Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients

Abstract

Background: Activation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease-related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients.

Methods: The effects of SMase on force production and intracellular Ca²⁺ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III-IV, ejection fraction 25 ± 1.0%, peak VO₂ 14.4 ± 0.6 mL × kg × min) and healthy age-matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO₂ 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure.

Results: Sphingomyelinase reduced muscle fibre force production (-30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca²⁺ release (P < 0.05) and reducing myofibrillar Ca²⁺ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up-regulated and down-regulated, respectively, at a FDR of 1%. Gene-set enrichment analysis identified 'proteasome degradation' as an up-regulated pathway (average fold-change 1.1, P = 0.008), while the pathway 'cytoplasmic ribosomal proteins' (average fold-change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold-change 0.8, P = 0.0002) where identified as down-regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age-matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF-receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow-up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease.

Conclusions: The present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure-related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease-related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Keywords: Ca2+ sensitivity RNAseq; Heart failure; Skeletal muscle; Sphingomyelinas.

Figure 1

SMase depresses force and tetanic [Ca²⁺]_i i in single intact fibres. (A) Representative [Ca²⁺]_i and force records during 20 Hz contractions in a single intact human intercostal muscle fibre exposed to SMase for up to 20 min. (B) Mean data of [Ca²⁺]_i and force as a percentage of control in single mouse toe muscle fibres exposed to SMase. Data are means ± SEM. Significant effects (P < 0.05): a = interaction; b = time; c = treatment, 40 or 100 Hz control versus 40 or 100 Hz SMase, respectively, with two‐way RM ANOVA.

Figure 2

SMase‐induced force depression is due to the combined effect of a reduction in SR Ca²⁺ release and myofibrillar Ca²⁺ sensitivity. (A,B) Representative [Ca²⁺]_i and force records in a single intact human intercostal and mouse toe muscle fibre, respectively, before and after exposure to SMase. (C,D) In human intercostal (n = 3) and mouse toe muscle (n = 8) fibres, SMase caused a shift of the mean force‐[Ca²⁺]_i values to the right of the control force‐[Ca²⁺]_i curve (i.e. an apparent decrease in myofibrillar Ca²⁺ sensitivity), and a shift downward to the left compared with frequency‐matched control force‐[Ca²⁺]_i values (i.e. an apparent decrease in tetanic force and [Ca²⁺]_i). (D) Increased SR Ca²⁺ release following application of caffeine fully offsets the SMase‐induced force depression in mouse toe muscle fibres. The hill plot in (D) was generated from the P _max, N and Ca₅₀ mean values of all mouse toe muscle fibres analysed (n = 8).

Figure 3

SMase activity induces the transcription of factors promoting protein degradation while transcription of ribosomal proteins is supressed. Volcano‐plot showing SMase‐induced changes in gene expression in isolated human muscle fibres with 471 differentially expressed genes (180 up‐regulated and 291 down‐regulated FDR <1%). Several genes of importance for muscle fibre structural integrity such as the transcription factor SP1 and eukaryotic translation initiation factor 4B, but also transcripts involved in calcium handling such as ATP2A2 were down‐regulated. Up‐regulated genes included transcripts associated with denervation (FRAT2, RRAD) and fibre‐type switching (TNNC2). (C) On the pathway level, the most notable up‐regulated pathway was proteasome degradation (P = 0.008) whereas cytoplasmic ribosomal proteins was down‐regulated (P < 0.0001). Transcripts belonging to these pathways are highlighted in red and blue on the volcano. The barcode plots illustrate the changes in expression of the members of these two pathways where most of all transcripts involved in ‘proteasome degradation’ and ‘cytoplasmic ribosomal proteins’ were up‐and down‐regulated respectively following SMase treatment.

Figure 4

Skeletal muscle SMase activity is elevated in human heart failure patients and the activity of SMase increases with disease duration. (A,B) Skeletal muscle nSMase and aSMase activity, respectively, in human heart failure patients (n = 61) and healthy age‐matched controls (n = 10). (C,D) Skeletal muscle nSMase and aSMase activity, respectively, in human heart failure patients at the time of the 1st biopsy (time of inclusion in the study) and at the 2nd biopsy (follow‐up). Mean follow‐up time 2.5 (±0.2) years, n = 16. Bars indicate mean value and circles represent individual data points. *P < 0.05, age‐matched control subjects versus heart failure patients with unpaired t‐test; 1st versus 2nd biopsy with paired t‐test.

Figure 5

Skeletal muscle SMase activity correlates to circulatory markers of inflammation and muscle atrophy. Correlation of skeletal muscle nSMase activity and serum protein quantities of factors analysed with targeted proteomics in human heart failure patients (n = 61). The figure depicts correlation coefficients of plasma proteins significantly (P < 0.05) correlated with skeletal muscle SMase activity. Several factors associated with systemic inflammation including TNF receptor 1 and 2 (TNF‐R1 and TNF‐R2) were significantly associated with skeletal muscle SMase activity in patients with heart failure.