Striatin knock out induces a gain of function of INa and impaired Ca2+ handling in mESC-derived cardiomyocytes.

AIM: Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. METHODS: We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn-KO and isogenic WT mouse embryonic stem cell lines. RESULTS: The spontaneous beating rate of Strn-KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2-8 Hz) Strn-KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn-KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn-KO CMs than in WT. Indeed, incubation of Strn-KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. CONCLUSION: Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn-related multi-protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement.

Dual role of miR-1 in the development and function of sinoatrial cells.

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

Cell-specific Dynamic Clamp analysis of the role of funny If current in cardiac pacemaking.

We used the Dynamic Clamp technique for i) comparative validation of conflicting computational models of the hyperpolarization-activated funny current, If, and ii) quantification of the role of If in mediating autonomic modulation of heart rate. Experimental protocols based on the injection of a real-time recalculated synthetic If current in sinoatrial rabbit cells were developed. Preliminary results of experiments mimicking the autonomic modulation of If demonstrated the need for a customization procedure to compensate for cellular heterogeneity. For this reason, we used a cell-specific approach, scaling the maximal conductance of the injected current based on the cell's spontaneous firing rate. The pacemaking rate, which was significantly reduced after application of Ivabradine, was restored by the injection of synthetic current based on the Severi-DiFrancesco formulation, while the injection of synthetic current based on the Maltsev-Lakatta formulation did not produce any significant variation. A positive virtual shift of the If activation curve, mimicking the Isoprenaline effects, led to a significant increase in pacemaking rate (+17.3 ± 6.7%, p < 0.01), although of lower magnitude than that induced by real Isoprenaline (+45.0 ± 26.1%). Similarly, a negative virtual shift of the activation curve significantly lowered the pacemaking rate (-11.8 ± 1.9%, p < 0.001), as did the application of real Acetylcholine (-20.5 ± 5.1%). The Dynamic Clamp approach, applied to the If study in cardiomyocytes for the first time and rate-adapted to manage intercellular variability, indicated that: i) the quantitative description of the If current in the Severi-DiFrancesco model accurately reproduces the effects of the real current on rabbit sinoatrial cell pacemaking rate and ii) a significant portion (50-60%) of the physiological autonomic rate modulation is due to the shift of the If activation curve.

Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels.

Ivabradine is a 'heart rate-reducing' agent able to slow heart rate, without complicating side-effects. Its action results from a selective and specific block of pacemaker f-channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f-channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, of which four isoforms (HCN1-4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose-dependent way with half-block concentrations of 0.94 microm for mHCN1 and 2.0 microm for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an 'open-channel' blocker of hHCN4, and a 'closed-channel' blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f-channel kinetics.

C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) family of "pacemaker" channels includes 4 isoforms, the kinetics and cAMP-induced modulation of which differ quantitatively. Because HCN isoforms are highly homologous in the central region, but diverge more substantially in the N and C termini, we asked whether these latter regions could contribute to the determination of channel properties. To this aim, we analyzed activation/deactivation kinetics and the response to cAMP of heterologously expressed isoforms mHCN1 and rbHCN4 and verified that mHCN1 has much faster kinetics and lower cAMP sensitivity than rbHCN4. We then constructed rbHCN4 chimeras by replacing either the N or the C terminus, or both, with the analogous domains from mHCN1. We found that: 1) replacement of the N terminus (chimera N1-4) did not substantially modify either the kinetics or cAMP dependence of wild-type channels; 2) replacement of the C terminus, on the contrary, resulted in a chimeric channel (4-C1), the kinetics of which were strongly accelerated compared with rbHCN4, and that was fully insensitive to cAMP; 3) replacement of both N and C termini led to the same results as replacement of the C terminus alone. These results indicate that the C terminus of rbHCN4 contributes to the regulation of voltage- and cAMP-dependent channel gating, possibly through interaction with other intracellular regions not belonging to the N terminus.

Integrated allosteric model of voltage gating of HCN channels.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

Crude oil spill in sea water: an assessment of the risk for bathers correlated to benzo(a)pyrene exposure.

In the spring of 1991, there was a shipwreck of the oil tanker "Haven" off the Ligurian coast of Italy. This resulted in the spillage of a very large amount of crude oil, some of which was burned off by fire. The accident caused several serious problems (sea and air pollution, damage to the marine fauna, risk of human exposure, etc.). In this context, an assessment was carried out at the Istituto Superior di Sanità with the aim of determining any possible risks to humans which might derive from bathing activities during the following summer season. The whole evaluation carried out after the accident demonstrated that the impacts induced were not serious enough to require bathing restrictions in the coastal areas involved. Assuming a benzo(a)pyrene (BaP) concentration in sea water of 1 microgram/m3 cancer risk is in the order of 10(-8) and in the case of 10-kg child, a 10(-6) risk level correspond to about 0.18 microgram/l of BaP in sea water.

Reproducibility of carcinogenic potencies estimated in different rodent species. Methylene chloride: a useful example.

