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Idiopathic ventricular fibrillation (IVF) is an unrefined diagnosis representing a heterogeneous patient group without a structural or genetic definition. IVF treatment is not mechanistic-based due to the lack of experimental patient-models. We sought to create a methodology to assess cellular arrhythmia mechanisms for IVF as a proof-of-concept study. Using IVF patient-specific induced pluripotent stem cell-derived cardiomyocytes, we integrate electrophysiological optical mapping with computational modeling to characterize the cellular phenotype. This approach flips the traditional paradigm using a biophysically detailed computational model to solve the problem inversely. Insight into the cellular mechanisms of this patient's IVF phenotype could also serve as a therapeutic testbed., Competing Interests: Funding Support and Author Disclosures This work was supported by NIH R01HL170521 (LLE, EG), NIH R01HL163987 (Dr Eckhardt), NIH R01HL139738 (Dr Eckhardt), 2R01HL141214 (Dr Glukhov), AHA846898, and AHA 24SCEFIA1255230 (Dr Lang), 24CDA1258695 (Dr Ni). This work was also funded in part by the Gary and Marie Weiner Professor in Cardiovascular Medicine Research (Dr Eckhardt). All authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cardiovascular disease is the leading cause of death in the USA and is known to be exacerbated by elevated mechanical stress from hypertension. Caveolae are plasma membrane structures that buffer mechanical stress but have been found to be reduced in pathological conditions associated with chronically stretched myocardium. To explore the physiological implications of the loss of caveolae, we used human engineered cardiac tissue (ECT) constructs, composed of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived cardiac fibroblasts, to develop a long-term cyclic stretch protocol that recapitulates the effects of hypertension on caveolae expression, membrane tension, and the β-adrenergic response. Leveraging this new stretch protocol, we identified neutral sphingomyelinases (nSMase) as mechanoregulated mediators of caveolae loss, ceramide production and the blunted β-adrenergic response in this human cardiac model. Specifically, in our ECT model, nSMase inhibition via GW4869 prevented stretch-induced loss of caveolae-like structures, mitigated nSMase-dependent ceramide production, and maintained the ECT contractile kinetic response to isoprenaline. These findings are correlated with a blood lipidomic analysis in middle-aged and older adults, which revealed an increase of the circulating levels of ceramides in adults with hypertension. Furthermore, we found that conduction slowing from increased pressure loading in mouse left ventricle was abolished in the context of nSMase inhibition. Collectively, these findings identify nSMase as a potent drug target for mitigating stretch-induced effects on cardiac function. KEY POINTS: We have developed a new stretch protocol for human engineered cardiac tissue that recapitulates changes in plasma membrane morphology observed in animal models of pressure/volume overload. Stretch of engineered cardiac tissue induces activation of neutral sphingomyelinase (nSMase), generation of ceramide, and disassembly of caveolae. Activation of nSMase blunts cardiac β-adrenergic contractile kinetics and mediates stretch-induced slowing of conduction and upstroke velocity. Circulating ceramides are increased in adults with hypertension, highlighting the clinical relevance of stretch-induced nSMase activity., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca 2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND)., Methods: Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca 2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking., Results: In both mouse and human SANs, caveolae compartmentalized HCN4, Ca v 1.2, Ca v 1.3, Ca v 3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca 2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca 2+ current, a negative shift of the HCN current ( I f ) activation curve, and a 40% reduction of Na + /Ca 2+ -exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND., Conclusions: SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

Atrial fibrillation (AF) is the most common cardiac rhythm disorder, often occurring in the setting of atrial distension and elevated myocardialstretch. While various mechano-electrochemical signal transduction pathways have been linked to AF development and progression, the underlying molecular mechanisms remain poorly understood, hampering AF therapies. In this review, we describe different aspects of stretch-induced electro-anatomical remodeling as seen in animal models and in patients with AF. Specifically, we focus on cellular and molecular mechanisms that are responsible for mechano-electrochemical signal transduction and the development of ectopic beats triggering AF from pulmonary veins, the most common source of paroxysmal AF. Furthermore, we describe structural changes caused by stretch occurring before and shortly after the onset of AF as well as during AF progression, contributing to longstanding forms of AF. We also propose mechanical stretch as a new dimension to the concept "AF begets AF", in addition to underlying diseases. Finally, we discuss the mechanisms of these electro-anatomical alterations in a search for potential therapeutic strategies and the development of novel antiarrhythmic drugs targeted at the components of mechano-electrochemical signal transduction not only in cardiac myocytes, but also in cardiac non-myocyte cells., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023. Published by Elsevier Ltd.)

