Rationale: Chronic elevation of pulmonary microvascular pressure in chronic heart failure results in compensatory changes in the lung that reduce alveolar fluid filtration and protect against pulmonary microvascular rupture.
Objectives: To determine whether these compensatory responses may have maladaptive effects on lung function.
Methods: Six weeks after myocardial infarction (chronic heart failure model) rat lung composition, both gross and histologic; air and saline mechanics; surfactant production; and immunological mediators were examined.
Measurements and Main Results: An increase in dry lung weight, due to increased insoluble protein, lipid and cellular infiltrate, without pulmonary edema was found. Despite this, both forced impedance and air pressure–volume mechanics were normal. However, there was increased tissue stiffness in the absence of surface tension (saline pressure–volume curve) with a concurrent increase in both surfactant content and alveolar type II cell numbers, suggesting a novel homeostatic phenomenon.
Conclusions: These studies suggest a compensatory reduction in pulmonary surface tension that attenuates the effect of lung parenchymal remodeling on lung mechanics, hence work of breathing.
Persisting elevation of pulmonary microvascular pressure in chronic heart failure leads to pulmonary remodeling and reduced diffusion coefficient for carbon monoxide. Although this remodeling appears to protect against pulmonary edema, other effects could be harmful.
Despite increased dry lung weight and stiffer lung tissue, mechanics are normal in a model of chronic heart failure. This paradox is due to a homeostatic response in which surface tension is reduced below normal with a concurrent increase in surface active surfactant content.
Long-standing elevation in Pmv leads to protective remodeling of the lung parenchyma and pulmonary circulation (3, 4). Using models of CHF, Huang and colleagues (5) and Townsley and colleagues (6) found that the capillary filtration coefficient was reduced, leading to a predicted 50% reduction in the water filtered when Pmv was elevated. Consistent with this, we (7) and others (8) have found a marked increase in dry lung weight with no increase in lung water, in a rat model of infarct-induced CHF.
Despite the potential clinical relevance there has been little investigation into the complex effects of acute and chronic heart failure on pulmonary function. Carbon monoxide diffusing capacity is reduced (1), consistent with thickening of the alveolocapillary barrier. In acute cardiogenic pulmonary edema, lung elastance is increased (9), consistent with the sudden increase in alveolar fluid and surfactant dysfunction from the associated plasma proteins. However, in CHF lung elastance has been variably reported as normal (10) or increased (1, 11), possibly reflecting a complex interplay between pulmonary remodeling, surfactant function, and the temporal evolution of the pulmonary manifestations of the CHF syndrome.
In the present study, we hypothesized that homeostatic responses in the CHF lung extend beyond protective remodeling to changes in respiratory mechanics. In a rat left coronary artery ligation infarct model of CHF, we demonstrate that by 6 weeks postinfarct there is increased dry lung weight and tissue stiffness. However, air pressure–volume (P–V) relations and forced impedance mechanics were normal, suggesting reduced surface tension, a previously unknown pathophysiological phenomenon in CHF. Elucidation of these lung homeostatic responses may open exciting new therapeutic avenues for the management of dyspnea in CHF.
The study protocol was approved by the Flinders University (Adelaide, Australia) Animal Welfare Committee. Six weeks after recovery from left coronary artery ligation (16), pulmonary effects were investigated in 69 male Sprague-Dawley rats. Please refer to the online supplement for greater detail.
Blind morphometric assessment (17, 18) was performed on selected tissues on the basis of maximal (large infarct; n = 4) and minimal (control; n = 4) dry lung weights (S.K.). Type II cells were identified by immunohistochemistry for surfactant protein (SP)-B. Upper lobes were homogenized and lipid and protein were separated (19). The nonaqueous fraction was separated into soluble and insoluble proteins, including the hydroxyproline content (20, 21).
Air, saline (22, 23), and then air after Tween 20 (24) P–V relations were measured to allow estimation of in vivo surface tension (23). Raw P–V expiration data were curve fitted (25) according to the Levenburg-Marquardt iterative algorithm (Sigma Plot 10.0; Systat Software, San Jose, CA). However, as this methodology (experiment 1) perturbs tissue and lavage, a second group of animals (experiment 2) was studied, and the impedance of their respiratory system was measured after a forced oscillation (26, 27).
