Cytoskeletal role in protection of the failing heart by ß-adrenergic blockade.

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac ß-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because ß-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on ß-adrenergic overdrive and thus could be reversed by ß-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, ß(1)- (but not ß(2)-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had ß-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB ß-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy.

Basis for MAP4 dephosphorylation-related microtubule network densification in pressure overload cardiac hypertrophy.

Increased activity of Ser/Thr protein phosphatases types 1 (PP1) and 2A (PP2A) during maladaptive cardiac hypertrophy contributes to cardiac dysfunction and eventual failure, partly through effects on calcium metabolism. A second maladaptive feature of pressure overload cardiac hypertrophy that instead leads to heart failure by interfering with cardiac contraction and intracellular transport is a dense microtubule network stabilized by decoration with microtubule-associated protein 4 (MAP4). In an earlier study we showed that the major determinant of MAP4-microtubule affinity, and thus microtubule network density and stability, is site-specific MAP4 dephosphorylation at Ser-924 and to a lesser extent at Ser-1056; this was found to be prominent in hypertrophied myocardium. Therefore, in seeking the etiology of this MAP4 dephosphorylation, we looked here at PP2A and PP1, as well as the upstream p21-activated kinase 1, in maladaptive pressure overload cardiac hypertrophy. The activity of each was increased persistently during maladaptive hypertrophy, and overexpression of PP2A or PP1 in normal hearts reproduced both the microtubule network phenotype and the dephosphorylation of MAP4 Ser-924 and Ser-1056 seen in hypertrophy. Given the major microtubule-based abnormalities of contractile and transport function in maladaptive hypertrophy, these findings constitute a second important mechanism for phosphatase-dependent pathology in the hypertrophied and failing heart.

Aging and susceptibility to toluene in rats: a pharmacokinetic, biomarker, and physiological approach.

Aging adults are a growing segment of the U.S. population and are likely to exhibit increased susceptibility to many environmental toxicants. However, there is little information on the susceptibility of the aged to toxicants. The toxicity of toluene has been well characterized in young adult rodents but there is little information in the aged. Three approaches were used: (1) pharmacokinetic (PK), (2) cardiac biomarkers, and (3) whole-animal physiology to assess whether aging increases susceptibility to toluene in the Brown Norway (BN) rat. Three life stages, young adult, middle aged, and aged (4, 12, and 24 mo, respectively), were administered toluene orally at doses of 0, 0.3, 0.65, or 1 g/kg and subjected to the following: terminated at 45 min or 4 h post dosing, and blood and brain toluene concentration were measured; terminated at 4 h post dosing, and biomarkers of cardiac function were measured; or monitor heart rate (HR), core temperature (Tc), and motor activity (MA) by radiotelemetry before and after dosing. Brain toluene concentration was significantly elevated in aged rats at 4 h after dosing with either 0.3 or 1 g/kg. Blood toluene concentrations were unaffected by age. There were various interactions between aging and toluene-induced effects on cardiac biomarkers. Most notably, toluene exposure led to reductions in mRNA markers for oxidative stress in aged but not younger animals. Toluene also produced a reduction in cardiac endothelin-1 in aged rats. Higher doses of toluene led to tachycardia, hypothermia, and a transient elevation in MA. Aged rats were less sensitive to the tachycardic effects of toluene but showed a prolonged hypothermic response. Elevated brain levels of toluene in aged rats may be attributed to their suppressed cardiovascular and respiratory responses. The expression of several cardiac biochemical markers of toluene exposure in the aged may also reflect differential susceptibility to this toxicant.

One-month diesel exhaust inhalation produces hypertensive gene expression pattern in healthy rats.