A biosensor based on field-effect transistors on silicon-on-insulator structures (SOI-biosensor) is a high-potential device for detection of biological molecules, for instance, such as troponin I; the biosensor allows conducting label-free real-time analysis. The aim of the study is the development of SOI-biosensor design for detection of acute myocardial infarction marker - troponin I. A notable feature of this design was the integration of two grounding electrodes directly onto the biosensor surface, which effectively nullified the static potential of the liquid sample and minimized physical breakdowns of biosensor elements., Materials and Methods: The highly specific anti-troponin I DNA aptamer was used as a receptor for specific detection of protein marker. Aptamer immobilization on the biosensor surface was carried out by physical adsorption. The analyzed range of target troponin I molecules concentration in the sample varied within 10 -11 to 10 -9 mol/L, mirroring clinical levels observed in myocardial infarction cases. During the experiment, a constant voltage of V ds =0.15 V was maintained., Results: The developed SOI-biosensor successfully detected target troponin I molecules at a concentration of 10 -11 mol/L. The detection process exhibited an effective time of approximately 200-300 s per sample. Moreover, analysis of the detection process revealed a noticeable decrease in current within the source-drain circuit, indicative of the negatively charged complex formed by troponin I and anti-troponin I DNA-aptamer at the "liquid sample-nanowire" phase interface., Competing Interests: Conflicts of interest. The authors have no conflicts of interest to declare.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca 2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca 2+ transients and caused a rise in Ca 2+ spark activity. Similar changes in Ca 2+ signaling were observed after chemical (methyl-β-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser 2030 and Ser 2808 ). Caveolae-mediated stimulatory effects on Ca 2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca 2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes., (Copyright © 2023. Published by Elsevier Ltd.)

Prostate cancer (PC) is one of the major causes of death among elderly men. PC is often diagnosed later in progression due to asymptomatic early stages. Early detection of PC is thus crucial for effective PC treatment. The aim of this study is the simultaneous highly sensitive detection of a palette of PC-associated microRNAs (miRNAs) in human plasma samples. With this aim, a nanoribbon biosensor system based on "silicon-on-insulator" structures (SOI-NR biosensor) has been employed. In order to provide biospecific detection of the target miRNAs, the surface of individual nanoribbons has been sensitized with DNA oligonucleotide probes (oDNA probes) complementary to the target miRNAs. The lowest concentration of nucleic acids, detectable with our biosensor, has been found to be 1.1 × 10 -17 M. The successful detection of target miRNAs, isolated from real plasma samples of PC patients, has also been demonstrated. We believe that the development of highly sensitive nanotechnology-based biosensors for the detection of PC markers is a step towards personalized medicine.

Ovarian cancer is a gynecological cancer characterized by a high mortality rate and tumor heterogeneity. Its early detection and primary prophylaxis are difficult to perform. Detecting biomarkers for ovarian cancer plays a pivotal role in therapy effectiveness and affects patients' survival. This study demonstrates the detection of microRNAs (miRNAs), which were reported to be associated with ovarian cancer tumorigenesis, with a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). The advantages of the method proposed for miRNA detection using the SOI-NW biosensor are as follows: (1) no need for additional labeling or amplification reaction during sample preparation, and (2) real-time detection of target biomolecules. The detecting component of the biosensor is a chip with an array of 3 µm wide, 10 µm long silicon nanowires on its surface. The SOI-NW chip was fabricated using the "top-down" method, which is compatible with large-scale CMOS technology. Oligonucleotide probes (oDNA probes) carrying sequences complementary to the target miRNAs were covalently immobilized on the nanowire surface to ensure high-sensitivity biospecific sensing of the target biomolecules. The study involved two experimental series. Detection of model DNA oligonucleotides being synthetic analogs of the target miRNAs was carried out to assess the method's sensitivity. The lowest concentration of the target oligonucleotides detectable in buffer solution was 1.1 × 10 -16 M. In the second experimental series, detection of miRNAs (miRNA-21, miRNA-141, and miRNA-200a) isolated from blood plasma samples collected from patients having a verified diagnosis of ovarian cancer was performed. The results of our present study represent a step towards the development of novel highly sensitive diagnostic systems for the early revelation of ovarian cancer in women.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish ( Danio rerio ) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411 , in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells., Competing Interests: YD, DL, JY, HB, HL, KJ, JY, HN, SM, TL, KC, JP, HC, YZ, JW, EG, ZL, YS, YL, AG, XX No competing interests declared, SE Reviewing editor, eLife, (© 2022, Ding et al.)

Caveola membrane structures harbor mechanosensitive chloride channels (MCCs; including chloride channel 2, chloride channel 3, and SWELL1, also known as LRRC8A) that form a swelling-activated chloride current (I Cl,swell ) and play an important role in cell volume regulation and mechanoelectrical signal transduction. However, the role of the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCC expression, activity, and contribution to membrane integrity in response to mechanical stress remains unclear. Here we showed that Cav3-transfected (Cav3-positive) HEK293 cells were significantly resistant to extreme (<20 milliosmole) hypotonic swelling compared with native (Cav3-negative) HEK293 cells; the percentage of cells with membrane damage decreased from 45% in Cav3-negative cells to 17% in Cav3-positive cells (p < 0.05). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the I Cl,swell -selective inhibitor 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (10 μM). These results were recapitulated in isolated mouse ventricular myocytes, where the percentage of cardiomyocytes with membrane damage increased from 47% in control cells to 78% in 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid-treated cells (p < 0.05). A higher resistance to hypotonic swelling in Cav3-positive HEK293 cells was accompanied by a significant twofold increase of I Cl,swell current density and SWELL1 protein expression, whereas ClC-2/3 protein levels remained unchanged. Förster resonance energy transfer analysis showed a less than 10-nm membrane and intracellular association between Cav3 and SWELL1. Cav3/SWELL1 membrane Förster resonance energy transfer efficiency was halved in mild (220 milliosmole) hypotonic solution as well as after disruption of caveola structures via cholesterol depletion by 1-h treatment with 10 mM methyl-β-cyclodextrin. A close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. Our findings indicate that, in the MCCs tested, SWELL1 abundance and activity are regulated by Cav3 and that their association relies on membrane tension and caveola integrity. This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca 2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher ( P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca 2+ transient synchronization and rise-up time. This region-specific difference in Ca 2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Ca v 1.2 proteins but similar levels of NCX1 and Ca v 1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca 2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca 2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling. NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca 2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

