Neurotoxic mechanisms of triclosan: The antimicrobial agent emerging as a toxicant.

Several scientific studies have suggested a link between increased exposure to pollutants and a rise in the number of neurodegenerative disorders of unknown origin. Notably, triclosan (an antimicrobial agent) is used in concentrations ranging from 0.3% to 1% in various consumer products. Recent studies have also highlighted triclosan as an emerging toxic pollutant due to its increasing global use. However, a definitive link is missing to associate the rising use of triclosan and the growing number of neurodegenerative disorders or neurotoxicity. In this article, we present systematic scientific evidence which are otherwise scattered to suggest that triclosan can indeed induce neurotoxic effects, especially in vertebrate organisms including humans. Mechanistically, triclosan affected important developmental and differentiation genes, structural genes, genes for signaling receptors and genes for neurotransmitter controlling enzymes. Triclosan-induced oxidative stress impacting cellular proteins and homeostasis which triggers apoptosis. Though the scientific evidence collated in this article unequivocally indicates that triclosan can cause neurotoxicity, further epidemiological studies may be needed to confirm the effects on humans.

Glyphosate induces toxicity and modulates calcium and NO signaling in zebrafish embryos.

Glyphosate, an herbicide used worldwide, has emerged as a pollutant. However, its toxic effects are debated by regulatory authorities. Therefore, it is essential to keep the use of such chemicals under continuous observation, and their effects must be re-evaluated. We used zebrafish embryos to evaluate the toxic effects of glyphosate and its mechanisms. We found that glyphosate induced significant toxicity in a time and concentration-dependent manner. We observed an LD50 of 66.04 ±â€¯4.6 µg/mL after 48 h of exposure. Glyphosate significantly reduced the heartbeat in a time and concentration-dependent manner indicating cardiotoxicity. Selective downregulation of Cacana1C (L-type calcium channel) and ryr2a (Ryanodine receptor) genes along with selective upregulation of hspb11 (heat shock protein) gene was observed upon exposure to glyphosate indicating alterations in the calcium signaling. A reduction in the nitric oxide (NO) generation was also observed in the zebrafish embryos upon exposure to glyphosate. Our results indicate that glyphosate induces significant toxicity including cardiotoxicity in zebrafish embryos in a time and concentration-dependent manner. Further, cardiotoxicity may be due to changes in calcium and NO signaling.

T-Type voltage gated calcium channels: a target in breast cancer?

PURPOSE: The purpose of this review article is to discuss the potential of T-type voltage gated calcium channels (VGCCs) as drug targets in breast cancer. Breast cancer, attributable to the different molecular subtypes, has a crucial need for therapeutic strategies to counter the mortality rate. VGCCs play an important role in regulating cytosolic free calcium levels which regulate cellular processes like tumorigenesis and cancer progression. In the last decade, T-type VGCCs have been investigated in breast cancer proliferation. Calcium channel blockers, in general, have shown an anti-proliferative and cytotoxic effects. T-type VGCC antagonists have shown growth inhibition owing to the inhibition of CaV3.2 isoform. CaV3.1 isoform has been indicated as a tumour-suppressor gene candidate and is reported to support anti-proliferative and apoptotic activity in breast cancer. The distribution of T-type VGCC isoforms in different breast cancer molecular subtypes is diverse and needs to be further investigated. The role of T-type VGCCs in breast cancer migration, metastasis and more importantly in epithelial to mesenchymal transition (EMT) is yet to be elucidated. In addition, interlaced therapy, using a combination of chemotherapy drugs and T-type VGCC blockers, presents a promising therapeutic approach for breast cancer but more validation and clinical trials are needed. Also, for investigating the potential of T-type VGCC blocker therapy, there is a need for isoform-specific agonists/antagonists to define and discover roles of T-type VGCC specific isoforms. CONCLUSION: Our article provides a review of the role of T-type VGCCs in breast cancer and also discusses future of the research in this area so that it can be ascertained whether there is any potential of T-type VGCCs as drug targets in breast cancer.

Sodium benzoate induced developmental defects, oxidative stress and anxiety-like behaviour in zebrafish larva.

