Anti-inflammatory nutrition with high protein attenuates cardiac and skeletal muscle alterations in a pulmonary arterial hypertension model

Abstract

Pulmonary arterial hypertension (PAH) is characterized by remodelling of the pulmonary arteries and right ventricle (RV), which leads to functional decline of cardiac and skeletal muscle. This study investigated the effects of a multi-targeted nutritional intervention with extra protein, leucine, fish oil and oligosaccharides on cardiac and skeletal muscle in PAH. PAH was induced in female C57BL/6 mice by weekly injections of monocrotaline (MCT) for 8 weeks. Control diet (sham and MCT group) and isocaloric nutritional intervention (MCT + NI) were administered. Compared to sham, MCT mice increased heart weight by 7%, RV thickness by 13% and fibrosis by 60% (all p < 0.05) and these were attenuated in MCT + NI mice. Microarray and qRT-PCR analysis of RV confirmed effects on fibrotic pathways. Skeletal muscle fiber atrophy was induced (P < 0.05) by 22% in MCT compared to sham mice, but prevented in MCT + NI group. Our findings show that a multi-targeted nutritional intervention attenuated detrimental alterations to both cardiac and skeletal muscle in a mouse model of PAH, which provides directions for future therapeutic strategies targeting functional decline of both tissues.

Figure 1

Total body weight development, lung weight and cardiac hypertrophy after 8 weeks of MCT injection. (A) Compared to sham mice, relative body weight was impaired (p < 0.05) in MCT and MCT + NI by 5%. (B) Both MCT groups showed an increase in lung weight (p < 0.05) compared to shams. (C) An increase in heart weight of MCT mice compared to shams was also seen, however these alterations were attenuated in MCT + NI mice. (D) Right ventricle thickness was increased only in MCT mice. (*p < 0.05, **p < 0.01) Scatterplots of the depicted data can be found in Supplementary Fig. S4.

Figure 2

Nutritional intervention prevents increased right ventricular fibrosis after MCT injection. Right ventricular fibrosis was increased in MCT mice (p < 0.05) compared to shams and attenuated in MCT + NI mice. (*p < 0.05) A scatterplot of the depicted data can be found in Supplementary Fig. S4.

Figure 3

Differential expression of genes in the right ventricle after MCT injection. (A) A total of 245 genes were differentially expressed in MCT compared to Sham and MCT + NI compared to MCT, using a cutoff of p < 0.05. (B) (A) Heatmap showing 32 genes that are differentially expressed in MCT compared to sham and in MCT + NI compared to MCT, using a cutoff of p < 0.01.

Figure 4

Fibrotic genes were upregulated in MCT, but not in MCT + NI goup. Compared to sham mice, mRNA levels of genes involved in fibrosis were upregulated in MCT mice, but not in MCT + NI mice (*p < 0.05, **p < 0.01, ***p < 0.001). (VCAN: versican; ADAMTS-1: a disintegrin and metalloproteinase with thrombospondin motifs 1; Col14a1: collagen type XIV alpha 1; TGFb1I1: transforming growth factor beta 1 induced transcript 1).

Figure 5

Nutritional intervention reduces an increase in TNFα mRNA levels in the right ventricle. (A) MCT injection induced a 5.5-fold increased level of TNFα mRNA in the right ventricle of MCT relative to sham mice. In MCT + NI mice this increase was only 2.26 fold. (B) COX-2 mRNA levels tended to be increased in right ventricle of MCT compared to sham mice (p = 0.068). TNFα: tumor necrosis factor alpha; COX2: cyclooxygenase-2. (*p < 0.05).

Figure 6

Changes in skeletal muscle weight and muscle fiber cross-sectional area. (A) Weight of the tibialis anterior (TA) muscle was unchanged, (B) but muscle fiber cross-sectional area (CSA) of the TA was reduced by 22% in MCT mice compared to sham, but unaltered in MCT + NI. (B) Weight of the EDL muscle was unchanged, but (D) skeletal muscle fiber CSA of the EDL was reduced by 29% in the MCT mice compared to sham and preserved in the MCT + NI group(*p < 0.05). (E) Weight of the soleus muscle was similar in all groups and (F) skeletal muscle fiber CSA was also unchanged.

Figure 7

An increase in E3 ubiquitin-protein ligases MuRF1 and MuRF2 in MCT mice is prevented by nutritional intervention. (A) Protein levels of the key E3 ligase MuRF1 was reduced by 30% in the group receiving nutritional intervention compared to MCT mice alone. (B) The E3 ligase MuRF2 was increased by 41% in the MCT group compared to sham. Nutritional intervention reduced MuRF2 protein levels by 28%. (*p < 0.05). Pictures are cropped from full-length pictures of blots. Full-length blots are presented in Supplementary Fig. S3. A MuRF1 knock-out (KO) sample was included to separate the MuRF1 bands from other bands due to nonspecific binding of the antibody.