In native heart tissue, cardiac fibroblasts provide the structural framework of extracellular matrix (ECM) while also influencing the electrical and mechanical properties of cardiomyocytes. Recent advances in the field of stem cell differentiation have led to the availability of human pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) in addition to cardiomyocytes (iPSC-CMs). Here we use a novel 2D in vitro micropatterned platform that provides control over ECM geometry and substrate stiffness. When cultured alone on soft micropatterned substrates, iPSC-CFs are confined to the micropatterned features and remodel the ECM into anisotropic fibers. Similar remodeling and ECM production occurs when cultured with iPSC-CMs in a co-culture model. In addition to modifications in the ECM, our results show that iPSC-CFs influence iPSC-CM function with accelerated Ca 2+ transient rise-up time and greater contractile strains in the co-culture conditions compared to when iPSC-CMs are cultured alone. These combined observations highlight the important role cardiac fibroblasts play in vivo and the need for co-culture models like the one presented here to provide more representative in vitro cardiac constructs., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Turner, Kang, Mesirca, Hong, Mangoni, Glukhov and Sah.)

The application of micro-Raman spectroscopy was used for characterization of structural features of the high-k stack (h-k) layer of "silicon-on-insulator" (SOI) nanowire (NW) chip (h-k-SOI-NW chip), including Al 2 O 3 and HfO 2 in various combinations after heat treatment from 425 to 1000 °C. After that, the NW structures h-k-SOI-NW chip was created using gas plasma etching optical lithography. The stability of the signals from the monocrine phase of HfO 2 was shown. Significant differences were found in the elastic stresses of the silicon layers for very thick (>200 nm) Al 2 O 3 layers. In the UV spectra of SOI layers of a silicon substrate with HfO 2 , shoulders in the Raman spectrum were observed at 480-490 cm -1 of single-phonon scattering. The h-k-SOI-NW chip created in this way has been used for the detection of DNA-oligonucleotide sequences (oDNA), that became a synthetic analog of circular RNA-circ-SHKBP1 associated with the development of glioma at a concentration of 1.1 × 10 -16 M. The possibility of using such h-k-SOI NW chips for the detection of circ-SHKBP1 in blood plasma of patients diagnosed with neoplasm of uncertain nature of the brain and central nervous system was shown.

Background : The mechanisms underlying dysfunction in the sinoatrial node (SAN), the heart's primary pacemaker, are incompletely understood. Electrical and Ca 2+ -handling remodeling have been implicated in SAN dysfunction associated with heart failure, aging, and diabetes. Cardiomyocyte [Na + ] i is also elevated in these diseases, where it contributes to arrhythmogenesis. Here, we sought to investigate the largely unexplored role of Na + homeostasis in SAN pacemaking and test whether [Na + ] i dysregulation may contribute to SAN dysfunction. Methods : We developed a dataset-specific computational model of the murine SAN myocyte and simulated alterations in the major processes of Na + entry (Na + /Ca 2+ exchanger, NCX) and removal (Na + /K + ATPase, NKA). Results : We found that changes in intracellular Na + homeostatic processes dynamically regulate SAN electrophysiology. Mild reductions in NKA and NCX function increase myocyte firing rate, whereas a stronger reduction causes bursting activity and loss of automaticity. These pathologic phenotypes mimic those observed experimentally in NCX- and ankyrin-B-deficient mice due to altered feedback between the Ca 2+ and membrane potential clocks underlying SAN firing. Conclusions : Our study generates new testable predictions and insight linking Na + homeostasis to Ca 2+ handling and membrane potential dynamics in SAN myocytes that may advance our understanding of SAN (dys)function.

The sinoatrial node (SAN), the primary pacemaker of the heart, consists of a heterogeneous population of specialized cardiac myocytes that can spontaneously produce action potentials, generating the rhythm of the heart and coordinating heart contractions. Spontaneous beating can be observed from very early embryonic stage and under a series of genetic programing, the complex heterogeneous SAN cells are formed with specific biomarker proteins and generate robust automaticity. The SAN is capable to adjust its pacemaking rate in response to environmental and autonomic changes to regulate the heart's performance and maintain physiological needs of the body. Importantly, the origin of the action potential in the SAN is not static, but rather dynamically changes according to the prevailing conditions. Changes in the heart rate are associated with a shift of the leading pacemaker location within the SAN and accompanied by alterations in P wave morphology and PQ interval on ECG. Pacemaker shift occurs in response to different interventions: neurohormonal modulation, cardiac glycosides, pharmacological agents, mechanical stretch, a change in temperature, and a change in extracellular electrolyte concentrations. It was linked with the presence of distinct anatomically and functionally defined intranodal pacemaker clusters that are responsible for the generation of the heart rhythm at different rates. Recent studies indicate that on the cellular level, different pacemaker clusters rely on a complex interplay between the calcium (referred to local subsarcolemmal Ca 2+ releases generated by the sarcoplasmic reticulum via ryanodine receptors) and voltage (referred to sarcolemmal electrogenic proteins) components of so-called "coupled clock pacemaker system" that is used to describe a complex mechanism of SAN pacemaking. In this review, we examine the structural, functional, and molecular evidence for hierarchical pacemaker clustering within the SAN. We also demonstrate the unique molecular signatures of intranodal pacemaker clusters, highlighting their importance for physiological rhythm regulation as well as their role in the development of SAN dysfunction, also known as sick sinus syndrome.