Sodium benzoate (SB) is a common food preservative. Its FDA described safety limit is 1000 ppm. Lately, increased use of SB has prompted investigations regarding its effects on biological systems. Data regarding toxicity of SB is divergent and controversial with studies reporting both harmful and beneficial effects. Therefore, we did a systematic dose dependent toxicity study of SB using zebrafish vertebrate animal model. We also investigated oxidative stress and anxiety-like behaviour in zebrafish larva treated with SB. Our results indicate that SB induced developmental (delayed hatching), morphological (pericardial edema, yolk sac edema and tail bending), biochemical (oxidative stress) and behavioural (anxiety-like behaviour) abnormalities in developing zebrafish larva. LC50 of SB induced toxicity was approximately 400 ppm after 48 h of SB exposure. Our study strongly supports its harmful effects on vertebrates at increasing doses. Thus, we suggest caution in the excessive use of this preservative in processed and convenience foods.

Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure.

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.

Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes.

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-ß-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.

A critical role for Telethonin in regulating t-tubule structure and function in the mammalian heart.

The transverse (t)-tubule system plays an essential role in healthy and diseased heart muscle, particularly in Ca(2+)-induced Ca(2+) release (CICR), and its structural disruption is an early event in heart failure. Both mechanical overload and unloading alter t-tubule structure, but the mechanisms mediating the normally tight regulation of the t-tubules in response to load variation are poorly understood. Telethonin (Tcap) is a stretch-sensitive Z-disc protein that binds to proteins in the t-tubule membrane. To assess its role in regulating t-tubule structure and function, we used Tcap knockout (KO) mice and investigated cardiomyocyte t-tubule and cell structure and CICR over time and following mechanical overload. In cardiomyocytes from 3-month-old KO (3mKO), there were isolated t-tubule defects and Ca(2+) transient dysynchrony without whole heart and cellular dysfunction. Ca(2+) spark frequency more than doubled in 3mKO. At 8 months of age (8mKO), cardiomyocytes showed progressive loss of t-tubules and remodelling of the cell surface, with prolonged and dysynchronous Ca(2+) transients. Ca(2+) spark frequency was elevated and the L-type Ca(2+) channel was depressed at 8 months only. After mechanical overload obtained by aortic banding constriction, the Ca(2+) transient was prolonged in both wild type and KO. Mechanical overload increased the Ca(2+) spark frequency in KO alone, where there was also significantly more t-tubule loss, with a greater deterioration in t-tubule regularity. In conjunction, Tcap KO showed severe loss of cell surface ultrastructure. These data suggest that Tcap is a critical, load-sensitive regulator of t-tubule structure and function.

Super-resolution scanning patch clamp reveals clustering of functional ion channels in adult ventricular myocyte.

RATIONALE: Compartmentation of ion channels on the cardiomyocyte surface is important for electric propagation and electromechanical coupling. The specialized T-tubule and costameric structures facilitate spatial coupling of various ion channels and receptors. Existing methods such as immunofluorescence and patch clamp techniques are limited in their ability to localize functional ion channels. As such, a correlation between channel protein location and channel function remains incomplete. OBJECTIVE: To validate a method that permits routine imaging of the topography of a live cardiomyocyte and study clustering of functional ion channels from a specific microdomain. METHODS AND RESULTS: We used scanning ion conductance microscopy and conventional cell-attached patch clamp with a software modification that allows controlled increase of pipette tip diameter. The sharp nanopipette used for topography scan was modified into a larger patch pipette that could be positioned with nanoscale precision to a specific site of interest (crest, groove, or T-tubules of cardiomyocytes) and sealed to the membrane for cell-attached recording of ion channels. Using this method, we significantly increased the probability of detecting activity of L-type calcium channels in the T-tubules of ventricular cardiomyocytes. We also demonstrated that active sodium channels do not distribute homogenously on the sarcolemma instead, they segregate into clusters of various densities, most crowded in the crest region, that are surrounded by areas virtually free of functional sodium channels. CONCLUSIONS: Our new method substantially increases the throughput of recording location-specific functional ion channels on the cardiomyocyte sarcolemma, thereby allowing characterization of ion channels in relation to the microdomain where they reside.

Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of ß2-adrenergic receptor (ß2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate ß2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of ß2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased ß2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, ß2AR response could only be generated in T-tubules. However, the normally compartmentalized ß2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial ß2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of ß2AR and compartmentation of ß2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.

The scanning ion conductance microscope for cellular physiology.

