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.

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Loss of muscular mechanical function occurs in several diseasesaffecting millions of people worldwide, including heart failure, stroke, and neuromuscular disorders. To date, no medical or surgical treatments can restore muscular contractility, and the development of artificial muscles is of extreme interest. Mimicking biological muscles, which are optimized systems displaying quick reaction times, is not trivial; only few examples are reported, mainly focused on the use of biomimetic smart materials. Among them, liquid crystalline elastomers (LCEs) can be biocompatible, show contraction parameters comparable to those of native striated muscles, and are able to effectively potentiate cardiac contraction in vitro. To go further and develop in vivo implantable devices, the integration of the stimulation system with the LCE material represents an essential step. Here, a light-stimulated biomimetic contractile unit (BCU), combining ultra-thin photoresponsive LCE films and mini-LED (mLED) matrixes is described. BCU performance (in terms of extent and kinetics of contractile force and shortening) can be fine-tuned by modulating both mLED light power and spatial stimulation patterns, allowing to reproduce mechanical dynamics of native muscles. These results pave the way for the development of novel LCE-based contraction assist devices for cardiac, skeletal, or smooth muscle support by assembling multiple BCUs.

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).

Mutations throughout cardiac troponin T (cTnT), a cardiac thin filament (CTF) component cause changes in protein structure and dynamics leading to pathologic cardiac remodeling observed in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Of note, mutations within the cTnT-linker region cause particularly severe and highly penetrant cardiomyopathies. Our understanding of the precise molecular mechanisms involved has been limited by the lack of a high-resolution structure in this highly flexible domain. We employed time-resolved fluorescence resonance energy transfer (TR-FRET) utilizing fully reconstituted CTFs with donor-labeled (IAEDANS) cTnT on one of 4 Cys-substituted residues (A168/177/192/198C) and acceptor-labeled (5-IAF) actin on Cys-374 to gain high-resolution insight into the cTnT-linker's positioning across the actin filament. Our data indicate the cTnT-linker is proximal to three adjacent actin monomers in both þ/-Ca 2þ conditions. To determine how mutations in the cTnT-linker region alter the native structure of this region, we investigated three cardiomyopathy-linked mutations: DCMassociated mutations R173W and R173Q and HCM-associated mutation D160E. We hypothesize that R173Q/W and D160E cause differential repositioning of the cTnT-linker in relationship to actin. Preliminary investigation of R173W and D160E effects on the positioning of the cTnT-linker þCa 2þ indicates a trending compaction of the linker towards actin. R173Q, however, exhibits both increases and decreases in the cTnT-linker to actin distances. Further TR-FRET studies are ongoing to confirm and extend these preliminary mutation-specific structural results. Lastly, in vivo studies functional studies are ongoing to compare our new cTnT R173W transgenic mouse model and previous D160E mouse model to begin to elucidate genotype-phenotype mechanisms of disease. Through this approach, we can craft a high-resolution structure of the flexible cTnT-linker region and gain an understanding of mutation-specific structural alterations in this region.