Polyaromatic hydrocarbons in pollution: a heart-breaking matter.

Air pollution is associated with detrimental effects on human health, including decreased cardiovascular function. However, the causative mechanisms behind these effects have yet to be fully elucidated. Here we review the current epidemiological, clinical and experimental evidence linking pollution with cardiovascular dysfunction. Our focus is on particulate matter (PM) and the associated low molecular weight polycyclic aromatic hydrocarbons (PAHs) as key mediators of cardiotoxicity. We begin by reviewing the growing epidemiological evidence linking air pollution to cardiovascular dysfunction in humans. We next address the pollution-based cardiotoxic mechanisms first identified in fish following the release of large quantities of PAHs into the marine environment from point oil spills (e.g. Deepwater Horizon). We finish by discussing the current state of mechanistic knowledge linking PM and PAH exposure to mammalian cardiovascular patho-physiologies such as atherosclerosis, cardiac hypertrophy, arrhythmias, contractile dysfunction and the underlying alterations in gene regulation. Our aim is to show conservation of toxicant pathways and cellular targets across vertebrate hearts to allow a broad framework of the global problem of cardiotoxic pollution to be established. AhR; Aryl hydrocarbon receptor. Dark lines indicate topics discussed in this review. Grey lines indicate topics reviewed elsewhere.

Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes.

The transverse (t-) tubules of cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. Many of the key proteins involved in excitation-contraction coupling appear to be located predominantly at the t-tubule membrane. Despite their importance, the fraction of cell membrane within the t-tubules remains unclear: measurement of cell capacitance following detubulation suggests approximately 32%, whereas optical measurements suggest up to approximately 65%. We have, therefore, investigated the factors that may account for this discrepancy. Calculation of the combinations of t-tubule radius, length and density that produce t-tubular membrane fractions of 32% or 56% suggest that the true fraction is at the upper end of this range. Assessment of detubulation using confocal and electron microscopy suggests that incomplete detubulation can account for some, but not all of the difference. High cholesterol, and a consequent decrease in specific capacitance, in the t-tubule membrane, may also cause the t-tubule fraction calculated from the loss of capacitance following detubulation to be underestimated. Correcting for both of these factors results in an estimate that is still lower than that obtained from optical measurements suggesting either that optical methods overestimate the fraction of membrane in the t-tubules, or that other, unknown, factors, reduce the apparent fraction obtained by detubulation. A biophysically realistic computer model of a rat ventricular myocyte, incorporating a t-tubule network, is used to assess the effect of the altered estimates of t-tubular membrane fraction on the calculated distribution of ion flux pathways.

T-tubules: a key structure of cardiac function and dysfunction.

The sarcolemmal membrane of mammalian ventricular cardiomyocytes are characterised by the presence of invaginations called transverse tubules (T-tubules) which constitute a network, the transverse tubule system. T-tubules occur at the Z line as transverse elements with also longitudinal extensions. While the existence of T-tubules has been known since a long time, recent studies have suggested their structure and function can be more complex than previously believed. Many of the proteins involved in excitation-contraction coupling process are concentrated within the T-tubular network, suggesting T-tubules are a highly specialized membrane system. Thus, T-tubules are a key determinant of cardiac cell function. The fundamental role of T-tubules is emphasized by changes in their structure and protein expression occurring during pathologies such as cardiac hypertrophy and heart failure. This review summarizes recent studies which highlight the key-role of the T-tubules in the regulation of cardiac function. Changes observed in pathological conditions are also discussed.

Quantification of calcium entry at the T-tubules and surface membrane in rat ventricular myocytes.

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.

beta-adrenergic stimulation restores the Ca transient of ventricular myocytes lacking t-tubules.

beta-adrenergic stimulation helps to synchronize Ca release in myocytes from failing hearts. Transverse (t-) tubules, which synchronize Ca release in normal cells and contain many of the elements of the beta-adrenergic pathway, may be depleted in such cells. The objective of the present study was to determine whether beta-adrenergic stimulation could reverse the desynchronization of Ca release observed in detubulated ventricular myocytes. The effect of isoprenaline (0.5 microM) on control and detubulated rat ventricular myocytes was investigated. Ca transients were monitored using whole-cell fluorescence and confocal microscopy, and Ca current recorded using the patch-clamp technique. Immunocytochemistry was used to investigate phospholamban (PLB) phosphorylation. Detubulation reduces and slows the Ca transient; these effects were reversed by isoprenaline. This restoration was associated with partial reversal of the desynchronization of Ca release that occurs in detubulated cells. Sarcoplasmic reticulum Ca load increased by the same amount in normal and detubulated cells, but Ca current increased less in detubulated cells (64%) than in control cells (124%) in response to isoprenaline. The pattern and extent of cAMP-dependent protein kinase and CaMKII-induced phosphorylation of PLB in response to isoprenaline was the same in both cell types. Thus, the beta-adrenergic pathway is functional in the absence of t-tubules; such stimulation appears to increase the speed of propagation of Ca via Ca-induced Ca release between adjacent clusters of ryanodine receptors, which may be relevant in pathological conditions, such as heart failure, in which t-tubules are depleted. The data also suggest that the Ca current responds to local signaling pathways, which are better coupled to the channel in the t-tubules than at the surface membrane, whereas PLB responds to whole-cell signaling.

Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes.

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.

Na+-Ca2+ exchange activity is localized in the T-tubules of rat ventricular myocytes.

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.

Enhancement of the T-type calcium current by hyposmotic shock in isolated guinea-pig ventricular myocytes.

It is known that swelling and shrinkage of cardiac cells can modulate their electrical activity. However, the effects of osmotic manipulation on cardiac T-type calcium current (I(CaT)) has not been previously reported. In this study, we have examined the effects of cell swelling on I(CaT), using the whole cell patch clamp configuration. Isolated guinea-pig ventricular myocytes were swollen by an external hypotonic challenge (0.7 T). We found that I(CaT)is enhanced during a hypotonic shock. This current has been determined to be the T type calcium current since it is rapidly activated and inactivated, its threshold was at negative potentials and was blocked by 40 microm Ni2+. Disruption of microfilaments by cytochalasin D and of microtubules by colchicine prevented the activation of I(CaT)during cell swelling. Taxol had no effect. These results indicate that I(CaT)is increased during cell swelling and this effect needs an intact cytoskeleton.

Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes.

The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD(90)), the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitude, and contraction increased, whereas the L-type Ca(2+) current (I(Ca, L)) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca(2+) release increased but SR Ca(2+) load did not. After a long exposure, I(Ca,L), APD(90), [Ca(2+)](i) transient amplitude, and contraction decreased. The abbreviation of APD(90) was partially reversed by 50 microM DIDS, which is consistent with the participation of Cl(-) current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I(Ca,L). After long exposure, Ca(2+) load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca(2+) release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca(2+) entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I(Ca,L) amplitude.

Mechanisms associated with the negative inotropic effect of deuterium oxide in single rat ventricular myocytes.

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.

Effects on L-type calcium current of agents interfering with the cytoskeleton of isolated guinea-pig ventricular myocytes.

We studied the effects of an external acute 10 min application of cytoskeletal interfering agents on cardiac L-type calcium current (ICa,L). We found that colchicine, taxol and cytochalasin D had no direct effect on the L-type calcium channel as indicated by the absence of effect on voltage-dependent parameters. Phalloidin induced a shift in the I-V curve which renders it difficult to use in excitation-contraction coupling studies. Microfilaments of actin did not seem to regulate cardiac ICa,L as indicated by the lack of effect of cytochalasin D on ICa,L amplitude and inactivation kinetics. On the contrary, microtubules seem to be involved in the calcium-dependent inactivation of ICa,L. This involvement might be direct, i.e. a physical link between the microtubules and some part of the channel protein, or it could be indirect, i.e. the calcium chelating properties and physical obstacle of microtubules in the space between the sarcolemma and the SR.

Health effects of attending a public swimming pool: follow up of a cohort of pupils in Paris.

OBJECTIVES: This study aimed to determine the health effects of attending a well-kept school swimming pool maintained according to French public health regulations. METHODS: This prospective month long study was carried out on a randomised sample of pupils aged 5 to 18 years who attended a private French school with two swimming pools. The children surveyed, helped by their parents, had to fill in questionnaires about their bathing habits and symptoms during the survey period. Inspections of the pool complex were made and these included physicochemical and bacteriological analyses of the pools' water. PARTICIPATION: The response rates achieved were 70% at primary and middle school levels but only 25% in the high school pupils. Because of this older teenagers were excluded from the final analysis (of 246 children). RESULTS: Compared with non-bathers, bathers experienced fatigue and eye irritation significantly more often (p < 0.001). The eyes were red (38% of bathers) and/or watery (16%) after swimming but this resolved spontaneously within 24 hours. Bathing behaviour (bath duration, head immersion, wearing swimming goggles) did not affect these incidence rates noticeably. There were no differences between bathers and non-bathers with regard to other symptoms, especially otolaryngological ones. This survey does not allow definite conclusions to be made about verrucas because 22% of non-bathers were exempted from swimming because of verrucas that they might have caught previously in a pool. CONCLUSIONS: Except for verrucas, the methodology was adequate and daily self reporting of symptoms was feasible. This college largely recruits pupils from higher social classes and is not therefore representative of schools in Paris.