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Cardiac magnetic resonance imaging (MRI) on small animals is possible but remains challenging and not well standardized. This publication aims to provide an overview of the current techniques, applications and challenges of cardiac MRI in small animals for researchers interested in moving into this field. Solutions have been developed to obtain a reliable cardiac trigger in both the rat and the mouse. Techniques to measure ventricular function and mass have been well validated and are used by several research groups. More advanced techniques like perfusion imaging, delayed enhancement or tag imaging are emerging. Regarding cardiac applications, not only coronary ischemic disease but several other pathologies or conditions including cardiopathies in transgenic animals have already benefited from these new developments. Therefore, cardiac MRI has a bright future for research in small animals.

The anion exchangers (AE) are encoded by a multigenic family that comprises at least three genes, AE1, AE2 and AE3, and numerous splicoforms. Besides regulating intracellular pH (pHi) via the Cl-/HCO3- exchange, the AEs exert various cellular functions including generation of a senescent antigen, anchorage of the cytoskeleton to the membrane and regulation of metabolism. Most cells express several AE isoforms. Despite the key role of this family of proteins, little is known about the function of specific AE isoforms in any tissue, including the heart. We therefore chose isolated cardiac cells, in which a tight control of pHi is mandatory for the excitation-contraction coupling process, to thoroughly investigate the expression of the AE genes at both the mRNA and protein levels. RT-PCR revealed the presence of AE1, AE2 and AE3 mRNAs in both neonatal and adult rat cardiomyocytes. AE1 is expressed both as the erythroid form (Band 3 or eAE1) and a novel alternate transcript (nAE1), which was more specifically characterized using a PCR mapping strategy. Two variants of AE2 (AE2a and AE2c) were found at the mRNA level. Cardiac as well as brain AE3 mRNAs were expressed in both neonatal and adult rat cardiomyocytes. Several AE protein isoforms were found, including a truncated form of AE1 and two AE3s, but there was no evidence of AE2 protein in adult rat cardiomyocytes. In cardiomyocytes transfected with an AE3 oligodeoxynucleotide antisense, AE3 immunoreactivity was dramatically decreased but the activity of the Cl-/HCO3- exchange was unchanged. In contrast, intracellular microinjection of blocking anti-AE1 antibodies inhibited the AE activity. Altogether, our findings suggest that a specific and novel AE1 splicoform (nAE1) mediates the cardiac Cl-/HCO3- exchange. The multiple gene and protein expression within the same cell type suggest numerous functions for this protein family.

1. To determine whether conductive pathways contribute to the H+ efflux from granulocytes, we used the whole‐cell patch‐clamp technique combined with microfluorimetric determinations of cytosolic pH (pHi) in single, dimethylsulphoxide‐differentiated HL‐60 cells. 2. In voltage‐clamp mode, depolarization of the cell from the resting potential (around ‐60 mV) to +60 mV caused an increase in pHi that was accompanied by a sizeable outward current. 3. Ion substitution experiments and analysis of the reversal potential of tail currents indicated that the outward current is carried largely by H+ ions. 4. Full activation of the H+ current occurred within 1‐2s after depolarization and deactivation within 100‐200 ms upon repolarization. 5. This H+ conductance was strongly dependent on pHi, being larger at acidic pH. In addition, at low pHi the threshold for voltage activation of the H+ conductance was shifted to more negative values. 6. Addition of millimolar concentrations of Cd2+ and Zn2+ to the bath solution reduced the maximum H+ conductance and shifted the voltage dependence of the H+ conductance to more positive potentials. The effects were reversible. 7. In conclusion, our results demonstrate that granulocytic HL‐60 cells possess a voltage‐gated and pHi‐sensitive H+ conductance. Because both a depolarization and a cytosolic acidification occur during the activation of granulocytes, this conductance may play a role in pHi homeostasis of granulocytes during microbial killing.

