In JoVE (1)

Other Publications (12)

Articles by Rafael Mejia-Alvarez in JoVE

 JoVE Biology

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

1School of Natural Sciences, University of California, Merced, 2Centro de Investigaciones Cardiovasculares, Universidad de la Plata and Conicet, 3Facultad de Ingenieria, Universidad Nacional de Entre Rios, 4Department of Physiology, Midwestern University, 5School of Engineering, University of California, Merced


JoVE 55202

Other articles by Rafael Mejia-Alvarez on PubMed

Gating Kinetics and Ligand Sensitivity Modified by Phosphorylation of Cardiac Ryanodine Receptors

Pflugers Archiv : European Journal of Physiology. May, 2002  |  Pubmed ID: 11976933

The effects of protein-kinase- (PKA-) dependent phosphorylation on the stationary gating kinetics of single ryanodine receptor (RyR) channels was defined. The single-channel activity from canine cardiac RyR was reconstituted into planar lipid bilayers. Exogenously applied PKA increased the single-channel open probability ( P(o)) of both native and purified cardiac RyR channels, after preincubation with ATP and Mg2+. The action of PKA on the RyR channel occurred only in the presence of ATP and adenosine 5'- O-(3-thiotriphosphate) (ATPgammaS), but not in the presence of 5'-adenylimidodiphosphate (AMP-PCP). Thus, the action of PKA requires the presence of a hydrolyzable ATP analog. PKA-induced channel activation was blocked by specific PKA inhibitors. All these results confirmed that the RyR channel can be phosphorylated by exogenous protein kinase. The gating kinetics of single RyR channels before PKA treatment were significantly altered by ATP and Mg2+ as physiological ligands. In contrast, after PKA treatment, neither ATP nor Mg2+ significantly alters the gating kinetics of these channels. PKA-dependent phosphorylation thus decreases the ATP and Mg2+ apparent sensitivity in most of the gating parameters of single RyR channels. The phosphorylated RyR channels open and close more frequently, stay open for longer, and stay closed for shorter periods. The dwell-time histograms obtained demonstrate that the phosphorylated and the dephosphorylated channels have strikingly different open and closed kinetics at physiological cytoplasmic concentrations of Mg and ATP.

Pulsed Local-field Fluorescence Microscopy: a New Approach for Measuring Cellular Signals in the Beating Heart

Pflugers Archiv : European Journal of Physiology. Mar, 2003  |  Pubmed ID: 12632197

In cardiac research, single-cell experimental models have been extensively used to study the molecular mechanisms of intracellular Ca(2+) homeostasis. The results of these studies are usually extrapolated to the tissue level assuming that the phenomena studied at the cellular level are either similar in the intact organ, or only slightly modified by variables that exist at the whole-heart level. The validity of these assumptions has rarely been confirmed experimentally. Common obstacles associated with the study of intracellular Ca(2+) signals in beating hearts include motion artifacts and spatio-temporal limitations of the recording system. In this work, action potentials and intracellular Ca(2+) signals were measured in beating hearts from young rats, with spatio-temporal resolutions similar to cellular studies using a novel pulsed local-field fluorescence technique. This method was based on maximizing emitted fluorescence to increase the signal-to-noise ratio (S/N). The fluorescence emission of the indicator molecules was synchronized with brief (<1 ns), high-power (400 W) laser pulses, and the common mode noise of the fluorescence signal was differentially cancelled. To follow rapidly evolving signals, a highly sensitive and fast detection system was used (10 kHz). The spatial resolution was improved using a small (50-200 microm diameter) multimode fiberoptic. Mechanical artifacts were effectively reduced by inserting the fiberoptic into a "floating" glass micropipette sealed to the heart wall with negative pressure. Our results demonstrate that local-field fluorescence microscopy offers an outstanding experimental approach for studying physiological signals at the whole-organ level with the high spatio-temporal resolution common to normal cellular approaches.

