SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy

Abstract

Background: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3).

Methods: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3^KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3^KO mice subjected to permanent nonreperfused myocardial infarction.

Results: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity.

Conclusions: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Keywords: cardiomyopathy; electron transport complex I; mitochondria; serine C-palmitoyltransferase; sphingolipids.

Figure 1:. Induction of SPTLC3 and SPTLC3-derived sphingolipids in ICM.

A. Western blot of human control (n = 3) and ICM (n = 9) cardiac tissue for SPT subunits SPTLC1, SPTLC2 and SPTLC3. ICM = ischemic cardiomyopathy. B. Schematic representation of permanent LAD ligation surgeries in mice. Western blot of heart homogenates from C57BL6/J sham (n = 4) and LAD-ligated (n = 4) mice 28 days post-op for SPT subunits SPTLC1, SPTLC2 and SPTLC3, the panel to the right is densitometry of the immunoblots using ImageJ. LAD = left anterior descending (coronary artery). [Image created with BioRender.com and published with permission.] C. Representative 1.04x (scale = 800 μm) magnified immunofluorescent images of heart sections from sham (n = 4) and LAD (n = 4) surgeries 28 days post-op for staining of nuclei (DAPI, blue), cardiac actin (green, 488) and SPTLC3 (red, 594). Inset areas at 63x (scale bar = 10 μm) magnification show the presence of SPTLC3 puncta in cardiac actin and near nuclei of infarcted areas in the LAD-ligated hearts. D. Schematic representation of the de novo synthesis pathways of canonical and non-canonical sphingolipids. SPT = serine palmitoyltransferase, DHS = dihydrosphingosine, DHS1P = dihydrosphingosine-1-phosphate, DHC = dihydroceramide, SM = sphingomyelin, Sph = sphingosine, S1P = sphingosine-1-phosphate, MHC = monohexosylceramide, LacCer = lactosylceramide. [Image created with BioRender.com and published with permission.] E. Targeted d16 and d18 lipidomics analysis of control (n = 4) and ICM (n = 8) heart tissue. F. Targeted d16 and d18 lipidomics analysis of sham (n = 7) and LAD-ligated (n = 7) heart tissue. Data are shown as mean ± SEM. Statistical tests used - B. densitometry is multiple unpaired t-tests with FDR-adjusted p-values shown. E., and F. unpaired Student’s t-test. *P<0.05, **P<0.01, ***P<0.001. Source data are provided as a Source Data file.

Figure 2:. HIF1α directly binds the SPTLC3 promoter under ischemic conditions.

A. mRNA expression of the SPT subunits SPTLC1, SPTLC2, and SPTLC3 in control (n = 4) and ICM (n = 8) heart tissue. Relative expression was determined by normalization to the geomean of mRNA expression of 18S RNA and Cyclophilin A. B. mRNA expression of the SPT subunits Sptlc1, Sptlc2, and Sptlc3 in sham (n = 7) and LAD-ligated (n = 7) heart tissue. Relative expression was determined by normalization to the geomean of mRNA expression of 18S RNA and Hprt. C. Schematic showing the three potential hypoxia response element (HRE) motifs within 2 kb upstream of the TSS of the SPTLC3 gene. TSS = transcription start site. D. Schematic showing in vitro simulated ischemia in AC16 cardiomyocytes. [Image created with BioRender.com and published with permission.] E. Western blot of AC16 cardiomyocytes treated with control siRNA (n = 3) or an HIF1α siRNA (n = 3) under normoxic or simulated ischemic conditions. (All samples were run on the same gel; non-adjacent samples are separated). F. ChIP-qPCR of HIF1α under normoxic and simulated ischemic conditions around the SPTLC3 gene HRE sites (n = 3–4/group). Data are shown as mean ± SEM. Statistical tests used - A., and B. multiple unpaired t-tests with FDR-adjusted p-values shown. F. Two-way ANOVA with Tukey’s multiple comparisons test with adjusted p-values shown. *P<0.05, ***P<0.001, ****P<0.0001. Source data are provided as a Source Data file

Figure 3:. cSPTLC3^KO mice are partially protected from non-reperfused myocardial infarction.

