Targeting MuRF1 by small molecules in a HFpEF rat model improves myocardial diastolic function and skeletal muscle contractility

Abstract

Background: About half of heart failure (HF) patients, while having preserved left ventricular function, suffer from diastolic dysfunction (so-called HFpEF). No specific therapeutics are available for HFpEF in contrast to HF where reduced ejection fractions (HFrEF) can be treated pharmacologically. Myocardial titin filament stiffening, endothelial dysfunction, and skeletal muscle (SKM) myopathy are suspected to contribute to HFpEF genesis. We previously described small molecules interfering with MuRF1 target recognition thereby attenuating SKM myopathy and dysfunction in HFrEF animal models. The aim of the present study was to test the efficacy of one small molecule (MyoMed-205) in HFpEF and to describe molecular changes elicited by MyoMed-205.

Methods: Twenty-week-old female obese ZSF1 rats received the MuRF1 inhibitor MyoMed-205 for 12 weeks; a comparison was made to age-matched untreated ZSF1-lean (healthy) and obese rats as controls. LV (left ventricle) function was assessed by echocardiography and by invasive haemodynamic measurements until week 32. At week 32, SKM and endothelial functions were measured and tissues collected for molecular analyses. Proteome-wide analysis followed by WBs and RT-PCR was applied to identify specific genes and affected molecular pathways. MuRF1 knockout mice (MuRF1-KO) SKM tissues were included to validate MuRF1-specificity.

Results: By week 32, untreated obese rats had normal LV ejection fraction but augmented E/e' ratios and increased end diastolic pressure and myocardial fibrosis, all typical features of HFpEF. Furthermore, SKM myopathy (both atrophy and force loss) and endothelial dysfunction were detected. In contrast, MyoMed-205 treated rats had markedly improved diastolic function, less myocardial fibrosis, reduced SKM myopathy, and increased SKM function. SKM extracts from MyoMed-205 treated rats had reduced MuRF1 content and lowered total muscle protein ubiquitination. In addition, proteomic profiling identified eight proteins to respond specifically to MyoMed-205 treatment. Five out of these eight proteins are involved in mitochondrial metabolism, dynamics, or autophagy. Consistent with the mitochondria being a MyoMed-205 target, the synthesis of mitochondrial respiratory chain complexes I + II was increased in treated rats. MuRF1-KO SKM controls also had elevated mitochondrial complex I and II activities, also suggesting mitochondrial activity regulation by MuRF1.

Conclusions: MyoMed-205 improved myocardial diastolic function and prevented SKM atrophy/function in the ZSF1 animal model of HFpEF. Mechanistically, SKM benefited from an attenuated ubiquitin proteasome system and augmented synthesis/activity of proteins of the mitochondrial respiratory chain while the myocardium seemed to benefit from reduced titin modifications and fibrosis.

Keywords: Diastolic dysfunction; HFpEF; MuRF1; Muscle atrophy; Skeletal muscle dysfunction; ZSF1.

Figure 1

Study design and animal characteristics at 20 weeks. A schematic drawing of the study design is depicted (A). ZSF1‐lean and ZSF1‐obese were included into the study. At an age of 20 weeks, 10 animals of each group were removed from the study to confirm the development of HFpEF. The remaining ZSF1‐lean animals served as controls (lean), whereas the ZSF1‐obese animals were randomized into a placebo group with no intervention (obese) or a group receiving rat chow containing MyoMed‐205 (obese‐MyoMed‐205). Twelve weeks after randomization, animals were subjected to echocardiography and invasive haemodynamic measurements, and collected tissues were used for functional, histological, and molecular analyses. Before randomization, skeletal muscle mass (B,C), skeletal muscle function (D,E), and endothelial function (F,G) was assessed in ZSF1‐lean and ZSF1‐obese animals. *P < 0.05, **P < 0.01 versus lean.

