Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

FAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULORelaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis361217171723FAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO95/0355-

Inhibition Of The Sarcoplasmic Reticulum Ca2+ Pump With Thapsigargin Estimate The Contribution Of Na+-ca2+ Exchange To Ventricular Myocyte Relaxation

Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 μM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 μM). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.361217171723Bers, D.M., Bassani, J.W.M., Bassani, R.A., Na/Ca exchange and Ca fluxes during contraction and relaxation in mammalian ventricular muscle (1996) Annals of the New York Academy of Sciences, 779, pp. 430-442Bers, D.M., (2001) Excitation-Contraction Coupling and Cardiac Contractile Force, , 2nd edn. Kluwer Press, Dordrecht, The NetherlandsBassani, J.W.M., Bassani, R.A., Bers, D.M., Relaxation in rabbit and rat cardiac cells: Species-dependent differences in cellular mechanisms (1994) Journal of Physiology, 476, pp. 279-293Bassani, R.A., Bassani, J.W.M., Bers, D.M., Relaxation in ferret ventricular myocytes: Unusual interplay among calcium transport systems (1994) Journal of Physiology, 476, pp. 295-308Negretti, N., O'Neill, S.C., Eisner, D.A., The relative contributions of different intracellular and sarcolemmal systems to relaxation in rat ventricular myocytes (1993) Cardiovascular Research, 27, pp. 1826-1830Rousseau, E., Meissner, G., Single cardiac sarcoplasmic reticulum Ca2+-release channel: Activation by caffeine (1989) American Journal of Physiology, 256, pp. H328-H333Bassani, R.A., Bassani, J.W.M., Contribution of Ca2+ transporters to relaxation in intact ventricular myocytes from developing rats (2002) American Journal of Physiology, 282, pp. H2406-H2413Li, L., Chu, G., Kranias, E.G., Bers, D.M., Cardiac myocyte calcium transport in phospholamban knockout mouse: Relaxation and endogenous CaMKII (1998) American Journal of Physiology, 274, pp. H1335-H1347McCall, E., Ginsburg, K.S., Bassani, R.A., Shannon, T.S., Qi, M., Samarel, A.M., Bers, D.M., Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes (1998) American Journal of Physiology, 274, pp. H1348-H1360Blaustein, M., Lederer, W.J., Sodium/calcium exchange: Its physiological implications (1999) Physiological Reviews, 79, pp. 763-854Zaniboni, M., Yao, A., Barry, W.H., Musso, E., Spitzer, K., Complications associated with rapid caffeine application to cardiac myocytes that are not voltage-clamped (1998) Journal of Molecular and Cellular Cardiology, 30, pp. 2229-2235Schlotthauer, K., Bers, D.M., Sarcoplasmic reticulum Ca2+ release causes myocyte depolarization: Underlying mechanism and threshold for triggered action potentials (2000) Circulation Research, 87, pp. 774-780Varro, A., Negretti, N., Hester, S.B., Eisner, D.A., An estimate of the calcium content of the sarcoplasmic reticulum in rat ventricular myocytes (1993) Pflügers Archives, 423, pp. 158-160Grynkiewicz, C., Poenie, M., Tsien, R.Y., A new generation of Ca2+ indicators with greatly improved fluorescence properties (1985) Journal of Biological Chemistry, 260, pp. 3440-3450Gomes, P.A.P., Bassani, R.A., Bassani, J.W.M., Measuring [Ca2+] with fluorescent indicators: Theoretical approach to the ratio method (1998) Cell Calcium, 24, pp. 17-26Bassani, J.W.M., Bassani, R.A., Bers, D.M., A method for calibration of indo-1 and resting [Ca]i in intact rabbit cardiac myocytes (1995) Biophysical Journal, 68, pp. 1453-1460Bassani, J.W.M., Bassani, R.A., Bers, D.M., Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes (1993) American Journal of Physiology, 265, pp. C533-C540Kirby, M.S., Sagara, Y., Gaa, S., Inesi, G., Lederer, W.J., Rogers, T.B., Thapsigargin inhibits contraction and Ca transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum calcium pump (1991) Journal of Biological Chemistry, 267, pp. 12545-12551Bassani, R.A., Shannon, T.S., Bers, D.M., Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats (1998) Cell Calcium, 23, pp. 433-442Pogwizd, S.M., Qi, M., Yuan, W., Samarel, A.M., Bers, D.M., Upregulation of Na+/Ca2+ exchange expression and function in an arrhythmogenic rabbit model of heart failure (1999) Circulation Research, 85, pp. 1009-101