BACKGROUND: Exposure to diesel exhaust (DE) is linked to vasoconstriction, endothelial dysfunction, and myocardial ischemia in compromised individuals. OBJECTIVE: We hypothesized that DE inhalation would cause greater inflammation, hematologic alterations, and cardiac molecular impairment in spontaneously hypertensive (SH) rats than in healthy Wistar Kyoto (WKY) rats. METHODS AND RESULTS: Male rats (12-14 weeks of age) were exposed to air or DE from a 30-kW Deutz engine at 500 or 2,000 microg/m3, 4 hr/day, 5 days/week for 4 weeks. Neutrophilic influx was noted in the lung lavage fluid of both strains, but injury markers were minimally changed. Particle-laden macrophages were apparent histologically in DE-exposed rats. Lower baseline cardiac anti-oxidant enzyme activities were present in SH than in WKY rats; however, no DE effects were noted. Cardiac mitochondrial aconitase activity decreased after DE exposure in both strains. Electron microscopy indicated abnormalities in cardiac mitochondria of control SH but no DE effects. Gene expression profiling demonstrated alterations in 377 genes by DE in WKY but none in SH rats. The direction of DE-induced changes in WKY mimicked expression pattern of control SH rats without DE. Most genes affected by DE were down-regulated in WKY. The same genes were down-regulated in SH without DE producing a hypertensive-like expression pattern. The down-regulated genes included those that regulate compensatory response, matrix metabolism, mitochondrial function, and oxidative stress response. No up-regulation of inflammatory genes was noted. CONCLUSIONS: We provide the evidence that DE inhalation produces a hypertensive-like cardiac gene expression pattern associated with mitochondrial oxidative stress in healthy rats.

Differential pulmonary and cardiac effects of pulmonary exposure to a panel of particulate matter-associated metals.

Biological mechanisms underlying the association between particulate matter (PM) exposure and increased cardiovascular health effects are under investigation. Water-soluble metals reaching systemic circulation following pulmonary exposure are likely exerting a direct effect. However, it is unclear whether specific PM-associated metals may be driving this. We hypothesized that exposure to equimolar amounts of five individual PM-associated metals would cause differential pulmonary and cardiac effects. We exposed male WKY rats (14 weeks old) via a single intratracheal instillation (IT) to saline or 1 micromol/kg body weight of zinc, nickel, vanadium, copper, or iron in sulfate form. Responses were analyzed 4, 24, 48, or 96 h after exposure. Pulmonary effects were assessed by bronchoalveolar lavage fluid levels of total cells, macrophages, neutrophils, protein, albumin, and activities of lactate dehydrogenase, gamma-glutamyl transferase, and n-acetyl glucosaminidase. Copper induced earlier pulmonary injury/inflammation, while zinc and nickel produced later effects. Vanadium or iron exposure induced minimal pulmonary injury/inflammation. Zinc, nickel, or copper increased serum cholesterol, red blood cells, and white blood cells at different time points. IT of nickel and copper increased expression of metallothionein-1 (MT-1) in the lung. Zinc, nickel, vanadium, and iron increased hepatic MT-1 expression. No significant changes in zinc transporter-1 (ZnT-1) expression were noted in the lung or liver; however, zinc increased cardiac ZnT-1 at 24 h, indicating a possible zinc-specific cardiac effect. Nickel exposure induced an increase in cardiac ferritin 96 h after IT. This data set demonstrating metal-specific cardiotoxicity is important in linking metal-enriched anthropogenic PM sources with adverse health effects.

Systemic translocation of (70)zinc: kinetics following intratracheal instillation in rats.

Mechanisms of particulate matter (PM)-induced cardiotoxicity are not fully understood. Direct translocation of PM-associated metals, including zinc, may mediate this effect. We hypothesized that following a single intratracheal instillation (IT), zinc directly translocates outside of the lungs, reaching the heart. To test this, we used high resolution magnetic sector field inductively coupled plasma mass spectrometry to measure levels of five stable isotopes of zinc ((64)Zn, (66)Zn, (67)Zn, (68)Zn, (70)Zn), and copper in lungs, plasma, heart, liver, spleen, and kidney of male Wistar Kyoto rats (13 weeks old, 250-300 g), 1, 4, 24, and 48 h following a single IT or oral gavage of saline or 0.7 micromol/rat (70)Zn, using a solution enriched with 76.6% (70)Zn. Natural abundance of (70)Zn is 0.62%, making it an easily detectable tracer following exposure. In IT rats, lung (70)Zn was highest 1 h post IT and declined by 48 h. Liver endogenous zinc was increased 24 and 48 h post IT. (70)Zn was detected in all extrapulmonary organs, with levels higher following IT than following gavage. Heart (70)Zn was highest 48 h post IT. Liver, spleen and kidney (70)Zn peaked 4 h following gavage, and 24 h following IT. (70)Zn IT exposure elicited changes in copper homeostasis in all tissues. IT instilled (70)Zn translocates from lungs into systemic circulation. Route of exposure affects (70)Zn translocation kinetics. Our data suggests that following pulmonary exposure, zinc accumulation and subsequent changes in normal metal homeostasis in the heart and other organs could induce cardiovascular injury.

