant catalytic properties, particularly in redox processes, either as ac atalyst

Since 30 years, liquid crystalline elastomers (LCEs) have been attracting the attention of many researchers thanks to their anisotropic molecular structure which allows them to build up artificial muscles. Possible applications span from soft robotics to biomedical or tunable optical devices. The power of thiol−yne click chemistry was recently demonstrated in the preparation of smart polymers, in particular LCEs, in which the mesogenic units are incorporated both in the main-chain and as pendant groups. To enrich the library of available LCE materials, in this work, several liquid crystalline dithiols and alkynes have been synthesized and copolymerized to obtain elastomers with different thermomechanical properties. The architecture of the main chain was found to play a prominent role in modulating the clearing point in a range of 60 °C, whereas only a minor contribution is given by the mesogens in the side chain. On the other hand, the mechanical response resulted highly sensitive to fine details of the side-chain structure. Accordingly, the present study not only improves the basic understanding of the chemical−physical properties of LCEs but paves the way to the preparation of multicomponent actuators able to deform in different temperature ranges, thus ultimately leading to complex soft robotic operations.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Heart failure (HF) is a complex disease characterized by abnormal contraction, metabolic imbalance, and increased propensity for arrhythmias. Dysregulation of intracellular Na þ handling is a major (yet understudied) aspect of HF-induced remodeling of cardiac myocytes. Elevated late Na þ current (I NaL ) in HF prolongs the action potential (AP), thereby facilitating the development of arrhythmogenic early afterdepolarizations. Increased Na þ loading limits the ability of the Na þ /Ca 2þ exchanger to remove Ca 2þ , which along with the reduced sarcoplasmic reticulum (SR) Ca 2þ uptake and increased diastolic SR Ca 2þ leak leads to Ca 2þ overload, thus contributing to diastolic dysfunction and triggered arrhythmias (i.e., via delayed afterdepolarizations). Increased Ca 2þ signals enhance the activity of the Ca 2þ /calmodulin-dependent protein kinase II (CaMKII), which is upregulated and chronically active in HF and directly promotes I NaL , diastolic Na þ influx and SR Ca 2þ leak. To investigate quantitatively this vicious cycle of positive feedback in HF, we updated our computational model of the failing rabbit ventricular myocyte. We modified the main repolarizing and depolarizing currents to reproduce the HF-induced changes measured during AP-clamp experiments performed with physiologic Ca 2þ handling 5 CaMKII inhibition. We validated the cellular model using data describing the frequency-dependence of AP and Ca 2þ transient properties assessed in normal condition and when various branches of the feedback loop are blocked. This updated model serves as a framework to investigate the role of the CaMKII-Na þ -Ca 2þ -CaMKII feedback in promoting Ca 2þ and AP instabilities. Analysis of the relative roles of the interacting components that form the feedback loop within the integrated AP-Ca 2þ cycling-signaling model will allow the identification of the key relationships in the signaling network that could be targeted therapeutically to limit arrhythmias in HF.