Potential Arrhythmogenic Role of TRPC Channels and Store-Operated Calcium Entry Mechanism in Mouse Ventricular Myocytes

Background and Purpose: Store-operated calcium entry (SOCE) is an important physiological phenomenon that extensively mediates intracellular calcium ion (Ca2+) load. It has been previously found in myocytes isolated from neonatal or diseased hearts. We aimed to determine its existence, molecular nature in undiseased hearts and its potential arrhythmogenic implications under hyperactive conditions.Experimental Approach: Ventricular myocytes isolated from adult FVB mice were studied by using Ca2+ imaging and whole-cell perforated patch-clamp recording. In addition, lead II ECGs were recorded in isolated Langendorff-perfused mice hearts. Functional TRPC channel antibodies and inhibitors, and TRPC6 activator hyperforin were used.Key Results: In this study, we demonstrate the existence and contribution of SOCE in normal adult mouse cardiac myocytes. For an apparent SOCE activation, complete depletion of sarcoplasmic reticulum (SR) Ca2+ by employing both caffeine (10 mM) and thapsigargin (1 μM) or cyclopiazonic acid (10 μM) was required. Consistent with the notion that SOCE may be mediated by heteromultimeric TRPC channels, SOCEs observed from those myocytes were significantly reduced by the pretreatment with anti-TRPC1, 3, and 6 antibodies as well as by gadolinium, a non-selective TRPC channel blocker. In addition, we showed that SOCE may regulate spontaneous SR Ca2+ release, Ca2+ waves, and triggered activities which may manifest cardiac arrhythmias. Since the spontaneous depolarization in membrane potential preceded the elevation of intracellular Ca2+, an inward membrane current presumably via TRPC channels was considered as the predominant cause of cellular arrhythmias. The selective TRPC6 activator hyperforin (0.1–10 μM) significantly facilitated the SOCE, SOCE-mediated inward current, and calcium load in the ventricular myocytes. ECG recording further demonstrated the proarrhythmic effects of hyperforin in ex vivo mouse hearts.Conclusion and Implications: We suggest that SOCE, which is at least partially mediated by TRPC channels, exists in adult mouse ventricular myocytes. TRPC channels and SOCE mechanism may be involved in cardiac arrhythmogenesis via promotion of spontaneous Ca2+ waves and triggered activities under hyperactivated conditions

Emerging technologies and their impact on regulatory science

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools

The contribution of the intracellular domains to P2X1 receptor regulation

P2X1 receptors are expressed throughout the body and contribute to a range of physiological process, e.g. thrombosis and smooth muscle tone. The intracellular terminals of the P2X1 receptor have been shown to be involved in channel regulation. The aim of this thesis was to explore the contribution of the intracellular amino and carboxy termini to channel regulation, using concatenation, site-directed mutagenesis and biochemical methods. Trimeric concatenated P2X1 receptors were non-functional.The interactions between the intracellular termini were revealed to be essential for subunit aggregation and channel expression. Subsequently, over 30 cysteine point mutations at both the amino and carboxy domains of the P2X1 receptors were generated and examined by electrophysiological studies with methanethiosulfonate modifications. The conserved TXTXK/R at the amino terminal and the residues around the YXXXK motif at the carboxy terminal were highlighted to be important for channel gating and protein trafficking. Residues close to the P2X1 channel pore were shown to be essential in ion conduction and implied interactions between the conserved regulatory motifs and the channel pore of adjacent residues. Roles of the intracellular domains of P2X1 receptor in regulation by protein kinase were revealed by co-expression of minigenes encoding the sequences of amino/carboxy terminus with WT receptors. The results indicated both amino and carboxy termini contribute to the phorbol ester PMA and GPCR mediated response regulations, and demonstrated the important roles of the conserved TXTXK/R motif and the residues around the YXXXK motif. This research sheds light on the possible interactions between the amino and carboxy termini to P2X1 receptor function. The results will be useful for understanding the intracellular topology and signaling modulation mechanism of P2X1 receptor

The contribution of the intracellular domains to P2X1 receptor regulation

P2X1 receptors are expressed throughout the body and contribute to a range of physiological process, e.g. thrombosis and smooth muscle tone. The intracellular terminals of the P2X1 receptor have been shown to be involved in channel regulation. The aim of this thesis was to explore the contribution of the intracellular amino and carboxy termini to channel regulation, using concatenation, site-directed mutagenesis and biochemical methods. Trimeric concatenated P2X1 receptors were non-functional.The interactions between the intracellular termini were revealed to be essential for subunit aggregation and channel expression. Subsequently, over 30 cysteine point mutations at both the amino and carboxy domains of the P2X1 receptors were generated and examined by electrophysiological studies with methanethiosulfonate modifications. The conserved TXTXK/R at the amino terminal and the residues around the YXXXK motif at the carboxy terminal were highlighted to be important for channel gating and protein trafficking. Residues close to the P2X1 channel pore were shown to be essential in ion conduction and implied interactions between the conserved regulatory motifs and the channel pore of adjacent residues. Roles of the intracellular domains of P2X1 receptor in regulation by protein kinase were revealed by co-expression of minigenes encoding the sequences of amino/carboxy terminus with WT receptors. The results indicated both amino and carboxy termini contribute to the phorbol ester PMA and GPCR mediated response regulations, and demonstrated the important roles of the conserved TXTXK/R motif and the residues around the YXXXK motif. This research sheds light on the possible interactions between the amino and carboxy termini to P2X1 receptor function. The results will be useful for understanding the intracellular topology and signaling modulation mechanism of P2X1 receptor.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

Recent studies have suggested that mitochondria may play important roles in the Ca(2+) homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+) flux can regulate the generation of Ca(2+) waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+) (Cai (2+)) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and CaWs were induced in the presence of high (4 mM) external Ca(2+) (Cao (2+)). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai (2+) levels even after depletion of SR Ca(2+) in the absence of Cao (2+) , suggesting Ca(2+) release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m ) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m ) or Ru360 (a mitochondrial Ca(2+) uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+) uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+) release and uptake exquisitely control the local Ca(2+) level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis

From Metal Thiobenzoates to Metal Sulfide Nanocrystals: An Experimental and Theoretical Investigation

nanomaterial

INVITED REVIEW Molecular properties of P2X receptors

Abstract P2X receptors for adenosine tri-phosphate (ATP) are a distinct family of ligand-gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven genes (P2X 1-7 ) have been cloned and the channels form as either homo or heterotrimeric channels giving rise to a wide range of phenotypes. This review aims to give an account of recent work on the molecular basis of the properties of P2X receptors. In particular, to consider emerging information on the assembly of P2X receptor subunits, channel regulation and desensitisation, targeting, the molecular basis of drug action and the functional contribution of P2X receptors to physiological processes

Action potentials (APs) were triggered by FCCP-enhanced Ca_i²⁺ waves.

<p><b>A</b>. A line-scan image along the long axis of the cell (A-a), whole-cell Ca fluorescence intensity (A-b), and membrane potential (A-c), were recorded simultaneously from a ventricular myocyte. CaWs (W) and sub-theshold depolarizations (SD) were exacerbated by FCCP (100 nM) so that triggered APs/activities (TA) and Ca transients (T) were induced. <b>B</b>. Summary data showing that FCCP (100 nM) markedly increased the incidence of TAs (**<i>p</i> < 0.01 vs. control, n = 4). </p

Effect of mCU Ca²⁺ flux on CaWs.

<p><b>A</b>. Kaempferol, a mitochondrial uniporter (mCU) opener, significantly attenuated CaWs induced by 100nM FCCP. A representative trace (A-<b>a</b>) and summarized data for CaW frequency (A-<b>b</b>) and basal [Ca_i²⁺] level (<b>c</b>) are shown (**<i>p</i> < 0.01 vs. control; #<i>p</i> < 0.05 vs. FCCP, n = 5). <b>B</b>. The effect of Ru360, a mCU blocker, on CaW frequency and basal [Ca_i²⁺] level in the presence of 50 nM FCCP. A representative trace (B-<b>a</b>) and summarized data (B-b & c) are shown. ^#<i>p</i> < 0.05 vs. FCCP (n = 5). <b>C</b>. The effect of Ru360 on CaW in the absence of FCCP. Note a persistent and fast oscillating status after Ru360 treatment. </p