The right upper lobe was resected for measurement of the wet-to-dry ratio; lavage of the remaining lung (28) was assayed for surfactant proteins (SP-A and SP-B) by Western analysis. The remainder was separated into the surface active tubular myelin–rich (Alv-1) and recycled tubular myelin–poor (Alv-2) fractions (29) before lipid extraction (30) and total and disaturated phospholipid (DSP) content determination (31, 32). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify gene expression of SP-A and SP-B from homogenized whole lung.
Statistical analyses were performed with SPSS 14.0 software (SPSS Inc, Chicago, IL). General linear model repeated measures analysis or one-way analysis of variance was used as appropriate to compare the three groups of animals, with between-group differences tested with either Tukey's honestly significant difference (HSD) (between three groups) or independent samples t test (between two groups). Linear relationships were examined using Pearson's correlation. Data are expressed as means ± SD, and P values not exceeding 0.05 were considered significantly different whereas P values less than 0.10 are discussed as potentially interesting trends, as suggested (33).
Six weeks after left coronary artery ligation the large infarct group had both increased LVEDP and right ventricular (RV) weight (Table 1), consistent with elevated pulmonary artery pressures due to left ventricle failure–induced CHF (16).
|Control (<25%)||Moderate (25–45%)||Large (>45%)||P Value*|
|Myocardial infarct, % LV||6.2 ± 8.1a||37.6 ± 5.9b||54.6 ± 6.1c||<0.001|
|RV, mg/g body weight||0.61 ± 0.05a||0.63 ± 0.04a||0.85 ± 0.24b||<0.001|
|mBP, mm Hg||123 ± 13a||121 ± 12ab||113 ± 17b||0.09|
|HR, beats/min||366 ± 35||377 ± 41||348 ± 26||0.11|
|LVEDP, mm Hg||8.3 ± 2.3a||12.0 ± 4.2a||25.3 ± 3.9b||<0.001|
|Dry lung, mg/g body weight||0.06 ± 0.01a||0.06 ± 0.01a||0.09 ± 0.03b||<0.001|
| Wet-to-dry weight ratio||4.8 ± 0.1||4.8 ± 0.2||4.9 ± 0.2||0.18|
Although the lung wet-to-dry weight ratio was unchanged, the dry weight was approximately 50% increased in the large infarct group (Table 1). Blinded histologic (Figure 1), morphometric (17, 18), and compositional analysis of lung tissue found no difference between large infarct and control animals (Table 2). However, immunohistochemical assessment found a 25% increase in alveolar type II cell prevalence in the large infarct group.
|Control (<25%)||Large (>45%)||P Value*|
|Type II count†||257 ± 47||318 ± 5||0.04|
|Surface density‡||960 ± 117||883 ± 84||0.33|
|Surface area, cm2||0.35 ± 0.08||0.32 ± 0.04||0.52|
|Tissue, %||29.5 ± 4.8||30.3 ± 2.4||0.80|
|Airspace, %||47.2 ± 3.4||51.5 ± 4.6||0.18|
|Vessels, %||4.6 ± 0.5||4.6 ± 1.9||0.98|
|Airways, %||9.2 ± 2.6||6.6 ± 1.7||0.13|
|Connective tissue, %||8.2 ± 4.1||6.9 ± 3.4||0.63|
|In situ lymphocytes, %||0.96 ± 0.67||0.48 ± 0.63||0.34|
Gross assessment of tissue lipid and protein fractions found a 30% increase in total lipid and 60% increase in insoluble protein in lungs of the large infarct group, with no increase in soluble protein (Table 3). Quantitation of insoluble collagen via hydroxyproline content (34) demonstrated strong relationships with total insoluble protein (see Figure E1C in the online supplement) and, remarkably, with right ventricular weight (R2 = 0.685; P ≤ 0.001).
|Control (<25%) (n = 13)||Moderate (25–45%) (n = 9)||Large (>45%) (n = 10)||P Value*|
|Total lipid†||15 ± 2a||14 ± 4a||20 ± 7b||0.009|
|Total soluble protein†||15 ± 7||17 ± 10||18 ± 8||0.59|
|Total insoluble protein†||45 ± 6a||49 ± 21a||73 ± 26b||0.004|
|Hydroxyproline†||0.87 ± 0.25||0.80 ± 0.11||1.15 ± 0.54||0.17|
In the large infarct group both static air P–V relations and impedance mechanics after forced oscillation measured at low, normal, and high lung volume were similar to control animals (Figure 2; and see Figure E3 in the online supplement).