Effect of ryanodine on sinus node recovery time determined in vitro

FAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOEvidence has indicated that the sarcoplasmic reticulum (SR) might be involved in the generation of spontaneous electrical activity in atrial pacemaker cells. We report the effect of disabling the SR with ryanodine (0.1 µM) on the sinus node recovery time (SNRT) measured in isolated right atria from 4-6-month-old male Wistar rats. Electrogram and isometric force were recorded at 36.5oC. Two methods for sinus node resetting were used: a) pulse: a single stimulus pulse interpolated at coupling intervals of 50, 65 or 80% of the regular spontaneous cycle length (RCL), and b) train: a 2-min train of pulses at intervals of 50, 65 or 80% of RCL. Corrected SNRT (cSNRT) was calculated as the difference between SNRT (first spontaneous cycle length after stimulation interruption) and RCL. Ryanodine only slightly increased RCL (<10%), but decreased developed force by 90%. When the pulse method was used, cSNRT (~40 ms), which represents intranodal/atrial conduction time, was independent of the coupling interval and unaffected by ryanodine. However, cSNRT obtained by the train method was significantly higher for shorter intervals between pulses, indicating the occurrence of overdrive suppression. In this case, ryanodine prolonged cSNRT in a rate-dependent fashion, with a greater effect at shorter intervals. These results indicate that: a) a functional SR, albeit important for force development, does not seem to play a major role in atrial automaticity in the rat; b) disruption of cell Ca2+ homeostasis by inhibition of SR function does not appear to affect conduction; however, it enhances overdrive-induced depression of sinusal automaticity.Evidence has indicated that the sarcoplasmic reticulum (SR) might be involved in the generation of spontaneous electrical activity in atrial pacemaker cells. We report the effect of disabling the SR with ryanodine (0.1 µM) on the sinus node recovery time (SNRT) measured in isolated right atria from 4-6-month-old male Wistar rats. Electrogram and isometric force were recorded at 36.5oC. Two methods for sinus node resetting were used: a) pulse: a single stimulus pulse interpolated at coupling intervals of 50, 65 or 80% of the regular spontaneous cycle length (RCL), and b) train: a 2-min train of pulses at intervals of 50, 65 or 80% of RCL. Corrected SNRT (cSNRT) was calculated as the difference between SNRT (first spontaneous cycle length after stimulation interruption) and RCL. Ryanodine only slightly increased RCL (<10%), but decreased developed force by 90%. When the pulse method was used, cSNRT (~40 ms), which represents intranodal/atrial conduction time, was independent of the coupling interval and unaffected by ryanodine. However, cSNRT obtained by the train method was significantly higher for shorter intervals between pulses, indicating the occurrence of overdrive suppression. In this case, ryanodine prolonged cSNRT in a rate-dependent fashion, with a greater effect at shorter intervals. These results indicate that: a) a functional SR, albeit important for force development, does not seem to play a major role in atrial automaticity in the rat; b) disruption of cell Ca2+ homeostasis by inhibition of SR function does not appear to affect conduction; however, it enhances overdrive-induced depression of sinusal automaticity32810391043FAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP – FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informação300069/95-2; 301905/84-3-N

Fine Splitting of Electron States in Silicon Nanocrystal with a Hydrogen-like Shallow Donor

Electron structure of a silicon quantum dot doped with a shallow hydrogen-like donor has been calculated for the electron states above the optical gap. Within the framework of the envelope-function approach we have calculated the fine splitting of the ground sixfold degenerate electron state as a function of the donor position inside the quantum dot. Also, dependence of the wave functions and energies on the dot size was obtained

The economic impact of moderate stage Alzheimer's disease in Italy: Evidence from the UP-TECH randomized trial

Background: There is consensus that dementia is the most burdensome disease for modern societies. Few cost-of-illness studies examined the complexity of Alzheimer's disease (AD) burden, considering at the same time health and social care, cash allowances, informal care, and out-of-pocket expenditure by families. Methods: This is a comprehensive cost-of-illness study based on the baseline data from a randomized controlled trial (UP-TECH) enrolling 438 patients with moderate AD and their primary caregiver living in the community. Results: The societal burden of AD, composed of public, patient, and informal care costs, was about �20,000/yr. Out of this, the cost borne by the public sector was �4,534/yr. The main driver of public cost was the national cash-for-care allowance (�2,324/yr), followed by drug prescriptions (�1,402/yr). Out-of-pocket expenditure predominantly concerned the cost of private care workers. The value of informal care peaked at �13,590/yr. Socioeconomic factors do not influence AD public cost, but do affect the level of out-of-pocket expenditure. Conclusion: The burden of AD reflects the structure of Italian welfare. The families predominantly manage AD patients. The public expenditure is mostly for drugs and cash-for-care benefits. From a State perspective in the short term, the advantage of these care arrangements is clear, compared to the cost of residential care. However, if caregivers are not adequately supported, savings may be soon offset by higher risk of caregiver morbidity and mortality produced by high burden and stress. The study has been registered on the website www.clinicaltrials.org (Trial Registration number: NCT01700556). Copyright � International Psychogeriatric Association 2015

