Biophysical Modulation Research Articles

Biophysical modulation and robustness of itinerant complexity in neuronal networks.

Transient synchronization of bursting activity in neuronal networks, which occurs in patterns of metastable itinerant phase relationships between neurons, is a notable feature of network dynamics observed in vivo. However, the mechanisms that contribute to this dynamical complexity in neuronal circuits are not well understood. Local circuits in cortical regions consist of populations of neurons with diverse intrinsic oscillatory features. In this study, we numerically show that the phenomenon of transient synchronization, also referred to as metastability, can emerge in an inhibitory neuronal population when the neurons' intrinsic fast-spiking dynamics are appropriately modulated by slower inputs from an excitatory neuronal population. Using a compact model of a mesoscopic-scale network consisting of excitatory pyramidal and inhibitory fast-spiking neurons, our work demonstrates a relationship between the frequency of pyramidal population oscillations and the features of emergent metastability in the inhibitory population. In addition, we introduce a method to characterize collective transitions in metastable networks. Finally, we discuss potential applications of this study in mechanistically understanding cortical network dynamics.

Abstract 17637: Lacking MicroRNA-1’s Biophysical Action Causes Heart Failure Revealed by a Newly Developed Biplane Echocardiography of Mouse Heart

MicroRNA-1 (miR1) is the most abundant microRNA in the heart and plays a key role in maintaining cardiac homeostasis through a well-known RNAi mechanism. Recently, we found a novel biophysical function of miR1 to physically bind to and modulate cardiac ion channels. A human single nucleotide polymorphism-hSNP14A/G (rs776480338) of miR1, in which the 14th nucleotide “A” is mutated to “G”, specifically abolishes the biophysical modulation while maintaining the RNAi. However, the physiological significance of microRNA’s biophysical action remains unknown. To study this, we created a single-nucleotide 14A/G mutation on miR1-1 and miR1-2 genes by using CRISPR/Cas9 and developed 14G-mutated homozygous (14G-Homo, miR1-1 14G/G:miR1-2 14G/G) transgenic mice. In addition, we also developed a new echocardiography methodology that can consistently record clear 4-chamber views of mouse hearts, allowing integration of 4-chamber and long axis views for the clinically recommended Biplane Simpson’s assessment of left ventricular (LV) function. Validation via Cardiac Magnetic Resonance (CMR) revealed that the Biplane method is more accurate than M-mode analysis for precise assessments of mouse LV functional parameters. These findings were highlighted in practice by monitoring both Wild Type (WT) and 14G-Homo mice at 2, 3, and 4 months of age, in which M-mode consistently overestimated LV parameters. Moreover, the clarity of the 4 chamber images enables many cardiac analyses that were previously not feasible for the mouse heart, such as the mitral valve color doppler that revealed turbulent blood flow into the LV of 14G-Homo mice. With the 4-chamber view methodology, we also studied the right ventricular (RV) function by assessing fractional area change (FAC) and tricuspid annular plane systolic excursion (TAPSE) and found that cardiac functional deterioration of 14G-Homo mice most likely originated in the LV, followed by RV deteriorations and development of heart failure. In conclusion, our study demonstrated that the biophysical action of miR1 is essential to cardiac homeostasis. Additionally, our new imaging methodology enables more clinically comparable analyses which significantly improve translational implications of mouse cardiac research.

Abstract P3039: The Biophysical Action Of Microrna-1 Is Critical To Maintaining The Homeostasis Of The Heart