Pyruvate Modulates Cardiac Sarcoplasmic Reticulum Ca2+ Release in Rats Via Mitochondria-dependent and -independent Mechanisms

The Journal of Physiology. Aug, 2003  |  Pubmed ID: 12824454

The glycolytic product pyruvate has beneficial effects on cardiac contractile function. The postulated cellular mechanisms underlying the positive inotropic effect of pyruvate, however, are contradictory or have remained elusive. Therefore, we studied the effects of pyruvate on cardiac Ca2+ regulation, intracellular pH (pHi) and flavoprotein oxidation using fluorescence confocal microscopy in intact and permeabilized rat ventricular myocytes and single channel recordings from rat cardiac ryanodine receptors (RyRs) incorporated into planar lipid bilayers. In intact cells extracellular pyruvate (10 mM) elevated diastolic [Ca2+]i, which was due, at least in part, to a concomitant acidification of the cytosol. Furthermore, pyruvate increased the amplitude and slowed the kinetics of the electrically evoked [Ca2+]i transient, and augmented sarcoplasmic reticulum (SR) Ca2+ content. Recording of flavoprotein (FAD) fluorescence indicated that pyruvate caused a reduction of mitochondrial redox potential, which is proportional to an increase of the rate of ATP synthesis. Inhibitors of mitochondrial monocarboxylate transport (alpha-cyano-4-hydroxycinnamate, 0.5 mM), adenine nucleotide translocation (atractyloside, 0.3 mM) and the electron transport chain (cyanide, 4 mM) abolished or attenuated the pyruvate-mediated increase of the amplitude of the [Ca2+]i transient, but did not change the effect of pyruvate on diastolic [Ca2+]i. Results from experiments with permeabilized myocytes indicated a direct correlation between ATP/ADP ratio and SR Ca2+ content. Furthermore, pyruvate (4 mM) reduced the frequency of spontaneous Ca2+ sparks by approximately 50%. Single RyR channel recordings revealed a approximately 60% reduction of the open probability of the channel by pyruvate (1 mM), but no change in conductance. This effect of pyruvate on RyR channel activity was neither Ca2+ nor ATP dependent. Taken together, these findings suggest that, in cardiac tissue, pyruvate has a dual effect on SR Ca2+ release consisting of a direct inhibition of RyR channel activity and elevation of SR Ca2+ content. The latter effect was most probably mediated by an enhanced SR Ca2+ uptake due to an augmentation of mitochondria-dependent ATP synthesis.

Developmental Changes of Intracellular Ca2+ Transients in Beating Rat Hearts

American Journal of Physiology. Heart and Circulatory Physiology. Mar, 2004  |  Pubmed ID: 14644760

Postnatal maturation of the rat heart is characterized by major changes in the mechanism of excitation-contraction (E-C) coupling. In the neonate, the t tubules and sarcoplasmic reticulum (SR) are not fully developed yet. Consequently, Ca(2+)-induced Ca(2+) release (CICR) does not play a central role in E-C coupling. In the neonate, most of the Ca(2+) that triggers contraction comes through the sarcolemma. In this work, we defined the contribution of the sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR to the Ca(2+) transient during the first 3 wk of postnatal development. To this end, intracellular Ca(2+) transients were measured in whole hearts from neonate rats by using the pulsed local field fluorescence technique. To estimate the contribution of each Ca(2+) flux to the global intracellular Ca(2+) transient, different pharmacological agents were used. Ryanodine was applied to evaluate ryanodine receptor-mediated Ca(2+) release from the SR, nifedipine for dihydropyridine-sensitive L-type Ca(2+) current, Ni(2+) for the current resulting from the reverse-mode Na(+)/Ca(2+) exchange, and mibefradil for the T-type Ca(2+) current. Our results showed that the relative contribution of each Ca(2+) flux changes considerably during the first 3 wk of postnatal development. Early after birth (1-5 days), the sarcolemmal Ca(2+) flux predominates, whereas at 3 wk of age, CICR from the SR is the most important. This transition may reflect the progressive development of the t tube-SR units characteristic of mature myocytes. We have hence directly defined in the whole beating heart the developmental changes of E-C coupling previously evaluated in single (acutely isolated or cultured) cells and multicellular preparations.