A. Schematic showing generation of the cSPTLC3^KO mouse by crossing the αMyHC-Cre and SPTLC3 homozygous flox mouse to excise exon 8 of the Sptlc3 gene containing the catalytic PLP region. PLP = pyridoxal 5-phosphate. [Image created with BioRender.com and published with permission.] B. Ejection fraction echocardiography analysis at baseline prior to surgery and days one, 7, 14, 21, and 28 after surgery in SPTLC3^fl/fl sham (n = 12), cSPTLC3^KO sham (n = 12), SPTLC3^fl/fl LAD (n = 7), and cSPTLC3^KO LAD-operated (n = 10) mice. C. Fractional shortening echocardiography analysis at baseline prior to surgery and days one, 7, 14, 21, and 28 after surgery in SPTLC3^fl/fl sham (n = 12), cSPTLC3^KO sham (n = 12), SPTLC3^fl/fl LAD (n = 7), and cSPTLC3^KO LAD-operated (n = 10) mice. D. Kaplan-Meier survival analysis with log-rank (Mantel-Cox) test of the SPTLC3^fl/fl and cSPTLC3^KO mice that underwent sham and LAD surgeries. E. Representative 1.04x (scale bar = 800 μm) magnified images of 28 day post-LAD ligated hearts stained with Picrosirius red and Fast Green FCF from SPTLC3^fl/fl (n = 3) and cSPTLC3^KO (n = 4) mice. LV = left ventricle. RV = right ventricle. F. Quantification of percent LV fibrotic area from Picrosirius red-stained and Fast Green FCF-stained 28 day post-LAD ligated hearts. G. Representative 1.04x (scale bar = 800 μm) magnified images of 28 day post-LAD ligated hearts stained with hematoxylin and eosin (H&E) from SPTLC3^fl/fl (n = 4) and cSPTLC3^KO (n = 4) mice. Inset areas at 40x (scale bar = 20 μm) magnification show cardiomyocytes in the infarcted regions of the myocardium. H. Quantification of cross-sectional area of cardiomyocytes from H&E-stained 40x magnified images of 28 day post-LAD ligated hearts. I. Changes in metabolites in hearts of SPTLC3^fl/fl (n = 5) and cSPTLC3^KO (n = 6) mice 24-hours post-LAD ligation portrayed in the glycolysis, ketogenesis, and TCA pathways. J. Overview of the top 25 enriched metabolite sets of 96 metabolites assayed in hearts of SPTLC3^fl/fl (n = 5) and cSPTLC3^KO (n = 6) mice 24-hours post-LAD ligation. Data are shown as mean ± SEM. Statistical tests used - B., and C. multiple unpaired t-tests with FDR-adjusted p-values shown. D. Log-rank (Mantel-Cox) test for SPTLC3^fl/fl sham vs. cSPTLC3^KO sham, SPTLC3^fl/fl LAD vs. cSPTLC3^KO LAD, SPTLC3^fl/fl sham vs. SPTLC3^fl/fl LAD, and cSPTLC3^KO sham vs. cSPTLC3^KO LAD. F., and I. unpaired Student’s t-test. H. Linear mixed-effects statistical modeling (LMEM). *P<0.05, **P<0.01, ***P<0.0001. Source data are provided as a Source Data file.

Figure 4:. Gene expression changes in hearts from cSPTLC3^KO mice are consistent with perturbation of mitochondrial function.

