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.

Small-Molecule Chemical Knockdown of MuRF1 in Melanoma Bearing Mice Attenuates Tumor Cachexia Associated Myopathy

“…Compounds may affect energy metabolism according to our earlier data in cardiac cachexia [ 22 , 23 ]. We, therefore, next assessed the enzymatic activity of mitochondrial citrate synthase (CS) as a rate-limiting enzyme in the trichloroacetic acid (TCA) cycle that locates downstream of Cpt2 with regards to Acetyl-CoA metabolism and of respiratory mitochondrial complex I.…”

“…Mass spectrometry (MS) based proteomic analysis was essentially performed as described earlier [ 22 , 23 ] using five EDL muscle tissues from compound-treated and non-treated Tu-muscles. MS raw data were processed as described by MaxQuant (1.6.6.0) using the Andromeda search engine and the Uniprot database for Mus musculus (as of 19 August 2019, containing 86,161 entries) [ 25 ].…”

“…Proteins were transferred to a polyvinylidene fluoride membrane (PVDF) and incubated overnight at 4 °C using the following primary antibodies: anti-MuRF1 (Abcam ab183094, 1:1000), anti-MuRF2 (Myomedix, 1:1600), anti-ubiquitin (linkage-specific K48; Abcam ab140601, 1:1000), anti-LC3 B (Abcam 48394, 1:1000), anti-Nox2 (Abcam ab80508, 1:1000), anti-nitro-tyrosine (Abcam ab42789, 1:500). Membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody and specific bands visualized by enzymatic chemiluminescence and densitometry quantified using a software package, essentially as described previously [ 22 , 23 ]. Measurements were normalized to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1/30000; HyTest Ltd., Turku, Finland).…”

“…We hypothesized that a recently discovered small molecule (ID#704946) from a titin-MuRF1 interaction high-throughput screen, attenuating MuRF1 and MuRF2 protein expression and skeletal muscle atrophy (prominently seen in the tibialis anterior muscle) and muscle dysfunction of the diaphragm muscle in murine cardiac cachexia models [ 22 , 23 ] might also be beneficial to skeletal muscle in CaCax: Skeletal muscles during cancer cachexia also upregulate the MuRF1-UPS (ubiquitin proteasome system) catabolic system [ 1 , 2 , 3 , 4 , 5 ]. For testing MuRF1 inhibitors in a cancer cachexia setting, we injected B16F10 melanoma cells into mouse thighs and monitored melanoma tumor outgrowth until severe cachexia was evident (“Tu mice”).…”

“…In the present study, two distinct inhibitors (MyoMed-946 and MyoMed-205) were fed to Tu mice, and muscle functions were compared between the different groups until sacrifice. MyoMed-946, although structurally identical to ID#704946 used in former studies [ 22 , 23 ], was obtained by a different large scale synthesis method developed by Enamine–Kiev. MyoMed-205 was obtained by a chemical modification where an ester bond in ID#704946 was replaced by an amide bond, in an attempt to enhance resistance to serum esterase.…”