Differential effects of right and left heart failure on skeletal muscle in rats

Abstract

Background: Exercise intolerance is a cardinal symptom in right (RV) and left ventricular (LV) failure. The underlying skeletal muscle contributes to increased morbidity in patients. Here, we compared skeletal muscle sarcopenia in a novel two-stage model of RV failure to an established model of LV failure.

Methods: Pulmonary artery banding (PAB) or aortic banding (AOB) was performed in weanling rats, inducing a transition from compensated cardiac hypertrophy (after 7 weeks) to heart failure (after 22-26 weeks). Cardiac function was characterized by echocardiography. Skeletal muscle catabolic/anabolic balance and energy metabolism were analysed by histological and biochemical methods, real-time PCR, and western blot.

Results: Two clearly distinguishable stages of left or right heart disease with a comparable severity were reached. However, skeletal muscle impairment was significantly more pronounced in LV failure. While the compensatory stage resulted only in minor changes, soleus and gastrocnemius muscle of AOB rats at the decompensated stage demonstrated reduced weight and fibre diameter, higher proteasome activity and expression of the muscle-specific ubiquitin E3 ligases muscle-specific RING finger 1 and atrogin-1, increased expression of the atrophy marker myostatin, increased autophagy activation, and impaired mitochondrial function and respiratory chain gene expression. Soleus and gastrocnemius muscle of PAB rats did not show significant changes in muscle weight and proteasome or autophagy activation, but mitochondrial function was mildly impaired as well. The diaphragm did not demonstrate differences in any model or disease stage except for myostatin expression, which was altered at the decompensated stage in both models. Plasma interleukin (IL)-6 and angiotensin II were strongly increased at the decompensated stage (AOB > > PAB). Soleus and gastrocnemius muscle itself demonstrated an increase in IL-6 expression independent from blood-derived cytokines only in AOB animals. In vitro experiments in rat skeletal muscle cells suggested a direct impact of IL-6 and angiotensin II on distinctive atrophic changes.

Conclusions: Manifold skeletal muscle alterations are more pronounced in LV failure compared with RV failure despite a similar ventricular impairment. Most of the catabolic changes were observed in soleus or gastrocnemius muscle rather than in the constantly active diaphragm. Mitochondrial dysfunction and up-regulation of myostatin were identified as the earliest signs of skeletal muscle impairment.

Keywords: Left heart failure; Mitochondria; Muscle wasting; Proteasome; Right heart failure.

Figure 1

Animal characteristics in the two‐stage ascending aortic banding (AOB) and pulmonary artery banding (PAB) models. (A) Left ventricular (LV) weight, right ventricular (RV) weight (weight in mg per tibia in mm), liver weight (weight in g × 10 per tibia in mm), and lung weight (weight in g × 100 per tibia in mm) in sham animals, AOB animals, and PAB animals 7 weeks after surgery (compensated stage) or 22/26 weeks after surgery (decompensated stage). At the decompensated stage, results from sham animals 22–26 weeks after surgery are shown. (B) LV ejection fraction (LVEF), LV fractional shortening (LVFS), tricuspid annular plane systolic excursion (TAPSE), and RV fractional area change (RV FAC). Groups are the same as in (A). All data are mean ± standard error of the mean. n = 10–14 animals per group. ^* P < 0.05; ^** P < 0.01; and ^*** P < 0.001.

Figure 2

Muscle weights (sarcopenia index) and muscle fibre types. (A) Differences in the weight of soleus muscle, diaphragm, and gastrocnemius muscle normalized per body weight (BW) in sham animals, ascending aortic banding (AOB) animals, and pulmonary artery banding (PAB) animals 7 weeks after surgery (compensated stage) or 22/26 weeks after surgery (decompensated stage). All data are mean ± standard error of the mean. n = 10–14 animals per group. ^* P < 0.05. (B) Representative results from ATPase staining in soleus muscle, diaphragm, and gastrocnemius muscle to distinguish between type I fibres (white), type IIA fibres (light blue), and type IIB fibres (dark blue). The size bar indicates 50 μm. (C) Analysis of the percentage of the according fibre types at the decompensated stage depicted as a pie chart. n = 6 animals per group.

Figure 3

Proteasome activation and expression. (A) Differences in chymotrypsin‐like and trypsin‐like proteasome activity in soleus muscle. (B) Differences in chymotrypsin‐like and trypsin‐like proteasome activity in diaphragm. (C) Differences in chymotrypsin‐like and trypsin‐like proteasome activity in gastrocnemius muscle. Data are normalized to 7‐week‐old sham animals. All data are mean ± standard error of the mean. n = 8 animals per group. ^* P < 0.05; ^** P < 0.01. (D) Representative western blots from soleus muscle at both disease stages. (E) Representative western blots from diaphragm at both disease stages. (F) Representative western blots from gastrocnemius muscle at both disease stages. Homogenates of skeletal muscle were probed with antibodies detecting muscle‐specific RING finger 1 (MuRF1), atrogin‐1, and myostatin. GAPDH served as a loading control. n = 6 samples per group. AOB, ascending aortic banding; PAB, pulmonary artery banding.