The quest for nonoptical imaging methods that can surmount light diffraction limits resulted in the development of scanning probe microscopes. However, most of the existing methods are not quite suitable for studying biological samples. The scanning ion conductance microscope (SICM) bridges the gap between the resolution capabilities of atomic force microscope and scanning electron microscope and functional capabilities of conventional light microscope. A nanopipette mounted on a three-axis piezo-actuator, scans a sample of interest and ion current is measured between the pipette tip and the sample. The feedback control system always keeps a certain distance between the sample and the pipette so the pipette never touches the sample. At the same time pipette movement is recorded and this generates a three-dimensional topographical image of the sample surface. SICM represents an alternative to conventional high-resolution microscopy, especially in imaging topography of live biological samples. In addition, the nanopipette probe provides a host of added modalities, for example using the same pipette and feedback control for efficient approach and seal with the cell membrane for ion channel recording. SICM can be combined in one instrument with optical and fluorescent methods and allows drawing structure-function correlations. It can also be used for precise mechanical force measurements as well as vehicle to apply pressure with precision. This can be done on living cells and tissues for prolonged periods of time without them loosing viability. The SICM is a multifunctional instrument, and it is maturing rapidly and will open even more possibilities in the near future.

Voltage-Gated T-Type Calcium Channel Modulation by Kinases and Phosphatases: The Old Ones, the New Ones, and the Missing Ones.

Calcium (Ca2+) can regulate a wide variety of cellular fates, such as proliferation, apoptosis, and autophagy. More importantly, changes in the intracellular Ca2+ level can modulate signaling pathways that control a broad range of physiological as well as pathological cellular events, including those important to cellular excitability, cell cycle, gene-transcription, contraction, cancer progression, etc. Not only intracellular Ca2+ level but the distribution of Ca2+ in the intracellular compartments is also a highly regulated process. For this Ca2+ homeostasis, numerous Ca2+ chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca2+. T-type Ca2+ channels (TTCCs) are one of those specialized proteins which play a key role in the signal transduction of many excitable and non-excitable cell types. TTCCs are low-voltage activated channels that belong to the family of voltage-gated Ca2+ channels. Over decades, multiple kinases and phosphatases have been shown to modulate the activity of TTCCs, thus playing an indirect role in maintaining cellular physiology. In this review, we provide information on the kinase and phosphatase modulation of TTCC isoforms Cav3.1, Cav3.2, and Cav3.3, which are mostly described for roles unrelated to cellular excitability. We also describe possible potential modulations that are yet to be explored. For example, both mitogen-activated protein kinase and citron kinase show affinity for different TTCC isoforms; however, the effect of such interaction on TTCC current/kinetics has not been studied yet.

Interactions between genes altered during cardiotoxicity and neurotoxicity in zebrafish revealed using induced network modules analysis.

As the manufacturing and development of new synthetic compounds increase to keep pace with the expanding global demand, adverse health effects due to these compounds are emerging as critical public health concerns. Zebrafish have become a prominent model organism to study toxicology due to their genomic similarity to humans, optical clarity, well-defined developmental stages, short generation time, and cost-effective maintenance. It also provides a shorter time frame for in vivo toxicology evaluation compared to the mammalian experimental systems. Here, we used meta-analysis to examine the alteration in genes during cardiotoxicity and neurotoxicity in zebrafish, caused by chemical exposure of any kind. First, we searched the literature comprehensively for genes that are altered during neurotoxicity and cardiotoxicity followed by meta-analysis using ConsensusPathDB. Since constant communication between the heart and the brain is an important physiological phenomenon, we also analyzed interactions among genes altered simultaneously during cardiotoxicity and neurotoxicity using induced network modules analysis in ConsensusPathDB. We observed inflammation and regeneration as the major pathways involved in cardiotoxicity and neurotoxicity. A large number of intermediate genes and input genes anchored in these pathways are molecular regulators of cell cycle progression and cell death and are implicated in tumor manifestation. We propose potential predictive biomarkers for neurotoxicity and cardiotoxicity and the major pathways potentially implicated in the manifestation of a particular toxicity phenotype.

Intercommunication between Voltage-Gated Calcium Channels and Estrogen Receptor/Estrogen Signaling: Insights into Physiological and Pathological Conditions.