The detection of influenza A virions with a nanoribbon detector (NR detector) has been demonstrated. Chips for the detector have been fabricated based on silicon-on-insulator nanoribbon structures (SOI nanoribbon chip), using a complementary metal-oxide-semiconductor (CMOS)-compatible technology-by means of gas-phase etching and standard optical photolithography. The surface of the SOI nanoribbon chip contains a matrix of 10 nanoribbon (NR) sensor elements. SOI nanoribbon chips of n-type conductance have been used for this study. For biospecific detection of target particles, antibodies against influenza virus have been covalently immobilized onto NRs. Influenza A virus detection was performed by real-time registration of the source-drain current through the NRs. The detection of the target viral particles was carried out in buffer solutions at the target particles concentration within the range from 10 7 to 10 3 viral particles per milliliter (VP/mL). The lowest detectable concentration of the target viral particles was 6 × 10 -16 M (corresponding to 10 4 VP/mL). The use of solutions containing ~10 9 to 10 10 VP/mL resulted in saturation of the sensor surface with the target virions. In the saturation mode, detection was impossible.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and the intact human myocardium. Fulfilling this potential is hampered by their relative immaturity, leading to poor physiological responsiveness. hiPSC-CMs grown in traditional two-dimensional (2D) culture lack a t-tubular system, have only rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as an energy substrate. Culturing hiPSC-CM in a variety of three-dimensional (3D) environments and the addition of nutritional, pharmacological, and electromechanical stimuli have proven, to various degrees, to be beneficial for maturation. We present a detailed assessment of a novel model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are cocultured in a 3D fibrin matrix to form engineered cardiac tissue constructs (hiPSC-ECTs). The hiPSC-ECTs are responsive to physiological stimuli, including stretch, frequency, and β-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Furthermore, transcript levels of various genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a relatively short period of time in culture (6 wk vs. 2 wk in hiPSC-ECTs). A comparison of the hiPSC-ECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to aid selection of the most appropriate platform for the research question at hand. Important and noteworthy aspects of this human cardiac model system are its reliance on "off-the-shelf" equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological, and electromechanical stimuli that may elicit unintended effects on function. NEW & NOTEWORTHY This study seeks to provide an in-depth assessment of contractile performance of human iPSC-derived cardiomyocytes cultured together with fibroblasts in a 3-dimensional-engineered tissue and compares performance both over time as cells mature, and with corresponding measures found in the literature using alternative 3D culture configurations. The suitability of 3D-engineered human cardiac tissues to model cardiac function is emphasized, and data provided to assist in the selection of the most appropriate configuration based on the target application.

Right ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca 2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca 2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca 2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca 2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.

Angiotensin II (AngII) is a key mediator of the renin-angiotensin system and plays an important role in the regulation of cardiac electrophysiology by affecting various cardiac ion currents, including transient outward potassium current, I to . AngII receptors and molecular components of I to , K v 4.2 and K v 4.3 channels, have been linked to caveolae structures. However, their functional interaction and the importance of such proximity within 50- to 100-nm caveolar nanodomains remain unknown. To address this, we studied the mechanisms of I to regulation by AngII in atrial myocytes of wild-type (WT) and cardiac-specific caveolin-3 (Cav3) conditional knockout (Cav3KO) mice. We showed that in WT atrial myocytes, a short-term (2 h) treatment with AngII (5 µM) significantly reduced I to density. This effect was prevented 1 ) by a 30-min pretreatment with a selective antagonist of AngII receptor 1 (Ang1R) losartan (2 µM) or 2 ) by a selective inhibition of protein kinase C (PKC) by BIM1 (10 µM). The effect of AngII on I to was completely abolished in Cav3-KO mice, with no change in a baseline I to current density. In WT atria, Ang1Rs co-localized with Cav3, and the expression of Ang1Rs was significantly decreased in Cav3KO in comparison with WT mice, whereas no change in K v 4.2 and K v 4.3 protein expression was observed. Overall, our findings demonstrate that Cav3 is involved in the regulation of Ang1R expression and is required for the modulation of I to by AngII in mouse atrial myocytes. NEW & NOTEWORTHY Angiotensin II receptor 1 is associated with caveolae and caveolar scaffolding protein caveolin-3 in mouse atrial myocytes that is required for the regulation of I to by angiotensin II. Downregulation of caveolae/caveolin-3 disrupts this regulation and may be implicated in pathophysiological atrial remodeling.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC-CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two-dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC-CMs., (© 2020 Wiley Periodicals LLC.)

The detection of CA 125 protein in a solution using a silicon-on-insulator (SOI)-nanowire biosensor with n-type chip has been experimentally demonstrated. The surface of nanowires was modified by covalent immobilization of antibodies against CA 125 in order to provide the biospecificity of the target protein detection. We have demonstrated that the biosensor signal, which results from the biospecific interaction between CA 125 and the covalently immobilized antibodies, increases with the increase in the protein concentration. At that, the minimum concentration, at which the target protein was detectable with the SOI-nanowire biosensor, amounted to 1.5 × 10 -16 M.