All panels are a comparison between SPTLC3^fl/fl and cSPTLC3^KO mice. A. Volcano plot of differentially expressed genes in the cSPTLC3^KO hearts compared to SPTLC3^fl/fl hearts (n = 3/group). The cut-off values are −log₁₀ p value of 1.3 (p≤0.05), and log₂fold change less than −0.5 (blue dots) or greater than 0.5 (red dots). B. Gene ontology enrichment analysis performed on differentially expressed genes ranked by log₂fold change. C. Representative TEM images of SPTLC3^fl/fl and cSPTLC3^KO cardiomyocytes and mitochondria at 6,000x (scale bar = 2 μm) magnification and of mitochondria at 40,000x (scale bar = 0.2 μm) magnification in hearts (n = 3/group). TEM = transmission electron microscopy. D. Quantification of mitochondrial area (n = 130–180 images/mouse) and cristae density (n = 5–10 images/mouse) from TEM images. E. qPCR of isolated DNA to determine mitochondrial copy number in SPTLC3^fl/fl and cSPTLC3^KO hearts (n = 6/group). Copy number was determined by the ratio of expression of mitochondrial gene mt-co2 to expression of nuclear gene 18s RNA. F. Western blot of isolated mitochondria from SPTLC3^fl/fl (n = 3) and cSPTLC3^KO (n = 3) mice for mitofission protein DRP1, phosphorylated (serine 616) DRP1, and the mitofusion proteins MFN2, and OPA1. G. Densitometry analysis of the immunoblots using ImageJ. H. Flow cytometry analysis of isolated heart mitochondria from SPTLC3^fl/fl (n = 3) and cSPTLC3^KO (n = 3) mice stained with TMRM for mitochondrial membrane potential (ΔΨm). I. Quantification of the percentage of mitochondria with low and high ΔΨm. Data are shown as mean ± SEM. Statistical tests used - D. Linear mixed-effects statistical modeling (LMEM). E. unpaired Student’s t-test. G., and I. Two-way ANOVA using Holm-Šídák’s multiple comparisons test with adjusted p-values shown. *P<0.05, **P<0.01, ***P<0.005. Source data are provided as a Source Data file.

Figure 5:. Complex I of the electron transport chain is inactivated in the absence of SPTLC3.

SSM and IFM mitochondria were isolated from SPTLC3^fl/fl and cSPTLC3^KO mice for panels A. to C. SSM = subsarcolemmal mitochondria, IFM = interfibrillar mitochondria. A. Measurement of CI, II, and IV OCRs using Oroboros (n = 3/group), normalized by nmol/minute to mg protein. CI = Complex I. CII = Complex II. CIV = Complex IV. OCR = oxygen consumption rate. B. Measurement of NADH-ubiquinone oxidoreductase activity (n = 3/group), normalized by enzyme activity mU to mg protein. mU = milliunit. C. The panels to the left are measurements of response to specific CI or II substrates of OCRs using Seahorse (n = 2 biological replicates with 5 technical replicates/group), normalized by O₂ pmol/minute/μg protein and cell count. The panels to the right are a quantification of the OCR rotenone-sensitive values for CI, and II (OCR_NADH or OCR_Pyr/Mal – OCR_ROT/AA), and azide-sensitive values for CIV (OCR_TMPD/Asc – OCR_Azide). AC16 cardiomyocytes were treated with control, SPTLC2, and SPTLC3 siRNA constructs for panels D. to F. D. The top panel is total NADH to NAD⁺ ratio. Values were normalized to cell count (n = 7/group) for graphing of the NADH to NAD⁺ ratio. The bottom panel is total ATP calculated and normalized to cell count (n = 4/group). E. Representative 20x (scale bar = 50 μm) magnified images of immunofluorescent staining for nuclei (Hoechst, blue), cellular ROS (far red CellROX^™, red) and merge (n = 3/group). H₂O₂ treatment was used as a positive control. F. The top panels are measurements of glycolytic rate for OCR, ECAR, and PER using Seahorse (n = 2 biological replicates with 4–5 technical replicates/group), normalized by pmol/minute/100 cells. The bottom panels are the quantification of the graph values. ECAR = extracellular acidification rate. PER = proton efflux rate. G. CI pulldown followed by targeted d16- and d18-sphingolipids for the bound CI fraction in mitochondria isolated from SPTLC3^fl/fl and cSTPCL3^KO mice (n = 3/group), normalized to pmol/mg protein. Data are shown as mean ± SEM. Statistical tests used - A. Two-way ANOVA using Tukey’s multiple comparisons test with adjusted p-values shown. B., D., and F. Ordinary one-way ANOVA using Tukey’s multiple comparisons test with adjusted p-values shown. C., and G. Two-way ANOVA using Holm-Šídák’s multiple comparisons test with adjusted p-values shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Source data are provided as a Source Data file.

Figure 6:. Summary figure.

[Image created with BioRender.com and published with permission.]