MicroRNAs (miRs) are evolutionarily-conserved small noncoding RNAs (~22 nucleotides) and are involved in most biological events through the classical mechanism of RNA interference (RNAi). Recently, we discovered a novel action of miR to biophysically modulate the function of bound proteins. miR1, the most predominant miR in the heart, physically binds to the inward rectifier potassium channel Kir2.1, directly represses the I K1 current, and biophysically modulates cardiac electrophysiology. An arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts biophysical modulation while retaining the RNAi function. However, the physiological significance of miR’s biophysical action on the heart remains unknown. To study this, we created hSNP14A/G single nucleotide mutation on miR1-1 and miR1-2 by using CRISPR/Cas9 technology and successfully generated 14G-mutated transgenic homozygous mice (14G-Homo, miR1-1 14G/G :miR1-2 14G/G ), in which the mutated miR1 maintains the RNAi while with a specific deletion of the biophysical action. We found that 14G-Homo mice had high mortality with impaired heart pumping function and some mice developed heart failure at 6 months of age. The whole transcriptome RNA-sequencing assays revealed cardiac electrical remodeling in 14G-Homo hearts. Consistently, 14G-Homo hearts had prolonged QRS and QT intervals with slower electrical conduction through the ventricle, and 14G-Homo mice are susceptible to arrhythmia. Our patch clamping showed that 14G-Homo cardiomyocytes had significantly prolonged action potential with bigger I K1 and L-type Ca 2+ current (I Ca,L ) than WT cells. We also performed a CLIP RNA pulldown-mass spectrometry assay and identified more ion channel proteins that interact with miR1. From the validation, miR1 biophysically suppresses I Ca,L via directly binding to Cavβ2, the β subunit of the voltage-gated Ca 2+ channel. We conclude that miR1 biophysically modulates the function of the heart, which is critical to maintaining heart homeostasis. Our discovery expands the biological significance of miR biology and broadens its implication of RNA medicine development for cardiovascular diseases.

Transdermal wires for improved integration in vivo

Alternative engineering approaches have led the design of implants with controlled physical features to minimize adverse effects in biological tissues. Similar efforts have focused on optimizing the design features of percutaneous VAD drivelines with the aim to prevent infection, omitting however a thorough look on the implant-skin interactions that govern local tissue reactions. Here, we utilized an integrated approach for the biophysical modification of transdermal implants and their evaluation by chronic sheep implantation in comparison to the standard of care VAD drivelines. We developed a novel method for the transfer of breath topographical features on thin wires with modular size. We examined the impact of implant's diameter, surface topography, and chemistry on macroscopic, histological, and physical markers of inflammation, fibrosis, and mechanical adhesion. All implants demonstrated infection-free performance. The fibrotic response was enhanced by the increasing diameter of implants but not influenced by their surface properties. The implants of small diameter promoted mild inflammatory responses with improved mechanical adhesion and restricted epidermal downgrowth, in both silicone and polyurethane coated transdermal wires. On the contrary, the VAD drivelines with larger diameter triggered severe inflammatory reactions with frequent epidermal downgrowth. We validated these effects by quantifying the infiltration of macrophages and the level of vascularization in the fibrotic zone, highlighting the critical role of size reduction for the benign integration of transdermal implants with skin. This insight on how the biophysical properties of implants impact local tissue reactions could enable new solutions on the transdermal transmission of power, signal, and mass in a broad range of medical devices.

Boolean modeling of mechanosensitive epithelial to mesenchymal transition and its reversal.

The significance of biophysical modulators of the epithelial to mesenchymal transition (EMT) is demonstrated by experiments that document full EMT on stiff, nano-patterned substrates in the absence of biochemical induction. Yet, current models focus on biochemical triggers of EMT without addressing its mechanosensitive nature. Here, we built a Boolean model of EMT triggered by mechanosensing- mitogen crosstalk. Our model reproduces epithelial, hybrid E/M and mesenchymal phenotypes, the role of autocrine TGFβ signaling in maintaining mesenchymal cells in the absence of external drivers, inhibition of proliferation by TGFβ, and its apoptotic effects on soft ECM. We offer testable predictions on the density-dependence of partial EMT, its molecular drivers, and the conflict between mitosis and hybrid E/M stability. Our model opens the door to modeling the effects of the biomechanical environment on cancer cell stemness linked to the hybrid E/M state, as well as the mutually inhibitory crosstalk between EMT and senescence.

