Research indicates that mentoring has been highly effective in promoting faculty success. Strong mentors in the area of scholarship are extremely valuable for junior faculty, not only because of their research and academic expertise but also for their role modeling behaviors. This paper highlights key components of research mentoring used by a senior nursing faculty member. The senior faculty mentor and junior faculty mentee developed a common vision, relating to research interests in health promotion for vulnerable populations. Impact at the individual, school, university, and society level is discussed, and benefits of mentoring to meet nursing faculty workforce needs are emphasized. In light of the shortage of nursing faculty, it is imperative that current faculty be retained. An essential part of retaining faculty involves fostering the professional growth of junior faculty members. The presence of an endowed chair is particularly vital to the success of junior faculty members: an endowed chair can provide research opportunities to gain high visibility for the nursing discipline, advance a focused research program, and create an environment for nurturing novice faculty members . Quality Fitzpatrick and Carnegie  describeMentoring involves a relationship between two people in which one person with greater experience and/or expertise teaches and counsels the other to develop professionally . The rel Typically one would expect a more senior nurse scientist in the role of mentor and a junior nursing faculty member as mentee, though peer-to peer mentoring is becoming increasingly common as the result of faculty shortages, increasing workloads and budget limitations . RegardlTrust is an essential ingredient for a successful mentoring relationship; in all advising settings, advisors seeking to guide and nurture others must first establish trust . Covey  notes thHeinrich  describeAs mentor and mentee, the authors recognized soon after meeting that they shared a common interest in health promotion and intervention research in community settings. The mentor's community health background and related research expertise were a good fit with the mentee's research interest in successful aging and health promotion of older adults, both in the community and assisted living facilities.Upon their initial meeting, the mentor and mentee began to identify shared interests in nursing research and discuss the mentee's career goals. They later discussed their academic institutions expectations of mentoring, outlined goals, and strategies for the upcoming academic year and made plans for how and when to meet. The process used for the mentorship was Dunham-Taylor et al.'s  proposedDunham-Taylor et al.  suggest Another element of mentoring is operation orientation , which eValidation and expectations are also components of mentoring . ValidatAnother important element of mentoring is transformation, encouraging professional growth through reading and attending educational conferences . The menExhibited behaviors have effects on the reputation of nursing; the mentor can discuss appropriate and inappropriate behaviors . The menAnother vital component of mentoring is documentation; faculty members need to know the importance of maintaining a method of organizing and tracking documentation necessary for licensure, promotion, and tenure. The mentor and mentee carefully planned research and service activities to prepare for the tenure review process, and how to document these. Further, the mentor worked with the mentee to develop a system for tracking manuscript plans, development, and submission status. Similarly, the mentor and mentee developed a system for documenting grant application status: from idea, to development progress, to submission. The mentee has obtained two externally funded grants while under the mentorship of the mentor.Generation, using one's wisdom and experience to help more junior faculty, is yet another element of mentoring . RegularLastly, Dunham-Taylor and colleagues  suggest Advantages of mentoring for both the mentors and the mentees might be the academic acceleration both parties receive. The mentees learn from the mentor, and, in turn, the mentor learns by teaching the mentees. Faculty mentoring of students can be especially rewarding for students learning vicariously through the mentor's experiences in nursing. This gives students a direct advantage in their class work and studies. At the authors' institution, graduate level nursing students experience faculty mentoring and are socialized to the researcher role as they work on their evidence-based capstone projects. Each student is paired with a project advisor, who guides the student through the steps of a research project. As mentors, both authors explained the steps of the research process to graduate students, socializing them to the role of nurse researcher, and demonstrating activities such as how to recruit research participants and obtain informed consent, and how to conduct statistical analysis and interpret these findings. The graduate students were able to observe the mentor and mentee collaborate on a research project and experience how collaboration occurs, and they became part of a team with a common interest, as the students assisted the mentor and mentee with manuscript critiques, data collection and entry, as suggested by the literature on mentoring students in research , 17.As a recipient of quality mentoring, the junior faculty author has practiced the vital skills the mentor implemented through her mentoring. The “passing on” of this quality mentoring has been productive; the mentee's former capstone advisees have had papers accepted for publication and presentation, and all have obtained advanced practice nursing positions. Riley and Fearing  note thaMentors also provide career information, for example, by providing feedback on strategic education enrichment opportunities such as essential professional organizations to join and allocation of time between research, teaching, and service, in order to optimize career success. Mentees are then better equipped to practice career enhancement mentoring with students. For example, the mentee has assisted her students to refine their resumes and interview skills in order to strengthen their job applications. This type of mentoring prepares students for the workforce and gives them the tools with which to seek employment after graduation. Thus, mentoring has multiple positive outcomes, for mentors, mentees, students, academic institutions, and healthcare organizations.Another outcome of mentoring is leadership development; according to a study by Young , particiGuided by the mentor's own experiences as a mentee and the literature on mentoring in nursing, the authors have experienced successful mentoring relationships. There have been positive outcomes at individual, school, university, and society levels. The mentee was successful in obtaining funding within the first year of the mentoring relationship  and thus was able to advance her program of research. The mentee has also been able to broaden her network of collaborators, through introductions to various nurse scientists and interdisciplinary research team members at national conferences, by the mentor, who has also role-modeled networking skills during these introductions. More broadly, successful mentoring relationships with several senior faculty members, including the current mentor, have contributed to faculty retention in the school of nursing and the university; the junior faculty mentee has remained at this university for 8 years now. The benefits of being well mentored have also contributed to the mentee's leadership skills, enabling her to make contributions on various college and university committees and serve as a member of dissertation committees. Beyond the university environment, mentoring has positive outcomes for the society: as mentees become more skilled in conducting research on health promotion for vulnerable populations, these research findings will have implications for clinical practice and the health of the community. For example, their research on successful aging with southern community-dwelling older adults  will add Skilled mentoring has also helped the junior faculty member establish partnerships with various members of the lay community, including local senior centers and federally qualified health centers, for research as well as service opportunities. Mentoring has facilitated dissemination of knowledge via presentations and papers, through support and encouragement the mentor provided.Mentoring can have limitations at times depending on demands on the mentor and the experience of the mentee . If the Although mentoring can be exhausting at times, seeing mentees receive grants to further their programs of research, get their manuscripts published, obtain tenure, and serve the university and the greater community is very rewarding. Mentoring is an important strategy that nursing leaders can use to facilitate the growth of our future nurse scientists. Mentoring is a way to continually pass on experience to the next generation of nurse scientists and meet nursing faculty workforce needs.


Although the molecular pathogenesis of CPVT is not entirely clear, it was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum Ca2+ leak, which initiates delayed afterdepolarizations (DADs) and triggered arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced pluripotent stem cells (iPSC) generated from somatic cells  now enables researches to investigate mutated cardiomyocytes generated from the patient’s iPSC. To this end, in the present article we review recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the mutated cells catecholamine-induced DADs and triggered arrhythmias.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias induced by physical or emotional stress, in the absence structural heart abnormalities. The arrhythmias may cause syncope or degenerate into cardiac arrest and sudden death which usually occurs during childhood. Recent studies have shown that CPVT is caused by mutations in the cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2 (CASQ2) genes. Both proteins are key contributors to the intracellular Ca Among the five major arrhythmogenic disorders occurring in the absence of structural heart diseases is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmogenic disease characterized by adrenergically mediated polymorphic ventricular tachycardia.In general, two genetic variants of CPVT have been identified. One is transmitted as an autosomal dominant trait caused by mutations in the gene encoding RyR2 (CPVT1) which is responsible for 50%–55% of all CPVT patients. Presently, more than 150 mutations have been identified in the RyR2 gene,42+ fluxes between the intracellular compartment and the extracellular space in cardiomyocytes is critical to ensure cellular viability, preserve normal contractile function, and to provide a stable heart rhythm.2+ enters the cardiomyocytes through the voltage-dependent L-type Ca2+ channels, causing Ca2+ release into the cytosol through the RyR2 channel located in the SR membrane. This process of CICR is the basis of cardiac E–C coupling.2+ causes activation of the contractile filaments of the cardiac sarcomere, which is followed by diminution of Ca2+ concentration to the diastolic level, thus causing relaxation. Ca2+ levels are lowered to diastolic values by means of three systems: 1) the sarcoplasmic endoplasmic reticulum calcium  which is responsible for reuptake of ∼75% of Ca2+ into the SR; 2) The sodium–calcium exchanger (NCX) which extrudes the remaining portion of cytosolic Ca2+ from the cytoplasm; and 3) a small portion of the Ca2+ is extruded to the extracellular space by means of an ATP-operated Ca2+ pump.11The delicate balance that regulates Ca2+ release channel located on the SR membrane. The RyR2 channels are composed of four pore-forming monomers, comprising a relatively small C-terminal transmembrane domain and a large N-terminal domain that protrudes into the cytosol. The cytoplasmic domain of RyR2 is stabilized by FKBP12.6 and is essential for channel closure during diastole.2+ storage protein in the SR and is capable of binding luminal Ca2+ (40–50 Ca2+ ions/molecule) during diastole in order to prevent Ca2+ precipitation and to reduce the ionic Ca2+ concentration.2+-buffering protein CASQ2,2+ release unit (CRU). The CRU is responsible for SR Ca2+ release, which is triggered by increased cytosolic Ca2+ resulting from opening of the L-type channel (CICR).2+ channels, thus causing gain of function of Ca2+ cycling in cardiomyocytes in response to adrenergic activation. FKBP12.6 stabilizes RyR2 in the closed state, and the hyperphosphorylation of RyR2 by PKA causes FKBP12.6 dissociation from RyR2, thereby increasing the open probability of RyR2.RyR2 is a large homotetrameric CaIn-vitro studies suggested that the RyR2 and CASQ2 mutations cause the CRU to open spontaneously without being triggered by voltage-gated Ca2+ influx, thereby leading to intracellular Ca2+ overload.2+ can trigger early or delayed afterdepolarizations  that can reach the threshold potential and cause triggered activity.2+ overload leads to NCX activation which extrudes Ca2+ in exchange for Na+ with a stoichiometry of 1:3, thereby generating a net inward current .2in-vitro and in-vivo models.in-vivo model proved the concept that RyR2 mutations may cause polymorphic and bidirectional ventricular tachycardia in a knock-in mouse model and mimic the clinical phenotype of CPVT patients.in vivo and in vitro.in-vitro modeling of the CASQ2 D307H mutation using infected rat myocytes with adenoviruses engineered to express the mutated CASQ2. In this study the authors demonstrated that the mutated myocytes developed DADs upon adrenergic stimulation while the control myocytes did not, as shown in in-vitro models of the CASQ2 R33Q and L167H mutations, respectively, and reported on the generation of DADs in the mutated myocytes.−/−) mice which were viable and exhibited the human-like arrhythmias. Despite the absence of CASQ2, these animals maintained relatively normal Ca2+ release and contractile function. However, myocytes isolated from these mice had exceptional increases in SR volume, increased gain of Ca2+-induced SR Ca2+ release, and increased diastolic SR Ca2+ leak.28The electrophysiological mechanisms of CPVT, which result from mutations in the RyR2 and CASQ2 genes, were investigated in several in vitro and opens new opportunities for investigating the diseases’ mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies  offers an innovative and practical approach to study inherited diseases including heart pathologies. Induced pluripotent stem cells are the product of somatic cell reprogramming to an embryonic-like state. Human iPSC have been generated from various types of somatic cells, most commonly skin fibroblasts, but also from hair keratinocytes isolated from plucked human hair which offers significant advantages over isolating skin fibroblasts through an invasive surgical procedure.rategies . Recentl+]i rise . This la+]i rise  for the 2+]i imaging Fatima et al. showed that CPVT1 cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous local Ca2+ release events already at basal state compared to the control cardiomyocytes. These findings are consistent with the irregular SR Ca2+ release observed in cells expressing the mutant RyR2 that underlie CPVT1.25More recently, Fatima and co-workers generated CPVT1-specific iPSC from a 46-year-old woman diagnosed with CPVT1, who carried the novel heterozygous autosomal dominant missense mutation F2483I in the RYR2 gene. This mutation is localized in the FKBP12.6-binding domain of the RYR2 protein.2+ handling in CPVT than in control cells. This frequency-induced stress is compatible with our findings2+ levels, as well as comparable SR Ca2+ content, determined by caffeine application. However, in the presence of isoproterenol the diastolic Ca2+ level was significantly elevated in CPVT1 cardiomyocytes compared to control cells, while systolic Ca2+ remained similar; these findings are in agreement with the effects of isoproterenol on CPVT2 cardiomyocytes.2+ load was not increased by isoproterenol in CPVT1 cells. Furthermore, under basal conditions, CPVT cardiomyocytes displayed abnormal Ca2+ sparks with a higher amplitude, prolonged (compared to control) plateau phase, longer duration at 50% peak amplitude, and longer decay time. In response to isoproterenol, in CPVT cardiomyocytes Ca2+ spark frequency increased compared to control cells, and the sparks had longer decay time. Finally, Jung et al. showed that in all CPVT1 cardiomyocytes stimulated with isoproterenol the DADs and triggered arrhythmias were abolished by the ryanodine antagonist dantrolene, suggesting that a defective inter-domain interaction within the RyR2 is the underlying arrhythmogenic mechanism of the S406L mutation.In a recent study Jung et al. reported on the generation of iPSC from a 24-year-old CPVT1 woman carrying the novel RyR2 S406L mutation.CPVT is a complex disease which poses several challenges in the management of affected patients.in vitro and in vivo in CPVT mouse models, the demonstration that these phenomena were responsible for arrhythmogenesis in humans was largely a consequence of genetic research.Although DADs were described


Treatment with triethylenetetramine (TETA), a divalent-copper-selective chelator, improves cardiac structure and function in patients and rats with diabetic cardiomyopathy, but the molecular basis of this action is uncertain. Here, we used TETA to probe potential linkages between left-ventricular (LV) copper and Ca2+ homeostasis, and cardiac function and structure in diabetic cardiomyopathy.Intracellular calcium (Capreventative protocol) or eight (restorative protocol) weeks after diabetes induction and continued thereafter for seven or eight weeks, respectively. Total copper content of the LV wall was determined, and simultaneous measurements of intracellular calcium concentrations and isometric contraction were made in LV trabeculae isolated from control, diabetic and TETA-treated diabetic rats.We treated streptozotocin-diabetic rats with a TETA-dosage known to ameliorate LV hypertrophy in patients with diabetic cardiomyopathy. Drug treatment was begun either one (2+]i. However, diabetic rats showed extensive cardiac remodelling and decreased myofibrillar calcium sensitivity, consistent with observed increases in phosphorylation of troponin I, whereas these changes were all prevented by TETA.Total myocardial copper levels became deficient in untreated diabetes but were normalized by TETA-treatment. Cardiac contractility was markedly depressed by diabetes but TETA prevented this effect. Neither diabetes nor TETA exerted significant effects on peak or resting [CaDiabetes causes cardiomyopathy through a copper-mediated mechanism that incorporates myocardial copper deficiency, whereas TETA treatment prevents this response and maintains the integrity of cardiac structure and myofibrillar calcium sensitivity. Altered calcium homeostasis may not be the primary defect in diabetic cardiomyopathy. Rather, a newly-described copper-mediated mechanism may cause this disease. Cardiovascular disease (CVD) is the main cause of mortality in diabetic patients, in whom it is frequently accompanied by heart failure . About tEffective treatments for established heart failure in diabetes are limited ,5 and itCopper is an essential trace element that plays key roles in many vital copper enzymes, e.g. cytochrome c oxidase, caeruloplasmin, copper/zinc superoxide dismutase (SOD1), and lysyl oxidase . Free diInsufficient dietary copper intake can cause systemic copper deficiency, myocardial copper deficiency, hypertrophic cardiomyopathy, and heart failure -14, all Copper accumulation in excess of bodily requirements is also cytotoxic ,18. AlteT2D patients acquire a hyperglycaemia-driven pathogenic abnormality of copper homeostasis that is reversible by TETA-treatment , which a2+ homeostasis have been implicated in the impaired mechanical performance of the diabetic heart iin left ventricular trabeculae were measured as previously described . Once the trabeculae were producing stable stimulus-evoked twitches, they were loaded with the fluorescent Ca2+ indicator fura-2/AM . Force and [Ca2+]i were then measured simultaneously at a fixed sarcomere length (2.1-2.2 μm), either at 37°C, 5-Hz, and 1.5 mmol/L [Ca2+]o, or other experimental conditions. The ratio of the emitted fluorescence at the two excitation wavelengths (340/380 ratio) of fura-2 was obtained  as a measurement of [Ca2+]i. The force measurements were normalized by the trabecular cross-sectional area and expressed as stress (mN/mm2).escribed . Briefly2+]i relationship was evaluated in LV trabeculae treated with 5 μmol/L of ryanodine, a plant alkaloid considered to block ryanodine receptor channels to generate a long-lived subconductance  state [2+]o values from 0.5 mmol/L to the concentration where the maximum force was obtained. Tetanus at different [Ca2+]o was separated by a “resting” period of 2 min. To avoid precipitation at high Ca2+ concentrations, solutions used in these experiments were switched to a phosphate-free composition including (in mmol/L): NaCl, 108; KCl, 5; MgCl2, 1; HEPES, 5; NaCH2COOH, 20; and D-glucose, 10. Calibration in terms of absolute values of [Ca2+]i and data analysis were performed following published procedures o and without stimulation, 10 mmol/L caffeine was added , and Ca2+ transients were recorded. After the [Ca2+]i had decreased to the resting level, cells were perfused with Tyrode’s solution incorporating 1.5 mmol/L [Ca2+]o and stimulated at 1 Hz (to restore the SR Ca2+) followed by a further series of solution changes in the following order: 0 mmol/L [Ca2+]o, 30 s; and then 10 mmol/L caffeine with 0 mmol/L [Na+]o and 10 mmol/L [Ni2+]o . Ca2+ transients were continuously recorded.escribed . The yieWestern Blotting was performed with proteins extracted from LV tissue as described o and 0 mmol/L [Na+]o were used to further eliminate the contribution of NCX to the decay of the Ca2+ transient. Results , whereas NCX was unaltered. These results could explain the slower decay of the Ca2+ transient in diabetic trabeculae. TETA had no significant effect on the expression of these proteins, consistent with the unchanged decay-rate of the Ca2+ transient in TETA-treated diabetic rats.Decay of the Caf total) . Caffeins Figure A showed y Figure B, in whi2+]i coupled with the slower kinetics of the Ca2+ transient cannot explain the depressed cardiac