The aim of this study was to investigate the reproducibility of carcinogenic potencies estimated by multistage model (and its linearized form) fitting to dose-response relationships referring to different rodent species and strains, different sexes, target organs and severity of prognosis. Methylene chloride represented a useful example since there were 11 single dose-response relationships available for the same administration route (inhalation) and for the above-mentioned variables. The multistage mathematical model and the 'linearized multistage' model (EPA's conservative procedure), which are the mainly adopted models for regulatory purposes, were used for the dose-response fitting of single trends and other different cumulative trends. The reproducibility of response frequencies at equal exposure doses was also analyzed. The mathematical function best describing each trend showed the constant presence of a linear component (for low doses) and, in the majority of cases, a quadratic second order component. Carcinogenic potencies were fairly consistent for the various parameters. A good interspecies reproducibility of carcinogenic potencies was observed. A high intraspecies reproducibility was also observed, even though obtained from different sexes, as well as varying degrees of severity of prognosis and, in the case of rats, different strains. The fluctuation of the estimated carcinogenic potencies was lower in mice than in rats.

The environmental behavior of chemicals in soil: atrazine as an example.

In this work a mathematical model has been developed and used to estimate the soil vertical distribution of Atrazine dispersed in the environment. Water transport, rise due to capillarity, and partition among soil-contained water, air, and organic matter, as well as degradation processes, are considered. As far as the vertical mobility in soil is concerned, the model has been derived from that proposed by P. H. Nichols, A. Walker, and R. J. Baker [1982). Pestic. Sci. 12, 484-494). Such a model has been extended to include a procedure which takes into account the Atrazine mobility due to gravitational water flow. The organic carbon (O.C.) concentration gradient in soil was also considered in the evaluation when assessing partition processes, according to models by P. J. McCall et al. [1983). Residue Rev. 13, 231-241) and D. McKay and S. Paterson [1982). Environ. Sci. Technol. 16, 12, 654). The degradation processes are assumed to be first order, linearly related with O.C. content in soil. The application of this model to two sets of soil data demonstrated that Atrazine requires a long time (2 years or more, depending on soil features) in order to percolate at a depth comparable with those of a groundwater source.

Reproducibility of low-dose extrapolation procedure: comparison of estimates obtained using different rodent species and strains.

Carcinogenic risk assessment based on low-dose extrapolation of dose-response relationships is characterized by a significant level of uncertainty. The aim of this study was to investigate the reproducibility of risk estimates carried out using different animal species and strains. The Weibull and multistage mathematical modes, as well as the "linearized multistage" model (EPA "conservative" procedure), have been used for dose-response relationship fitting. Whenever necessary, the Michaelis-Menten function was introduced in the models to account for a possible metabolic process. The analysis was directed to dietary sodium saccharin and vinyl chloride inhalation carcinogenicity (nine total dose-response relationships). Data from different strains, species, and researchers were available for these substances. The general pattern of two sodium saccharin curves was an upward curve in both cases with a high consistency among parameters and risk estimates (the latter ranging within a factor 3). For vinyl chloride the trend was clearly downward for six carcinogenic dose-response relationships from different species; low-dose risk estimates, based on the upper confidence limits of the linear component, range within a factor 3. The study has indicated the reproducibility of the low-dose extrapolation process using different strains and species.

Dose-response relationships in rodents of promoter carcinogens: a tentative interpretation of some downward trends.

This study analyzes a set of dose-response curves relative to promoter carcinogens administered to experimental animals with or without initiator pretreatment. In the case of initiator plus promoter treatment, as well as for low experimental doses, the experimental data and their mathematical fitting indicate a downward shape of the dose-response curves as a general feature. In this study, the following hypothesis, according to the two-stage carcinogenic action of initiator plus promoter treatment, is presented to interpret this downward shape: (i) an initiating action, involving a subset of available "targets" (the number of "initiated" targets is assumed to depend on initiating treatment level), and (ii) a subsequent dose-dependent promoting process, mainly involving the initiated targets. This process reaches a saturation level when all the initiated targets have been promoted. This kind of process may be quantitatively described by a saturated exponential model: P(d) = K(1 - exp(-K0 + K1d)] which fits the experimental data fairly well. No indication supporting the hypothesis of a threshold for the chemicals examined resulted from the analysis. In the specific case of sodium saccharin, experimental dose-response relationships were available with or without initiator pretreatment (BBN and MNU). While in the first case the shape of the dose-response curve is downward, in the second case, it is upward and significantly different.

Animal species and strains used to test chemical carcinogens: results of a preliminary study.

The selection of animal species sufficiently representative of human physiological processes and sensitive to a wide spectrum of toxic agents is one of the major problems in toxicology. The aim of this study, based on animal experimental carcinogenesis data available from the literature, was the identification of animal strains most often used in experimental carcinogenesis. The analysis was limited to experiments on chemicals or groups of chemicals classified by IARC under Group 1 of carcinogenic risk (sufficient evidence). For each experiment considered in the study, data concerning animal species, strains, and substrains (whenever possible) were collected. The data analysis has shown that approximately 83 and 61% of the considered carcinogens were tested positively in mouse and rat, respectively, and that A, Swiss, and C3H mouse strains and Sprague-Dawley and Wistar rat strains were used most often. The study has shown that practically all decisions regarding the carcinogenicity of substances are based on the response of only two animal species (mouse and rat). Therefore, a clear and complete understanding of carcinogenicity mechanisms in these species becomes essential in order to extrapolate results to man.