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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
Calcium (Ca
) can regulate a wide variety of cellular fates, such as proliferation, apoptosis, and autophagy. More importantly, changes in the intracellular Ca
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 Ca
level but the distribution of Ca
in the intracellular compartments is also a highly regulated process. For this Ca
homeostasis, numerous Ca
chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca
. T-type Ca
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 Ca
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
RATIONALE:Disruption in subcellular targeting of Ca 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 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.
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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 transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca 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 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.
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 ± 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.
•Glyphosate induces significant toxicity in zebrafish embryos.•Glyphosate lowers the heartbeat in zebrafish embryos.•Glyphosate modulates calcium signaling in zebrafish embryos.•Glyphosate reduces nitric oxide generation in zebrafish embryos.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Arrhythmogenesis during heart failure is a major clinical problem. Regional electrical gradients produce arrhythmias, and cellular ionic transmembrane gradients are its originators. We investigated ...whether the nanoscale mechanosensitive properties of cardiomyocytes from failing hearts have a bearing upon the initiation of abnormal electrical activity. Hydrojets through a nanopipette indent specific locations on the sarcolemma and initiate intracellular calcium release in both healthy and heart failure cardiomyocytes, as well as in human failing cardiomyocytes. In healthy cells, calcium is locally confined, whereas in failing cardiomyocytes, calcium propagates. Heart failure progressively stiffens the membrane and displaces sub-sarcolemmal mitochondria. Colchicine in healthy cells mimics the failing condition by stiffening the cells, disrupting microtubules, shifting mitochondria, and causing calcium release. Uncoupling the mitochondrial proton gradient abolished calcium initiation in both failing and colchicine-treated cells. We propose the disruption of microtubule-dependent mitochondrial mechanosensor microdomains as a mechanism for abnormal calcium release in failing heart.
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•Nanomechanical pressure application changes mechanosensitivity in failing heart cells•Microtubular network disorganization mediates the change in mechanosensitivity•Mitochondria are displaced from their original location and trigger calcium release•Uncoupling the mitochondrial proton gradient completely abolishes the phenomena
Miragoli et al. show that failing heart cells have altered sensitivity to nanomechanical stimuli mediated by changes in the alignment of microtubules. The microtubule network disorganization leads to displacement of mitochondria and alterations in calcium release.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•Altered expression of TTCCs and ESRs in breast cancer is subtype-dependent.•Alterations in ESR2 co-occur with the alterations in CACNA1H and CACNA1I.•CACNA1G, CACNA1H and ESR2 emerge ...as potential novel targets in luminal type breast cancer.•Downregulation of ESR2 in breast cancer reduces overall patient survival.
T-type voltage-gated calcium channels (TTCCs) have been implicated as novel targets in breast cancer treatment; however, the knowledge of isoform-specific expression of TTCCs in the pathology of breast cancer remains limited. Although estrogen receptor blockers are clinically beneficial, patients often show resistance to these compounds. Interestingly, TTCCs are modulated by estrogen in the brain and heterologous expression systems; however, whether TTCCs and estrogen receptors (ERs) interact in breast tissue is unknown. In addition, the isoform specific expression of TTCCs and ERs in breast cancer subtypes has not been determined.
We investigated the alterations in TTCC and ER gene isoforms, their co-occurrence and the association of alterations with patient survival in breast cancer and its subtypes by using The Cancer Genome Atlas (TCGA) dataset. To understand the functional roles of TTCC and ESR genes in breast cancer, we studied cell proliferation in the presence of a TTCC inhibitor and ER activator.
Our results from the TCGA dataset indicated differential alterations in TTCC isoform expression in patients with breast cancer, including downregulation of CACNA1G and upregulation of CACNA1H, and CACNA1I. Among ER isoforms, upregulation of ESR1 and downregulation of ESR2 were observed. Survival analysis indicated that ESR2 may be a potential target, because its downregulation was associated with shorter overall survival. Subtype specific survival analysis revealed CACNA1G, CACNA1H and ESR2 as potential novel targets in luminal type breast cancer. Alterations in ESR2 co-occurred with alterations in CACNA1H and CACNA1I, thus warranting investigation of potential crosstalk. Experimental data indicated that targeting both TTCCs and ESR2 may be beneficial for luminal type breast cancer.
Our data provide novel insights into the altered expression of TTCC and ESR gene isoforms in breast cancer subtypes and suggest novel therapeutic targets.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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