contractility in diabetic rats [2+]i handling may not be the primary contributor to diabetes-evoked cardiac dysfunction, and that diabetic rats may have intrinsic myocardial defects that contribute to their depressed cardiac contractility. Our observations invited further investigation of myofibrillar Ca2+ sensitivity and cardiac remodelling in diabetic rat hearts.The unchanged levels of peak and resting i in diabetic rats  to induce cardiac tetanus for investigation of the steady-state force-[Ca2+]i relationship  whereas TETA-treatment preserved the maximum contractility in treated-diabetic animals (P = 0.03). Untreated-diabetic rats also showed increased K1/2 , consistent with decreased myofibrillar Ca2+ sensitivity (P = 0.012). TETA prevented this alteration of K1/2, suggesting that drug-treated diabetic rats have normal myofibrillar Ca2+ sensitivity compared to untreated ones (P = 0.05). Furthermore, the phase-plot between [Ca2+]i and tetanus at 20 mmol/L [Ca2+]o from a number of trabeculae in each experimental group , and TETA prevented this reduction . This finding is consistent with that obtained using the conventional Hill equation fitting method  but also affected by the phosphorylation status of the troponin complex , by the cooperation between the troponin complex and F-actin, and by force generation via binding of cross-bridges. Alterations in protein expression of TnT i[2+ transient , therefore exposing the myosin binding sites on actin to enable cross-bridge formation. As a result of the cooperative nature of this process, a decreased propensity of Ca2+ binding to evoke the conformational changes of these proteins and cross-bridge formation would eventually affect force development. Alterations in the relative expression of TnT isoforms have been demonstrated, coincident with diminished Ca2+ sensitivity of skinned cardiac-muscle contractility in diabetic rats [2+ sensitivity [2+ sensitivity in a cardiac actomyosin system reconstituted with regulatory-protein complexes from diabetic hearts [2+ sensitivity. TETA treatment maintained the phosphorylation level of TnI in treated diabetic animals, therefore contributing to their normal Ca2+ sensitivity. The altered phosphorylation states of one or more of the contractile proteins perhaps reflect increased protein kinase C (PKC) activity [δ gene expression is increased by ~30% in diabetic myocardium [δ is uniquely able to phosphorylate Ser23/Ser24 in troponin I; and consequently, like protein kinase A (PKA), it can reduce myofilament Ca2+ sensitivity [Normally, tension development is initiated by Catic rats . Here, wsitivity , consistc hearts . This fuactivity . This isocardium . PKCδ issitivity .We have previously reported that, in this animal model, the expression of mRNA corresponding to β-myosin heavy-chain undergoes a ~50% increase in LV tissues, whereas that of α-myosin heavy chain is decreased to ~35% of control values . However2+ sensitivity [2+ sensitivity.Further, we report here that diabetes decreases total myocardial copper to markedly-lowered values, whereas we previously showed increases in the systemic and cardiac Cu(II) in the same diabetic model, as can be extracted by intra-arterial infusion of TETA. The excess Cu(II) is likely to be bound in the ECM ,25 sincesitivity , which isitivity . Thus TEsitivity , therebyCtr1, cardiomyopathy induced by lack of myocardial copper transport led to decreased copper accumulation in cardiac tissue, accompanied by an unexpected increase in serum copper levels [Myocardial copper deficiency is a known cause of myocardial fibrosis and cardiac failure ,13,15,16r levels . The autprevented the development of cardiomyopathy and remodelling when administered ab initio. The increased collagen content and disrupted F-actin in diabetic hearts could in part account for the depressed cardiac contractility and decreased myofibrillar Ca2+ sensitivity observed therein : Divalent copper; EC: Extracellular; ECM: Extracellular matrix; F-actin: Filamentous actin; LV: Left ventricular; Na+: Sodium; NCX: NaThe authors declare no duality of interest associated with this manuscript.LZ participated in study design, acquisition and analysis of data, interpretation of results, and wrote the first draft of the manuscript; GC participated in study concept and design, data analysis and interpretation of results, and wrote manuscript drafts; AP and SZ participated in study concept and design, data analysis and interpretation of results; MW and MC participated in study design, data analysis and interpretation of results. All authors participated in critical revision and approved the final version of the manuscript. LZ and GC are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.


A number of ion channels regulate Ca2+ influx from the extracellular space in T cells, either by conducting Ca2+ ions or by modulating the membrane potential that provides the driving force for Ca2+ influx  channel, which mediates store-operated Ca2+ entry (SOCE) in response to T cell receptor (TCR) activation and is composed of ORAI and stromal interaction molecules (STIM) family proteins. Several other channels may also mediate Ca2+ influx directly in T cells including members of the transient receptor potential (TRP) family, P2X receptors, and voltage-gated Ca2+ (Cav) channels. Compared to CRAC channels, however, their contribution to TCR-induced Ca2+ influx and immunity is less well defined.Ca2+ release-activated Ca2+ channels were first identified in T cells (and mast cells) over 20 years ago  responses to recall antigens in patients and mice  cells  in the gut , cyclic ADPR (cADPR), and Nicotinic acid adenine dinucleotide phosphate (NAADP)   channels are highly Ca2+ selective channels that play an important role in Ca2+ influx and the function of electrically excitable cells such as neurons following cell depolarization  were reported to be expressed but their contribution to Ca2+ influx has remained controversial  used to measure Ca2+ influx in most studies, P2X7 channels form a large pore  ATP concentrations (Junger, in vivo remains poorly understood.P2X receptors are not, unlike the Ca Junger,  that is  Junger, . Similar Junger, . However Junger,  and thei2+ signals have long been recognized as essential for T cell function and several channels may contribute to Ca2+ influx in T cells. Whereas the role of CRAC channels to T cell function and adaptive immunity is well documented by findings in ORAI1 and STIM1-deficient patients and mice, the contributions of TRP, CaV1, and P2X receptor channels remain to be more clearly defined. These channels could contribute to Ca2+ influx in specific T cell subsets, at distinct stages of T cell development or following stimuli other than TCR engagement. A better understanding of the contributions of different Ca2+ influx pathways in T cells will be essential to define potential drug targets for the modulation of T cell function in a variety of diseases caused by aberrant T cell function.Ca


The substance triggers tumor cell apoptosis. Side effects of carmustine include myelotoxicity with anemia. At least in theory, anemia could partly be due to stimulation of eryptosis, the suicidal death of erythrocytes, characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i). The present study tested whether carmustine triggers eryptosis. To this end [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. As a result a 48 h exposure to carmustine (≥25 µM) significantly increased [Ca2+]i, decreased forward scatter and increased annexin V binding. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca2+. In conclusion, carmustine stimulates eryptosis at least partially by increasing cytosolic Ca2+ activity. The nitrosourea alkylating agent, carmustine, is used as chemotherapeutic drug in several malignancies As a result, carmustine increases [Ca2+]i, decreases erythrocyte volume and enhances the PS abundance at the erythrocyte surface. The present study explored the effect of carmustine on erythrocyte [Ca2+ activity ([Ca2+]i), Fluo3 fluorescence was employed to estimate [Ca2+]i. To this end, the erythrocytes were incubated in Ringer solution without or with carmustine (10–100 µM), loaded with Fluo3 AM and Fluo3 fluorescence quantified by FACS analysis. As illustrated in 2+]i in human erythrocytes.The present study was designed to explore whether carmustine stimulates eryptosis, the suicidal death of erythrocytes. As eryptosis is triggered by increase of cytosolic Ca2+]i is expected to activate Ca2+ sensitive K+ channels leading to cellular loss of KCl together with osmotically obliged water and thus to cell shrinkage. Accordingly, cell volume was estimated from forward scatter in FACS analysis. As shown in An increase of [Ca2+]i is further expected to trigger cell membrane scrambling with breakdown of PS asymmetry of the cell membrane and appearance of phosphatidsylserine at the cell surface. Accordingly, PS abundance at the cell surface was estimated utilizing annexin V binding in FACS analysis. As shown in An increase of i is well known to stimulate cell membrane scrambling with PS translocation from the inner leaflet of the cell membrane to the outer leaflet of the cell membrane [2+ entry include Ca2+ permeable nonselective cation channels involving the transient receptor potential channel TRPC6 [2+ permeable erythrocyte cation channels are activated by oxidative stress [The breakdown of PS asymmetry of the erythrocyte cell membrane was significantly blunted in the absence of extracellular Camembrane . Mechaniel TRPC6 . The Ca2e stress , a well-e stress .In vivo, eryptotic erythrocytes are mainly trapped in the spleen and thus rapidly removed from circulating blood [Consequences of enhanced eryptosis include anemia. ng blood . As soonng blood . During ng blood ,8 is expConsequences of enhanced eryptosis further include adhesion of PS, exposing erythrocytes to endothelial CXCL16/SR PSO . The adhin vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to carmustine  at the indicated concentrations. Carmustine was dissolved in 50% ethanol. The final concentration of ethanol did not exceed 0.1%. Annexin V binding was not significantly different in the absence  and presence of 0.1% ethanol . In Ca2+ free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Leukocyte depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated 2 and then stained with Annexin V FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin V fluorescence intensity was measured in FL 1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and 2 µM Fluo 3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo 3/AM loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+ dependent fluorescence intensity was measured in fluorescence channel FL 1 in FACS analysis.After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo 3/AM  in Ringer solution containing 5 mM CaClg, room temperature) after incubation, and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.For the determination of hemolysis the samples were centrifuged (3 min at 400t test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and 2+ entry with subsequent eryptosis, the suicidal erythrocyte death. Enhanced eryptosis may contribute to the development of anemia following carmustine treatment.Exposure of erythrocytes from healthy volunteers to carmustine triggers Ca


Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH.2+]i) levels were evaluated by flow cytometry using the JC-1  and fluo-4/AM probes respectively. Anti-oxidant and Ca2+ transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively.Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca2+]i levels were found to be higher (p<0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca2+/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca2+/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H202) reduced expression of Na+/Ca2+  exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT–PCR.Intracellular ROS production was increased in GLC compared to NLC . Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p=0.02) compared to NLC control. MMP was lower in GLC (57.5±6.8%) compared to NLC (41.8±5.3%). [Ca2+]i.Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca Glaucoma is a neurodegenerative disease of the eye that is one of the leading causes of visual impairment and blindness worldwide ,2. It isOxidative stress is defined as an increase over physiologic values of the intracellular concentration of reactive oxygen species (ROS). These ROS are free radicals containing one or more unpaired electrons which can damage a wide variety of biomolecules and cell structures. An imbalance between pro-oxidative and anti-oxidant capacity has been postulated to be a crucial feature in early retinal injury and glaucoma pathology ,9 as wel2+ homeostasis, mitochondrial dysfunction and oxidative cell injury are known to be associated with a variety of neurodegenerative diseases, including glaucoma i in NLC and GLC using flow cytometry after the cells were preloaded with the intracellular free [Ca2+]i indicator fluo-4/AM in NLC and GLC cells. Addition of 2 mM extracellular Ca2+ elevates [Ca2+]i in control LC cells and the increase in cytosolic Ca2+ (fluo-4/AM fluorescence) is more intense and has greater variability in GLC cells compared with NLC cells (p<0.001).While ROS frequently function as cellular second messengers, at elevated levels they can be extremely damaging and disturb normal cellular physiologic function. We measured the [Ca2+ levels are elevated in GLC cells  was observed in GLC compared to NLC cells. No significant differences were observed in the mRNA expression of either the detected . Westerndetected .NCX genes  failed to show a steady pattern of expression in NLC, thereby preventing reliable comparison of expression rates between NLC and GLC  can alter the expression of Cachannels ,37,39 th to H202 . Cells w2+ homeostasis  on calcium transporter expression (202) induced down-regulation of Ca2+ extrusion systems  in NLC cells. This decrease in expression mimics the endogenous decrease in expression of these channels found in GLC compared with NLC cells . It is a Cane, PC12  and SERCne, PC12 . This in tissues . SERCA3  tissues . Therefo2+ homeostasis which correlate with elevated cytosolic Ca2+. Whether anomalous Ca2+ homeostasis in GLC cells is a primary pathological event or a consequence of other pathological changes has yet to be elucidated. Future anti-glaucoma therapies may target oxidative stress and Ca2+ overload in the LC cell.In summary, we have clearly shown that conditions of oxidative stress prevail in GLC cells. These cells also exhibit impaired mitochondrial function and marked abnormalities of Ca


Depletion of Ca2+ and accumulation of K+ occurring in extracellular spaces slightly affected the transmembrane Ca2+ flux, but not the action potential duration (APD90). The [Ca2+]i-transient was reduced (by 2%–9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1–2.5 Hz) caused a temporal decrease of [Ca2+] in both extracellular spaces, a reduction of [Ca2+]i-transient (by 15%) and APD90 (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca2+ turnover.We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca Their differences demonstrate luminal ion concentration gradients in the radial direction. The alterations of [Ca2+] in clefts ([Ca2+]c) were induced by the activation of the main Ca2+ currents in the surface membrane  and by Ca2+ diffusion between t-tubules and clefts. Small changes of [K+]c were induced by activation of IKto,s, IK1,s and INaK,s and by K+ diffusion between t-tubules and clefts. In addition, concentrations of all ions in the cleft space were affected by diffusion of ions between the cleft space and the external bulk space.The depletion of t-tubular Ca2.2.2+]t1, [Ca2+]c, [K+]t1, [K+]c) described in 2+ load in NSR ([Ca2+]NSR) and on the intracellular Ca2+-transient during a steady-state stimulation cycle at 1 and 2.5 Hz. The differences between traces representing NSR Ca2+ load and cytosolic [Ca2+]-transient in the basic model before (red lines) and after fixing all extracellular ionic concentrations at external bulk values (black lines) reflect a cumulative effect of small changes in sarcolemmal Ca2+ transport  that are induced by variations of extracellular ion concentrations.2+ turnover in cardiac cells at steady state. The time integral of intracellular Ca2+-transients was reduced by 2.5% and 3.4% at 1 and 2.5 Hz, respectively. A deeper analysis also showed that this effect is predominantly induced by depletions of Ca2+ concentration in the vicinity of the t-tubular membrane ([Ca2+]t1); the relative contribution of changes of [Ca2+]c, [K+]c and [K+]t1 in total was below 0.3%.Though small, this effect shows a potential of these variations to slightly affect the Ca2+ concentration changes in t-tubules on cytosolic Ca2+-transient for potentially different combinations of critical fractions fCaL,t, fNaCa,t and fpCa,t in human ventricular cells, we performed a series of simulations enabling us to construct these relations in 3D graphs , the repeated depletions of Ca2+ in human t-tubules might result in a reduction of Ca2+-transient by 3% at resting heart rate (1 Hz) and by 4% during exercise (2.5 Hz) , this effect would further increase to more than 9% at 2.5 Hz (not illustrated). While the dependency of this effect on the value of fNaCa,t appeared to be small, high values of fpCa,t would even reverse it, as they would lead to accumulation of Ca2+ in the tubular space and, consequently, to a slight increase of the Ca2+-transient.To explore the effect of CaD graphs . The resinea-pig  (basic smyocytes ) would l2.3.2+ load and intracellular [Ca2+], [Na+] and [K+] during transient events after a sudden change of stimulation frequency. The results from the basic model (blue dots) are compared with the results after fixing ion concentrations in the cleft space (red dots) or in both t-tubular and cleft spaces (black dots) at external bulk levels. The right panels illustrate progression toward the steady state in an expanded time scale.2+ flux into the cell decaying within several seconds  induced a marked transient reduction of mean Ca2+ concentration in the cleft space  that was also reflected by a similar change in the mean Ca2+ concentration in the first tubular segment . Note that this reduction of [Ca2+] in both extracellular spaces was substantially larger (blue dots) than the reduction of [Ca2+]t1 in the model after fixing ion concentrations in clefts space (red dots). This initial rapid reduction in extracellular [Ca2+] decays with a time constant of approximately 50 s due to equilibration between the clefts and bulk space .The fast transient increase of Ca2+ in both cleft and t-tubular spaces was a parallel decrease of Ca2+ concentration in NSR and a significant reduction of cytosolic Ca2+-transient by 15% at 10 s after the onset of faster stimulation . This deflection decays towards steady-state values in parallel with [Ca2+] in cleft space . The slow cumulative changes in [Na+]i and [K+]i (time constant of about 250 s) related to sodium and potassium currents, Na/Ca exchange and Na/K ATPase activity are also affected by changes in extracellular Ca2+ concentrations (panels f and g). The underlying processes lead to the final steady state in which the net total transmembrane Ca2+ transport (across the surface and tubular membrane) equals zero in the course of each stimulation period [see Condition  was reduced by 13 ms in accordance with a decreased influx of Ca2+ ions in the course of AP. [Ca2+]i-transient dropped by 15%. If the stimulation frequency returned from 2.5 to 1 Hz, the opposite deviations in AP and [Ca2+]i-transient appeared (not shown).These results suggest that, while the role of ion concentration changes in cleft spaces in modulating cellular electrical activity and the Ca+] and [Ca2+] in the clefts that were documented in the literature, we simulated, from steady-state stimulation at 1 Hz, the application of a train of stimuli at 3 Hz for durations from 34 up to 700 s and a subsequent return to 1 Hz for 700 s.To verify the ability of our model to reproduce the changes in c,end during the depletion depended on the duration of the stimulation train at high frequency.+] done with a K+-selective microelectrode by Kunze [+ depletion upon returning to 1 Hz depended on the duration of the stimulation at 3 Hz.These features compare well with the measurements of interstitial [Kby Kunze . Another+] changes in the clefts: the membrane was depolarized during K+ accumulation and hyperpolarized during K+ depletion, by about 4 mV. The onset of the depolarization at the application of 3 Hz stimulation and that of the hyperpolarization upon returning to 1 Hz stimulation were as fast as from the microelectrode recordings of Attwell et al. [+] signal in c,end at the beginning of the 3 Hz stimulation train was fast and amounted to 0.25 mM  reported by Hilgemann and Langer  changes occurring in the limited space of t-tubules are potentiated by [Ca2+] changes in the intercellular clefts, primarily during transients caused by changes in stimulation frequency.This study demonstrates that ion concentration changes in the t-system of human cardiomyocytes, though small compared to those observed in skeletal muscle ,24, are inea-pig  and rat inea-pig . In addi3.1.ICaL[et al. [et al. [et al. [+]i and [K+]i were fixed at 10 and 130 mM, respectively (ion concentrations used in the pipette solution). Surprisingly, the frequency-dependent changes of AP duration in the models )ICaL[et et al. [2+]i) and Pieske et al. [+]i) is illustrated in 2+]i in our model (63.6 nM) agrees well with the experimental data of Beuckelmann et al. [+]i (9.3 mM) is in the range of the experimentally observed values of Pieske et al. i, but, to a certain degree, also by changes in [Ca2+]t and [Ca2+]c. Those modulations represent a complex feedback system maintaining SR Ca2+ load and, hence, the magnitude of the [Ca2+]i-transient, at a level that guarantees steady-state Condition (All currents transporting Caondition  at any fondition  is distu3.3.2+ concentration changes ([Ca2+]i and [Ca2+]NSR) at steady state . In contrast, in the t-tubules, the mean Ca2+ concentration differs from the bulk concentration as a consequence of the sustained flux, JCa,net,t  at the t-tubular membrane than at the surface membrane.Under steady state at any frequency, the mean ion concentrations in the intercellular clefts are equilibrated with concentrations in the bulk. Not surprisingly, inclusion of the clefts into the model only minutely affected the simulated results of intracellular Cady state . When exmembrane ). In add2+ transporters in the t-tubular membrane on the modulation of intracellular Ca2+-transient . The graphs in fCaL,t assessed in guinea-pig (0.64) and rat (0.8) cardiomyocytes. As ICaL,t causes Ca2+ depletion in the t-tubules, it is not surprising that a higher fCaL,t corresponds to a lower amount of Ca2+ in the intracellular compartments reflected by Δ[Ca2+]i,area,rel. Conversely, raising fractions fNaCa,t and fpCa,t attenuates changes in Cai-transient, because INaCa,t and IpCa,t tend to enhance the mean value of [Ca2+]t. The simulations suggest that the changes in intracellular Ca2+-transient may be significant if, simultaneously, fCaL,t was high (≥0.6) and fpCa,t low (≤0.2).Although the fractions of the main ion transporting proteins localized in the tubular membrane have been quantified in some experimental animals , sim2+ handling and APs , since, during each shortened stimulation period after transition to 2.5 Hz, Ca2+ input via ICaL transiently exceeded Ca2+ output via INaCa and IpCa. Restoration of equilibrium between the clefts and the bulk space required tens of seconds.In contrast, under non-steady-state conditions, when sudden changes in heart rate were simulated, the intercellular cleft space significantly affected Ca and APs  and 6. T+ in response to sudden changes of stimulation frequency (2+-transient (not shown), but its effect was substantially smaller than the effect of Ca2+ depletions. The effects of minute relative variations of [Na+]i were negligible.Transient accumulation of extracellular Krequency  was evid3.4.2+ turnover and EC coupling can be expected. Intercellular clefts can also be modified by, e.g., hypertrophic cardiomyopathy [As documented by numerous studies, cardiac diseases, like ischemic or dilated heart failure, affect the structure and function of the t-tubular system, both in animal –35 and hmyopathy .2+ handling in normal human cardiomyocytes at different frequencies. Our aim in a future work will be to utilize the present model to investigate the consequences of the above-mentioned changes of ion concentrations in extracellular spaces occurring under pathological conditions.The purpose of the present model study was to estimate the impact of ionic changes in extracellular spaces on APs, ionic currents and intracellular Ca3.5.3.5.1.2+ channels, at the limited region of the t-tubules that contribute to the dyads, would result in larger local depletions in luminal Ca2+, as modeled using partial differential equations [The distribution of ion channels along the t-tubules is considered uniform in our model. However, the tubular system comprises both transverse and longitudinal extensions, and ion channels are probably unevenly distributed among these two membrane pools . Furtherquations .3.5.2.Deformation of the t-tubules during activity  is likel3.5.3.Subtypes of ventricular cardiac myocytes  with different ion channel densities were not considered in our study, as their description in human cardiac ventricular myocytes is not fully documented. It was also shown that the amount of t-tubules-SR junctions (but not the volume fraction of t-tubules) varied among different regions of rabbit ventricular myocardium that displayed a difference in AP configuration ,49. For 4.The structure of the model  is similINa, ICaL and IK1 by the formulations used by Iyer et al. [IKr, IKs and IKto by the simpler formulations of Ten Tusscher et al. [2+ handling mainly on the basis of its description in [The principal modifications include: (i) replacement of the description of r et al.  and thosr et al. ; (ii) reption in ; (iii) pption in  and readption in ; and , Na+-Ca2+ exchangers  and the sarcolemmal SR Ca2+ pump , were, however, considered as variable in some simulations to evaluate their importance. All details related to the development of the model and its full mathematical description are given in the Because of the paucity of experimental data from human ventricular myocytes, the time constants describing ion exchange between t-tubular and cleft spaces and also t-tubular fractions of ion transporters related to inea-pig . The t-tThe model was implemented in MATLAB 7.2 , and the numerical computation of the system of 98 nonlinear differential equations was performed using the solver for stiff system ODE-15s. The stability of the model with time is demonstrated in Figures 1To compare the model results with the behavior of other models of human ventricular cells published so far ,6,8, we 5.2+-transient) were explored as a function of a few crucial parameters, still not quantified in human cardiac cells. In the present state, the model visualizes the activity-related accumulation-depletion of Ca2+ and K+ ions in t-tubular and interstitial extracellular spaces. The results suggest that cumulative calcium depletion in the limited extracellular spaces can affect transmembrane Ca2+ currents, the duration of AP and the cytosolic calcium-transients that govern cellular contraction. Further experimental work is needed to evaluate the missing values describing the distribution of ion transporters in the cell membrane and the rate of ion exchanges between the extracellular compartments of human cardiomyocytes.The available data related to human cardiac ventricular cells were used to design the first mathematical model of human ventricular cardiomyocyte incorporating t-tubular and intercellular cleft spaces. The model was used to study the physiological consequences of activity-related ion concentration changes in these extracellular spaces. Some of them (particularly, the changes of the intracellular Ca


It is well established that atrial fibrillation (AF) is far more common in elderly humans. Autonomic activation is thought to be an operative mechanism for AF propensity. The aim of the study was to investigate the impact of age on atrial tachyarrhythmia induction in a rabbit model. Six old (aged 4–6 years) and 9 young (aged 3-4 months) New Zealand white rabbits were subjected to a catheter-based electrophysiological study. Atrial tachyarrhythmia susceptibility was tested by burst pacing before and after infusion of increasing concentrations of acetylcholine. Both young and old rabbits were in normal sinus rhythm at the beginning of the infusion/burst pacing protocol. The old rabbits had faster heart rates and a marked increase in atrial tachyarrhythmias compared to the young rabbits. Nonsustained and sustained AF events were more frequent in the old rabbits. No significant fibrosis was observed in the atria of either young or old rabbits. In conclusion, the old rabbits have a greater propensity for induction of AF. The significantly faster heart rates in the old rabbits suggest that dominant sympathetic activity may play an important role in the propensity for AF in this group. The mececeptors . In a prreceptor . The resμM, 100 μM, and 1 mM) at a rate of 1 mL/min. Nonsustained (<10 sec) and sustained (≥10 sec) arrhythmia occurrence was determined in response to burst pacing at baseline and with each of the 3 concentrations of ACh infusions for 2 minutes before initiating burst pacing. Rhythm disturbances were characterized from the ECG by their rate and duration and from the morphology of the recorded atrial electrograms  and 9 young (aged 3-4 months) New Zealand white rabbits of either sex fed standard rabbit chow were studied. Each animal was anesthetized with ketamine/xylazine (35 mg/5 mg/kg) and subjected to a catheter-based electrophysiologicalstudy . Standartrograms .Portions of the atria and ventricles from the hearts fixed in formalin were prepared for histological study. Specimens were processed in paraffin-embedded histological sections cut along the long axis of the atria and ventricles. Sections were stained with hematoxylin-eosin and Masson's trichrome for detection of fibrosis. The degree of fibrosis was quantified on the basis of the following scale: 0, no fibrosis; 1+, stainable collagen adjacent to capillaries and/or between myocytes (excluding large vessels) less than half of myocyte diameter; 2+, greater than half to equal to myocyte diameter; 3+, greater than myocyte diameter or fibrosis with features of replacement.t-test was used to compare heart rates between young and old rabbits. Chi-square analysis (2 × 2 contingency table) followed by a two-tailed Fisher's exact was used to determine the difference in occurrence of a sustained arrhythmia (≥10 sec) for each rabbit at all concentrations of ACh infusion. A P value of <0.05 was considered statistically significant.Data are expressed as mean ± standard deviation (SD). A standard n = 6) was 200 ± 23/min, compared to the young rabbits (n = 9) whose heart rate averaged 160 ± 13/min (P = 0.003). In addition, there were a significantly greater number of AF events in the old versus young rabbits (P = 0.0004). As for sustained AF, there were 7 of 24 inductions in the old rabbits and 0 of 36 in the young rabbits (P = 0.0009). The average duration of AF in the old rabbits was 258 seconds. In the baseline state, the mean heart rate for the old rabbits  were used to initiate arrhythmias. Previous studies in ambulatory dogs with a propensity for inducing atrial tachyarrhythmias were based on the simultaneous interaction of the neural activity of both arms of the autonomic nervous system . In thisβ2-adrenergic receptor, sustained atrial tachycardias were consistently evoked [Clinical studies have shown that humans aged 60–80 years have diminished autonomic cardiovascular responses , 14. We y evoked . There iy evoked . These ny evoked , both ofOne of the first questions which arises when considering susceptibility to AF in old animals is the presence of atrial fibrosis. Whether there is a direct cause or effect relationship between atrial fibrosis and AF is still open to question. A histological study of lone AF patients found that 25% of patients did not have atrial fibrosis . On the Appropriate animal models for AF are problematic. Issues include changes in innervation, size of the heart, and differences in aging. Recent pressures in the United States and elsewhere have led to significant restrictions on use of the common canine model. Substitution with small rodents is difficult owing to their small cardiac size and notable differences in biological function from humans. These difficulties are even greater when there is a need to study the effect of aging. The telescoped life span of rodents is advantageous for aging research but may not reflect the pathophysiology of larger mammals. The present study presents evidence that the rabbit may represent a convenient intermediate species wherein such studies of aging are possible within a reasonable time frame and the pathophysiological changes in the heart are not insurmountable.Using a relatively new experimental model, we have demonstrated that rabbits 4–6 years of age have an increased susceptibility to develop AF compared to the young rabbits. This was associated with evidence supportive of increased autonomic activity and was induced by the combination of ACh and burst pacing. Atrial fibrosis was not observed in the atria and could be ruled out as a potential explanation for the greater propensity for AF in the old rabbits. This rabbit model presents several advantages for the study of immunological issues that affect atrial or ventricular tachyarrhythmias.


