Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo. METHODS: In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts. RESULTS: We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs. CONCLUSION: The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

Fabrication and Optimization of Poly(ε-caprolactone) Microspheres Loaded with S-Nitroso-N-Acetylpenicillamine for Nitric Oxide Delivery.

Heart disease is one of the leading causes of death in the United States and throughout the world. While there are different techniques for reducing or preventing the impact of heart disease, nitric oxide (NO) is administered as nitroglycerin for reversing angina or chest pain. Unfortunately, due to its gaseous and short-lived half-life, NO can be difficult to study or even administer. Therefore, controlled delivery of NO is desirable for therapeutic use. In the current study, the goal was to fabricate NO-releasing microspheres (MSs) using a donor molecule, S-Nitroso-N-Acetyl penicillamine, (SNAP), and encapsulating it in poly(ε-caprolactone) (PCL) using a single-emulsion technique that can provide sustained delivery of NO to cells over time without posing any toxicity risks. Optimization of the fabrication process was performed by varying the duration of homogenization (5, 10, and 20 min) and its effect on entrapment efficiency and size. The optimized SNAP-MS had an entrapment efficiency of ˃50%. Furthermore, we developed a modified method for NO detection by using NO microsensors to detect the NO release from SNAP-MSs in real time, showing sustained release behavior. The fabricated SNAP-MSs were tested for biocompatibility with HUVECs (human umbilical vein endothelial cells), which were found to be biocompatible. Lastly, we tested the effect of controlled NO delivery to human induced pluripotent stem-derived cardiomyocytes (hiPSC-CMs) via SNAP-MSs, which showed a significant improvement in the electrophysiological parameters and alleviated anoxic stress.

Thromboinflammatory Biomarkers of Cardiorenal Syndrome in Patients With End-Stage Renal Disease.

Cardiovascular disease is a prevalent complication in patients with end-stage renal disease (ESRD) on maintenance hemodialysis. In the ESRD patient population, cardiovascular mortality is 20 times higher compared to the general population. The strong relationship between both illnesses can be explained through cardiorenal syndrome (CRS). CRS encompasses a spectrum of disorders involving both the heart and kidneys in which acute or chronic dysfunction in one organ may induce a similar effect in the other organ. Current literature reveals that inflammation and thrombosis are integral to CRS development. Hence, this study aims to demonstrate whether thromboinflammatory biomarkers and laboratory parameters correlate with ESRD progression and the development of CRS. Ninety-five ESRD patients were recruited at Loyola University Medical Center hemodialysis unit. Epic chart analysis was used to determine patients with CRS. Biomarkers (C-reactive protein, tumor necrosis factor alpha, interleukin-6, Annexin V, L-fatty acid binding protein, monocyte chemoattractant protein 1, nitric oxide, von Willebrand factor, D-dimer, and plasminogen activator inhibitor-1) were profiled using the enzyme-linked immunosorbent assay method in patients with and without CRS in the ESRD cohort. All biomarkers were significantly elevated in ESRD patients compared to normal controls (P < .05) and laboratory parameters, ferritin (521.99 ± 289.33) and PTH (442.91 ± 1.50). Through EPIC chart analysis 47% of ESRD patients have CRS. D-dimer and TNF-α were significantly elevated in patients with CRS compared to patients without CRS. This study suggests that biomarkers, D-dimer, and TNF-α, can be good predictors of CRS in ESRD patients.

Preclinical Large Animal Porcine Models for Cardiac Regeneration and Its Clinical Translation: Role of hiPSC-Derived Cardiomyocytes.

Myocardial Infarction (MI) occurs due to a blockage in the coronary artery resulting in ischemia and necrosis of cardiomyocytes in the left ventricular heart muscle. The dying cardiac tissue is replaced with fibrous scar tissue, causing a decrease in myocardial contractility and thus affecting the functional capacity of the myocardium. Treatments, such as stent placements, cardiac bypasses, or transplants are beneficial but with many limitations, and may decrease the overall life expectancy due to related complications. In recent years, with the advent of human induced pluripotent stem cells (hiPSCs), newer avenues using cell-based approaches for the treatment of MI have emerged as a potential for cardiac regeneration. While hiPSCs and their derived differentiated cells are promising candidates, their translatability for clinical applications has been hindered due to poor preclinical reproducibility. Various preclinical animal models for MI, ranging from mice to non-human primates, have been adopted in cardiovascular research to mimic MI in humans. Therefore, a comprehensive literature review was essential to elucidate the factors affecting the reproducibility and translatability of large animal models. In this review article, we have discussed different animal models available for studying stem-cell transplantation in cardiovascular applications, mainly focusing on the highly translatable porcine MI model.