Voltage-gated calcium channels (VGCCs) and estrogen receptors are important cellular proteins that have been shown to interact with each other across varied cells and tissues. Estrogen hormone, the ligand for estrogen receptors, can also exert its effects independent of estrogen receptors that collectively constitute non-genomic mechanisms. Here, we provide insights into the VGCC regulation by estrogen and the possible mechanisms involved therein across several cell types. Notably, most of the interaction is described in neuronal and cardiovascular tissues given the importance of VGCCs in these electrically excitable tissues. We describe the modulation of various VGCCs by estrogen known so far in physiological conditions and pathological conditions. We observed that in most in vitro studies higher concentrations of estrogen were used while a handful of in vivo studies used meager concentrations resulting in inhibition or upregulation of VGCCs, respectively. There is a need for more relevant physiological assays to study the regulation of VGCCs by estrogen. Additionally, other interacting receptors and partners need to be identified that may be involved in exerting estrogen receptor-independent effects of estrogen.

New insights into inhibitory nature of triclosan on acetylcholinesterase activity.

The antimicrobial agent, triclosan, has been designated as a "contaminant of emerging concern (CEC)". Previous in vivo studies have shown that triclosan exposure can inhibit acetylcholinesterase (AChE) activity. However, mechanistic insights into AChE inhibition by triclosan are missing. Here, using in vitro activity assay with purified AChE, we show that triclosan can directly inhibit AChE. In vivo, triclosan exposure resulted in reduced total antioxidant capacity concomitant with reduced AChE activity in the adult zebrafish brain. Adult zebrafish when pre-treated with antioxidant melatonin, resulted in attenuated oxidative stress and attenuated inhibitory effect of triclosan on the AChE activity. Our results indicate that triclosan can affect AChE activity both by direct binding and indirectly through increased oxidative stress and therefore, provide important mechanistic insights into triclosan induced neurotoxicity.

Triclosan affects motor function in zebrafish larva by inhibiting ache and syn2a genes.

The widespread use of triclosan in personal care products as an antimicrobial agent is leading to its alarming tissue-bioaccumulation including human brain. However, knowledge of its potential effects on the vertebrate nervous system is still limited. Here, we hypothesized that sublethal triclosan concentrations are potent enough to alter motor neuron structure and function in zebrafish embryos exposed for prolonged duration. In this study, zebrafish embryos were used as vertebrate-animal model. Prolonged exposure (up to 4 days) of 0.6 mg/L (LC50, 96 h) and 0.3 mg/L (

Triclosan alters adult zebrafish behavior and targets acetylcholinesterase activity and expression.

Triclosan is widely used in consumer products as an antimicrobial agent. Epidemiological studies have reported the association of triclosan with adverse birth outcomes. The toxic effects of triclosan on the developing stages of zebrafish are reported, however, its role as behavioral modifier is limited. In the present study, adult zebrafish were exposed to triclosan (0.3 and 0.6 mg/L) for 48 h and the exploratory behavior was analyzed using ZebraTrack. Triclosan exposed group showed significantly reduced locomotion concomitant with increased freezing duration. They also showed erratic movements suggesting that triclosan induced anxiety-like behavior in adult zebrafish. Next, we tested the hypothesis that the anxiety-like behavior is linked to altered acetylcholinesterase activity. We found that the triclosan exposure decreased acetylcholinesterase activity in the brain and skeletal muscle but acetylcholinesterase (ache) gene was significantly down-regulated only in the skeletal muscle of the adult zebrafish exposed to triclosan. In addition, we also observed a down-regulation of myelin basic protein (mbp) gene in the skeletal muscle of adult zebrafish treated with triclosan. Thus, our data indicates that even short exposure of triclosan is potent enough to induce behavioral anomalies in adult zebrafish that appear to involve acetylcholinesterase and other structural proteins especially in the skeletal muscle.

T-type calcium channel antagonist, TTA-A2 exhibits anti-cancer properties in 3D spheroids of A549, a lung adenocarcinoma cell line.

