Small-molecule-mediated chemical knock-down of MuRF1/MuRF2 and attenuation of diaphragm dysfunction in chronic heart failure

Abstract

Background: Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently discovered small-molecule inhibitor of MuRF1 in treating CHF-induced diaphragm myopathy and loss of contractile function.

Methods: Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham-operated animals (sham) served as controls. One week post-left anterior descending coronary artery ligation animals were randomized into two groups-one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946-a recently described MuRF1-interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed.

Results: Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P < 0.05). Treatment with compound ID#704946 in CHF mice had beneficial effects on the diaphragm: contractile function was protected, while mitochondrial enzyme activity and up-regulation of the MuRF1 and MuRF2 was attenuated after infarct.

Conclusions: Our murine CHF model presented with diaphragm fibre atrophy, impaired contractile function, and reduced mitochondrial enzyme activities. Compound ID#704946 rescued from this partially, possibly by targeting MuRF1/MuRF2. However, at this stage of our study, we refrain to claim specific mechanism(s) and targets of compound ID#704946, because the nature of changes after 12 weeks of feeding is likely to be complex and is not necessarily caused by direct mechanistic effects.

Keywords: Cardiac cachexia; Chronic heart failure; Diaphragm; Mitochondrial metabolism; MuRF1; Muscle wasting.

Figure 1

An overview of the study design (A). Animals at an age of 12 weeks were either subjected to myocardial infarction to induce chronic heart failure (CHF) or sham operated. One week after myocardial infarction echocardiography was performed to confirm reduced contractility. Animals with a left ventricular ejection fraction (LVEF) <20% were randomized into groups either receiving normal mouse chow (CHF) or chow supplemented with compound ID#704946 (CHF + 704946). Nine weeks after randomization echocardiography was repeated, animals were sacrificed, and tissue was collected for subsequent analysis. Echocardiographic analyses 1 week after sham operation or myocardial infarction and prior randomization revealed a significant reduction in LVEF (B) and fractional shortening (FS) (C) in CHF and CHF + 704946 mice. Representative images of myocardial sections stained with Picosirius red after the treatment period are shown (D) documenting clear fibrosis in the CHF and CHF + 704946 animals. LAD, left anterior descending coronary artery.

Figure 2

In vitro contractile function of diaphragm fibre bundles, as assessed during isometric (A, B) and isotonic contractions (C, D). Comparing maximal specific force (B) and maximal power (D), chronic heart failure (CHF) animals demonstrated a 21 and 28% reduction, respectively, which was significantly attenuated by ID#704948. Data are presented as mean ± standard error of the mean. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. CHF, ^§ P < 0.05, and ^§§ P < 0.01 vs. CHF + 704946.

Figure 3

Cross‐sectional area (A, B) and fibre‐type distribution (C, D) was evaluated in diaphragm sections from all three groups. Paraffin sections of the diaphragm were stained with periodic acid–Schiff (A), and the cross‐sectional area was determined by ImageJ (B). The distribution of type I and type II was quantified in diaphragm samples from all three groups, after staining paraffin sections with a myosin heavy chain slow isoform antibody (dark brown fibres = type I fibres; non‐stained fibres = type II fibres) (D). Data are presented as mean ± standard error of the mean. CHF, chronic heart failure.

Figure 4

Diaphragm tissues from mice sacrificed after completion of the myocardial infarction‐compound feeding intervention study was characterized by mass spectrometry‐based quantitative proteomics analysis. (A) Mrps5, a mitochondrial‐cytosolic shuttle protein in charge of protein initiation and elongation in the mitochondrial ribosome, is decreased significantly in the chronic heart failure (CHF) group in comparison with both the sham + compound control group and the CHF + compound group, respectively. Each data point represents protein ratio data between two mice. Relative changes in protein ratio are shown on the y‐axis as log2. By Bayesian moderated t‐testing as implemented in the R package limma and after multiple hypothesis testing correction (Benjamini–Hochberg), corrected P values are 0.02 for CHF + compound vs. CHF or for sham + compound vs. CHF. Proteins with P < 0.05 before Benjamini–Hochberg correction were not included at this stage. (B) Western blot analysis was performed for Mrps5 in the diaphragm of all animals included into the study. A significant reduction of Mrps5 expression was noted in CHF, which was reversed by compound ID#704946 feeding. A representative western blot is shown, and data are presented as mean ± standard error of the mean.

Figure 5

Enzyme activities of citrate synthase (A), succinate dehydrogenase (B), mitochondrial complex I (C), and creatine kinase (D). In addition, protein expression of mitochondrial porin of the outer mitochondrial membrane (E) and TOM‐20 (F) was quantified in diaphragm samples from all animals in the three groups [sham, chronic heart failure (CHF), and CHF + 704946]. A representative western blot for the detection of porin, TOM‐20, and GAPDH is shown (G). Our data revealed a significant down‐regulation of citrate synthase and succinate dehydrogenase activity and porin and TOM‐20 expression in CHF when compared with sham, but this was attenuated in mice fed the compound ID#704946. No difference in creatine kinase activity was observed between the three groups. Data are presented as mean ± standard error of the mean.

Figure 6

Enzyme activities of lactate dehydrogenase (A), pyruvate kinase (B), and β‐hydroxyacyl‐COA dehydrogenase (C) were quantified in diaphragm samples from all animals in the three groups [sham, chronic heart failure (CHF), and CHF + 704946]. No difference for these enzymes was observed between the three groups. Data are presented as mean ± standard error of the mean.

Figure 7

Protein expression of MuRF1 (A), MuRF2 (B), and telethonin (C) was quantified by western blot analysis. MuRF1 and MuRF2 were significantly up‐regulated in the chronic heart failure (CHF) group (A, B), which was attenuated in the CHF‐704946 group. A bigger effect of compound ID#704946 was seen for MuRF2 when compared with MuRF1. For telethonin expression, a trend towards lower expression was evident in the CHF group, which was attenuated after compound feeding (C) Data are presented as mean ± standard error of the mean.

Figure 8

The effect of compound ID#704946 on MuRF1 protein stability was determined in vitro by differential scanning fluorimetry. Changes in the intrinsic protein fluorescence in a thermal gradient were monitored at 350 and 330 nm, respectively. The first derivative of the fluorescence wavelength ratio of 350/330 nm upon thermal protein unfolding was used to calculate transition midpoint (Tm) of single and multiple transition states. Tm of MuRF1 central in phosphate‐buffered saline (solid line) was 65.2 °C and was only negligible changed to 65.8 °C by the addition of 1% DMSO (dashed line). In contrast, a strong effect on the thermal unfolding of MuRF1 was observed by the addition of compound ID#704946 (dotted line). Compound ID#704946 destabilized MuRF1 as indicated by the significantly reduced main Tm of 52.5 °C.