Antonio Zaza

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor κB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca2+/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cγ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

Baroreflex sensitivity and its evolution during the first year after myocardial infarction.

Experimental data have indicated that baroreflex sensitivity is often depressed in dogs after myocardial infarction and that this depression correlates strongly with subsequent mortality during episodes of acute myocardial ischemia. This finding has several clinical implications. The present study was undertaken with the objectives of assessing the potential existence of differences in baroreflex sensitivity between men with and without myocardial infarction and the time course during the 1st year after infarction of these potential changes in baroreflex sensitivity. Fifty-three subjects entered the study: 32 postinfarction patients and 21 control subjects. Baroreflex sensitivity was assessed by increasing mean blood pressure by aphenylephrine infusion (70 micrograms/ml) and recording the consequent RR interval changes. Baroreflex sensitivity, expressed as the slope of the regression line relating mean blood pressure to RR interval changes, was evaluated 18 days (n = 32), 3 months (n = 17) and 13 months (n = 10) after infarction. Baroreflex sensitivity was lower in the patients than in the control subjects (8.2 +/- 3.7 versus 12.3 +/- 2.9 ms/mm Hg, p = 0.0001). Moreover, 13 (41%) of 32 patients had a baroreflex slope less than 6.5 ms/mm Hg, which was 2 SD below the mean value of the control subjects. The internal control follow-up study showed that baroreflex sensitivity increased 3 months after infarction to values quite similar to those observed in the control subjects (11.1 +/- 5.3 versus 8.7 +/- 3.5 ms/mm Hg, p = 0.02). No further change was observed between 3 and 13 months after myocardial infarction. These data indicate that baroreflex sensitivity is lower in a proportion of postinfarction patients than in control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Autonomic indexes based on the analysis of heart rate variability: a view from the sinus node

OBJECTIVE Clinical indexes of autonomic activity are based on the analysis of sinus cycle length and of its variability. A common assumption underlying this practice is that neural activity and cycle length may be linearly related. Recent experimental evidence suggests that such an assumption may not be correct; indeed, the relation linking autonomic agonist concentration to cycle length was found to be strongly non-linear in single sinoatrial myocytes. In the present work, we (i) test compatibility of non-linearity of neural modulation of cycle length (CL) with previous experimental and clinical observations; (ii) evaluate its implications for what concerns the interpretation of time- and frequency-domain parameters of heart rate variability (HRV) and baroreflex sensitivity (BRS). CONCLUSION Non-linearity of neural modulation of CL may result in an intrinsic rate-dependency of autonomic indexes, with the exception of normalised frequency-domain indexes (e.g. the low frequency/high frequency (LF/HF) ratio), which appear to be devoid of intrinsic rate-dependency. This characteristic may not limit the value of HRV indexes and BRS in risk stratification, but has to be taken into account in their pathophysiological interpretation.

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether‐a‐go‐go‐related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long‐QT syndrome and Drosophila ‘seizure’ mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50 = 16±1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure‐function relationships and novel pharmacological tools with potential therapeutic efficacy.—Gurrola, G. B., Rosati, B., Rocchetti, M., Pimienta, G., Zaza, A., Arcangeli, A., Olivotto, M., Possani, L. D., Wanke, E. A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom. FASEB J. 13, 953–962 (1999)

Basal responses of the L‐type Ca2+ and hyperpolarization‐activated currents to autonomic agonists in the rabbit sino‐atrial node.