AIMS: Despite the advanced cancer treatments, there is increased resistance to chemotherapy and subsequent mortality. In lack of reliable data in monolayer cultures and animal models, researchers are shifting to 3D cancer spheroids, which represents the in vivo robust tumour morphology. Calcium is essential in cell signalling and proliferation. It is found that T-type calcium channels (TTCCs) are overexpressed in various cancer cells, supporting their increased proliferation. Many of the TTCCs blockers available could target other channels besides TTCCs, which can cause adverse effects. Therefore, we hypothesise that TTA-A2, a highly selective blocker towards TTCCs, can inhibit the growth of cancer spheroids, and provide an anti-cancer and an adjuvant role in cancer therapy. METHODS: We studied TTA-A2 and paclitaxel (PTX-control drug) in lung adenocarcinoma cell line- A549, cancer cells and human embryonic kidney cell line- HEK 293, control cell, in their monolayer and spheroids forms for viability, proliferation, morphology change, migration, and invasion-after 48-96 h of treatment. KEY FINDINGS: Though the results varied between the monolayer and spheroids studies, we found both anti-cancer as well as adjuvant effect of TTA-A2 in both the studies. TTA-A2 was able to inhibit the growth, viability, and metastasis of the cancer cells and spheroids. Differences in the results of two modes might explain that why drugs tested successfully in monolayer culture fail in clinical trials. SIGNIFICANCE: This study establishes the role of TTA-A2, a potent TTCC blocker as an anti-cancer and adjuvant drug in reducing the viability and metastasis of the cancer cells.

Cardiac voltage gated calcium channels and their regulation by ß-adrenergic signaling.

Voltage-gated calcium channels (VGCCs) are the predominant source of calcium influx in the heart leading to calcium-induced calcium release and ultimately excitation-contraction coupling. In the heart, VGCCs are modulated by the ß-adrenergic signaling. Signaling through ß-adrenergic receptors (ßARs) and modulation of VGCCs by ß-adrenergic signaling in the heart are critical signaling and changes to these have been significantly implicated in heart failure. However, data related to calcium channel dysfunction in heart failure is divergent and contradictory ranging from reduced function to no change in the calcium current. Many recent studies have highlighted the importance of functional and spatial microdomains in the heart and that may be the key to answer several puzzling questions. In this review, we have briefly discussed the types of VGCCs found in heart tissues, their structure, and significance in the normal and pathological condition of the heart. More importantly, we have reviewed the modulation of VGCCs by ßARs in normal and pathological conditions incorporating functional and structural aspects. There are different types of ßARs, each having their own significance in the functioning of the heart. Finally, we emphasize the importance of location of proteins as it relates to their function and modulation by co-signaling molecules. Its implication on the studies of heart failure is speculated.

ZebraPace: An Open-Source Method for Cardiac-Rhythm Estimation in Untethered Zebrafish Larvae.

For the assessment of cardiac function, heartbeat represents one key parameter. Current methods of heartbeat measurements in the zebrafish larvae usually require larval immobilization, fluorescent transgenic strains and a confocal microscope, costly commercial software for analysis, or strong programming skills if the software is open-source. Here, we present a simple yet powerful method of heartbeat analysis using untethered, unlabeled zebrafish larva using ImageJ (open-source software), which does not require programming skills. We named it as ZebraPace for Zebrafish Precise Algorithm for Cardiac-rhythm Estimation. ZebraPace works directly with AVI videos and requires no image processing steps. ZebraPace uses pixel intensity change in a grayscale video to count the number of beats. We have validated the ZebraPace method by pharmacological alterations of the heartbeat in zebrafish larvae of 48 and 72 hpf stages. We have also determined beat-to-beat interval, which relates to rhythmicity of heartbeat. The results obtained by using ZebraPace corroborates well with the heartbeat values previously reported for similarly aged larvae as determined by using specialized software. We believe that the ZebraPace method is simple, cost-effective, and easy to grasp as it involves fewer steps. It not only reduces the manual workload but also eliminates sample preparation time and researcher subjectivity.

T-tubule remodelling disturbs localized ß2-adrenergic signalling in rat ventricular myocytes during the progression of heart failure.

AIMS: Cardiomyocyte ß2-adrenergic receptor (ß2AR) 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), ß2ARs 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 ß2AR-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), ß2ARs 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 ß2AR-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 ß2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell ß2AR-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 ß2AR-mediated signal compartmentation or reduce cAMP diffusion. CONCLUSION: Although changes in T-tubule structure and ß2AR-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 ß2AR-mediated cAMP is temporarily reversed. Complete disorganization of ß2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.