Modulation of Mitochondrial Bioenergetics by Polydopamine Nanoparticles in Human iPSC-Derived Cardiomyocytes.

Myocardial infarction (MI) leads to the formation of an akinetic scar on the heart muscle causing impairment in cardiac contractility and conductance, leading to cardiac remodeling and heart failure (HF). The current pharmacological approaches for attenuating MI are limited and often come with long-term adverse effects. Therefore, there is an urgent need to develop novel multimodal therapeutics capable of modulating cardiac activity without causing any major adverse effects. In the current study, we have demonstrated the applicability of polydopamine nanoparticles (PDA-NPs) as a bioactive agent that can enhance the contractility and beat propagation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Treatment of hiPSC-CMs with PDA-NPs demonstrated accumulation of the latter into mitochondria and significantly enhanced time-dependent adenosine triphosphate (ATP) production in these cells, indicating improved mitochondrial bioenergetics. Furthermore, the effect of PDA-NPs on hiPSC-CM activity was evaluated by measuring calcium transients. Treatment with PDA-NPs increased the calcium cycling in hiPSC-CMs in a temporal manner. Our results demonstrated a significant reduction in peak amplitude, transient duration, time to peak, and transient decay time in the PDA-NPs-treated hiPSC-CMs as compared to untreated hiPSC-CMs. Additionally, treatment of isolated perfused rat heart ex vivo with PDA-NPs demonstrated cardiotonic effects on the heart and significantly improved the hemodynamic function, suggesting its potential for enhancing whole heart contractility. Lastly, the gene expression analysis data revealed that PDA-NPs significantly upregulated cardiac-specific genes (ACADM, MYL2, MYC, HCN1, MYL7, GJA5, and PDHA1) demonstrating the ability to modulate genetic expression of cardiomyocytes. Taken together, these findings suggest PDA-NPs capability as a versatile nanomaterial with potential uses in next-generation cardiovascular applications.

Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury.

BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channel's activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes, which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL-positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.

Assessment of mitophagy in human iPSC-derived cardiomyocytes.

Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.Abbreviations: AAVS1: adeno-associated virus integration site 1; AKT/protein kinase B: AKT serine/threonine kinase; CAG promoter: cytomegalovirus early enhancer, chicken ACTB/ß-actin promoter; CIS: cisplatin; CRISPR: clustered regularly interspaced short palindromic repeats; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; hiPSC: human induced pluripotent stem cell; hiPSC-CMs: human induced pluripotent stem cell-derived cardiomyocytes; ISO: isoproterenol; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RT: room temperature; SB: SBI-0206965; ULK1: unc-51 like autophagy activating kinase 1.

A One-Stop Protocol to Assess Myocardial Fibrosis in Frozen and Paraffin Sections.

Masson's Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert's Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections.

De novo Drug Delivery Modalities for Treating Damaged Hearts: Current Challenges and Emerging Solutions.

Cardiovascular disease (CVD) is the leading cause of mortality, resulting in approximately one-third of deaths worldwide. Among CVD, acute myocardial infarctions (MI) is the leading cause of death. Current treatment modalities for treating CVD have improved over the years, but the demand for new and innovative therapies has been on the rise. The field of nanomedicine and nanotechnology has opened a new paradigm for treating damaged hearts by providing improved drug delivery methods, specifically targeting injured areas of the myocardium. With the advent of innovative biomaterials, newer therapeutics such as growth factors, stem cells, and exosomes have been successfully delivered to the injured myocardial tissue, promoting improvement in cardiac function. This review focuses on three major drug delivery modalities: nanoparticles, microspheres, and hydrogels, and their potential for treating damaged hearts following an MI.

Adenosine deaminase modulates metabolic remodeling and orchestrates joint destruction in rheumatoid arthritis.