Figure 4

Autophagy and apoptosis. (A) Representative western blots and densitometry of protein data from soleus muscle, diaphragm, and gastrocnemius muscle at both disease stages. Homogenates of skeletal muscle were probed with an antibody detecting the autophagy marker light chain 3 (LC3, isoform A + B). The conversion of the LC3‐I form to the lower migrating form, LC3‐II, was used as an autophagy indicator. GAPDH served as a loading control. n = 6 samples per group. ^** P < 0.01. (B) Ten micrograms of tissue homogenate was transferred to a 96‐well microplate, and caspase 3/7 activity was detected in sham animals, ascending aortic banding (AOB) animals, and pulmonary artery banding (PAB) animals 7 weeks after surgery (compensated stage) or 22/26 weeks after surgery (decompensated stage). All data are mean ± standard error of the mean. n = 8 samples per group.

Figure 5

Mitochondrial function in soleus muscle. (A) Citrate synthase activity and the activity of complex I, complex II, complex III, and complex IV of the respiratory chain were measured in soleus muscle lysates. All values were normalized to total protein content of the samples. (B) Active rates of respiration (state 3) were measured in saponin‐skinned fibres of soleus muscle in the presence of 5 mM ADP and either 10 mM pyruvate + 2 mM malate or 10 mM succinate in the presence of 5 μM rotenone. All data are mean ± standard error of the mean. n = 6 samples per group. ^* P < 0.05. AOB, ascending aortic banding; PAB, pulmonary artery banding.

Figure 6

Mitochondrial respiratory chain protein expression. (A) Representative blots and densitometry of selected protein data (complex I and complex IV) from soleus muscle at both disease stages. (B) Representative blots and densitometry of selected protein data (ND1 and COX I) from soleus muscle at both disease stages. Homogenates of skeletal muscle were probed with antibodies detecting mitochondrial respiratory chain complexes I–V (total OXPHOS), the complex I proteins ND1 and NDUFS1, and the complex IV proteins COX I and COX IV. GAPDH served as a loading control. n = 6 samples per group. ^** P < 0.01; ^*** P < 0.001. AOB, ascending aortic banding; PAB, pulmonary artery banding.

Figure 7

Impact of interleukin‐6 (IL‐6) and angiotensin II (Ang II) on mediators involved in muscular atrophy and autophagy. (A) Rat skeletal muscle cell (RSkMC) myoblasts (left panel) or RSkMC myotubes (right panel) were treated with increasing concentrations of IL‐6 as indicated. Homogenates of RSkMC were probed with antibodies detecting muscle‐specific RING finger 1 (MuRF1), atrogin‐1, myostatin, and light chain 3 (LC3) (isoform A + B). GAPDH served as a loading control. (B) RSkMC myoblasts (left panel) or RSkMC myotubes (right panel) were treated as described previously and probed with an antibody detecting ubiquitin. A molecular weight marker to estimate the size of the ubiquitinated proteins is included. GAPDH served as a loading control. (A, B) n = 8 samples per group, four independent experiments. (C) RSkMC myoblasts (left panel) or RSkMC myotubes (right panel) were treated with increasing concentrations of Ang II as indicated. Homogenates of RSkMC were probed with antibodies detecting MuRF1, atrogin‐1, myostatin, and LC3 (isoform A + B). GAPDH served as a loading control. (D) RSkMC myoblasts (left panel) or RSkMC myotubes (right panel) were treated as described previously and probed with an antibody detecting ubiquitin. A molecular weight marker to estimate the size of the ubiquitinated proteins is included. GAPDH served as a loading control. (C, D) n = 8 samples per group, four independent experiments.

Figure 8

Role of interleukin‐6 (IL‐6) and angiotensin II (Ang II) pathway activation in rat skeletal myoblasts and myotubes in vitro. (A) Rat skeletal muscle cells (RSkMC) myoblasts (left panel) or RSkMC myotubes (right panel) were treated with Stattic (1 μM) or losartan (10 μM) for 1 h before adding IL‐6 (10 ng/mL) or Ang II (100 nM). Homogenates of RSkMC were probed with antibodies detecting muscle‐specific RING finger 1 (MuRF1), atrogin‐1, myostatin, light chain 3 (LC3) (isoform A + B), and pSTAT3. GAPDH served as a loading control. (B) RSkMC myoblasts (left panel) or RSkMC myotubes (right panel) were treated as described previously and probed with an antibody detecting ubiquitin. A molecular weight marker to estimate the size of the ubiquitinated proteins is included. GAPDH served as a loading control. (A, B) n = 8 samples per group, four independent experiments.