Figure 2

MyoMed‐205 increased muscle weight and cross sectional area in TA muscle. Muscle wet weight normalized to tibia length was measured in the soleus (A), EDL (B), and TA (C) muscle of ZSF1‐lean (lean), ZSF1 obese (obese), and ZSF1‐obese rats treated with MyoMed‐205 (obese + MyoMed‐205) (n = 14–15 per group). In addition, cross sectional area (CSA) was quantified in the TA muscle of each group (n = 10 per group) (D). Representative histological images (nuclei stained with haemalaun) for CSA are depicted. Results are expressed as mean ± SEM.

Figure 3

MyoMed‐205 improved muscle function of EDL and soleus muscle. Absolute (A–D) and specific (E–H) muscle force were measured in vitro from ZSF1‐lean (lean, black squares), ZSF1 obese (obese, open circles), and ZSF1‐obese rats treated with MyoMed‐205 (obese + MyoMed‐205, grey triangles) in the EDL and soleus muscle. Results are expressed as mean ± SEM (n = 9–11 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. lean; ^§ P < 0.05, ^§§ P < 0.01, ^§§§ P < 0.001 vs. obese + MyoMed‐205.

Figure 4

Impact of MyoMed‐205 on protein synthesis of atrophy related proteins in TA muscle. Protein synthesis of atrophy related proteins (A–D) was quantified by western blot analysis in TA muscle homogenates obtained from ZSF1‐lean (lean), ZSF1 obese (obese), and ZSF1‐obese rats treated with MyoMed‐205 (obese + MyoMed‐205). As atrophy related proteins MuRF1 (A), MAFBx (B), Trim72 (C), and ubiquitinylated proteins (D) were measured. Results are expressed as mean ± SEM (n = 10–14 per group). Representative western blots are depicted.

Figure 5

Proteome analysis of TA muscle. The proteomes expressed in TA muscle samples were analysed to identify differentially expressed proteins in the different groups (n = 5 per group). The set of 81 differentially expressed proteins between lean and obese and of 31 proteins of obese versus obese + MyoMed‐205 share a group of eight proteins that are differentially expressed in both sets and exhibit no difference between lean and obese + MyoMed‐205 (A). The eight proteins are listed in the table. Four out of these eight proteins were further investigated by western blot analysis (B,C) or RT‐PCR (D,E) in the TA muscle of ZSF1‐lean (lean), ZSF1 obese (obese) and ZSF1‐obese rats treated with MyoMed‐205 (obese + MyoMed‐205). Results are expressed as mean ± SEM (n = 11–15 per group).

Figure 6

Impact of MyoMed‐205 on mitochondrial protein synthesis and enzyme activity in TA muscle. Protein synthesis of mitochondrial proteins or enzyme activities (A–E) were quantified by western blot analysis or specific enzyme assays in TA muscle homogenates obtained from ZSF1‐lean (lean), ZSF1 obese (obese) and ZSF1‐obese rats treated with MyoMed‐205 (obese + MyoMed‐205). Porin (A), mitochondrial complex I (C), mitochondrial complex II (D), mitochondrial complex III (E), mitochondrial complex IV (F), and mitochondrial complex V (G) synthesis were assessed by western blot and mitochondrial complex I enzyme activity was determined (B). Moreover, the enzymatic activity of citrate synthase (CS) in TA (I) and the protein synthesis of UCP3 was quantified (J). Results are expressed as mean ± SEM (n = 10–14 per group). A representative western blot shows the detection of mitochondrial complex I–V (H).

Figure 7

Impact of MyoMed‐205 on molecular parameters in the myocardium. The myocardium from ZSF1‐lean (lean), ZSF1 obese (obese), and ZSF1‐obese rats treated with MyoMed‐205 (obese + MyoMed‐205) was used to quantify the extent of perivascular fibrosis (A), the activity of matrix metalloproteinase 2 (MMP2) (B), the ratio of phosphorylated titin/total titin (C) and the synthesis of advanced glycation end products (AGE) modified proteins (D). Moreover, the mRNA expression of atrial natriuretic peptide (ANP) (E), B‐type natriuretic peptide (BNP) (F), collagen‐1a1 (Col1a1) (G), and collagen‐3a1 (Col3a1) (H) were quantified. Results are expressed as mean ± SEM (n = 10–14 per group). Representative western blots are depicted.