The role of particulate matter-associated zinc in cardiac injury in rats.

BACKGROUND: Exposure to particulate matter (PM) has been associated with increased cardiovascular morbidity; however, causative components are unknown. Zinc is a major element detected at high levels in urban air. OBJECTIVE: We investigated the role of PM-associated zinc in cardiac injury. METHODS: We repeatedly exposed 12- to 14-week-old male Wistar Kyoto rats intratracheally (1x/week for 8 or 16 weeks) to a) saline (control); b) PM having no soluble zinc (Mount St. Helens ash, MSH); or c) whole-combustion PM suspension containing 14.5 microg/mg of water-soluble zinc at high dose (PM-HD) and d ) low dose (PM-LD), e) the aqueous fraction of this suspension (14.5 microg/mg of soluble zinc) (PM-L), or f ) zinc sulfate (rats exposed for 8 weeks received double the concentration of all PM components of rats exposed for 16 weeks). RESULTS: Pulmonary inflammation was apparent in all exposure groups when compared with saline (8 weeks > 16 weeks). PM with or without zinc, or with zinc alone caused small increases in focal subepicardial inflammation, degeneration, and fibrosis. Lesions were not detected in controls at 8 weeks but were noted at 16 weeks. We analyzed mitochondrial DNA damage using quantitative polymerase chain reaction and found that all groups except MSH caused varying degrees of damage relative to control. Total cardiac aconitase activity was inhibited in rats receiving soluble zinc. Expression array analysis of heart tissue revealed modest changes in mRNA for genes involved in signaling, ion channels function, oxidative stress, mitochondrial fatty acid metabolism, and cell cycle regulation in zinc but not in MSH-exposed rats. CONCLUSION: These results suggest that water-soluble PM-associated zinc may be one of the causal components involved in PM cardiac effects.

Subchronic inhalation of zinc sulfate induces cardiac changes in healthy rats.

Zinc is a common metal in most ambient particulate matter (PM), and has been proposed to be a causative component in PM-induced adverse cardiovascular health effects. Zinc is also an essential metal and has the potential to induce many physiological and nonphysiological changes. Most toxicological studies employ high levels of zinc. We hypothesized that subchronic inhalation of environmentally relevant levels of zinc would cause cardiac changes in healthy rats. To address this, healthy male WKY rats (12 weeks age) were exposed via nose only inhalation to filtered air or 10, 30 or 100 microg/m(3) of aerosolized zinc sulfate (ZnSO(4)), 5 h/day, 3 days/week for 16 weeks. Necropsies occurred 48 h after the last exposure to ensure effects were due to chronic exposure rather than the last exposure. No significant changes were observed in neutrophil or macrophage count, total lavageable cells, or enzyme activity levels (lactate dehydrogenase, n-acetyl beta-D-glucosaminidase, gamma-glutamyl transferase) in bronchoalveolar lavage fluid, indicating minimal pulmonary effect. In the heart, cytosolic glutathione peroxidase activity decreased, while mitochondrial ferritin levels increased and succinate dehydrogenase activity decreased, suggesting a mitochondria-specific effect. Although no cardiac pathology was seen, cardiac gene array analysis indicated small changes in genes involved in cell signaling, a pattern concordant with known zinc effects. These data indicate that inhalation of zinc at environmentally relevant levels induces cardiac effects. While changes are small in healthy rats, these may be especially relevant in individuals with pre-existent cardiovascular disease.