Multiprotein Complex Containing Succinate Dehydrogenase Confers Mitochondrial ATP-sensitive K+ Channel Activity

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2004  |  Pubmed ID: 15284438

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel plays a central role in protection of cardiac and neuronal cells against ischemia and apoptosis, but its molecular structure is unknown. Succinate dehydrogenase (SDH) is inhibited by mitoK(ATP) activators, fueling the contrary view that SDH, rather than mitoK(ATP), is the target of cardioprotective drugs. Here, we report that SDH forms part of mitoK(ATP) functionally and structurally. Four mitochondrial proteins [mitochondrial ATP-binding cassette protein 1 (mABC1), phosphate carrier, adenine nucleotide translocator, and ATP synthase] associate with SDH. A purified IM fraction containing these proteins was reconstituted into proteoliposomes and lipid bilayers and shown to confer mitoK(ATP) channel activity. This channel activity is sensitive not only to mitoK(ATP) activators and blockers but also to SDH inhibitors. These results reconcile the controversy over the basis of ischemic preconditioning by demonstrating that SDH is a component of mitoK(ATP) as part of a macromolecular supercomplex. The findings also provide a tangible clue as to the structural basis of mitoK(ATP) channels.

Ryanodine Receptor Function in Newborn Rat Heart

American Journal of Physiology. Heart and Circulatory Physiology. May, 2005  |  Pubmed ID: 15626694

The role of ryanodine receptor (RyR) in cardiac excitation-contraction (E-C) coupling in newborns (NB) is not completely understood. To determine whether RyR functional properties change during development, we evaluated cellular distribution and functionality of sarcoplasmic reticulum (SR) in NB rats. Sarcomeric arrangement of immunostained SR Ca(2+)-ATPase (SERCA2a) and the presence of sizeable caffeine-induced Ca2+ transients demonstrated that functional SR exists in NB. E-C coupling properties were then defined in NB and compared with those in adult rats (AD). Ca2+ transients in NB reflected predominantly sarcolemmal Ca2+ entry, whereas the RyR-mediated component was approximately 13%. Finally, the RyR density and functional properties at the single-channel level in NB were compared with those in AD. Ligand binding assays revealed that in NB, RyR density can be up to 36% of that found in AD, suggesting that some RyRs do not contribute to the Ca2+ transient. To test the hypothesis that RyR functional properties change during development, we incorporated single RyRs into lipid bilayers. Our results show that permeation and gating kinetics of NB RyRs are identical to those of AD. Also, endogenous ligands had similar effects on NB and AD RyRs: sigmoidal Ca2+ dependence, stronger Mg(2+)-induced inhibition at low cytoplasmic Ca2+ concentrations, comparable ATP-activating potency, and caffeine sensitivity. These observations indicate that NB rat heart contains fully functional RyRs and that the smaller contribution of RyR-mediated Ca2+ release to the intracellular Ca2+ transient in NB is not due to different single RyR channel properties or to the absence of functional intracellular Ca2+ stores.

Cell Culture Modifies Ca2+ Signaling During Excitation-contraction Coupling in Neonate Cardiac Myocytes

Cell Calcium. Jan, 2007  |  Pubmed ID: 16908061

In heart, the excitation-contraction coupling (ECC) mechanism changes during development. Primary cell culture has been used to study Ca(2+) signaling in newborn (NB) rat heart. In this work, the effects of cell culture on the action potential (AP) and ECC Ca(2+) signaling during development were investigated. Specifically, AP, Ca(2+) currents (I(Ca)), and ryanodine receptor (RyR) properties (i.e. density, distribution, and contribution to Ca(2+) transients and Ca(2+) sparks) were defined in cultured myocytes (CM) from 0-day-old NB rat at different times in culture (1-4 days). Compared with acutely dissociated myocytes (ADM) from NB of equivalent ages (1-4 days), CM showed lower RyR density (50% at 1 day, 25% at 4 days), but larger RyR contribution to the Ca(2+) transient (25% at 1 day, 57% at 4 days). Additionally, Ca(2+) sparks were larger, longer, wider, and more frequent in CM than in ADM. RyR cellular distribution also showed different arrangement. While in CM, RyRs were located peripherally, in ADM of equivalent ages a sarcomeric arrangement was predominant. Finally, CM showed a two-fold increase in sarcolemmal Ca(2+) entry during the AP. These results indicated that primary culture is a feasible model to study Ca(2+) signaling in heart; however, it does not precisely reproduce what occurs in ECC during development.

The Relevance of Non-excitable Cells for Cardiac Pacemaker Function

The Journal of Physiology. Dec, 2007  |  Pubmed ID: 17932143

Age-dependent changes in the architecture of the sinus node comprise an increasing ratio between fibroblasts and cardiomyocytes. This change is discussed as a potential mechanism for sinus node disease. The goal of this study was to determine the mechanism through which non-excitable cells influence the spontaneous activity of multicellular cardiomyocyte preparations. Cardiomyocyte monolayers (HL-1 cells) or embryonic stem cell-derived cardiomyocytes were used as two- and three-dimensional cardiac pacemaker models. Spontaneous activity and conduction velocity (theta) were monitored by field potential measurements with microelectrode arrays (MEAs). The influence of fibroblasts (WT-fibs) was determined in heterocellular cultures of different cardiomyocyte and fibroblast ratios. The relevance of heterocellular gap junctional coupling was evaluated by the use of fibroblasts deficient for the expression of Cx43 (Cx43(-/-)-fibs). The beating frequency and of heterocellular cultures depended negatively on the fibroblast concentration. Interspersion of fibroblasts in cardiomyocyte monolayers increased the coefficient of the interbeat interval variability. Whereas Cx43(-/-)-fibs decreased theta significantly less than WT-fibs, their effect on the beating frequency and the beat-to-beat variability seemed largely independent of their ability to establish intercellular coupling. These results suggest that electrically integrated, non-excitable cells modulate the excitability of cardiac pacemaker preparations by two distinct mechanisms, one dependent and the other independent of the heterocellular coupling established. Whereas heterocellular coupling enables the fibroblast to depolarize the cardiomyocytes or to act as a current sink, the mere physical separation of the cardiomyocytes by fibroblasts induces bradycardia through a reduction in frequency entrainment.

T-tubule Formation in Cardiacmyocytes: Two Possible Mechanisms?

Journal of Muscle Research and Cell Motility. 2007  |  Pubmed ID: 17940841

We have followed the differentiation of transverse (T) tubules and of the associations between sarcoplasmic reticulum (SR) and either the plasmalemma (peripheral couplings) or the T tubules (dyads) in postnatal rat ventricular myocytes using electron microscopy. Dyads and peripheral couplings are collectively called Ca(2+) Release Units (CRUs) because they are the sites at which Ca(2+) is released from the SR. Profiles of T tubules, caveolae and dyads are mostly at the cell edge in early postnatal days and are found with increased frequency in the cell interior during the first two postnatal weeks. Using ferritin to trace continuity of T tubules lumen with the extracellular space, we find that some of T tubules (between approximately 6 and 25%), either singly or within dyads, lack ferritin in their lumen. The percentage of tubules that do not contain ferritin decreases slightly during postnatal differentiation and is not very different at the cells' edges and interior. We propose that T tubules form as invaginations of the plasmalemma that penetrate inward driven by accrual of membrane lipids and specific proteins. This occurs by a dual mechanism: either by the independent flow of SR and T tubule proteins into the two separate membranes or by the fusion of preformed vesicle tandems into the dyads. Most of the CRUs (approximately 86%) are constituted by peripheral couplings and ferritin containing dyads, thus constituting CRUs in which Ca(2+ )release from the SR is initiated by a membrane depolarization. In the remaining CRUs, activation of Ca(2+) release must be dependent on some other mechanisms.