1. To study Ca(2+)‐activated K+ currents in dimethyl sulphoxide (DMSO)‐differentiated HL‐60 cells (HL‐60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N‐formyl‐L‐methionyl‐L‐phenylalanine (f‐MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole‐cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2+]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca(2+)‐activated current was a function of the extracellular K+ concentration (56.1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole‐cell current was inwardly rectifying. No time‐dependent activation or inactivation of the current could be observed within the range of voltages tested (‐100 to +100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half‐maximal activation at approximately 350 nM [Ca2+]i, and a highly co‐operative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half‐maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca(2+)‐activated single‐channel events in cell‐attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca(2+)‐activated whole‐cell current suggested an underlying single‐channel conductance of a similar size (28 pS). 8. In summary, we describe a charybdotoxin‐sensitive, intermediate‐conductance Ca(2+)‐activated K+ channel in HL‐60 granulocytes. The characteristics of the Ca2+ activation of this current (i.e. sensitivity to submicromolar [Ca2+]i, high co‐operativity and voltage independence) are similar to the Ca2+ activation of the apamin‐sensitive small‐conductance K+ channel. Our results also suggest that [Ca2+]i elevations are the predominant, if not the only, activators of this channel during physiological stimulation of HL‐60 granulocytes.

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In “silent” cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.

To study the role of cytosolic free calcium, [Ca2+]i, in cell activation, in particular during adhesion and movement on a surface in response to chemotactic peptide stimulation and during phagocytosis, we monitored [Ca2+]i in single human neutrophils. The neutrophils were loaded with fura-2 and allowed to adhere to albumin-coated glass coverslips. [Ca2+]i was monitored with a dual excitation microfluorimeter. Half of the cells showed spontaneous [Ca2+]i transients that lasted up to 15 min with an amplitude averaging 77±10 nM above basal levels (mean basal value of 110±20 nM) and a mean duration of 28±5 s. These repetitive [Ca2+]i elevations depended on the continuous presence of extracellular Ca2+ and could be dissociated from those triggered by the chemotactic peptide N-formyl-methionyl-leucylphenylalanine (fMLP). Cell morphology was monitored in parallel by recording fluorescent images with a high sensitivity charge coupled device (CCD) camera. The majority of the cells studied showed visible changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. Removal of extracellular Ca2+ abolished [Ca2+]i transients without impairing cell movement and spreading. Blockade of adherence and cell movement with cytochalasin B markedly inhibited [Ca2+]i transients. Monoclonal antibodies directed against the leucocyte integrin CR3 (CD11b/CD18 αmβ2) blocked adherence, spreading and most of the [Ca2+]i activity. Total [Ca2+]i activity was assessed during phagocytosis of C3bi-opsonized yeast particles and correlated with fusion of secondary granules with the phagosomal membrane (P-L fusion). In Ca2+-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks. P-L fusion was detected in 80% of the cells after 5–10 min. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to <20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. Taken together these results indicate that: 1. The action of leucocyte integrins is necessary for the generation of the multiple [Ca2+]i transient observed in surface adherent human neutrophils. These [Ca2+]i transients do not preclude and can be dissociated from the response to fMLP and they prime the cell to subsequent stimulation. 2. Although the ingestion step of phagocytosis is a Ca2+-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of granules with the phagosomal membrane. 3. Studies at a single cell level will be necessary to detect subtle deficiencies in intracellular mediators during human diseases.

In granulocytes, emptying of intracellular Ca2+ stores activates Ca2+ influx across the plasma membrane. To study the putative role of GTP-binding proteins in this process, we have introduced non-hydrolyzable guanosine phosphate analogues into the cytosol of non-permeabilized HL-60 granulocytes using an endocytosis-hypoosmotic shock procedure. At the cytosolic concentrations obtained (100-500 microM), neither guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) nor guanosine 5'-3-O-(thio)diphosphate (GDP beta S) affected basal [Ca2+]i. Ca2+ release in response to the receptor agonist fMet-Leu-Phe, the Ca(2+)-ATPase inhibitor thapsigargin, or the Ca2+ ionophore ionomycin was also unaffected by GTP gamma S or GDP beta S. In contrast, the activation of the Ca2+ influx pathway by fMet-Leu-Phe or by thapsigargin was blocked by GTP gamma S but not by GDP beta S. The GTP gamma S effect was mimicked by NaF. The GTP gamma S and NaF effects were independent of protein kinase C activation and actin polymerization. Our results demonstrate that a GTP-sensitive element is involved in the signaling between intracellular Ca2+ stores and plasma membrane Ca2+ channels. The identical effects of GTP gamma S and NaF suggest that the GTP-sensitive element is a heterotrimeric G-protein.

Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.