Systemic translocation of particulate matter-associated metals following a single intratracheal instillation in rats.

Respirable ambient particulate matter (PM) exposure has been associated with an increased risk of cardiovascular disease. Direct translocation of PM-associated metals from the lungs into systemic circulation may be partly responsible. We measured elemental content of lungs, plasma, heart, and liver of healthy male WKY rats (12-15 weeks old) 4 or 24 h following a single intratracheal (IT) instillation of saline or 8.33 mg/kg of oil combustion PM (HP-12) containing a variety of transition metals with differing water and acid solubility. Tissues were digested with a combination of quaternary acid, amine, and nitric acid and analyzed using inductively coupled plasma-atomic emission spectroscopy. Lung levels of metals were lower at 24 h than at 4 h. Metals with high water solubility and relatively high concentration in HP-12 were increased in extrapulmonary organs. Water-soluble nonessential metals, like vanadium and nickel, were increased in plasma, hearts, and livers of exposed animals at both time points. Exposure-related small increases in essential metals, like zinc and manganese, were also noted in extrapulmonary tissues at both time points. Lead, with low water solubility but high acid solubility, was detected in liver only at 24-h postinstillation. Elements with low water or acid solubility, like silicon and aluminum, were not detected in extrapulmonary tissues despite decreased levels in the lung suggesting mucociliary clearance. We have shown that HP-12-associated metals translocate to systemic circulation and extrapulmonary organs following IT exposure. This translocation is dependent upon their relative levels and water solubility. Thus, following inhalation, PM-associated metals deposited in the lung may be released into systemic circulation at different rates depending on their water/acid solubility, thereby providing a means by which metals may elicit direct extrapulmonary effects.

Cardiopulmonary responses of Wistar Kyoto, spontaneously hypertensive, and stroke-prone spontaneously hypertensive rats to particulate matter (PM) exposure.

Humans with underlying cardiovascular disease, including stroke, are more susceptible to ambient particulate matter (PM)-induced morbidity and mortality. We hypothesized that stroke-prone spontaneously hypertensive rats (SHRSP) would be more susceptible than healthy Wistar Kyoto (WKY) rats to PM-induced cardiac oxidative stress and pulmonary injury. We further postulated that PM-induced injury would be greater in SHRSP than in spontaneously hypertensive rats (SHR) based on the greater disease severity in SHRSP than SHR. First, male WKY and SHRSP were intratracheally (IT) instilled with saline or 1.11, 3.33, or 8.33 mg/kg of oil combustion PM and responses were analyzed 4 or 24 h later. Second, SHR and SHRSP were IT instilled with saline or 3.33 or 8.33 mg/kg of the same PM and responses were analyzed 24 h later. Pulmonary injury and inflammation were assessed in bronchoalveolar lavage fluid (BALF) and cardiac markers in cytosolic and mitochondrial fractions. BALF neutrophilic inflammatory response was induced similarly in all strains following PM exposure. BALF protein leakage, gamma-glutamyl transferase, and N-acetylglucosaminidase activities, but not lactate dehydrogenase activity, were exacerbated in SHRSP compared to WKY or SHR. Pulmonary cytosolic and cardiac mitochondrial ferritin levels decreased, and cardiac cytosolic superoxide dismutase (SOD) activity increased in SHRSP only. Pulmonary SOD activity decreased in WKY and SHRSP. Cardiac mitochondrial isocitrate dehydrogenase (ICDH) activity decreased in PM-exposed WKY and SHR; control levels were lower in SHRSP than SHR or WKY. In summary, strain-related differences exist in pulmonary protein leakage and oxidative stress markers. PM-induced changes in cardiac oxidative stress sensitive enzymes are small, and appear only slightly exacerbated in SHRSP compared to WKY or SHR. Multiple biological markers may be differentially affected by PM in genetic models of cardiovascular diseases. Preexisting cardiovascular disease may influence susceptibility to PM pulmonary and cardiac health effects in a disease-specific manner.

Vascular and cardiac impairments in rats inhaling ozone and diesel exhaust particles.