1. The dose dependence of the cholinergic agonist acetylcholine (ACh) and the beta‐adrenergic agonist isoprenaline (Iso) were determined for the hyperpolarization‐activated current (If) and the L‐type Ca2+ current (ICa,L) in single cells isolated from the rabbit sino‐atrial (SA) node. 2. ACh inhibited If by a negative shift of its activation curve with a maximal effect of ‐9.9 mV; half‐maximal effect was produced by 0.019 microM ACh. High ACh concentrations were required to inhibit ICa,L only partially (31% inhibition at 300 microM). 3. In contrast, If and ICa,L responded to Iso over a similar dose range, with concentrations for half‐maximal enhancement of 0.0136 and 0.0070 microM, respectively. 4. The effects on spontaneous activity of ACh (range 0.001‐0.03 microM) and Iso (range 0.001‐1 microM) were investigated. ACh decreased the slope of diastolic depolarization at concentrations similar to those inhibiting If (> 50% at 0.03 microM). Iso enhanced diastolic depolarization at concentrations similar to those affecting both If and ICa,L (half‐maximal effect at 0.027 microM). 5. In a ramp‐clamp protocol simulating diastolic depolarization, the threshold for activation of inward nifedipine‐sensitive current was ‐41.22 +/‐ 0.68 mV. Although enhancing ICa,L, Iso did not affect this threshold. 6. Half‐maximal ACh concentrations for inhibition of automaticity and If are similar and are lower than the threshold concentrations for modulation of ICa,L; this argues against a role of ICa,L in direct muscarinic modulation of pacemaking. In contrast, modulation of If, ICa,L and automaticity occur at similar Iso concentrations. The difference between maximum diastolic potential (‐61.95 +/‐ 0.93 mV) and the threshold for Iso‐stimulated ICa,L (‐39.54 +/‐ 1.03 mV) suggests that this current plays a role only at later stages of diastolic depolarization.

The effect of antiarrhythmic drugs on life-threatening arrhythmias induced by the interaction between acute myocardial ischemia and sympathetic hyperactivity

Transient myocardial ischemia, with attendant sympathetic hyperactivity, seems to play a major role in sudden cardiac death among patients with ischemic heart disease. Ventricular tachycardia (VT) and fibrillation (VF) are consistently and repeatedly elicited in cats by the interaction between a 2-minute occlusion of the left descending coronary artery and a 30-second stimulation of the left stellate ganglion. When three consecutive trials yield almost identical results, time alone will not modify the response and a given drug can be injected to test its efficacy with an internal control analysis. In 90 cats the efficacy of the following drugs was assessed: lidocaine (n = 11), mexiletine (n = 12), propafenone (n = 12), propranolol (n = 19), prazosin (n = 10), amiodarone (n = 14), and verapamil (n = 12). Class I antiarrhythmic drugs completely failed to afford protection and worsening of arrhythmia was observed in several instances. Propranolol and prazosin showed efficacy in approximately 80% and 60% of the animals, respectively. Amiodarone and verapamil completely prevented the onset of VT and VF. Protection from arrhythmias seems to be related to the combined presence of a noncompetitive adrenergic blockade associated with salutary effects on coronary circulation. These findings correlate with and help to explain the results of clinical trials in postmyocardial infarction patients. This model may help to provide a rational choice of antiarrhythmic drugs to be tested in clinical trials.

Rate dependency of delayed rectifier currents during the guinea‐pig ventricular action potential

1 The action potential clamp technique was exploited to evaluate the rate dependency of delayed rectifier currents (IKr and IKs) during physiological electrical activity. IKr and IKs were measured in guinea‐pig ventricular myocytes at pacing cycle lengths (CL) of 1000 and 250 ms. 2 A shorter CL, with the attendant changes in action potential shape, was associated with earlier activation and increased magnitude of both IKr and IKs. Nonetheless, the relative contributions of IKr and IKs to total transmembrane current were independent of CL. 3 Shortening of diastolic interval only (constant action potential shape) enhanced IKs, but not IKr. 4 I Kr was increased by a change in the action potential shape only (constant diastolic interval). 5 In ramp clamp experiments, IKr amplitude was directly proportional to repolarization rate at values within the low physiological range (< 1.0 V s−1); at higher repolarization rates proportionality became shallower and finally reversed. 6 When action potential duration (APD) was modulated by constant current injection (I‐clamp), repolarization rates > 1.0 V s−1 were associated with a reduced effect of IKr block on APD. The effect of changes in repolarization rate was independent of CL and occurred in the presence of IKs blockade. 7 In spite of its complexity, the behaviour of IKr was accurately predicted by a numerical model based entirely on known kinetic properties of the current. 8 Both IKr and IKs may be increased at fast heart rates, but this may occur through completely different mechanisms. The mechanisms identified are such as to contribute to abnormal rate dependency of repolarization in prolonged repolarization syndromes.