The biophysical modulation and RNA interference are two independent actions of microRNA.

MicroRNAs (miRs) are evolutionarily conserved small noncoding RNAs (∼22 nucleotides) and are involved in most biological events through an RNA interference (RNAi) mechanism. Recently, we discovered a novel action of miRs to biophysically modulate the function of bound proteins. miR1, the most predominant miR in the heart, directly binds to the inward rectifier potassium channel Kir2.1 and biophysically represses the IK1 current. However, the relationship between miR's biophysical and RNAi mechanisms has not been studied.

New insights into the molecular basis of allosteric activation of the TMEM16A channel and consequences for the control of vascular tone.

The TMEM16A chloride channel represents a fundamental depolarising mechanism in arterial smooth muscle cells (SMCs) and cerebral contractile pericytes. The channel is a proposed target for diseases of impaired vascular tone including stroke, vascular dementia and hypertension; however, the mechanisms of modulation of the channel by synthetic inhibitors and activators are incompletely understood. Here, we provide a functional characterization of a recently disclosed positive allosteric modulator (PAM) of the TMEM16A channel. In the presence of sub-maximal (300 nM) intracellular free Ca2+ concentration [Ca2+]i, PAM activated the heterologously expressed TMEM16A channels at positive and negative potentials (EC50≈4 nM), while being almost ineffective on the closely related TMEM16B channel. PAM did not activate the TMEM16A currents in either the absence of intracellular Ca2+ or in the presence of saturating [Ca2+]i (12 μM). Mutant TMEM16A channels with the intracellular gate constitutively open were much less sensitive to PAM, suggesting that PAM may act as a modifier of TMEM16A channel gating. Consistent with the effects observed in heterologously expressed TMEM16A channels, PAM activated endogenous TMEM16A currents in isolated rat aortic SMCs, promoted contraction of isolated aortic rings and enhanced capillary (pericyte) constriction evoked by endothelin-1 or oxygen-glucose deprivation (OGD) to stimulate cerebral ischemia. Conversely, inhibiting TMEM16A pharmacologically facilitated arterial and pericyte relaxation, and protected against OGD-mediated pericyte cell death. In summary, this work (i) enhances our understanding of fundamental mechanisms of biophysical modulation of the TMEM16A channel and (ii) supports the view that TMEM16A is a key determinant of SMCs and pericytes tone.

Biophysical Kv channel alterations dampen excitability of cortical PV interneurons and contribute to network hyperexcitability in early Alzheimer’s

AbstractBackgroundIn Alzheimer’s disease (AD), a multitude of genetic risk factors and early biomarkers are known. Nevertheless, the causal factors responsible for initiating cognitive decline in AD remain controversial. Toxic plaques and tangles correlate with progressive neuropathology, yet disruptions in circuit activity emerge before their deposition in AD models and patients. Parvalbumin (PV) interneurons are potential candidates for dysregulating cortical excitability, as they display altered AP firing before neighboring excitatory neurons in prodromal AD.MethodWe used an interneuron‐enhancer AAV labeling technique to specifically target PV interneurons for whole‐cell and outside‐out patch clamp recordings in young 5xFAD mice. Our findings were complemented by PV dyanmic clamp recordings, PV‐specific qPCR, quantitative proteomics, and NEURON modeling.ResultWe report a novel mechanism responsible for cortical PV hypoexcitability in young adult familial AD mice. We found that biophysical modulation of K+ channels, but not changes in mRNA expression, are responsible for dampened excitability. These K+ conductances could efficiently regulate near‐threshold AP firing, resulting in gamma‐frequency specific network hyperexcitability.ConclusionOur findings suggest that posttranslational modulation of ion channels can reshape cortical network activity prior to changes in their gene expression in early AD.

Biophysical Kv3 channel alterations dampen excitability of cortical PV interneurons and contribute to network hyperexcitability in early Alzheimer's.