Background: Arrhythmia syndromes associated with KCNJ2 mutations have been described clinically; however, little is known of the underlying arrhythmia mechanism. We create the first patient inspired KCNJ2 transgenic mouse and study effects of this mutation on cardiac function, I K1 , and Ca 2+ handling, to determine the underlying cellular arrhythmic pathogenesis., Methods: A cardiac-specific KCNJ2 -R67Q mouse was generated and bred for heterozygosity (R67Q +/- ). Echocardiography was performed at rest, under anesthesia. In vivo ECG recording and whole heart optical mapping of intact hearts was performed before and after adrenergic stimulation in wild-type (WT) littermate controls and R67Q +/- mice. I K1 measurements, action potential characterization, and intracellular Ca 2+ imaging from isolated ventricular myocytes at baseline and after adrenergic stimulation were performed in WT and R67Q +/- mice., Results: R67Q +/- mice (n=17) showed normal cardiac function, structure, and baseline electrical activity compared with WT (n=10). Following epinephrine and caffeine, only the R67Q +/- mice had bidirectional ventricular tachycardia, ventricular tachycardia, frequent ventricular ectopy, and/or bigeminy and optical mapping demonstrated high prevalence of spontaneous and sustained ventricular arrhythmia. Both R67Q +/- (n=8) and WT myocytes (n=9) demonstrated typical n-shaped I K1 IV relationship; however, following isoproterenol, max outward I K1 increased by ≈20% in WT but decreased by ≈24% in R67Q +/- ( P <0.01). R67Q +/- myocytes (n=5) demonstrated prolonged action potential duration at 90% repolarization and after 10 nmol/L isoproterenol compared with WT (n=7; P <0.05). Ca 2+ transient amplitude, 50% decay rate, and sarcoplasmic reticulum Ca 2+ content were not different between WT (n=18) and R67Q +/- (n=16) myocytes. R67Q +/- myocytes (n=10) under adrenergic stimulation showed frequent spontaneous development of early afterdepolarizations that occurred at phase 3 of action potential repolarization., Conclusions: KCNJ2 mutation R67Q +/- causes adrenergic-dependent loss of I K1 during terminal repolarization and vulnerability to phase 3 early afterdepolarizations. This model clarifies a heretofore unknown arrhythmia mechanism and extends our understanding of treatment implications for patients with KCNJ2 mutation.

Rapid firing from pulmonary veins (PVs) frequently initiates atrial fibrillation, which is a common comorbidity associated with hypertension, heart failure, and valvular disease, i.e., conditions that pathologically increase cardiomyocyte stretch. Autonomic tone plays a crucial role in PV arrhythmogenesis, while its interplay with myocardium stretch remains uncertain. Two-microelectrode technique was used to characterize electrophysiological response of Wistar rat PV to adrenaline at baseline and under mild (150 mg of applied weight that corresponds to a pulmonary venous pressure of 1 mmHg) and moderate (10 g, ∼26 mmHg) stretch. Low concentrations of adrenaline (25-100 nmol/L) depolarized the resting membrane potential selectively within distal PV (by 26 ± 2 mV at baseline, by 18 ± 1 mV at 150 mg, P < 0.001, and by 5.9 ± 1.1 mV at 10 g, P < 0.01) suppressing action potential amplitude and resulting in intra-PV conduction dissociation and rare episodes of spontaneous activity (arrhythmia index of 0.4 ± 0.2, NS vs. no activity at baseline). In contrast, 1-10 μmol/L of adrenaline recovered intra-PV propagation. While mild stretch did not affect PV electrophysiology at baseline, moderate stretch depolarized the resting potential within distal PV (-56 ± 2 mV vs. -82 ± 1 mV at baseline, P < 0.01), facilitated the triggering of rapid PV firing by adrenaline (arrhythmia index: 4.4 ± 0.2 vs. 1.3 ± 0.4 in unstretched, P < 0.001, and 1.7 ± 0.8 in mildly stretched preparations, P < 0.005, at 10 μmol/L adrenaline) and induced frequent episodes of potentially arrhythmogenic atrial "echo" extra beats. Our findings demonstrate complex interactions between the sympathetic tone and mechanical stretch in the development of arrhythmogenic activity within PVs that may impact an increased atrial fibrillation vulnerability in patients with elevated blood pressure., (Copyright © 2020 Egorov, Rosenshtraukh and Glukhov.)

