Wayne E. Cascio

Air pollution and cardiovascular disease: A statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association

Air pollution is a heterogeneous, complex mixture of gases, liquids, and particulate matter. Epidemiological studies have demonstrated a consistent increased risk for cardiovascular events in relation to both short- and long-term exposure to present-day concentrations of ambient particulate matter. Several plausible mechanistic pathways have been described, including enhanced coagulation/thrombosis, a propensity for arrhythmias, acute arterial vasoconstriction, systemic inflammatory responses, and the chronic promotion of atherosclerosis. The purpose of this statement is to provide healthcare professionals and regulatory agencies with a comprehensive review of the literature on air pollution and cardiovascular disease. In addition, the implications of these findings in relation to public health and regulatory policies are addressed. Practical recommendations for healthcare providers and their patients are outlined. In the final section, suggestions for future research are made to address a number of remaining scientific questions.

THE MITOCHONDRIAL PERMEABILITY TRANSITION IN CELL DEATH : A COMMON MECHANISM IN NECROSIS, APOPTOSIS AND AUTOPHAGY

Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reyes syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reyes syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.

Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death.

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca2+ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca2+, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-α.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.

Role of Mitochondrial Inner Membrane Permeabilization in Necrotic Cell Death, Apoptosis, and Autophagy

Inhibition of mitochondrial oxidative phosphorylation progresses to uncoupling when opening of cyclosporin A-sensitive permeability transition pores increases permeability of the mitochondrial inner membrane to small solutes. Involvement of the mitochondrial permeability transition (MPT) in necrotic and apoptotic cell death is implicated by demonstrations of protection by cyclosporin A against oxidative stress, ischemia/reperfusion, tumor necrosis factor-alpha exposure, Fas ligation, calcium overload, and a variety of toxic chemicals. Confocal microscopy directly visualizes the MPT in single mitochondria within living cells from the translocation of impermeant fluorophores, such as calcein, across the inner membrane. Simultaneously, mitochondria release potential-indicating fluorophores. Subsequently, mitochondria swell, causing outer membrane rupture and release of cytochrome c and other proapoptotic proteins from the intermembrane space. In situ a sequence of decreased NAD(P)H, increased free calcium, and increased reactive oxygen species formation within mitochondria promotes the MPT and subsequent cell death. Necrotic and apoptotic cell death after the MPT depends, in part, on ATP levels. If ATP levels fall profoundly, glycine-sensitive plasma membrane permeabilization and rupture ensue. If ATP levels are partially maintained, apoptosis follows the MPT. The MPT also signals mitochondrial autophagy, a process that may be important in removing damaged mitochondria. Cellular features of necrosis, apoptosis, and autophagy frequently occur together after death signals and toxic stresses. A new term, necrapoptosis, describes such death processes that begin with a common stress or death signal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmed cellular resorption (apoptosis), depending on modifying factors such as ATP.

Heart rate variability in patients with coronary artery disease: Differences in patients with higher and lower depression scores.

Objective This study tested the hypothesis that coronary artery disease patients with higher depression scores have lower heart rate variability during daily life. Method: Thirty-three men and nine women, ranging in age from 46 to 79, with coronary artery disease and exercise-induced ischemia were studied. The standard deviation of normal R-R intervals (SDNN) and average heart rate were obtained from 24-hour ambulatory electrocardiographic monitoring. Patients were grouped by a median split of the Minnesota Multiphasic Personality Inventory (MMPI-D) score. Results: SDNN was lower (p =.009) and average heart rate was higher (p =.003) in patients with higher depression scores. These relationships remained substantially unaltered after statistically adjusting for the only demographic/clinical factor that varied between the groups: gender. Conclusions: In comparison to the lower depression score group, those with higher depression scores had lower heart rate variability during daily life. These findings may be related to the reported relationship between depression and survival risk in patients with coronary artery disease.

The Ib Phase of Ventricular Arrhythmias in Ischemic In Situ Porcine Heart Is Related to Changes in Cell-to-Cell Electrical Coupling

BACKGROUND This study was designed to test the hypothesis that the loss of cell-to-cell electrical interaction during ischemia modulates the amplitude of ischemia-induced TQ-segment depression (ie, the injury potential) and the occurrence of ventricular fibrillation (VF) during the so-called Ib phase of ventricular arrhythmias. METHODS AND RESULTS Regional ischemia was induced by 60 minutes of mid-left anterior descending coronary artery ligation in open-chest swine (n = 10). Cell-to-cell electrical uncoupling was defined as the onset of the terminal rise in whole-tissue resistivity (Rt). Local activation times and TQ-segment changes (injury potential) were determined from unipolar electrograms. Extracellular K+ ([K+]e) and pH (pHe) were measured with plunge-wire ion-selective electrodes. VF occurred in 6 of 10 pigs during regional no-flow ischemia between 19 and 30 minutes after the arrest of perfusion. The occurrence of VF was positively correlated to the onset of cell-to-cell electrical uncoupling (R2 = .885). Cell-to-cell electrical uncoupling superimposed on changes of [K+]e and pHe contributed to the failure of impulse propagation between 19 and 30 minutes after the arrest of perfusion. During ischemia, maximum TQ-segment depression was -10 mV at 19 minutes, after which TQ-segment depression slowly recovered. The onset of the TQ-segment recovery was correlated to the second rise in Rt (R2 = .886). CONCLUSIONS In the regionally ischemic in situ porcine heart, loss of cell-to-cell electrical interaction is related to the occurrence of VF and changes in the amplitude of the injury current. Cellular electrical uncoupling contributes to failure of impulse propagation in the setting of altered tissue excitability as a result of elevated [K+]e and low pHe. These data indicate that Ib arrhythmias and ECG changes during ischemia are influenced by the loss of cell-to-cell electrical interaction.

