Exposure to inhaled particulate matter impairs cardiac function in senescent mice

Abstract

Daily exposure to particulate matter (PM) is known to adversely affect cardiac function and is also known to be exaggerated with senescence. This study tests the hypothesis that cardiac function is uniquely altered by PM exposure in senescent mice. A mechanism for PM-induced cardiac effects is also postulated by examining the activity of nitric oxide synthase (NOS) and the generation of reactive oxygen species (ROS) in heart tissue. Echocardiography is performed in awake 18- and 28-mo-old mice at baseline and immediately following 3-h exposures to either filtered air or carbon black (CB; approximately 400 microg/m3) on 4 days. At 28 mo, left ventricular diameter at end-systole and end-diastole is significantly (P < 0.05) elevated, and fractional shortening is significantly reduced (49 +/- 3% vs. 56 +/- 3%) with CB exposure. In vivo hemodynamic measurements at 28 mo also demonstrate significant (P < 0.05) reductions in ejection fraction and increases in right ventricular and pulmonary vascular pressures following CB exposure. Functional changes at 28 mo are associated with increased ROS production as suggested by enhanced luminol activity. This elevated ROS production with aging and CB exposure is attributable to NOS uncoupling. Measurements of natriuretic peptide (atrial and brain) transcription and matrix metalloproteinase (MMP2 and MMP9) activity in heart tissue are significantly (P < 0.05) amplified with senescence and exposure to CB, pointing to increased cardiac stress and remodeling. These results demonstrate that acute PM exposure reduces cardiac contractility in senescent mice, and this decline in function is associated with increased ROS production linked to NOS uncoupling.

Fig. 1.

Representative echocardiographic images for 18- and 28-mo-old mice prior to and following 4 days of carbon black (CB) exposure for 3 h/day. The image at 28 mo shows noticeable right ventricular (RV) dilation that is not apparent in the 18-mo-old mice prior to CB exposure. Also, the intraventricular septal wall appears to be flattened in the older mice before exposure and appears desynchronized during left ventricular (LV) contraction with the posterior wall following CB exposure.

Fig. 2.

Average values for LV end-diastolic (LVEDD; A) and end-systolic (LVESD; B) diameters are depicted for 18- and 28-mo-old mice (n = 15 mice/age group) before and after 4 exposure days each for 3 h/day to either filtered air (FA) or CB. *P < 0.05, vs. preexposure; †P < 0.05, 28 mo vs. 18 mo.

Fig. 3.

Average values for relative wall thickness (A) and fractional shortening (B) are depicted for 18- and 28-mo-old mice (n = 15 mice per age group) before and after 4 exposure days each for 3 h/day to either FA or CB. *P < 0.05, vs. preexposure; ††P < 0.01, 28 mo vs. 18 mo.

Fig. 4.

A: Ca²⁺-dependent and Ca²⁺-independent NOS activity as measured by the rate of citrulline formation from heart tissue samples obtained from 18- and 28-mo-old mice exposed to either FA or CB for 4 days each for 3 h. Left: nitric oxide synthase (NOS) uncoupling was evident by a decline in Ca²⁺-dependent NOS activity (NOS3 and NOS1) in both age groups following CB exposure. Right: Ca²⁺-independent activity (NOS2) was upregulated in only the 28-mo-old mice exposed to CB. B: luminol activity at baseline and during N^G-nitro-l-arginine methyl ester (l-NAME) coincubation indicates an increase in reactive oxygen species (ROS) generation with CB exposure in both age groups, and the origin of ROS formation is associated with an increase in NOS activity. The coincubation with the selective NOS2 inhibitor GW274150 was not significantly (P > 0.05) different from baseline. C: representative blot comparing NOS3 dimer and monomer from isolated cardiac myocytes from 28-mo-old mice exposed to either FA or CB demonstrating a reduction of NOS3 dimer with CB exposure. D: increase in nitrotyrosine concentration as an indicator of peroxynitrite (ONOO⁻ species) formation further suggest that greater ROS generation occurs in hearts of 28-mo-old animals following CB exposure. E: significant reduction in PKG activity in 28-mo-old mice exposed to FA or CB indicates lower second messenger activation by NOS or natriuretic peptide release (n = 10 mice per age group). *P < 0.05 and **P < 0.01, CB vs. FA exposure; †P < 0.05, 28 mo vs. 18 mo; §P < 0.05, l-NAME vs. baseline.

Fig. 5.

A: activity levels of MMP2 and MMP9 are increased in heart tissue samples with age after FA exposure and in both 18- and 28-mo-old mice exposed to CB (n = 10 mice per age group). *P < 0.05, CB vs. FA exposure; †P < 0.05, 28 mo vs. 18 mo. B: representative zymogram performed comparing 28-mo-old mice exposed to FA and mice exposed to CB. These data suggest that both total and activated MMP9 and MMP2 are increased following exposure to CB.

Fig. 6.

Gene expression levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are increased in heart tissue samples with age after FA exposure and in both 18- and 28-mo-old mice exposed to CB (n = 10 mice per age group). *P < 0.05, CB vs. FA exposure; †P < 0.05, 28 mo vs. 18 mo.

Fig. 7.

Effects of tetrahydrobiopterin (BH4) treatment significantly improved cardiac function in CB-exposed 28-mo-old mice as shown by left ventricular ejection fraction (LVEF; A) and right ventricular contractility (dP/dt_max/IP, where IP is instantaneous pressurse; B) and relaxation time (Tau; C). Improvements in LV and RV function with BH4 treatment were coincident with reversing a fall in Ca²⁺-dependent and a rise in Ca²⁺-independent NOS activity (D), and a fall in PKG-1 activity (E). Likewise, BH4 treatment led to a lowered ROS generation derived from NOS-uncoupling in cardiac tissue from older CB-exposed mice (F). G: representative NOS3 dimer blot from isolated cardiac myocytes from 18- and 28-mo-old mice exposed to either FA or CB shows a preservation of the NOS3 dimer in older mice following CB exposure. ‡P < 0.05, BH4 treatment vs. control; *P < 0.05, CB vs. FA exposure; †P < 0.05, 28 mo vs. 18 mo; §P < 0.05, l-NAME vs. baseline.