Ca2+ Sparks and Cellular Distribution of Ryanodine Receptors in Developing Cardiomyocytes from Rat

Journal of Molecular and Cellular Cardiology. Jun, 2008  |  Pubmed ID: 18468619

Although abundant ryanodine receptors (RyRs) exist in cardiomyocytes from newborn (NB) rat and despite the maturity of their single-channel properties, the RyR contribution to excitation-contraction (E-C) coupling is minimal. Immature arrangement of RyRs in the Ca(2+) release site of the sarcoplasmic reticulum and/or distant RyRs location from the sarcolemmal Ca(2+) signal could explain this quiescence. Consequently, Ca(2+) sparks and their cellular distribution were studied in NB myocytes and correlated with the formation of dyads and transverse (T) tubules. Ca(2+) sparks were recorded in fluo-4-loaded intact ventricular myocytes acutely dissociated from adult and NB rats (0-9 days old). Sparks were defined/compared in the center and periphery of the cell. Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyad formation, while the development of T tubules was studied using di-8-ANEPPS and diIC12. Our results indicate that in NB cells, Ca(2+) sparks exhibited lower amplitude (1.7+/-0.5 vs. 3.6+/-1.7 F/F(0)), shorter duration (47+/-3.2 vs. 54.1+/-3 ms), and larger width (1.7+/-0.8 vs. 1.2+/-0.4 microm) than in adult. Although no significant changes were observed in the overall frequency, central sparks increased from approximately 60% at 0-1 day to 82% at 7-9 days. While immunolocalization revealed many central release sites at 7-8 days, fluorescence labeling of the plasma membrane showed less abundant internal T tubules. This could imply that although during the first week, release sites emerge forming dyads with DHPR-containing T tubules; some of these T tubules may not be connected to the surface, explaining the RyR quiescence during E-C coupling in NB.

Cushingoid Lipodystrophy Can Be Prevented by Thiazolidinediones

Endocrine. Dec, 2012  |  Pubmed ID: 22801989

Role of Inositol 1,4,5-trisphosphate in the Regulation of Ventricular Ca(2+) Signaling in Intact Mouse Heart

Journal of Molecular and Cellular Cardiology. Dec, 2012  |  Pubmed ID: 22960455

Inositol 1,4,5-trisphosphate (InsP(3)R)-mediated Ca(2+) signaling is a major pathway regulating multiple cellular functions in excitable and non-excitable cells. Although InsP(3)-mediated Ca(2+) signaling has been extensively described, its influence on ventricular myocardium activity has not been addressed in contracting hearts at the whole-organ level. In this work, InsP(3)-sensitive intracellular Ca(2+) signals were studied in intact hearts using laser scanning confocal microscopy and pulsed local-field fluorescence microscopy. Intracellular [InsP(3)] was rapidly increased by UV flash photolysis of membrane-permeant caged InsP(3). Our results indicate that the basal [Ca(2+)] increased after the flash photolysis of caged InsP(3) without affecting the action potential (AP)-induced Ca(2+) transients. The amplitude of the basal [Ca(2+)] elevation depended on the intracellular [InsP(3)] reached after the UV flash. Pretreatment with ryanodine failed to abolish the InsP(3)-induced Ca(2+) release (IICR), indicating that this response was not mediated by ryanodine receptors (RyR). Thapsigargin prevented Ca(2+) release from both RyR- and InsP(3)R-containing Ca(2+) stores, suggesting that these pools have similar Ca(2+) reuptake mechanisms. These results were reproduced in acutely isolated cells where photorelease of InsP(3) was able to induce changes in endothelial cells but not in AP-induced transients from cardiomyocytes. Taken together, these results suggest that IICR does not directly regulate cardiac excitation-contraction coupling. To our knowledge, this is the first demonstration of IICR in intact hearts. Consequently, our work provides a reference framework of the spatiotemporal attributes of the IICR under physiological conditions.

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