Angiotensin II infusion induces marked diaphragmatic skeletal muscle atrophy

Abstract

Advanced congestive heart failure (CHF) and chronic kidney disease (CKD) are characterized by increased angiotensin II (Ang II) levels and are often accompanied by significant skeletal muscle wasting that negatively impacts mortality and morbidity. Both CHF and CKD patients have respiratory muscle dysfunction, however the potential effects of Ang II on respiratory muscles are unknown. We investigated the effects of Ang II on diaphragm muscle in FVB mice. Ang II induced significant diaphragm muscle wasting (18.7±1.6% decrease in weight at one week) and reduction in fiber cross-sectional area. Expression of the E3 ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) and of the pro-apoptotic factor BAX was increased after 24 h of Ang II infusion (4.4±0.3 fold, 3.1±0.5 fold and 1.6±0.2 fold, respectively, compared to sham infused control) suggesting increased muscle protein degradation and apoptosis. In Ang II infused animals, there was significant regeneration of injured diaphragm muscles at 7 days as indicated by an increase in the number of myofibers with centralized nuclei and high expression of embryonic myosin heavy chain (E-MyHC, 11.2±3.3 fold increase) and of the satellite cell marker M-cadherin (59.2±22.2% increase). Furthermore, there was an increase in expression of insulin-like growth factor-1 (IGF-1, 1.8±0.3 fold increase) in Ang II infused diaphragm, suggesting the involvement of IGF-1 in diaphragm muscle regeneration. Bone-marrow transplantation experiments indicated that although there was recruitment of bone-marrow derived cells to the injured diaphragm in Ang II infused mice (267.0±74.6% increase), those cells did not express markers of muscle stem cells or regenerating myofibers. In conclusion, Ang II causes marked diaphragm muscle wasting, which may be important for the pathophysiology of respiratory muscle dysfunction and cachexia in conditions such as CHF and CKD.

Figure 1. Diaphragm Muscle Wasting.

(A) Diaphragm weight of sham at day 1 (Sham-D1), Ang II at day 1 (Ang II-D1), sham at day 7 (Sham-D7) and Ang II at day 7 (Ang II-D7). N = 8, Mean±SEM, ^###P<0.001. (B) Cross sectional area of diaphragm at day 7 (Sham, blue; Ang II, red). (C–E) Quantitative RT-PCR of Atrogin-1 (C), MuRF-1 (D) and BAX (E).

Figure 2. Regeneration of Diaphragm Muscles.

(A) Representitive hematoxylen and Eosin staining sections at days 1 and 7 for sham and for Ang II. White arrows show centralized nuclei. (B) Quantitative RT-PCR of embryonic MyHC (E-MyHC). (C) Immunostaining of E-MyHC (green) and Laminin (magenta). Nuclei were stained with DAPI (blue). First row shows no expression of E-MyHC in the Sham infused mice at day 7(20× magnification). Diaphragm of Ang II infused mice (second row, 20× magnification) shows high expression of E-MyHC (green). Higher magnification (100×) of cross sections (third row) clearly shows the centralized nucleus in the newly formed myofiber. Fourth row represented the longitudinal section of a newly formed myofiber showing the location of satellite cells (whit arrows). (D, E) Quantitative RT-PCR of IGF-1 (D) and IGF-1R (E). (F) Immunoblot analysis showed a slight increase of M-cadherin in diaphragm of Ang II infused mice. N = 4, Mean±SEM, p = 0.08.

Figure 3. Recruitment of bone marrow derived cells.

(A) Fluorescence activated cell sorter (FACS) analysis data at day 7 (Sham and Ang II infused mice) of recruited bone marrow derived cells (GFP⁺), and skeletal muscle stem cells (SMSC). N = 7, Mean±SEM, **P<0.01. (B–D) Bone marrow derived GFP positive cells recruited to diaphragm were costained with the satellite cell markers MyoD (B) and M-cadherin (C), and a marker of regenerating myofibers, E-MyHC (D).