Rheumatoid Arthritis (RA) is a chronic autoimmune disease associated with inflammation and joint remodeling. Adenosine deaminase (ADA), a risk factor in RA, degrades adenosine, an anti-inflammatory molecule, resulting in an inflammatory bias. We present an integrative analysis of clinical data, cytokines, serum metabolomics in RA patients and mechanistic studies on ADA-mediated effects on in vitro cell culture models. ADA activity differentiated patients into low and high ADA sets. The levels of the cytokines TNFα, IFNγ, IL-10, TGFß and sRANKL were elevated in RA and more pronounced in high ADA sets. Serum metabolomic analysis shows altered metabolic pathways in RA which were distinct between low and high ADA sets. Comparative analysis with previous studies shows similar pathways are modulated by DMARDs and biologics. Random forest analysis distinguished RA from control by methyl-histidine and hydroxyisocaproic acid, while hexose-phosphate and fructose-6-phosphate distinguished high ADA from low ADA. The deregulated metabolic pathways of High ADA datasets significantly overlapped with high ADA expressing PBMCs GEO transcriptomics dataset. ADA induced the death of chondrocytes, synoviocyte proliferation, both inflammation in macrophages and their differentiation into osteoclasts and impaired differentiation of mesenchymal stem cells to osteoblasts and mineralization. PBMCs expressing elevated ADA had increased expression of cytokines and P2 receptors compared to synovial macrophages which has low expression of ADA. Our data demonstrates increased cytokine levels and distinct metabolic signatures of RA based on the ADA activity, suggests an important role for ADA in the pathophysiology of RA joints and as a potential marker and therapeutic target in RA patients.

Emerging Roles of Extracellular Vesicles Derived Non-Coding RNAs in the Cardiovascular System.

Cardiovascular disease is the leading cause of morbidity and mortality all over the world. Emerging evidence emphasize the importance of extracellular vesicles (EVs) in the cell to cell communication in the cardiovascular system which is majorly mediated through non-coding RNA cargo. Advancement in sequencing technologies revealed a major proportion of human genome is composed of non-coding RNAs viz., miRNAs, lncRNAs, tRNAs, snoRNAs, piRNAs and rRNAs. However, our understanding of the role of ncRNAs-containing EVs in cardiovascular health and disease is still in its infancy. This book chapter provides a comprehensive update on our understanding on the role of EVs derived ncRNAs in the cardiovascular pathophysiology and their therapeutic potential.

In situ differentiation of human-induced pluripotent stem cells into functional cardiomyocytes on a coaxial PCL-gelatin nanofibrous scaffold.

Human-induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs) have been explored for cardiac regeneration and repair as well as for the development of in vitro 3D cardiac tissue models. Existing protocols for cardiac differentiation of hiPSCs utilize a 2D culture system. However, the efficiency of hiPSC differentiation to cardiomyocytes in 3D culture systems has not been extensively explored. In the present study, we investigated the efficiency of cardiac differentiation of hiPSCs to functional cardiomyocytes on 3D nanofibrous scaffolds. Coaxial polycaprolactone (PCL)-gelatin fibrous scaffolds were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. hiPSCs were cultured and differentiated into functional cardiomyocytes on the nanofibrous scaffold and compared with 2D cultures. To assess the relative efficiencies of both the systems, SEM, immunofluorescence staining and gene expression analyses were performed. Contractions of differentiated cardiomyocytes were observed in 2D cultures after 2 weeks and in 3D cultures after 4 weeks. SEM analysis showed no significant differences in the morphology of cells differentiated on 2D versus 3D cultures. However, gene expression data showed significantly increased expression of cardiac progenitor genes (ISL-1, SIRPA) in 3D cultures and cardiomyocytes markers (TNNT, MHC6) in 2D cultures. In contrast, immunofluorescence staining showed no substantial differences in the expression of NKX-2.5 and α-sarcomeric actinin. Furthermore, uniform migration and distribution of the in situ differentiated cardiomyocytes was observed in the 3D fibrous scaffold. Overall, our study demonstrates that coaxial PCL-gelatin nanofibrous scaffolds can be used as a 3D culture platform for efficient differentiation of hiPSCs to functional cardiomyocytes.

Electrospun Aligned Coaxial Nanofibrous Scaffold for Cardiac Repair.

Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide and a number one killer in the USA. Cell-based approaches to treat CVDs have only shown modest improvement due to poor survival, retention, and engraftment of the transplanted cells in the ischemic myocardium. Recently, tissue engineering and the use of 3D scaffolds for culturing and delivering stem cells for ischemic heart disease are gaining rapid potential. Here, we describe a protocol for the fabrication of aligned coaxial nanofibrous scaffold comprising of a polycaprolactone (PCL) core and gelatin shell. Furthermore, we describe a detailed protocol for the efficient seeding and maintenance of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on these nanofibrous scaffolds, which could have a potential application in the generation of functional "cardiac patch" for myocardial repair applications as well as an in vitro 3D cardiac tissue model to evaluate the efficacy of cardiovascular drugs and cardiac toxicities.

Systems analysis of avascular necrosis of femoral head using integrative data analysis and literature mining delineates pathways associated with disease.

Avascular necrosis of femoral head (AVNFH) is a debilitating disease, which affects the middle aged population. Though the disease is managed using bisphosphonate, it eventually leads to total hip replacement due to collapse of femoral head. Studies regarding the association of single nucleotide polymorphisms with AVNFH, transcriptomics, proteomics, metabolomics, biophysical, ultrastructural and histopathology have been carried out. Functional validation of SNPs was carried out using literature. An integrated systems analysis using the available datasets might help to gain further insights into the disease process. We have carried out an analysis of transcriptomic data from GEO-database, SNPs associated with AVNFH, proteomic and metabolomic data collected from literature. Based on deficiency of vitamins in AVNFH, an enzyme-cofactor network was generated. The datasets are analyzed using ClueGO and the genes are binned into pathways. Metabolomic datasets are analyzed using MetaboAnalyst. Centrality analysis using CytoNCA on the data sets showed cystathionine beta synthase and methylmalonyl-CoA-mutase to be common to 3 out of 4 datasets. Further, the genes common to at least two data sets were analyzed using DisGeNET, which showed their involvement with various diseases, most of which were risk factors associated with AVNFH. Our analysis shows elevated homocysteine, hypoxia, coagulation, Osteoclast differentiation and endochondral ossification as the major pathways associated with disease which correlated with histopathology, IHC, MRI, Micro-Raman spectroscopy etc. The analysis shows AVNFH to be a multi-systemic disease and provides molecular signatures that are characteristic to the disease process.

Scalable Biomimetic Coaxial Aligned Nanofiber Cardiac Patch: A Potential Model for "Clinical Trials in a Dish".

Recent advances in cardiac tissue engineering have shown that human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a three-dimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D in vitro model for "clinical trials in a dish" and for in vivo cardiac repair applications for treating myocardial infarction.

Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients.

We earlier established the mouse embryonic stem (ES) cell "GS-2" line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420-495), green (~510-575), and red (~600-700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

The Drosophila protein, Nausicaa, regulates lamellipodial actin dynamics in a Cortactin-dependent manner.

Drosophila CG10915 is an uncharacterized protein coding gene with sequence similarity to human Cortactin-binding protein 2 (CTTNBP2) and Cortactin-binding protein 2 N-terminal-like (CTTNBP2NL). Here, we have named this gene Nausicaa (naus) and characterize it through a combination of quantitative live-cell total internal reflection fluorescence microscopy, electron microscopy, RNAi depletion and genetics. We found that Naus co-localizes with F-actin and Cortactin in the lamellipodia of Drosophila S2R+ and D25c2 cells and this localization is lost following Cortactin or Arp2/3 depletion or by mutations that disrupt a conserved proline patch found in its mammalian homologs. Using permeabilization activated reduction in fluorescence and fluorescence recovery after photobleaching, we find that depletion of Cortactin alters Naus dynamics leading to a decrease in its half-life. Furthermore, we discovered that Naus depletion in S2R+ cells led to a decrease in actin retrograde flow and a lamellipodia characterized by long, unbranched filaments. We demonstrate that these alterations to the dynamics and underlying actin architecture also affect D25c2 cell migration and decrease arborization in Drosophila neurons. We present the hypothesis that Naus functions to slow Cortactin's disassociation from Arp2/3 nucleated branch junctions, thereby increasing both branch nucleation and junction stability.