Rabbit hepatitis E virus (HEV) is a novel genotype of HEV, and is considered to pose a risk of zoonotic transmission. Research into the systemic distribution of rabbit HEV in rabbits during different periods of infection has rarely been reported. To better understand this virus, we infected rabbits with second-passage rabbit HEV via an intraperitoneal route. After inoculation, the infection showed two types, temporary and constant infection. The detection of HEV RNA in the feces varied with time, and serum antigen correlated with fecal HEV RNA. Viremia only appeared 72 days after inoculation. The rabbits remained antibody negative throughout the experimental period. When HEV was localized, several organs besides the liver were HEV RNA positive. Tissue antigen was observed immunohistochemically in the different cells of various organs, especially in parts of the small intestine and the characteristic rabbit gut-associated lymphoid tissue. These data provide valuable information for future research into the pathogenesis of HEV. Hepatitis E (HE) is an acute self-limiting disease caused by a nonenveloped, positive-sense, single-stranded RNA virus designated “hepatitis E virus” (HEV) A recent study demonstrated that HE is a zoonotic disease. As well as human HEV, some animal strains of HEV has been discovered, including swine HEV from pigs in 1997 and avian HEV from chickens in 2001 Although rabbit HEV is not highly similar to other mammalian HEV genotypes, a recent experimental study suggested that rabbits can be successfully infected with HEV genotypes 1–4 as well as rabbit HEV The animal experiments were approved by the Animal Care and Use Committee of China Agricultural University (CAU) (permit number: 20121110-178). We followed the guidelines of the CAU Animal Care and Use Committee in handling the experimental animals during this study.7 genome equivalents per mL. The viral suspension was stored at −86°C for later use.The strain of rabbit HEV was derived from the second passage of a fecal sample from a rabbit infected with rabbit HEV . A 10% suspension of positive feces was prepared 5′-AATTATGCYCAGTAYCGRGTTG-3′ and 5′-CCCTTRTCYTGCTGMGCATTCTC-3′, and inner primers 5′-GTWATGCTYTGCATWCATGGCT-3′ and 5′-AGCCGACGAAATCAATTCTGTC-3′.Sixteen 80-day-old rabbits were purchased from Xing Long Experimental Animal Center, Beijing, China. Before inoculation, the sera of the rabbits were confirmed to be negative for HEV antibodies with reverse transcription-nested PCR (RT-nPCR) Sixteen rabbits were randomly divided into two groups, with eight rabbits in each group. Each rabbit in the inoculated group was inoculated intraperitoneally with 5 mL of viral suspension, whereas each rabbit in the control group was injected with an equal volume of phosphate-buffered saline (PBS).Serum and feces were collected at 1, 4, 7, 14, 21, 28, 35, 42, 52, 62, and 72 days postinoculation (dpi). The serum samples were collected from the marginal ear veins and tested for anti-HEV IgM and IgG antibodies with an enzyme-linked immunosorbent assay (ELISA) based on the HEV ORF2 protein All the serum samples were collected and tested for HEV antigen and antibodies, including IgM and IgG antibodies, with an HEV ELISA kits. All assays were performed according to the manufacturer's instructions .Livers, lungs, kidneys, spleens, lymph nodes, duodenums, jejunums, ileums, SR, and appendices were collected and fixed in 4% paraformaldehyde for 48 h. The fixed tissues were then processed routinely in paraffin and 4-mm sections were prepared. Monoclonal mouse anti-HEV ORF2 antibody  was used as the primary antibody. The primary antibody was added to the sections and incubated at 37°C for 2 h. Immunohistochemical staining was performed according to the instructions of the Histostain™-Plus Kit . 3,3′- Diaminobenzidine tetrahydrochloride  was applied for 10 min to visualize the antigen–antibody reaction, and then Gill's hematoxylin was applied as the background stain. The primary antibody was replaced with phosphate-buffered saline in the negative control. The slides were then observed under an Olympus microscope (Japan).No clinical symptoms were observed in the HEV-infected rabbits. However, one rabbit died during the study, which may be attributable to the stress caused by the regular collection of blood.All the sera and feces from the rabbits in the control group were negative for HEV RNA. Viremia was detected in only one rabbit (72B) at 72 dpi . The fecThe rabbits in both the control group and the inoculated group remained negative for anti-HEV IgM and IgG antibodies throughout the study. However, one rabbit (14B) was positive for serum HEV antigen from before 7 dpi to 14 dpi . Four raAll the tissues from the rabbits in the control group tested negative for HEV RNA. At 14 dpi, the liver, spleen, ileum, and lymph-node samples from only two rabbits  with HEV-RNA-positive feces were positive for HEV RNA. HEV was detected in the lung and duodenum of one of these two rabbits (14D). At 72 dpi, HEV was detected in all the tissues collected from the rabbit (72B) whose feces was constantly positive for HEV RNA, whereas the results were negative for the transiently HEV-positive rabbits .With immunohistochemical staining, HEV antigen was detected in the duodenums, jejunums, ileums, SR, and appendices of two rabbits  in the inoculated group at 14 dpi and in one rabbit (72B) at 72 dpi, whereas antigen was detected in the livers, spleens, lungs, and lymph nodes of all the rabbits in the inoculated group at 14 dpi and 72 dpi. Positive signals were observed in the cytoplasm of hepatocytes and biliary epithelial cells, the epithelium of the bronchioles and alveoli, various cells in spleen and lymph node, the intestinal epithelium, and the follicle-associated epithelium (FAE) of the SR and appendix  [24]. WeHE is recognized as a zoonosis because HEV has been detected in different kinds of animals, including the pig, boar, deer, chicken, rabbit, and rat In contrast to the intravenous injections reported previously, we infected the rabbits intraperitoneally. Although HEV is considered to be transmitted via a fecal–oral route HEV RNA was first detected in the rabbit feces at 4 dpi and persisted until 72 dpi, and thus appeared earlier than in a previous study of rabbits injected intravenously (detected 7 days post-injection) All rabbits were negative for anti-HEV IgM and IgG antibodies throughout the experimental period. In previous studies of other animals, serum antibodies have often been detected before 4 or 5 weeks Previous studies have shown that HEV RNA might be replicated in the liver and many extrahepatic tissues in different animals Rabbits were successfully infected with rabbit HEV via an intraperitoneal route. In HEV infected rabbits, HEV RNA and antigens distributed in different organs according to the different infection types. These data provide valuable information for future research into the pathogenesis of HEV.


SHG) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression.Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue's morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (B These require invasive extraction of tissue samples, thin slicing by microtomes (1–10 µm sections), embedding, fixation, and staining procedure in vivo and hence understand how changes in its production affect the progression of cardiac dysfunction. Recently non-linear optical microscopy has been shown to be a powerful tool for non-invasive imaging of thick specimens The importance of increased fibrosis in heart pathology and dysfunction has been increasingly noted in diseases including dilated, ischaemic and hypertrophic cardiomyopathies. In healthy tissue the extracellular fibrillar collagen forms a dynamic scaffold with important functions for tissue integrity and efficiency of systolic contraction and diastolic relaxation SHG) alone, as only backward-propagating SHG is accessible for future in vivo applications. In particular we propose a 3D intensity-based and volumetric method which can be easily implemented in any laser scanning microscope equipped with multiphoton excitation with no modifications to the light path, thus enabling and enhancing its applicability. To validate the proposed methodology we evaluate collagen presence in the non-infarcted, viable portion of the left ventricle in a rat model of myocardial infarction in vivo.Our aim is to combine the use of TPE  with the simultaneous collection of SHG from collagen fibrils (type I) in thick cardiac tissues to quantitatively evaluate the increase in fibrosis by analyzing the backward SHG  Act 1986, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health under assurance number A5634-01 . The protocol was approved by the Imperial College Ethical Review Committee (PPL: 70/6568 and 70/7399). Adult male Sprague-Dawley rats (250–300 g) underwent proximal left anterior descending coronary artery ligation to induce chronic myocardial infarction For histological analysis, left ventricular tissues were washed in sterile phosphate-buffered saline, fixed in 4% paraformaldehyde, processed for paraffin embedding. 4 µm thick sections were stained with picrosirus red to highlight interstitial collagen As controls, rat tail (∼100% collagen) and permeabilised skeletal muscle fibres (∼0% collagen) were used. Rat tail sections were dissected in relaxing solution and collagenous strands were isolated, T-clipped and stored in relax and glycerol solution at −20°C. Skeletal muscle fibers were isolated from the psoas muscle of adult male New Zealand white rabbits. Bundles were removed and permeabilized as described previously SHG and TPE autofluorescence. Lambda stacks for spectral analysis were collected from 390 nm to 700 nm (10 nm increments) exciting at different two-photon excitation wavelengths from 800 nm to 940 nm (20 nm increments). BSHG versus excitation power were performed by varying the neutral filter at the laser output to increase the excitation power: power was measured after the objective (directly onto the samples) with a power meter (Thorlabs), ranging from 10 mW up to 70 mW. Forward vs Backward SHG measurements were performed by collecting 3D sectioning at different NaCl concentrations  compared to relaxing solution. Polarization measurements were performed by rotating the stage in 10degree increments. Intensity and volumetric analysis are performed on SHG and autofluorescence stacks using a custom-designed macro within the Java-based program ImageJ (http://rsb.info.nih.gov/ij/).All samples were mounted in relaxing solution between hooks on a custom-made set-up optimized for fitting the microscope stage. Two-photon excitation and SHG imaging was performed using a Leica TCS SP5 upright laser scanning system (Leica Microsystem), coupled to a titanium∶sapphire laser . All imaging was performed with an excitation wavelength of 900 nm (except for the spectra collection) with an average power of ∼10–12 mW  at the specimen using a long working distance water immersion objective 25×0.9NA. Typical acquisition parameters are the following: 512×512 pixels, zoom 2.5, pixel size 0.53×0.53 µm, Kalman average of four images, pixel dwell time 18.75 µs, z-step size 0.4 µm. The SHG was collected in the backward direction by selecting the 440–460 nm range while autofluorescence light was collected in the 500–700 nm range. Typical z-stack of 100–150 µm have been acquired simultaneously collecting BIn this study 16 animals (8MI and 8AMC) were used. From each animal 2–3 trabecular preparations were excised for imaging analysis. For each