However, the large infarct saline P–V curve was significantly right shifted, indicating a “stiffer” lung (Figure 2D). The difference in pressure between air and saline P–V relations during expiration tended to be less in large infarct animals compared with control animals (Figure 2E), particularly at high lung volumes. Consistent with this, using the method of Valberg and Brain (24), the surface tension due to surfactant over decreasing lung volume in the large infarct group tended to be less than that of control animals (Figure 2F).
Although whole lung tissue SP-A and SP-B mRNAs were unchanged (see the online supplement), whole lung lavage from the large infarct group had both increased SP-A and SP-B protein (Figure 3) and surfactant phospholipid (Table 4). Consistent with a specific increase, when these relations were examined relative to dry lung weight, rather than per lung, only the surface active fraction (Alv-1) had increased phospholipid and DSP in large infarct animals (Table 4).
|Control (<25%)||Moderate (25–45%)||Large (>45%)||P Value*||Control (<25%)||Moderate (25–45%)||Large (>45%)||P Value*|
|Total†||6.0 ± 1.1a||6.7 ± 1.1a||11.4 ± 5.3b||<0.001||2.5 ± 0.5a||2.6 ± 0.3a||4.8 ± 2.3b||<0.001|
|Alv-1†||1.2 ± 0.4a||1.4 ± 0.4a||3.6 ± 2.5b||<0.001||0.5 ± 0.2a||0.5 ± 0.1a||1.5 ± 1.0b||<0.001|
|Alv-2†||4.7 ± 1.0a||5.3 ± 0.8a||7.8 ± 3.1b||<0.001||2.0 ± 0.4a||2.1 ± 0.3a||3.3 ± 1.5b||0.002|
|Total‡||106 ± 24||114 ± 22||123 ± 32||0.33||45 ± 9||45 ± 7||52 ± 16||0.27|
|Alv-1‡||22 ± 7a||24 ± 7a||36 ± 14b||0.003||8.7 ± 3.4a||8.7 ± 2.4a||14.5 ± 5.9b||0.003|
|Alv-2‡||84 ± 20||90 ± 17||87 ± 25||0.79||36 ± 7||36 ± 6||37 ± 13||0.94|
Chronic heart failure is a major public health burden in developed countries (35). Despite management advances, its natural history continues to follow a relapsing course superimposed on a progressive decline. This leads to recurrent hospital admission, frequently due to the cardinal clinical symptom of dyspnea (36), which accounts for about 70% of the total health care cost of CHF and about 2% of total health care expenditure (35).
Chronic elevation of Pmv in CHF results in structural lung parenchymal changes that serve to reduce alveolar fluid filtration and protect against pulmonary microvascular rupture. However, these adaptive responses might be expected to have maladaptive effects on lung function. Our studies suggest a compensatory reduction in pulmonary surface tension that attenuates the effect of lung parenchymal remodeling on lung mechanics, hence work of breathing. We found, in this model of CHF, that the lung is both dry and heavy despite prolonged elevation of Pmv, consistent with previous reports (7, 8). The increase in dry lung weight is accompanied by increased insoluble protein, total lipid, surface active pulmonary surfactant content, and cellular infiltrate. In addition to variably contributing to or potentially regulating increased tissue density and pulmonary mechanics, these changes may also contribute to altered homeostatic control of lung water in CHF (6), and extend the current paradigm of the lung's response to raised Pmv.
As expected, the heavy, dry lung showed increased intrinsic tissue stiffness. Using saline instead of air removes the air–fluid interface and associated surface tension. By this technique we found a right-shifted saline P–V curve. However, both impedance mechanics at low, mid, and high lung volume and static air P–V relations were normal in the CHF lung. In considering this paradox, it is important to recognize that the elastic properties of the lung reflect both surface tension at the gas–liquid interface and parenchymal tissue effects. Depending on lung volume, surface tension, which is largely determined by surfactant function, normally comprises 60 to 70% of elastic recoil (37). In bleomycin-induced pulmonary fibrosis, in which there is a marked increase in lung elastance, surfactant dysfunction makes a significant contribution to increased elastic recoil (38). Normal elastic mechanics despite increased intrinsic stiffness of the lung suggests lower than normal surface tension in the air-filled state, which is supported by our subsequent finding of a trend to decreased in vivo surface tension in the CHF lung. Further, the direct correlation between increasing surfactant content and dry lung weight suggests surface tension compensation through regulation of surfactant content and function as an important homeostatic factor that compensates for increased lung tissue stiffness, normalizing lung mechanics in CHF.