Ionic currents during sustained pacemaker activity in rabbit sino-atrial myocytes

1 The contribution of various ionic currents to diastolic depolarization (DD) in rabbit sino‐atrial myocytes was evaluated by the action potential clamp technique. Individual currents were identified, during sustained pacemaking activity reproduced under voltage clamp conditions, according to their sensitivity to supecific channel blockers. 2 The current sensitive to dihydropyridines (DHPs), blockers of L‐type Ca2+ current (ICa,L), was small and outward during most of DD. Diastolic DHP‐sensitive current was affected by changes in the driving force for K+, but it was insensitive to E‐4031, which blocks the current termed IK,r; it was abolished by cell dialysis with a Ca2+ chelator. 3 The current sensitive to 2 mm Cs+ (ICs), a blocker of hyperpolarization‐activated current (If), was inward during the whole DD and it was substantially larger than the net inward current flowing during this phase. However, diastolic IK,r, identified in the same cells as the current sensitive to the blocker E‐4031, exceeded ICs 2‐fold. 4 These findings suggest that: (a) Ca2+ influx during the pacemaker cycle increases a K+ conductance, thus inverting the direction of the net current generated by L‐type Ca2+ channel activity during DD; (b) the magnitude of If would be adequate to account fully for DD; however, the coexistence of a larger IK,r suggests that other channels besides If contribute inward current during this phase.

Sympathetic modulation of the relation between ventricular repolarization and cycle length.

Sympathetic influences on ventricular repolarization are not yet fully elucidated, despite their relevance to arrhythmogenesis. The sympathetic control of repolarization, measured from an endocardial monophasic action potential duration (APD) and from the QT interval, was investigated in 24 anesthetized cats. The effects of right and left stellectomy and of subsequent bilateral stellectomy or beta-blockade on the relation between APD (or QT) and cycle length (CL) at steady state, and on the kinetics of adaptation of APD to a sudden change in cycle length were studied separately. Steady-state APD/CL (or QT/CL) relations were obtained by atrial pacing at different cycle lengths. The kinetics of APD adaptation were evaluated for a sudden decrease of approximately 100 msec in pacing cycle length. The steady-state APD/CL (QT/CL) relation was fitted by the hyperbolic function APD = CL/[(a. CL) + b]. From this, two parameters were computed: 1) 1/a, that is, APD (QT) extrapolated at infinite cycle length (APDmax or QTmax) and 2) the cycle length at which 50% of the total change in APD (or QT) occurred (CL50 = b/a). Right stellectomy reduced APDmax and CL50, an effect reversed by subsequent left stellectomy or beta-blockade (propranolol, 0.5 mg/kg). Left stellectomy prolonged APDmax and CL50. Bilateral stellectomy, in both groups, caused a further increase in these variables. Results were similar for the QT/CL relation. The adaptation kinetics of APD to cycle length was described by the sum of two exponentials. The first time constant (tau fast, about three beats) was unchanged by any intervention; the second (tau slow) was shortened by right stellectomy and prolonged by left stellectomy. The further removal of the remaining stellate ganglion had the same effect in both groups, that is, an increase in tau slow. Thus, sympathetic innervation modulates both the steady-state dependence on cycle length and the kinetics of adaptation to sudden rate changes of ventricular repolarization. The effects of sympathetic influence are asymmetrical. Right stellectomy shortens APDmax and QTmax, reduces CL50, and accelerates APD adaptation to a new steady state. Because these effects are reversed by beta-blockade or left stellectomy, they are likely to be due to a reflexly enhanced sympathetic outflow to the ventricles through the left-sided nerves.

miR669a and miR669q prevent skeletal muscle differentiation in postnatal cardiac progenitors

miR669a and miR669q inhibit postnatal cardiac progenitor differentiation by directly targeting the 3′UTR of MyoD.