trabecula, four z-stacks (each nearly 100 µm depth) were collected in different areas (248 µm×248 µm) of the sample in order to control for inter sample variability. Both the intensity and the volume analysis are presented as mean and standard deviation.SHG from autofluorescence. As shown in SHG peaks at exactly half the excitation wavelength, as expected from theory, correspondingly shifting with it, while autofluorescence spectra neither change shape or shift, showing a weaker intensity in a much broader λ-range. This spectral analysis represents the identity card of the collected light allowing for optimisation of excitation-emission settings. In experiments shown here, 900 nm excitation is a convenient compromise between BSHG intensity, low autofluorescence (but still useful to outline the sample volume), penetration depth and Ti-sapphire laser performance.Histological sections of left ventricular tissues stained with Picrosirius Red (PR) are used to determine the biological origin of the SHG and autofluorescence signal collection. As shown in Excluding myosin-SHG. In biological tissue, SHG has been shown to arise from collagen, microtubules as well as myosin SHG signal generated in Type II skeletal muscle fibres arises only from myosin while the one generated in cardiac tissues arises from both myosin and collagen: in this case, despite both collagen-SHG and myosin-SHG generate light at the same wavelength, the different localization within the muscle tissue allows for discriminating the two signals. In SHG generation by myosin requires very much higher laser intensity at 900 nm than collagen. Other excitation wavelengths were tested at increasing powers inducing a similar response (data not shown). In the power range 10 to 20 mW, the myosin-SHG signal can be ignored as it contributes to less than 5% of the total with a good signal to noise ratio for the collagen-SHG collection. All the measurements on infarcted and healthy cardiac tissues were performed at 10–12 mW, thus also limiting the induction of photodamage.Maximising Backward Collection. While fluorescence is isotropic and hence emitted in all directions, SHG is anisotropic because of phase matching considerations and hence highly directional SHG on rat tail fibrils at different NaCl concentrations in comparison with the relaxing solution used to image fresh cardiac tissues, at an ionic strength comparable to physiological saline concentrations . SHG with increasing NaCl concentration, reaching saturation above 50 mM NaCl; measurements performed in relaxing solution were also at the plateau, demonstrating that in our experimental conditions, we maximize the BSHG collection. It is worth noting however that the BSHG we refer to, collected in the epi-mode, is partly due to true backward-SHG and partly due to back-scattered forward-SHG. This means that even though the truly backward SHG light intensity is usually quite small, with the contribution from the back-scatter a measurable SHG signal is obtained in tissue. Optimizing the ionic strength of the solution further maximizes the signal collection.Polarization effect. SHG intensity is dependent on the angle between the collagen fibrils and the polarization of incident light SHG seen in MI samples compared to AMC. As an added precaution, the infarcted or healthy samples were imaged in the same orientation under the microscope. The experimental observation suggests that collagen fibres in both healthy and in diseased samples are mainly parallel to the main muscle axis. It is worth noting that increased fibrosis in diseased samples could lead to the appearance of collagen fibres which are not parallel to the main muscle axis, in the worse case perpendicular to it. In this case the BSHG intensity evaluated would be underestimated by 6%. The use of circularly-polarized light would eliminate polarization-induced intensity differences.Detection Range. Measurements to determine the BSHG detection range were performed on rat tail samples, skeletal muscle fibres and cardiac trabeculae. We used rat tail samples as positive control because they are mainly composed of collagen type I, and single permeabilised skeletal muscle fibres as negative control because the permeabilisation process removes the outer membrane and the thin collagen sheath surrounding it. SHG collected for rat tail (black), three trabeculae (dark grey) and two skeletal fibres (light grey), normalized to the tail BSHG intensity. The rat tail BSHG response represents indeed the upper bound of detection; permeabilised skeletal fibres show little collagen and define the lower limit of detection ; cardiac trabeculae from healthy hearts show a small but detectable intensity (5–6%) of BSHG with respect to the tail, thus ensuring a maximal range (95%) allowance for the increased fibrosis expected in samples from infarcted hearts.Penetration Depth. SHG intensity is limited by penetration depth. Depth attenuation is ruled by bulk optical properties: absorption coefficient, scattering, refractive index, etc. Experimentally the excitation light at the range of wavelengths used here (800–950 nm) is limited to 200–300 µm depth, depending on the tissue turbidity and scatterers distribution. The backward light collected (BSHG convolved with scattering) rapidly decays within the first 100 µm as shown in SHG arising from collagen and autofluorescence from elastin and other myocyte proteins. The autofluorescence image is used to define the edges of the sample, by thresholding the autofluorescence intensity as depicted in SHG image  of 3 MI samples is presented in comparison with 2 AMC samples as an example. Clearly an increased BSHG intensity can be observed due to the increased fibrosis. But even more interestingly, a different spatial distribution through the sample thickness can be outlined. Control samples show a more superficial presence of collagen (the first 10 µm), while in the MI samples the BSHG intensity increases also in central layers (see Video S1 and S2 for the entire z-stack and 3D rendering examples). To quantitatively evaluate the BSHG intensity increase in the MI samples with respect to the controls the integral over 10 µm steps is performed and the mean and standard deviation is evaluated for the two groups. SHG intensity . More interestingly, at higher depth the BSHG intensity ratio (MI/AMC) increases up to 6-fold, demonstrating the appearance of new collagen, not present in the AMC samples.To quantitatively analyze the increase in fibrosis, optical sectioning measurements were performed on non-infarcted trabeculae extracted from rats sacrificed 20 weeks following myocardial infarction (8MI samples) in comparison with age-match controls (8AMC samples), by simultaneously acquiring BHG image  in orderSHG to assess the volume of collagen in our samples. The autofluorescence image : these data are normalized so that the area under the autofluorescence-related curves is equal to unity, hence representing the total volume of that sample, and the BSHG-related curves representing the percentage of collagen in that volume. Red and orange curves refer to MI samples; black and grey lines represent AMC samples. By integrating orange and grey curves the fraction of collagen present in that volume is then calculated. Finally we used autofluorescence and Bce image  is used in vivo. In particular, to quantitatively evaluate the collagen presence in the myocardium, a careful calibration of the acquisition settings is presented by performing measurements on rat tail samples and permeabilised skeletal muscle fibres as controls. In particular we show how to specifically separate SHG arising from collagen and SHG arising from myosin, by utilizing powers ranging from 10 to 20 mW  is observed because the polarization status at the focus is elliptical and the tail samples presented fibrils at different orientation, thus flattening the response However, this result ensures that in our experimental conditions, the polarization direction doesn't affect the evaluation of the increase in BSHG seen in MI samples compared to AMC.(TIF)Click here for additional data file.Video S1SHG in fire pseudo scale colour and autofluorescence in green.Optical sectioning example of a MI sample showing B(AVI)Click here for additional data file.Video S2SHG in fire pseudo scale colour and autofluorescence in green.3D rendering of the MI sample in movie1 showing B(AVI)Click here for additional data file.


Induced pluripotent stem cells (iPSC) provide means to study the pathophysiology of genetic disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant inherited ion channel disorder predominantly caused by mutations in the cardiac ryanodine receptor (RyR2). In this study the cellular characteristics of CPVT are investigated and whether the electrophysiological features of this mutation can be mimicked using iPSC -derived cardiomyocytes (CM).P2328S mutation in RyR2 and from two healthy controls. Calcium (Ca2+) cycling and electrophysiological properties were studied by Ca2+ imaging and patch-clamp techniques. Monophasic action potential (MAP) recordings and 24h-ECGs of CPVT-P2328S patients were analyzed for the presence of afterdepolarizations. We found defects in Ca2+ cycling and electrophysiology in CPVT CMs, reflecting the cardiac phenotype observed in the patients. Catecholaminergic stress led to abnormal Ca2+ signaling and induced arrhythmias in CPVT CMs. CPVT CMs also displayed reduced sarcoplasmic reticulum (SR) Ca2+ content, indicating leakage of Ca2+ from the SR. Patch-clamp recordings of CPVT CMs revealed both delayed afterdepolarizations (DADs) during spontaneous beating and in response to adrenaline and also early afterdepolarizations (EADs) during spontaneous beating, recapitulating the changes seen in MAP and 24h-ECG recordings of patients carrying the same mutation.Spontaneously beating CMs were differentiated from iPSCs derived from a CPVT patient carrying a 2+ cycling characteristic of CPVT and in addition to DADs it displays EADs. This cell model for CPVT provides a platform to study basic pathology, to screen drugs, and to optimize drug therapy.This cell model shows aberrant Ca Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe inherited cardiac disorder characterized by stress-induced polymorphic ventricular tachycardia in a structurally normal heart. Approximately 30% of CPVT patients have symptoms before the age of 10 and the mortality rate is 30–35% by the age of 30. β-blockers are recommended for CPVT, but this treatment often fails to prevent even fatal arrhythmias RyR2) or calsequestrin (CASQ2) gene. RyR2 is involved in the release of calcium (Ca2+) from the sarcoplasmic reticulum (SR) and thus plays a key role in excitation-contraction coupling. Calsequestrin is a regulatory calcium-buffering protein associated with RyR2 in the SR. RyR2 mutations can be detected in about 70% of patients with CPVT. These mutations are thought to result in increased release, or leak, of Ca2+ from the SR potentially leading to diastolic oscillations of intracellular Ca2+, delayed afterdepolarizations (DAD), and polymorphic ventricular tachycardia CPVT is caused by mutations in the cardiac ryanodine receptor  technology where pluripotent stem cells are generated by reprogramming differentiated cells into a pluripotent state provides a way to study the pathophysiology of various disorders in human cells. iPSCs can be differentiated into the desired cell type, retaining the original genotype. Recently CPVT-specific iPSCs -derived cardiomyocytes (CMs) from individuals carrying