BACKGROUND: Mechanisms of cardiovascular injuries from exposure to gas and particulate air pollutants are unknown. OBJECTIVE: We sought to determine whether episodic exposure of rats to ozone or diesel exhaust particles (DEP) causes differential cardiovascular impairments that are exacerbated by ozone plus DEP. METHODS AND RESULTS: Male Wistar Kyoto rats (10-12 weeks of age) were exposed to air, ozone (0.4 ppm), DEP (2.1 mg/m(3)), or ozone (0.38 ppm) + DEP (2.2 mg/m(3)) for 5 hr/day, 1 day/week for 16 weeks, or to air, ozone (0.51 or 1.0 ppm), or DEP (1.9 mg/m(3)) for 5 hr/day for 2 days. At the end of each exposure period, we examined pulmonary and cardiovascular biomarkers of injury. In the 16-week study, we observed mild pulmonary pathology in the ozone, DEP, and ozone + DEP exposure groups, a slight decrease in circulating lymphocytes in the ozone and DEP groups, and decreased platelets in the DEP group. After 16 weeks of exposure, mRNA biomarkers of oxidative stress (hemeoxygenase-1), thrombosis (tissue factor, plasminogen activator inhibitor-1, tissue plasminogen activator, and von Willebrand factor), vasoconstriction (endothelin-1, endothelin receptors A and B, endothelial NO synthase) and proteolysis [matrix metalloprotease (MMP)-2, MMP-3, and tissue inhibitor of matrix metalloprotease-2] were increased by DEP and/or ozone in the aorta, but not in the heart. Aortic LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) mRNA and protein increased after ozone exposure, and LOX-1 protein increased after exposure to ozone + DEP. RAGE (receptor for advanced glycation end products) mRNA increased in the ozone + DEP group. Exposure to ozone or DEP depleted cardiac mitochondrial phospholipid fatty acids (DEP > ozone). The combined effect of ozone and DEP exposure was less pronounced than exposure to either pollutant alone. Exposure to ozone or DEP for 2 days (acute) caused mild changes in the aorta. CONCLUSIONS: In animals exposed to ozone or DEP alone for 16 weeks, we observed elevated biomarkers of vascular impairments in the aorta, with the loss of phospholipid fatty acids in myocardial mitochondria. We conclude that there is a possible role of oxidized lipids and protein through LOX-1 and/or RAGE signaling.

Cardiovascular and blood coagulative effects of pulmonary zinc exposure.

Cardiovascular damage induced by pulmonary exposure to environmental chemicals can result from direct action or, secondarily from pulmonary injury. We have developed a rat model of pulmonary exposure to zinc to demonstrate cardiac, coagulative, and fibrinolytic alterations. Male Wistar Kyoto rats were instilled intratracheally with saline or zinc sulfate, 131 microg/kg (2 micromol/kg); the alterations were determined at 1, 4, 24, and 48 h postexposure. High-dose zinc enabled us to show changes in circulating levels of zinc above normal and induce significant pulmonary inflammation/injury such that cardiac impairments were likely. At 1-24 h postexposure, plasma levels of zinc increased to nearly 20% above the base line. Significant pulmonary inflammation and injury were determined by analysis of bronchoalveolar lavage fluid and histopathology in zinc-exposed rats at all time points. Starting at 4 h postexposure, pulmonary damage was accompanied by persistently increased gene expressions of tissue factor (TF) and plasminogen activator-inhibitor-1 (PAI-1), but not thrombomodulin (TM). Cardiac tissues demonstrated similar temporal increases in expressions of TF, PAI-1, and TM mRNA following pulmonary instillation of zinc. In contrast to extensive pulmonary edema and inflammation, only mild, and focal acute, myocardial lesions developed in a few zinc-exposed rats; no histological evidence showed increased deposition of fibrin or disappearance of troponin. At 24 and 48 h postexposure to zinc, increases occurred in levels of systemic fibrinogen and the activated partial thromboplastin time. These data suggest that cardiovascular blood coagulation impairments are likely following pulmonary zinc exposure and associated pulmonary injury and inflammation.