Adjustment of Dysregulated Ceramide Metabolism in a Murine Model of Sepsis-Induced Cardiac Dysfunction

Abstract

Cardiac dysfunction, in particular of the left ventricle, is a common and early event in sepsis, and is strongly associated with an increase in patients' mortality. Acid sphingomyelinase (SMPD1)-the principal regulator for rapid and transient generation of the lipid mediator ceramide-is involved in both the regulation of host response in sepsis as well as in the pathogenesis of chronic heart failure. This study determined the degree and the potential role to which SMPD1 and its modulation affect sepsis-induced cardiomyopathy using both genetically deficient and pharmacologically-treated animals in a polymicrobial sepsis model. As surrogate parameters of sepsis-induced cardiomyopathy, cardiac function, markers of oxidative stress as well as troponin I levels were found to be improved in desipramine-treated animals, desipramine being an inhibitor of ceramide formation. Additionally, ceramide formation in cardiac tissue was dysregulated in SMPD1^+/+ as well as SMPD1^-/- animals, whereas desipramine pretreatment resulted in stable, but increased ceramide content during host response. This was a result of elevated de novo synthesis. Strikingly, desipramine treatment led to significantly improved levels of surrogate markers. Furthermore, similar results in desipramine-pretreated SMPD1^-/- littermates suggest an SMPD1-independent pathway. Finally, a pattern of differentially expressed transcripts important for regulation of apoptosis as well as antioxidative and cytokine response supports the concept that desipramine modulates ceramide formation, resulting in beneficial myocardial effects. We describe a novel, protective role of desipramine during sepsis-induced cardiac dysfunction that controls ceramide content. In addition, it may be possible to modulate cardiac function during host response by pre-conditioning with the Food and Drug Administration (FDA)-approved drug desipramine.

Keywords: acid sphingomyelinase; cardiac dysfunction; ceramide; de novo synthesis; desipramine; sepsis.

Figure 1

Evaluation of desipramine treatment on cardiac function. Transthoracic echocardiography measurements at 6 and 24 h following sepsis (n = 4 per strata/time point). (A) Cardiac output was calculated by stroke volume (B) and heart rate (C). SMPD1^+/+ and SMPD1^−/− animals displayed impaired cardiac output, whereas measurements of desipramine-pretreated (d) strata demonstrated no changes during sepsis; (D) Ejection fraction (EF) was significantly increased in dSMPD1^+/+ and dSMPD1^−/− animals compared to SMPD1^+/+ at 6 h following polymicrobial sepsis; (E) Data of mitral valve E/A (MV E/A) as well as (F) E’ revealed impaired diastolic ventricular function in SMPD1^+/+ animals and SMPD1^−/− animals (p = 0.057) following polymicrobial sepsis, but showed less pronounced altered function in dSMPD1^+/+ as well as in dSMPD1^−/−. * p ≤ 0.05 versus corresponding baseline values; # p ≤ 0.05 versus SMPD1^+/+ at corresponding time points. Abbreviations: p. sepsis = post sepsis.

Figure 2

Reduced oxidative stress in desipramine-pretreated animals. Measurement of reduced glutathione (GSH) and glutathione disulfide (GSSG) as surrogates of oxidative stress in cardiac tissue homogenates (n = 4 animals per strata/time point). (A) Total glutathione and (B) GSH was decreased in all four strata, however changes were less pronounced in dSMPD1^+/+, dSMPD1^−/− and SMPD1^−/− animals compared to SMPD1^+/+ at 24 h following septic insult; (C) GSH/GSSG ratio decreased in SMPD1^+/+ and SMPD1^−/− animals, but remained unchanged in dSMPD1^+/+ and dSMPD1^−/− strata as compared to baseline values following sepsis. * p ≤ 0.05; versus corresponding baseline values; # p ≤ 0.05 versus SMPD1^+/+ at corresponding time points. Abbreviations: p. sepsis = post sepsis.

Figure 3

Desipramine pretreatment improves cardiac integrity in the acute phase of sepsis. Lactate dehydrogenase (LDH) as a global marker of tissue injury and troponin I levels as a cardiomyocyte specific marker in serum samples. (A) LDH was significantly increased in all strata, although less pronounced following desipramine pretreatment (dSMPD1^+/+, dSMPD1^−/−); (B) Troponin I levels increased in SMPD1^+/+ as well as SMPD1^−/− animals (24 h), whereas desipramine-pretreated animals (dSMPD1^+/+) demonstrated less pronounced enhancement as compared to SMPD1^+/+. dSMPD1^−/− animals displayed no change in the values over time, but had significantly increased baseline levels. Data were obtained from n ≥ 4 at baseline and at least n ≥ 6 at 24 h. * p ≤ 0.05; ** p ≤ 0.01 versus corresponding baseline values; # p ≤ 0.05 versus SMPD1^+/+ at the corresponding time points. Abbreviations: p. sepsis = post sepsis.

Figure 4

Dysregulated C16/C18-ceramide metabolism during sepsis. Ceramide specimens were analyzed in cardiac tissue homogenates. Data are obtained from n = 4 per strata/time point. (A) C16-ceramide and (B) C18-ceramide demonstrating increased generation in SMPD1^+/+ and SMPD1^−/− animals, respectively, whereas dSMPD1^+/+ as well as dSMPD1^−/− revealed enhanced levels at baseline, but unchanged values during sepsis. * p ≤ 0.05 versus corresponding baseline values; # p ≤ 0.05 versus SMPD1^+/+ at corresponding time points. Abbreviations: p. sepsis = post sepsis.

Figure 5

Analysis of differentially expressed transcripts from heart tissue homogenates comparing SMPD1^+/+ and desipramine-pretreated SMPD1^+/+ animals and sepsis. The transcriptional expression of 11,620 detected transcripts sampled at 24 h after polymicrobial sepsis were analyzed with the Illumina iScan microarray system. (A) Heatmap of differentially expressed transcripts in heart homogenates is demonstrated comparing SMPD1^+/+ and dSMPD1^+/+ (n = 4 each, columns). Relative expression changes due to sepsis are visualized by color code; (B) Subset of transcripts related to mitochondrial activity are visualized comparing SMPD1^+/+ and dSMPD1^+/+ following sepsis (n = 4 each, columns).

Figure 6

Ceramide metabolism in desipramine-pretreated mice undergoing septic cardiomyopathy. Data of transcripts related to biosynthesis and metabolism of ceramide were extracted from micro array experiments for schematic depiction of gene expression changes comparing mice with polymicrobial sepsis with subsequent cardiac dysfunction. Desipramine pretreatment not only resulted in inhibition of rapid and transient ceramide generation by acid sphingomyelinase Smpd1, but also in blockade of metabolism by ceramidases (Asah1, Asah2, Asah3l), which were all moderately down-regulated. The increase of ceramide levels could also be controlled by an up-regulation of transcripts related to de novo synthesis of ceramide: three ceramide synthases (Cers2, Cers4, Cers5) were found to be up-regulated. Interestingly, the desaturase Degs1 catalyzing the last step in the ceramide biosynthetic pathway and a catalytic subunit of the rate limiting enzyme serine-palmitoyl transferase (Sptlc) were also significantly up-regulated. Development of ceramide overload might also be affected by strong and significant down-regulation of sphingomyelin synthase (Sms). Transcripts encoding enzymes for the metabolism of sphingosine (sphingosine kinases Sphk1/2 as well as sphingosine-1-phosphate phosphatase, Sgpp1) were unaffected. There was also no change in transcript levels of enzymes regulating (de-)phosphorylation of ceramide (Ppp2ca, Lpin). Change of expression level is represented by a color code (blue down-regulation, red up-regulation), significant changes are indicated by an arrow (Abbreviations: C1P: ceramide-1-phosphate).