P2328S mutation in RyR2 has been found in families with CPVT. Here we introduce a functional cell model for CPVT caused by this mutation. We investigated the mechanistic characteristics of this disease in vitro using iPSC –derived CMs. Importantly, we demonstrate the presence of EADs in addition to DADs as a pathophysiological mechanism of CPVT.The RyR2-P2328S mutation. iPSC lines UTA.00112.hFF (derived from foreskin fibroblasts) and UTA.04602.WT  were used as controls.The study was approved by the ethical committee of Pirkanmaa Hospital District (R08070) and written informed consent was obtained from all the participants. Patient-specific iPSC lines were established as described earlier RyR2-P2328S mutation was assayed with PCR amplification of genomic DNA with primers for RyR2 exon 46  and HaeIII digestion . DNA for wild type was 170 and 87 and for P2328S heterozygote 257, 170 and 87 base pairs long. For confirmation of the mutation by direct sequencing, the RyR2 exon 46 PCR products were sequenced with BigDye Terminator v3.1 and ABI 3730xl DNA Analyzer .The Karyotypes of the cell lines were determined using standard G-banding chromosome analysis .Endogenous and exogenous gene expressions were studied from iPSCs by RT-PCR. The PCR reaction consisted of 1 µl cDNA and 500 nmol/L of each primer. PCR primers for iPSC characterization and detailed reaction conditions have been described earlier The iPSCs were fixed with 4% paraformaldehyde . Primary antibodies anti-SOX2, anti-NANOG, anti-stage-specific embryonic antigen (SSEA)4, and anti-tumour-related antigen (TRA)1–81 , anti-OCT3/4  and anti-TRA1–60  were used. Cells were mounted with Vectashield  containing 4′, 6-diamidino-2-phenylindole (DAPI) for staining nuclei.Nestin), endoderm (AFP), and mesoderm (α-cardiactin) development in EBs were studied  for 5 weeks. The expression of markers characteristic of ectoderm . iPSCs were injected into nude mice under the testis capsule and tumor samples collected 8 weeks after injection. This was followed by fixation with 4% paraformaldehyde and staining of the sections with haematoxylin and eosin.Differentiation into cardiomyocytes (CMs) was carried out by co-culturing iPSCs with murine visceral endoderm-like (END-2) cells  as described earlier Single beating CMs were immunostained with anti-cardiac-troponin-T , anti-α-actinin  and anti-connexin-43 .TNTT2), RyR2, SR Ca2+ ATPase (SERCA2a), L-type Ca2+ channel (v1.2Ca), phospholamban (PLN) and sodium-calcium exchanger (NCX) were assessed. Primer sequences are listed in GAPDH served as a housekeeping gene.RNA was isolated from iPSC-derived CMs. The expression of troponin T .Dissociated CMs on a coverslip were loaded with 4 µmol/L Fura-2 AM  for 30 minutes in HEPES based medium, followed by a 30-minute de-esterification. The coverslip was then transferred to an RC-27NE or an RC-25 recording chamber  and continuously perfused with 37°C HEPES based perfusate. The perfusate was preheated by an SH-27B inline-heater controlled by a TC-324B unit  and consisted of (in mmol/L): 137 NaCl, 5 KCl, 0.44 KH2+ measurements were conducted on an inverted IX70 microscope  where spontaneously beating CMs were visualized with a UApo/340 x20 air objective (Olympus). Images were acquired with an ANDOR iXon 885 CCD camera  synchronized with a Polychrome V light source by a real time DSP control unit and TILLvisION software . Fura-2 in CMs was excited at 340 nm and 380 nm light and the emission was recorded at 505 nm. For Ca2+ analysis, regions of interests were selected for spontaneously beating cells and background noise was subtracted before further processing. The Ca2+ levels are presented as ratiometric values of F340/F380 or as ΔF/F0.Ca2+ were recorded during spontaneous baseline beating, electrical pacing, spontaneous beating during 1 µmol/L adrenaline (Sigma-Aldrich) perfusion and electrical pacing during adrenaline perfusion. The SR Ca2+ content was measured by releasing all the SR Ca2+ with instantaneous and high concentration (40 mmol/L) caffeine (Sigma-Aldrich) puffs after each measurement, after which the relative amplitude change in calcium release in CPVT versus control CMs was quantified. Viability of CMs was confirmed after the experimental protocol. Amplitudes, beating frequency and caffeine induced Ca2+ peaks were analyzed with Clampfit version 9.2 . Analysis was performed blinded to genotype of CMs.CMs were paced with 10–50 ms pulses (27–32 mA)  at a frequency of 0.1–0.3 Hz higher than the spontaneous beating rate. The changes in Ca2, 1.2 MgCl2, 5 glucose, 10 HEPES, pH was adjusted to 7.4 with NaOH and the osmolarity set to 300±2 mOsm . The intracellular solution consisted of (in mmol/L): 122 KMeSO4, 30 KCl, 1 MgCl2, 10 HEPES. KOH was used to set pH to 7.15 and the osmolarity was set to 295±2 mOsm. Amphotericin B (Sigma-Aldrich) was used as membrane perforation agent and dissolved in DMSO to a final concentration in the patch pipette of 0.24 mg/ml. Spontaneously beating CMs were patched in same bath conditions as in Ca2+ imaging. Patch pipettes  were pulled with a PC-10 puller and flame polished with Microforge MF-900  to a resistance of 2.0–2.5 MΩ measured in the bath perfusate. APs were recorded in gab-free mode with pClamp 10.2 using the Axopatch 200B patch-clamp amplifier connected to an acquisition computer via AD/DA Digidata 1440 . Current-clamp recordings were digitally sampled at 20 kHz and filtered at 5 kHz using the lowpass Bessel filter on the recording amplifier. AP duration at 50% and 90% of repolarization (ADP50 and ADP90), AP amplitude (APA), maximum diastolic potential (MDP) and beats per minute (BPM) were extracted from AP recordings using an automated script in Microcal OriginTM 8.6.Action potentials (APs) were recorded in current-clamp mode using the standard patch-clamp technique in the perforated patch configuration Monophasic action potentials (MAPs) were previously recorded from CPVT patients and healthy controls as reported EADs were defined as low-amplitude depolarizations that occur during phase 2 or 3 of the AP, before completion of repolarization, and have an amplitude of ≥3% of the preceding AP. DADs were defined as low-amplitude depolarizations that occur after completion of repolarization, and have an amplitude of ≥3% of the preceding AP 24h-ECGs were previously recorded from CPVT patients and healthy controls as reported The first peak during repolarization was considered as a T1-wave. The second peak was considered as a T2-wave if it occasionally merged with the T1-wave, or as a U-wave if it never merged with the T1-wave. The third peak, which never merged with the T1-wave, was also considered as a U-wave t-test. The significance of changes within a group was evaluated with the paired Student’s t-test. P<0.05 was considered statistically significant, where (*) represents P<0.05 and (**) P<0.01. Data are expressed as means ± S.E.M. and n (where indicated) refers to the number of cells or experiments.The significance of differences between two groups was evaluated with the unpaired Student’s P2328S mutation was confirmed in the two CPVT-P2328S iPSC lines (in vitro embryoid body (EB) formation expressing all three germ layers . Pacing stabilized Ca2+ cycling partially in CPVT CMs and completely in control CMs. Adrenaline increased Ca2+ cycling abnormalities to 30% of the CPVT CMs. In control CMs adrenaline had no effect on Ca2+ cycling. Pacing with adrenaline abolished all Ca2+ cycling abnormalities in controls but did not have an effect in CPVT CMs.Car rhythm . Ca2+ cyondition . At base2+ levels  DADs were randomly observed during baseline recordings (1–2 DADs/60 APs). At baseline DADs were observed in 6/14 (42%) of CPVT CMs. In 6/11 CPVT CMs exposed to adrenaline, wash-out recovered the beating rate to normal as in control CMs. However, in the remaining 5/11 CPVT CMs, adrenaline subsequently evoked DADs and resulted in decreased beating rate  increases SR Ca2+ load. When the fractional SR Ca2+ release is measured immediately after the pacing, increased values are observed. As expected, in this case the fractional SR Ca2+ release is greater in the RyR2 mutant CMs, which are more sensitive to luminal SR Ca2+ release.Fractional SR Ca2+ channel (LTCC) during conditions of prolonged APD, such as LQT2 2+ overload and spontaneous Ca2+ release as the main triggers behind EADs 2+ overload include heart failure, digitalis toxicity, and CPVT. Under conditions of SR Ca2+ overload or leaky RyR2s, spontaneous release of Ca2+ from the SR leads to activation of the sodium-calcium-exchanger (NCX), which results in a depolarizing current that reactivates the LTCC, leading to an EAD. It has been shown that early Ca2+ aftertransients are the primary events that induce EADs, and not vice versa 2+ by reducing NCX-mediated Ca2+ influx 2+ will less likely cause SR Ca2+ leak, which would lead to forward-mode NCX activation and afterdepolarizations. Accordingly, we must acknowledge the potential role of the late sodium current in EAD provocation.Traditionally, EADs have been thought to result from spontaneous reactivation of the L-type CaCPVT-P2328S patients show both DADs and EADs. Although not experimentally shown, it has previously been suggested that CPVT patients with RyR2 mutations are susceptible to both EAD and DAD-mediated arrhythmia mechanisms Our results support this emerging consensus on the role of NCX-mediated generation of EADs. We found that CPVT CMs display irregular spontaneous calcium release events, DADs, and EADs. Furthermore, MAP recordings in RyR2 mutation P2328S-RyR2 mutation presents arrhythmias only in the presence of catecholaminergic stimulation, but not if increased beating rate is generated by pacing.We could not demonstrate increased arrhythmogenicity with pacing. At baseline, pacing stabilized beating in both control and CPVT CMs. When arrhythmias were provoked in CPVT CMs with adrenaline, pacing on top of that did not have any affect on the recorded arrhythmias. This is contrary to previous CPVT reports using either spontaneously beating CMs with a CASQ2 mutation CPVT-P2328S CMs display EADs which may be involved in arrhythmogenesis in these patients. This broadens the mechanistic understanding of arrhythmias linked to RyR2 mutations and helps to direct efforts to optimize therapy in these patients. Our iPSC-derived CM model offers a promising platform for further research into the pathophysiological mechanisms of CPVT, as well as a safe tool for screening and optimizing drug therapy with patient-specific CMs.Our findings demonstrate that in addition to DADs, We studied iPSC-derived CMs from two CPVT and two control iPS cell lines. However, both CPVT lines were from the same patient. The control lines were from two healthy controls. It is therefore unclear whether the changes we saw are typical of all CPVT patients, of this specific mutation, or only this specific patient. In the future we need to extend our studies, looking at various RyR2 mutations and several cell lines from multiple patients harboring a specific mutation.2+ imaging studies. Part of the variability in the results may stem from differences between the CM types. To address this in the future, simultaneous recording of APs and intracellular Ca2+ will help to distinguish cell type and additionally give temporally synchronized info on the interplay between Ca2+ handling and APs.The type of CM  under investigation was unclear in the CaAdditionally, further development of cardiac differentiation and maturation procedures will hopefully improve the homogeneity of iPS cell lines.