In Alzheimer's disease (AD), a multitude of genetic risk factors and early biomarkers are known. Nevertheless, the causal factors responsible for initiating cognitive decline in AD remain controversial. Toxic plaques and tangles correlate with progressive neuropathology, yet disruptions in circuit activity emerge before their deposition in AD models and patients. Parvalbumin (PV) interneurons are potential candidates for dysregulating cortical excitability as they display altered action potential (AP) firing before neighboring excitatory neurons in prodromal AD. Here, we report a novel mechanism responsible for PV hypoexcitability in young adult familial AD mice. We found that biophysical modulation of Kv3 channels, but not changes in their mRNA or protein expression, were responsible for dampened excitability in young 5xFAD mice. These K+ conductances could efficiently regulate near-threshold AP firing, resulting in gamma-frequency-specific network hyperexcitability. Thus, biophysical ion channel alterations alone may reshape cortical network activity prior to changes in their expression levels. Our findings demonstrate an opportunity to design a novel class of targeted therapies to ameliorate cortical circuit hyperexcitability in early AD.

Biophysical Modulation of Mesenchymal Stem Cell Differentiation in the Context of Skeletal Repair.

A prominent feature of the skeleton is its ability to remodel in response to biophysical stimuli and to repair under varied biophysical conditions. This allows the skeleton considerable adaptation to meet its physiological roles of stability and movement. Skeletal cells and their mesenchymal precursors exist in a native environment rich with biophysical signals, and they sense and respond to those signals to meet organismal demands of the skeleton. While mechanical strain is the most recognized of the skeletal biophysical stimuli, signaling phenomena also include fluid flow, hydrostatic pressure, shear stress, and ion-movement-related electrokinetic phenomena including, prominently, streaming potentials. Because of the complex interactions of these electromechanical signals, it is difficult to isolate the significance of each. The application of external electrical and electromagnetic fields allows an exploration of the effects of these stimuli on cell differentiation and extra-cellular matrix formation in the absence of mechanical strain. This review takes a distinctly translational approach to mechanistic and preclinical studies of differentiation and skeletal lineage commitment of mesenchymal cells under biophysical stimulation. In vitro studies facilitate the examination of isolated cellular responses while in vivo studies permit the observation of cell differentiation and extracellular matrix synthesis.

A Review Article: Protein Engineering of Therapeutic Enzymes

Through the development of advanced, stimulus-responsive pharmacological systems, protein engineering has the potential to alter the metabolic drug landscapes. Protein therapies are a fast growing category of FDA-approved medications that have the potential to improve clinical consequences in the long run. Protein therapeutics engineering is still in its preliminary phase; however recent advancements in protein engineering skills are being used to gain direct monitoring over pharmacodynamics. Drugs that are intended to be metabolized under specific conditions are known as stimulus-responsive protein medicines. Protein engineering is being used to create biochemically based smart therapies that are personalized to the patient's needs. Protein engineering brings a new features and functionality to the bio-chemical and bio-physical modification of proteins to meet the necessities of therapeutic applications. Provided the enormous capability of protein engineering approaches to modify the activity of bio-catalysts, this review summarizes the current studies are showing the advancements and limitations in using these methods to therapeutic enzyme engineering. The current review will concentrate mainly on three types of therapeutic based enzymes: Diagnostic enzymes,  Fibrinolytic enzymes, and pharmaceutical enzymes, in which the protein is the restorative agent, prodrug-activating enzymes, provokes a therapeutic effects, and diagnostic enzymes, in which the remarkable specificity and selectivity  of a protein offer advantages compared to conventional analytical techniques.

Engineered Magnetic Nanocomposites to Modulate Cellular Function.