Socioeconomic Predictors of the Employment of Migrant Care Workers by Italian Families Assisting Older Alzheimer's Disease Patients: Evidence from the Up-Tech Study

Background: The availability of family caregivers of older people is decreasing in Italy as the number of migrant care workers (MCWs) hired by families increases. There is little evidence on the influence of socioeconomic factors in the employment of MCWs. Method: We analyzed baseline data from 438 older people with moderate Alzheimer's disease (AD), and their family caregivers enrolled in the Up-Tech trial. We used bivariate analysis and multilevel regressions to investigate the association between independent variables - education, social class, and the availability of a care allowance - and three outcomes - employment of a MCW, hours of care provided by the primary family caregiver, and by the family network (primary and other family caregivers). Results: The availability of a care allowance and the educational level were independently associated with employing MCWs. A significant interaction between education and care allowance was found, suggesting that more educated families are more likely to spend the care allowance to hire a MCW. Discussion: Socioeconomic inequalities negatively influenced access both to private care and to care allowance, leading disadvantaged families to directly provide more assistance to AD patients. Care allowance entitlement needs to be reformed in Italy and in countries with similar long-term care and migration systems. � 2015 The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved

Serca Upregulation: Breaking The Positive Feedback In Heart Failure?

[No abstract available]674581582Hasenfuss, G., Alterations of calcium-regulatory proteins in heart failure (1998) Cardiovasc Res, 37, pp. 279-289Periasamy, M., Huke, S., SERCA pump level is a critical determinant of Ca2+ homeostasis and cardiac contractility (2001) J Mol Cell Cardiol, 33, pp. 1053-1063Pogwizd, S.M., Schlotthauer, K., Li, L., Yuan, W., Bers, D.M., Arrhythmogenesis and contractile dysfunction in heart failure: Roles of sodium-calcium exchanger, inward rectifier potassium channels and β-adrenergic responsiveness (2001) Circ Res, 88, pp. 1159-1169Kaprielian, R., Del Monte, F., Hajjar, R.J., Targeting Ca2+ cycling proteins and the action potential in heart failure by gene transfer (2002) Basic Res Cardiol, 97 (1 SUPPL.), pp. 136-I/145Frank, K.F., Bolck, B., Erdmann, E., Schwinger, R.H.G., Sarcoplasmic reticulum Ca-ATPase modulates cardiac contraction and relaxation (2003) Cardiovasc Res, 57, pp. 20-27Sipido, K., Volders, P.G.A., Vos, M.A., Verdonck, F., Altered Na/Ca exchange activity in cardiac hypertrophy and heart failure: A new target for therapy? (2002) Cardiovasc Res, 53, pp. 782-805Bristow, M.R., Why does the myocardium fail? Insights from basic sciences (1998) Lancet, 352 (1 SUPPL.), pp. 8-SI14Kiriazis, H., Kranias, E.G., Genetically engineered models with alterations in cardiac membrane calcium-handling proteins (2000) Annu Rev Physiol, 62, pp. 321-351Maier, L.S., Wahl-Schott, C., Horn, W., Weichert, S., Pagel, C., Wagner, S., Increased SR Ca2+ cycling contributes to improved contractile performance in SERCA2a-overexpressing transgenic rats (2005) Cardiovasc Res, 67, pp. 636-646Yao, A., Su, Z., Dillman, W.H., Barry, W.H., SR function in murine ventricular myocytes overexpressing SR Ca 2+-ATPase (1998) J Mol Cell Cardiol, 30, pp. 2711-2718Bassani, J.W.M., Yuan, W., Bers, D.M., Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes (1995) Am J Physiol, 268, pp. 1313-C1319Del Monte, F., Lebeche, D., Guerrero, J.L., Tsuji, T., Doye, A.A., Gwathmey, J.K., Abrogation of ventricular arrhythmias in a model of ischemia and reperfusion by targeting myocardial calcium cycling (2004) Proc Natl Acad Sci U S a, 101, pp. 5622-5627Ziolo, M.T., Martin, J.L., Bossuyt, J., Bers, D.M., Pogwizd, S.M., Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function (2005) Circ Res, 96, pp. 815-817Shorofsky, S.R., Aggarwal, R., Corretti, M., Baffa, J.M., Strum, J.M., Al-Seikhan, B.A., Cellular mechanisms of altered contractility in the hypertrophied heart. Big hearts, big sparks (1999) Circ Res, 84, pp. 424-434Bassani, R.A., Carvalho, B.M.R., Franchini, K.G., Bassani, J.W.M., Greater sarcoplasmic reticulum (SR) Ca2+ release in early ventricular hypertrophy induced by pressure overload (2005) Biophys J, 88 (1 SUPPL.), pp. 1556-PosJanczewski, A.M., Zahid, N., Lemster, B.H., Frye, C.S., Gibson, G., Higuchi, Y., Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model (2004) Cardiovasc Res, 62, pp. 468-48

Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis