Diaphragm dysfunction in heart failure is accompanied by increases in neutral sphingomyelinase activity and ceramide content

Abstract

Aims: Chronic heart failure (CHF) causes inspiratory (diaphragm) muscle weakness and fatigue that contributes to dyspnoea and limited physical capacity in patients. However, the mechanisms that lead to diaphragm dysfunction in CHF remain poorly understood. Cytokines and angiotensin II are elevated in CHF and stimulate the activity of the enzyme sphingomyelinase (SMase) and accumulation of its reaction product ceramide. In the diaphragm, SMase or ceramide exposure in vitro causes weakness and fatigue. Thus, elevated SMase activity and ceramide content have been proposed as mediators of diaphragm dysfunction in CHF. In the present study, we tested the hypotheses that diaphragm dysfunction was accompanied by increases in diaphragm SMase activity and ceramide content.

Methods and results: Myocardial infarction was used to induce CHF in rats. We measured diaphragm isometric force, SMase activity by high-performance liquid chromatography, and ceramide subspecies and total ceramide using mass spectrometry. Diaphragm force was depressed and fatigue accelerated by CHF. Diaphragm neutral SMase activity was increased by 20% in CHF, while acid SMase activity was unchanged. We also found that CHF increased the content of C18 -, C20 -, and C24 -ceramide subspecies and total ceramide. Downstream of ceramide degradation, diaphragm sphingosine was unchanged, and sphingosine-1-phosphate level was increased in CHF.

Conclusion: Our major novel finding was that diaphragm dysfunction in CHF rats was accompanied by higher diaphragm neutral SMase activity, which is expected to cause the observed increase in diaphragm ceramide content.

Keywords: Dyspnoea; Force; Myocardial infarction; Skeletal muscle; Sphingolipids.

Figure 1

Diaphragm weakness and fatigue in rats with chronic heart failure (CHF). A) Force-frequency relationship. Specific force, force normalized for bundle cross sectional area. Data are mean ± SE from Sham (n = 10) and CHF (n = 13) rats. Twitch (1 Hz) and maximal tetanic (200 Hz) forces were lower in CHF rats (* P < 0.05, t-test). Dotted and solid lines are line of best fit of mean data using Hill equation. B) Diaphragm fatigue during submaximal tetanic contractions. Forces are normalized within each animal to percentage of peak value for the first contraction during the fatigue protocol. The increase in normalized force seen in Sham after 25 contractions reflect post-tetanic potentiation. Data are mean ± SE for Sham (n = 5) and CHF rats (n = 8). * P < 0.05 for Sham vs. CHF (repeated measures ANOVA and Tukey's post-hoc test). C) Diaphragm fibre type composition based on myosin heavy chain (MHC) isoforms determined by SDS-PAGE. Images are representative profile of MHC in gels. Data are mean ± SE from n = 5 animals per group.

Figure 2

Heart failure increases diaphragm sphingomyelinase (SMase) activity. A-SMase, acid sphingomyelinase; N-SMase, neutral sphingomyelinase. A) Measurements of enzyme activity were made in whole-tissue homogenates. Data are shown in nmol ceramide/mg protein/hour for Sham (n = 7) and CHF (n = 10) rats. All values are expressed as mean ± SE. *P < 0.05 by t-test.

Figure 3

Heart failure increases diaphragm ceramide content. A) Content of each ceramide subspecies measured in rat diaphragm. Inset, data from select ceramide subspecies shown in different scale for clarity of visualization. Open bars, Sham (n = 16); closed bars, CHF (n = 21). B) Total ceramide content calculated as the sum of individual subspecies shown in panel A. * P < 0.05 by ANOVA and Bonferroni post-hoc test for planned comparison (Panel A) and t-test (Panel B) C) Sphingosine content. D) Sphingosine-1-phosphate content. Data from one Sham animal (2.28 pmole/mg protein) was identified as an outlier by Grubb's test and was not included in graph and statistical analysis shown above. * P < 0.005 by Mann-Whitney test. In healthy skeletal muscle, S1P promotes growth and regeneration (34, 35). In CHF, elevation of S1P in vascular smooth muscle causes vasoconstriction (36).

Figure 4

Diaphragm expression of select sphingolipid metabolizing enzymes. A) Relative mRNA abundance of sphingomyelinases, acid (A-SMase) and neutral (N-SMase) isoforms, sphingosine kinase 1 (SK1), sphingosine-1-phosphaste (S1P) lyase, and S1P phosphatase 1 (SPP1). Data are from n = 5 rats per group. B) Protein abundance of SK1 is diminished in CHF diaphragm. Data are normalized to α-tubulin (loading control) and expressed as mean ± SE. * P < 0.05 by t-test