AT1 receptor blocker losartan protects against mechanical ventilation-induced diaphragmatic dysfunction

Abstract

Mechanical ventilation is a life-saving intervention for patients in respiratory failure. Unfortunately, prolonged ventilator support results in diaphragmatic atrophy and contractile dysfunction leading to diaphragm weakness, which is predicted to contribute to problems in weaning patients from the ventilator. While it is established that ventilator-induced oxidative stress is required for the development of ventilator-induced diaphragm weakness, the signaling pathway(s) that trigger oxidant production remain unknown. However, recent evidence reveals that increased plasma levels of angiotensin II (ANG II) result in oxidative stress and atrophy in limb skeletal muscles. Using a well-established animal model of mechanical ventilation, we tested the hypothesis that increased circulating levels of ANG II are required for both ventilator-induced diaphragmatic oxidative stress and diaphragm weakness. Cause and effect was determined by administering an angiotensin-converting enzyme inhibitor (enalapril) to prevent ventilator-induced increases in plasma ANG II levels, and the ANG II type 1 receptor antagonist (losartan) was provided to prevent the activation of ANG II type 1 receptors. Enalapril prevented the increase in plasma ANG II levels but did not protect against ventilator-induced diaphragmatic oxidative stress or diaphragm weakness. In contrast, losartan attenuated both ventilator-induced oxidative stress and diaphragm weakness. These findings indicate that circulating ANG II is not essential for the development of ventilator-induced diaphragm weakness but that activation of ANG II type 1 receptors appears to be a requirement for ventilator-induced diaphragm weakness. Importantly, these experiments provide the first evidence that the Food and Drug Administration-approved drug losartan may have clinical benefits to protect against ventilator-induced diaphragm weakness in humans.

Keywords: muscle atrophy; reactive oxygen species; respiratory muscles; weaning.

Fig. 1.

Plasma angiotensin II (ANG II) levels were measured before the beginning of mechanical ventilation (MV; 0 h) and following 12 h of MV. The reported plasma ANG II levels are the differences between these two time points. SB, spontaneous breathing; MVL, mechanical ventilation treated with losartan; MVE, mechanical ventilation treated with enalapril. Values are means ± SE. P < 0.05, significantly different vs. MV (*) and MVE (‡).

Fig. 2.

Plasma interleukin-6 (IL-6) levels were measured at the completion of both 6 (A) and 9 (B) h of prolonged MV. Note that plasma IL-6 levels were not measured following 12 h of MV because of inadequate sample availability. Values are means ± SE. Note that no significant group differences existed in plasma IL-6 levels at either time point.

Fig. 3.

Plasma corticosterone levels were measured at the completion of 12 h of prolonged MV. Values are means ± SE. Note that no significant group differences existed in plasma corticosterone levels.

Fig. 4.

Immunohistochemistry was used to identify the presence of ANG II type 1 (AT₁) receptors in rat diaphragm muscle. Blue represents DAPI staining of myonuclei. A: photograph of the presence of AT₁ receptors within diaphragm muscle fibers in rat. AT₁ receptors are stained in red. B: photograph illustrating the presence of AT₁ receptors in smooth muscle of the carotid artery in rat. AT₁ receptors are stained in red.

Fig. 5.

The relative abundance of 4-hydroxynonenal (4-HNE)-modified proteins (index of lipid peroxidation) in the diaphragm were determined via Western blot. Values are means ± SE and normalized to α-tubulin. *Significantly different from MV (P < 0.05).

Fig. 6.

Mitochondrial respiratory control ratio (RCR) measured in mitochondria within permeabilized diaphragm muscle fibers. Values are expressed as means ± SE. P < 0.05, significantly different vs. MV (*) and SB (†).

Fig. 7.

Diaphragm-specific force production as a function of the stimulation frequency (i.e., force-frequency curve) measured in vitro in costal diaphragm muscle strips following 12 h of SB or MV. Values are means ± SE. Note that no significant force differences existed between SB animals and MVL animals at any stimulation frequency. P < 0.05, SB significantly different vs. MV and MVE (*) and MVL significantly different vs. MVE (‡).

Fig. 8.

Diaphragm muscle fiber cross-sectional area. Values are means ± SE. P < 0.05, significantly different vs. MV (*) and significantly different from SB (†).

Fig. 9.

A: calpain 1 activity in diaphragm was determined via Western blotting. B: values are means ± SE. P < 0.05, significantly different vs. MV (*) and significantly different from SB (†).