Multiple Metabolic Syndrome Is Associated With Lower Heart Rate Variability: The Atherosclerosis Risk in Communities Study

OBJECTIVE To test at the population level whether people with multiple metabolic syndrome (MMS) disorders have reduced cardiac autonomic activity (CAA). RESEARCH DESIGN AND METHODS We examined the association between the level of CAA and MMS disorders, at the degree of clustering and the segregate combination levels, using a random sample of 2,359 men and women aged 45–64 years from the biracial, population-based Atherosclerosis Risk in Communities (AR1C) Study. Supine resting 2-min beat-to-beat heart rate data were collected. High-frequency (HF) (0.15–0.35 Hz) and low-frequency (LF) (0.025–0.15 Hz) spectral powers, the ratio of LF to HF, and the SD of all normal R-R intervals (SDNN) were used as the conventional indices of heart rate variability (HRV) to measure CAA. The MMS disorders included hypertension, type 2 diabetes, and dyslipidemia. RESULTS HRV indices were significantly lower in individuals with MMS disorders. The multivariable adjusted mean HF was 0.85 (beat/min)2 in subjects with all three MMS disorders, in contrast to 1.31 beat/min)2 in subjects without any MMS disorder. At the segregated combination level, the multivariable adjusted means ± SEM of HF were 1.34 ± 0.05, 1.16 ± 0.05, 1.01 ± 0.17, and 1.34 ± 0.05 (beat/min)2, respectively, for subjects without any MMS disorder, with hypertension only, with diabetes only, and with dyslipidemia only, and the means ± SEM of HF were 0.93 ± 0.04,0.70 ± 0.15, and 1.20 ± 0.05 eat/min)2, respectively, for subjects with diabetes and hypertension, diabetes and dyslipidemia, and hypertension and dyslipidemia. An increase in fasting insulin of 1 SD was associated with 88% higher odds of having a lower HF The pattern of associations was similar for LF and SDNN. CONCLUSIONS These findings suggest that MMS disorders adversely affect cardiac autonomic control and a reduced cardiac autonomic control may contribute to the increased risk of subsequent cardiovascular events in individuals who exhibit MMS disorders.

Exposure to Concentrated Ambient Air Particles Alters Hematologic Indices in Humans

Descriptions of changes in hematological indices have contested the premise that the biological effects of suspended particulate matter (PM) are restricted to the lung. Employing approximately 40 hematologic parameters reflecting blood cells, chemistries, mediators, and coagulation factors, we tested the hypothesis that exposure to concentrated ambient air particles (CAPs) can be associated with changes in hematologic indices in normal humans. Twenty healthy young volunteers were exposed to either filtered air (n = 5) or CAPs (n = 15) with a mean PM mass of 120.5 ± 14.0 µg/m3 and a range from 15.0–357.6 µg/m3. Hematologic indices were measured. Changes in all parameters are expressed as the absolute value either immediately after or 24 h after exposure. Differences between responses of those individuals exposed to filtered air and CAPs were tested using the T-test of independent means. If significant differences between the two groups were suggested by the T-test (p < .10), the relationship was further evaluated employing linear regression techniques. Regression analysis verified significant linear relationships between particle mass the individual was exposed to and (1) decrements in WBC count 24 h later, (2) decreases in lactate dehydrogenase (LDH) concentration 24 h later, and (3) elevations in fibrinogen levels 24 h later. There were no changes in either inflammatory mediators in the blood or indices of coagulation/fibrinolysis other than fibrinogen. We conclude that exposure of healthy volunteers to CAPs can be associated with decreases of both white blood cell (WBC) count and LDH and increased concentrations of fibrinogen in the blood.

CARDIOVASCULAR EFFECTS ASSOCIATED WITH AIR POLLUTION: POTENTIAL MECHANISMS AND METHODS OF TESTING

A recent series of epidemiologic reports have shown associations between fine particulate matter (PM) levels and increased cardiovascular morbidity and mortality. Elevated PM levels have been linked with cardiac events, including serious ventricular arrhythmias and myocardial infarction. A workshop brought together epidemiologists, cardiologists, and toxicologists from academia, government, and industry to examine plausible mechanisms that could be responsible for such effects, and to consider the armamentarium of noninvasive tests available to examine these relationships. Possible mechanisms considered by the participants include: (a) effects on the autonomic nervous system; (b) alterations on ion channel function in myocardial cells; (c) ischemic responses in the myocardium; and (d) inflammatory responses triggering endothelial dysfunction, atherosclerosis, and thrombosis. A large number of tests were identified to assess specific mechanistic pathways underlying the cardiovascular effects of air pollution and include: (a) autonomic control of the cardiovascular system assessed primarily by heart-rate variability; (b) myocardial substrate and vulnerability assessed by the electrocardiogram and estimations of ejection fraction and wall motion abnormalities in imaging studies; and (c) endothelial function, atherosclerosis, and thrombosis assessed by clotting parameters, cytokines, lipid profiles, and forearm blood flow. A variety of approaches ranging from molecular and genetic investigations to human clinical studies were recommended to further investigate the important epidemiologic associations.

Mitochondrial calcium transients in adult rabbit cardiac myocytes: Inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca2+-indicating fluorophores

A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca(2+) uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.