Magnetic nanoparticles (MNPs) have various applications in biomedicine, including imaging, drug delivery and release, genetic modification, cell guidance, and patterning. By combining MNPs with polymers, magnetic nanocomposites (MNCs) with diverse morphologies (core-shell particles, matrix-dispersed particles, microspheres, etc.) can be generated. These MNCs retain the ability of MNPs to be controlled remotely using external magnetic fields. While the effects of these biomaterials on the cell biology are still poorly understood, such information can help the biophysical modulation of various cellular functions, including proliferation, adhesion, and differentiation. After recalling the basic properties of MNPs and polymers, and describing their coassembly into nanocomposites, this review focuses on how polymeric MNCs can be used in several ways to affect cell behavior. A special emphasis is given to 3D cell culture models and transplantable grafts, which are used for regenerative medicine, underlining the impact of MNCs in regulating stem cell differentiation and engineering living tissues. Recent advances in the use of MNCs for tissue regeneration are critically discussed, particularly with regard to their prospective involvement in human therapy and in the construction of advanced functional materials such as magnetically operated biomedical robots.

Biophysical modulation as a technological stage of in vitro studies of the biochemical composition of biological objects

Data describing the effects of ultrasound upon biological objects of blood serum is analyzed in the paper. Biophysical as well as medical aspects of tissue reactions caused by ultrasound are considered in fine detail including the immune complexes’ dissociation processes accompanied by releasing of antibodies and antigens that used to be present in bound form and have been inaccessible for revelation by means of laboratory tests. The prospects of using ultrasonic techniques in laboratory diagnostics are presented using HIV-infection as an example.

Biophysical Modulation of the Mitochondrial Metabolism and Redox in Bone Homeostasis and Osteoporosis: How Biophysics Converts into Bioenergetics.

Bone-forming cells build mineralized microstructure and couple with bone-resorbing cells, harmonizing bone mineral acquisition, and remodeling to maintain bone mass homeostasis. Mitochondrial glycolysis and oxidative phosphorylation pathways together with ROS generation meet the energy requirement for bone-forming cell growth and differentiation, respectively. Moderate mechanical stimulations, such as weight loading, physical activity, ultrasound, vibration, and electromagnetic field stimulation, etc., are advantageous to bone-forming cell activity, promoting bone anabolism to compromise osteoporosis development. A plethora of molecules, including ion channels, integrins, focal adhesion kinases, and myokines, are mechanosensitive and transduce mechanical stimuli into intercellular signaling, regulating growth, mineralized extracellular matrix biosynthesis, and resorption. Mechanical stimulation changes mitochondrial respiration, biogenesis, dynamics, calcium influx, and redox, whereas mechanical disuse induces mitochondrial dysfunction and oxidative stress, which aggravates bone-forming cell apoptosis, senescence, and dysfunction. The control of the mitochondrial biogenesis activator PGC-1α by NAD+-dependent deacetylase sirtuins or myokine FNDC/irisin or repression of oxidative stress by mitochondrial antioxidant Nrf2 modulates the biophysical stimulation for the promotion of bone integrity. This review sheds light onto the roles of mechanosensitive signaling, mitochondrial dynamics, and antioxidants in mediating the anabolic effects of biophysical stimulation to bone tissue and highlights the remedial potential of mitochondrial biogenesis regulators for osteoporosis.

An Efficient Pipeline for Biophysical Modeling of Neurons.

Automation of the process of developing biophysical conductance-based neuronal models involves the selection of numerous interacting parameters, making the overall process computationally intensive, complex, and often intractable. A recently reported insight about the possible grouping of currents into distinct biophysical modules associated with specific neurocomputational properties also simplifies the process of automated selection of parameters. The present paper adds a new current module to the previous report to design spike frequency adaptation and bursting characteristics, based on user specifications. We then show how our proposed grouping of currents into modules facilitates the development of a pipeline that automates the biophysical modeling of single neurons that exhibit multiple neurocomputational properties. The software will be made available for public download via our site cyneuro.org.

MicroRNA Biophysically Modulates Cardiac Action Potential by Direct Binding to Ion Channel.

MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms. We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice. We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed IK1 at sub-pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo. Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs' biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.

Deciphering Biophysical Modulation in Ovarian Cancer Cells.