The increase in both phospholipid and DSP, with concurrent type II cell hyperplasia, suggests a source for the increased surfactant content, and reduced surface tension. Alternatively, altered surfactant turnover or composition could explain this finding. Increased type II prevalence might also tend toward increasing surfactant recycling while chemokine-driven recruitment of circulating leukocytes, resulting in an increase in in situ alveolar macrophages, would predispose to an increase in surfactant clearance (39, 40). Associated secretagogues may additionally account for, or contribute to, increased surfactant content. Nevertheless, the finding of increased SP-A and SP-B without a concurrent increase in RNA supports type II cell hyperplasia as the most likely mechanism.
In the premature lung, reduced surfactant content results in increased surface tension (41). In considering the possibility that increased surfactant content reduced surface tension below normal, there are limited data. Yamashita and colleagues (42) reduced surface tension below normal through depletion of alveolar macrophages. This resulted in an increase in pulmonary surfactant and reduction in ventilator-induced lung injury. Verbrugge and colleagues (43) found a dose-dependent reduction in surface tension, and lung injury, after exogenous surfactant administration consistent with the notion that greater than normal surfactant content reduces surface tension further. Together, these data support the notion that the increase in surfactant content we found in CHF may have beneficial effects on lung mechanics.
Although there is some clinical evidence for remodeling of the pulmonary circulation and lung parenchyma, including type II cell hyperplasia when Pmv is persistently elevated due to mitral stenosis (3), and CHF (4, 8, 44, 45), previous reports have tended to report autopsy findings or illustrative sections from models of CHF. Neither of these provides an accurate description of lung remodeling in CHF. In autopsy specimens there are likely to be changes in the lung reflecting a sudden worsening of heart failure immediately before death, and the lung may not be optimally prepared for careful morphologic analysis. Illustrative sections are prone to bias. Consequently, we blindly performed morphological analysis of appropriately prepared specimens. Apart from hyperplasia of type II cells, there was no morphologic evidence of the increase in insoluble protein or lung remodeling to explain the increase in dry lung weight. However, careful light microscopy may not be sensitive enough to detect subtle changes in the lung parenchyma. For example, despite a significant reduction in lung hydroxyproline content after type II cell transplantation, even illustrative light microscopy sections appear little different (46). Morphometric electron microscopy examining collagen content along with other structural proteins may provide further insight into these subtle yet significant changes.
We found a normal wet-to-dry weight ratio in the CHF lung in the face of chronically elevated Pmv. A reduced capillary filtration coefficient, resulting in a 50% reduction in water filtered at high Pmv, has been reported in models of CHF due to thickening of the basement membrane (6). Although we did not measure the thickness of the basement membrane we did find increased insoluble protein with strongly correlated increases in hydroxyproline, consistent with this mechanism. Further, increased alveolar fluid clearance has been reported in a model of CHF (47). Verghese and coworkers (48) measured alveolar fluid clearance in 65 ventilated patients with hydrostatic pulmonary edema, and noted that patients with CHF were more likely to have higher rates of alveolar fluid clearance. This may represent up-regulation of basal sodium transport, or the observation that there are a greater number of type II cells in CHF (3, 4, 45). Finally, reduced surface tension, as suggested here in the CHF lung, may reduce the tendency to develop alveolar edema because of reduction in the microvascular transmural pressure (40, 49).
Acute pulmonary edema due to elevated Pmv occurs immediately after myocardial infarction in the rat model. As disease progression to CHF ensues there are secondary changes in the lung as a result of chronic Pmv elevation. Because the 6-week time point used in the current study is arbitrary the development and progression of these changes are unknown, and will likely yield important insights into the natural history of the lung in CHF, and possibly the development of novel pulmonary therapeutic targets.
The authors thank Ms. Heather Barr, Ms. Malgosia Krupa, Ms. Kim Griggs, Mr. Denzil Paul, Ms. Tamara Crittenden, and Ms. Ceilidh Marchant for technical assistance.
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