Various experimental mouse models are extensively used to research human diseases, including atrial fibrillation, the most common cardiac rhythm disorder. Despite this, there are no comprehensive mathematical models that describe the complex behavior of the action potential and [Ca 2+ ] i transients in mouse atrial myocytes. Here, we develop a novel compartmentalized mathematical model of mouse atrial myocytes that combines the action potential, [Ca 2+ ] i dynamics, and β-adrenergic signaling cascade for a subpopulation of right atrial myocytes with developed transverse-axial tubule system. The model consists of three compartments related to β-adrenergic signaling (caveolae, extracaveolae, and cytosol) and employs local control of Ca 2+ release. It also simulates ionic mechanisms of action potential generation and describes atrial-specific Ca 2+ handling as well as frequency dependences of the action potential and [Ca 2+ ] i transients. The model showed that the T-type Ca 2+ current significantly affects the later stage of the action potential, with little effect on [Ca 2+ ] i transients. The block of the small-conductance Ca 2+ -activated K + current leads to a prolongation of the action potential at high intracellular Ca 2+ . Simulation results obtained from the atrial model cells were compared with those from ventricular myocytes. The developed model represents a useful tool to study complex electrical properties in the mouse atria and could be applied to enhance the understanding of atrial physiology and arrhythmogenesis. NEW & NOTEWORTHY A new compartmentalized mathematical model of mouse right atrial myocytes was developed. The model simulated action potential and Ca 2+ dynamics at baseline and after stimulation of the β-adrenergic signaling system. Simulations showed that the T-type Ca 2+ current markedly prolonged the later stage of atrial action potential repolarization, with a minor effect on [Ca 2+ ] i transients. The small-conductance Ca 2+ -activated K + current block resulted in prolongation of the action potential only at the relatively high intracellular Ca 2+ .

Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy., Competing Interests: Declaration of competing interest TJK is a consultant for FujiFilm Cellular Dynamics Inc. RAA is a consultant for Cell Reprogramming and Therapeutics LLC. Pending patent applications are related to iCPC generation (TJK, PAL). EGS and ANR have ownership in Cellular Logistics Inc., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background Atrial fibrillation often occurs in the setting of hypertension and associated atrial dilation with pathologically increased cardiomyocyte stretch. In the setting of atrial dilation, mechanoelectric feedback has been linked to the development of ectopic beats that trigger paroxysmal atrial fibrillation mainly originating from pulmonary veins (PVs). However, the precise mechanisms remain poorly understood. Methods and Results We identify mechanosensitive, swelling-activated chloride ion channels ( I C l,swell ) as a crucial component of the caveolar mechanosensitive complex in rat and human cardiomyocytes. In vitro optical mapping of rat PV, single rat PV, and human cardiomyocyte patch clamp studies showed that stretch-induced activation of I Cl,swell leads to membrane depolarization and decreased action potential amplitude, which trigger conduction discontinuities and both ectopic and reentrant activities within the PV. Reverse transcription quantitative polymerase chain reaction, immunofluorescence, and coimmunoprecipitation studies showed that I Cl,swell likely consists of at least 2 components produced by mechanosensitive ClC-3 (chloride channel-3) and SWELL1 (also known as LRRC8A [leucine rich repeat containing protein 8A]) chloride channels, which form a macromolecular complex with caveolar scaffolding protein Cav3 (caveolin 3). Downregulation of Cav3 protein expression and disruption of caveolae structures during chronic hypertension in spontaneously hypertensive rats facilitates activation of I Cl,swell and increases PV sensitivity to stretch 10- to 50-fold, promoting the development of atrial fibrillation. Conclusions Our findings identify caveolae-mediated activation of mechanosensitive I Cl,swell as a critical cause of PV ectopic beats that can initiate atrial arrhythmias including atrial fibrillation. This mechanism is exacerbated in the setting of chronically elevated blood pressures.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923., (© 2019 AlphaMed Press.)

Key Points: Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Ca v 1.2-encoded L-type Ca 2+ channels responsible for I Ca,L and also cause loss of function effects on heterologously expressed K v 4.2 and K v 4.3 channels responsible for I to . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating I Ca,L but, for Cav3-S141R, both increased I Ca,L and increased late Na + current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches., Abstract: Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Ca v 1.2+Ca v β 2cN4 channels, as well as K v 4.2 and K v 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased I Ca,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca 2+ -dependent inactivation of I Ca,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of I Kv4.2 and I Kv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of I Kv4.2 but had no effect on I Kv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in I to are predicted to have negligible effect on APD, whereas blunted Ca 2+ -dependent inactivation of I Ca,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased I Ca,L and late I Na by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Spontaneous beating of the sinoatrial node (SAN), the primary pacemaker of the heart, is initiated, sustained, and regulated by a complex system that integrates ion channels and transporters on the cell membrane surface (often referred to as "membrane clock") with subcellular calcium handling machinery (by parity of reasoning referred to as an intracellular "Ca 2+ clock"). Stable, rhythmic beating of the SAN is ensured by a rigorous synchronization between these two clocks highlighted in the coupled-clock system concept of SAN timekeeping. The emerging results demonstrate that such synchronization of the complex pacemaking machinery at the cellular level depends on tightly regulated spatiotemporal signals which are restricted to precise sub-cellular microdomains and associated with discrete clusters of different ion channels, transporters, and regulatory receptors. It has recently become evident that within the microdomains, various proteins form an interacting network and work together as a part of a macromolecular signaling complex. These protein-protein interactions are tightly controlled and regulated by a variety of neurohormonal signaling pathways and the diversity of cellular responses achieved with a limited pool of second messengers is made possible through the organization of essential signal components in particular microdomains. In this review, we highlight the emerging understanding of the functionality of distinct subcellular microdomains in SAN myocytes and their functional role in the accumulation and neurohormonal regulation of proteins involved in cardiac pacemaking. We also demonstrate how changes in scaffolding proteins may lead to microdomain-targeted remodeling and regulation of pacemaker proteins contributing to SAN dysfunction.