It has been long known that the oncogenic extracellular environment plays an indispensable role in developing and nurturing cancer cell progression and in resistance to standard treatments. However, by how much the biophysical components of tumour extracellular environment contribute to these processes is uncertain. In particular, the topographic environment is scarcely explored. The biophysical modulation of cell behaviour is primarily facilitated through mechanotransduction-associated mechanisms, including focal adhesion and Rho/ROCK signalling. Dysregulation of these pathways is commonly observed in ovarian cancer and, therefore, biophysical modulation of these mechanisms may be of great importance to ovarian cancer development and progression. In this work, aspects of the biophysical environment were explored using a bioimprinting technique. The study showed that topography-mediated substrate sensing delayed cell attachment, however, cell-cell interactions overrode the effect of topography in some cell lines, such as OVCAR-5. Also, 3D topographical cues were shown to modulate the inhibition of focal adhesion and Rho signalling, which resulted in higher MAPK activity in cells on the bioimprints. It was revealed that c-Src is vital to the biophysical modulation of cell proliferation and inhibition of c-Src could downregulate biophysically modulated MAPK activity. This study provides evidence that the biophysical extracellular environment affects key intracellular mechanisms associated with tumourigenicity in ovarian cancer cells.

PSXI-2 An update on the development of the Ruminant Farm Systems (RuFaS) model

Abstract Research, extension, and education communities often focus on the adoption of single strategies because they lack the tools to integrate opportunities at a farm level. Modeling is the best approach to synchronize the complex linkages between animal health, animal-based protein production, water resources, and soil health on dairy farms. Available farm systems models have limitations that prevent scalability and adaptation to evolving technologies and scientific knowledge. Participatory modeling with corporate partners and farmers is being used to create the Ruminant Farm Systems (RuFaS) model. RuFaS tracks the flows of nitrogen, carbon, and phosphorus through four biophysical modules: soil and crop, feed storage, animal herd, and manure. Users input farm variables through tiered files, from general farm information (e.g., crop type, animal numbers, field characteristics) to advanced inputs (e.g., soil characteristics, milk production parameters). The soil and crop module predicts alfalfa, corn, and soybean growth. The animal module uses a Monte-Carlo approach to determine individual animal characteristics and production, incorporating breed traits, reproduction protocols, and animal management decisions. RuFaS calculates animal nutrient requirements from NRC equations, which are averaged on a pen basis to reflect industry practices. A nutritional grouping algorithm can allocate lactating cows to different pens. Manure processing and feed storage modules are currently being developed. RuFaS is coded in python, a general-purpose programming language emphasizing readability. Pool and flux values are stored daily and transferred to an output generator before being cleared annually. Simulation data can be summarized to determine resource use, greenhouse gas emissions, and the cost of production. RuFaS will be available as open-source software to radically change users’ access to dairy farm systems models. The completed RuFaS model will be an essential tool for researchers to evaluate strategies for farm sustainability, and we anticipate the first version of RuFaS will be available in 2021.

Abstract 15963: Microrna Biophysically Modulates Cardiac Physiology via Directly Binding to Ion Channel

Background: Cardiac arrhythmias are a leading cause of morbidity and mortality. MicroRNAs (miRs) regulate the (electro) physiology of the heart and remodeling by canonical RNAi mechanism. Hypothesis: miRs maintain cardiac physiology also through noncanonical mechanisms. Methods: The physical binding between cardiac predominant miR--miR1 and ion channels was explored by EMSA, in situ PLA, RNA pull down and RIP assays. Electrophysiology of cardiomyocytes (CMs) and ex vivo miR1-deficient hearts were studied to reveal the functional outcome and the pathophysiological significance. Results: miR1 physically binds with an inward rectifier K + channel Kir2.1, which exists endogenously in CMs. This miR1-Kir2.1 binding is evolutionarily conserved. Functionally, miR1, at sub-pmol/L concentration, significantly suppresses IK1, depolarizes resting membrane potential, and prolongs final repolarization of action potentials in CMs. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 though the core sequence AAGAAG, which is outside the seed region. This biophysical modulation is involved in the dysregulation of a gain-of-function mutation Kir2.1-M301K in short-QT/AF patients. An AF-associated miR1-hSNP14A/G specifically disrupts the biophysical modulation while maintains miR1’s RNAi function. Significantly, miR1 but not hSNP14A/G eliminates the high inducibility of arrhythmia in miR1-deficient hearts. Conclusion: We reveal a novel function of miRs and develop a ground-breaking concept that endogenous miRs can physically bind with ion channels and rapidly modulate cardiac electrophysiology before its long-term effect of conventional RNAi mechanism. Our study provides more comprehensive understanding of ion-channel dysregulation associated with cardiac arrhythmias.

