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.

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits

“…Like in our study, effects were limited or absent in muscle types that express slow type I fiber types (e.g., soleus), but an increase in muscle size was present in fast type II muscle types (e.g., EDL) [ 56 ]. However, unlike in the present study, EDL muscles from inhibitor-treated animals produced higher forces at both the absolute force and the specific force levels [ 56 ]. A possible explanation could be that the pharmacological targeting of MuRF1 was started in adult animals where the musculature was mature and under those conditions a positive effect on muscle function can be found.…”

“…Finally, it is important to discuss the recent discovery of small-molecule inhibitors that downregulate MuRF1 function by interfering with its recognition of titin A168–170 domains [ 55 , 56 ]. In multiple animal studies of muscle disease (cachexia, myocardial infarction, or heart failure with preserved ejection fraction), these small-molecule compounds were shown to protect muscles from wasting [ 55 , 56 ].…”

“…Finally, it is important to discuss the recent discovery of small-molecule inhibitors that downregulate MuRF1 function by interfering with its recognition of titin A168–170 domains [ 55 , 56 ]. In multiple animal studies of muscle disease (cachexia, myocardial infarction, or heart failure with preserved ejection fraction), these small-molecule compounds were shown to protect muscles from wasting [ 55 , 56 ]. Like in our study, effects were limited or absent in muscle types that express slow type I fiber types (e.g., soleus), but an increase in muscle size was present in fast type II muscle types (e.g., EDL) [ 56 ].…”

“…In multiple animal studies of muscle disease (cachexia, myocardial infarction, or heart failure with preserved ejection fraction), these small-molecule compounds were shown to protect muscles from wasting [ 55 , 56 ]. Like in our study, effects were limited or absent in muscle types that express slow type I fiber types (e.g., soleus), but an increase in muscle size was present in fast type II muscle types (e.g., EDL) [ 56 ]. However, unlike in the present study, EDL muscles from inhibitor-treated animals produced higher forces at both the absolute force and the specific force levels [ 56 ].…”

Protein Quality Control at the Sarcomere: Titin Protection and Turnover and Implications for Disease Development

“…In skeletal muscle, MuRF-1 and 2 are reported to ubiquitinate titin fragments from the M-line-A-band transition zone ( Witt et al, 2005 ; Higashikuse et al, 2019 ). Inhibition of MuRF-1 by small molecules improves diastolic function in heart failure with preserved ejection fraction (HFpEF) and attenuates skeletal muscle wasting in cardiac cachexia ( Bowen et al, 2017 ; Adams et al, 2022 ). Recently, the involvement of MuRF-1, -2, -3, Fbx32 and CHIP in proteasome- and autophagosome dependent titin filament ubiquitination has been demonstrated ( Section 4.1 and Figure 2 ) ( Müller et al, 2021 ).…”