Application of micro-Raman spectroscopy for the monitoring of quality of high-k (h-k) dielectric protective layer deposition onto the surface of a nanowire (NW) chip has been demonstrated. A NW chip based on silicon-on-insulator (SOI) structures, protected with a layer of high-k dielectric ((h-k)-SOI-NW chip), has been employed for highly sensitive detection of microRNA (miRNA) associated with oncological diseases. The protective dielectric included a 2-nm-thick Al₂O₃ surface layer and a 8-nm-thick HfO₂ layer, deposited onto a silicon SOI-NW chip. Such a chip had increased time stability upon operation in solution, as compared with an unprotected SOI-NW chip with native oxide. The (h-k)-SOI-NW biosensor has been employed for the detection of DNA oligonucleotide (oDNA), which is a synthetic analogue of miRNA-21 associated with oncological diseases. To provide biospecificity of the detection, the surface of (h-k)-SOI-NW chip was modified with oligonucleotide probe molecules (oDVA probes) complementary to the sequence of the target biomolecule. Concentration sensitivity of the (h-k)-SOI-NW biosensor at the level of DL ~10 -16 M has been demonstrated.

Background: Increased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated., Methods: High-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes., Results: TC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (-10.2±1.5 versus -5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (-22.3±1.1 versus -9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L., Conclusion: Our findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA.

Aims: Cardiomyocyte β2-adrenergic receptor (β2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors' subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), β2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon β2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown., Methods and Results: Rat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), β2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local β2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the β2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell β2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve β2AR-mediated signal compartmentation or reduce cAMP diffusion., Conclusion: Although changes in T-tubule structure and β2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of β2AR-mediated cAMP is temporarily reversed. Complete disorganization of β2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI., (© The Author 2017 Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: The PGC-1α/PPAR axis has been proposed as a potential therapeutic target for several metabolic disorders. The aim was to evaluate the efficacy of the pan-PPAR agonist, bezafibrate, in tafazzin knockdown mice (TazKD), a mouse model of Barth syndrome that exhibits age-dependent dilated cardiomyopathy with left ventricular (LV) dysfunction., Results: The effect of bezafibrate on cardiac function was evaluated by echocardiography in TazKD mice with or without beta-adrenergic stress. Adrenergic stress by chronic isoproterenol infusion exacerbates the cardiac phenotype in TazKD mice, significantly depressing LV systolic function by 4.5 months of age. Bezafibrate intake over 2 months substantially ameliorates the development of LV systolic dysfunction in isoproterenol-stressed TazKD mice. Without beta-adrenergic stress, TazKD mice develop dilated cardiomyopathy by 7 months of age. Prolonged treatment with suprapharmacological dose of bezafibrate (0.5% in rodent diet) over a 4-month period effectively prevented LV dilation in mice isoproterenol treatment. Bezafibrate increased mitochondrial biogenesis, however also promoted oxidative stress in cardiomyocytes. Surprisingly, improvement of systolic function in bezafibrate-treated mice was accompanied with simultaneous reduction of cardiolipin content and increase of monolysocardiolipin levels in cardiac muscle., Conclusions: Thus, we demonstrate that bezafibrate has a potent therapeutic effect on preventing cardiac dysfunction in a mouse model of Barth syndrome with obvious implications for treating the human disease. Additional studies are needed to assess the potential benefits of PPAR agonists in humans with Barth syndrome.

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Rationale: Disruption in subcellular targeting of Ca(2+) signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias., Objective: To explore microdomain-targeted remodeling of ventricular L-type Ca(2+) channels (LTCCs) in HF., Methods and Results: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium-calmodulin kinase II-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias., Conclusions: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease., (© 2016 The Authors.)

Heart failure (HF) claims 250,000 lives per year in the US, and nearly half of these deaths are sudden and presumably due to ventricular tachyarrhythmias. QT interval and action potential (AP) prolongation are hallmark proarrhythmic changes in the failing myocardium, which potentially result from alterations in repolarizing potassium currents. Thus, we aimed to examine whether decreased expression of the rapid delayed rectifier potassium current, IKr, contributes to repolarization abnormalities in human HF. To map functional IKr expression across the left ventricle (LV), we optically imaged coronary-perfused LV free wall from donor and end-stage failing human hearts. The LV wedge preparation was used to examine transmural AP durations at 80% repolarization (APD80), and treatment with the IKr-blocking drug, E-4031, was utilized to interrogate functional expression. We assessed the percent change in APD80 post-IKr blockade relative to baseline APD80 (∆APD80) and found that ∆APD80s are reduced in failing versus donor hearts in each transmural region, with 0.35-, 0.43-, and 0.41-fold reductions in endo-, mid-, and epicardium, respectively (p=0.008, 0.037, and 0.022). We then assessed hERG1 isoform gene and protein expression levels using qPCR and Western blot. While we did not observe differences in hERG1a or hERG1b gene expression between donor and failing hearts, we found a shift in the hERG1a:hERG1b isoform stoichiometry at the protein level. Computer simulations were then conducted to assess IKr block under E-4031 influence in failing and nonfailing conditions. Our results confirmed the experimental observations and E-4031-induced relative APD80 prolongation was greater in normal conditions than in failing conditions, provided that the cellular model of HF included a significant downregulation of IKr. In human HF, the response to IKr blockade is reduced, suggesting decreased functional IKr expression. This attenuated functional response is associated with altered hERG1a:hERG1b protein stoichiometry in the failing human LV, and failing cardiomyoctye simulations support the experimental findings. Thus, of IKr protein and functional expression may be important determinants of repolarization remodeling in the failing human LV., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs)., Methods and Results: Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈ 40% greater current. Optical mapping of Ca(2+) transients revealed that rat AMs presented ≈ 3-fold as many spontaneous Ca(2+) release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca(2+) transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈ 30% whole-cell ICa,L reduction. Heart failure (16 weeks post-myocardial infarction) in rats resulted in a T-tubule degradation (by ≈ 40%) and significant elevation of spontaneous Ca(2+) release events. Although heart failure did not affect LTCC occurrence, it led to ≈ 25% decrease in T-tubule LTCC amplitude., Conclusions: We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner., (© 2015 The Authors.)