Abstract 3656: Development of novel, selective SMARCA2 (BRM) degraders for treatment of SMARCA4 (BRG1) mutated tumors

Abstract Background: Synthetic lethality is one of the most innovative approaches for selective targeting of cancer cells with defined genetic background. SMARCA2 and SMARCA4 are two mutually exclusive helicase/ATPase catalytic subunits belonging to SWI/SNF chromatin remodeling complex. It is estimated that one in five tumors possess a mutation in proteins of this complex. The Cancer Genome Atlas (TCGA) and the Catalog of Somatic Mutation in Cancer (Cosmic) shows that SMARCA4 is one of the most mutated genes in lung, colorectal, breast, melanoma and CNS tumors. Herein, we present development of a potent and selective SMARCA2 degrader, structurally unrelated to known chemotypes with first in class potential selectively targeting SMARCA4 mutant cells. Methods: ADP-Glo assay was used to identify SMARCA2 inhibitors through HTS. Binding was confirmed using MST, FTS and SPR methods. Biochemical and biophysical investigation guided rational optimization. Identified ligands were used to build a hybrid PROTAC by linking to a ligand for the E3 ligase. Western blotting was used to assess degradation efficiency and selectivity. Target engagement was confirmed using qPCR and AlphaLISA methods. On target activity was confirmed using SMARCA4 isogenic cells and a panel of SMARCA4 WT and mutant cell lines. Results: High throughput screening allowed identification of novel inhibitors of SMARCA2 ATPase activity. Medicinal chemistry efforts improved potency and affinity to the target over 100 fold. Target engagement was confirmed using biophysical methods and biomarker modulation. Proprietary ligand was used to build a hybrid PROTAC. Western blotting confirmed selective and long-lasting degradation of SMARCA2. Co-treatment with Epoxomicin confirmed proteasomal dependent degradation of targeted protein. Specific SMARCA2 depletion in SMARCA4 mutated cancer cell lines induced apoptosis, growth inhibition and cell death. Observed mechanism of action is consistent with a phenotype seen with perturbation through inhibition of ATPase activity of SMARCA2 and genetic knock-down. Conclusion: Treatment with Ryvu's PROTACs led to selective, proteasomal dependent degradation of SMARCA2 protein and in consequence to a targeted cell death of SMARCA4 mutated cancers. Fine-tuning of available compounds will allow for proof-of-concept experiments in animal models as a single agent or in combinations with radio- or immuno-therapies. Citation Format: Andrzej Mazan, Anna Wróbel, Agnieszka Dreas, Adam Radzimierski, Kinga Michalik, Anna Kowal, Katarzyna Wiklik, Katarzyna Wnuk-Lipińska, Paulina Niedziejko, Kamila Kozłowska, Magdalena Łośko, Joanna Szczęśniak, Agnieszka Sroka-Porada, Justyna Martyka, Urszula Kulesza, Karol Zuchowicz, Magdalena Zastawna, Jose Alvarez, Luigi Stasi, Peter Littlewood, Tomasz Rzymski, Krzysztof Brzózka. Development of novel, selective SMARCA2 (BRM) degraders for treatment of SMARCA4 (BRG1) mutated tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3656.