Introduction: Despite the importance of neurogenic initiation of rapid firing from pulmonary veins (PVs), the mechanism of autonomic modulation of electrophysiological properties of the PV myocardium to form a substrate for atrial arrhythmia remains poorly understood., Methods and Results: A 2-microelectrode technique was used to characterize electrophysiological properties of rat PV myocardium and to explore PV arrhythmogenesis, at baseline, during electrical stimulation and/or under autonomic modulation. PV myocardium was characterized by prolonged action potential duration (APD), high degree of APD alternans, and spontaneous depolarizations. Autonomic stimulation resulted in significantly enhanced APD dispersion within the PV, which dynamically changed over time and was associated with intra-PV and atria-PV conduction blocks and could lead to spontaneous fibrillation-like high-frequency activity. In the distal part of the PV we found an unexcitable area that was characterized by depolarized resting potential (-50 ± 4 mV vs. -75 ± 2 mV vs. PV mouth, P < 0.01). This region could be activated during autonomic stimulation or fast pacing that led to multiple conduction discontinuities (uni- and bi-directional conduction blocks, Wenckebach periodicity, electrotonic modulation conduction block, echo phenomenon) in 17/23 preparations, including those occurring under norepinephrine superfusion (14/17) and during pacing frequency changes (3/17). PV echoes (unstable reentrant circuits) were found in 8/23 preparations. In some experiments, several types of conduction abnormalities were observed., Conclusion: The PV myocardium demonstrates distinct electrophysiological characteristics, which could be considerably exaggerated by electrical stimulation and/or autonomic nervous system to dynamically form a functional substrate to support re-entry as well as focal activity., (© 2015 Wiley Periodicals, Inc.)

Aims: Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2., Methods and Results: In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex., Conclusions: Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2013. For permissions please email: journals.permissions@oup.com.)

Cardiac excitation involves the generation of action potential by individual cells and the subsequent conduction of the action potential from cell to cell through intercellular gap junctions. Excitation of the cellular membrane results in opening of the voltage-gated L-type calcium ion (Ca 2+ ) channels, thereby allowing a small amount of Ca 2+ to enter the cell, which in turn triggers the release of a much greater amount of Ca 2+ from the sarcoplasmic reticulum, the intracellular Ca 2+ store, and gives rise to the systolic Ca 2+ transient and contraction. These processes are highly regulated by the autonomic nervous system, which ensures the acute and reliable contractile function of the heart and the short-term modulation of this function upon changes in heart rate or workload. It has recently become evident that discrete clusters of different ion channels and regulatory receptors are present in the sarcolemma, where they form an interacting network and work together as a part of a macro-molecular signalling complex which in turn allows the specificity, reliability and accuracy of the autonomic modulation of the excitation-contraction processes by a variety of neurohormonal pathways. Disruption in subcellular targeting of ion channels and associated signalling proteins may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure and certain arrhythmias. Recent methodological advances have made it possible to routinely image the topography of live cardiomyocytes, allowing the study of clustering functional ion channels and receptors as well as their coupling within a specific microdomain. In this review we highlight the emerging understanding of the functionality of distinct subcellular microdomains in cardiac myocytes (e.g. T-tubules, lipid rafts/caveolae, costameres and intercalated discs) and their functional role in the accumulation and regulation of different subcellular populations of sodium, Ca 2+ and potassium ion channels and their contributions to cellular signalling and cardiac pathology.

Atrial fibrillation (AF) associated with fibrosis is characterized by the appearance of interstitial myofibroblasts. These cells are responsible for the uncontrolled deposition of the extracellular matrix, which pathologically separate cardiomyocyte bundles. The enhanced fibrosis is thought to contribute to arrhythmias "indirectly" because a collagenous septum is a passive substrate for propagation, resulting in impulse conduction block and/or zigzag conduction. However, the emerging results demonstrate that myofibroblasts in vitro also promote arrhythmogenesis due to direct implications upon cardiomyocyte electrophysiology. This electrical interference may be considered beneficial as it resolves any conduction blocks; however, the passive properties of myofibroblasts might cause a delay in impulse propagation, thus promoting AF due to discontinuous slow conduction. Moreover, low-polarized myofibroblasts reduce, via cell-density dependence, the fast driving inward current for cardiac impulse conduction, therefore resulting in arrhythmogenic uniformly slow propagation. Critically, the subsequent reduction in cardiomyocytes resting membrane potential in vitro significantly increases the likelihood of ectopic activity. Myofibroblast densities and the degree of coupling at cellular border zones also impact upon this likelihood. By considering future in vivo studies, which identify myofibroblasts "per se" as a novel targets for cardiac arrhythmias, this review aims to describe the implications of noncardiomyocyte view in the context of AF.