A PKB-SPEG signaling nexus links insulin resistance with diabetic cardiomyopathy by regulating calcium homeostasis

Abstract

Diabetic cardiomyopathy is a progressive disease in diabetic patients, and myocardial insulin resistance contributes to its pathogenesis through incompletely-defined mechanisms. Striated muscle preferentially expressed protein kinase (SPEG) has two kinase-domains and is a critical cardiac regulator. Here we show that SPEG is phosphorylated on Ser²⁴⁶¹/Ser²⁴⁶²/Thr²⁴⁶³ by protein kinase B (PKB) in response to insulin. PKB-mediated phosphorylation of SPEG activates its second kinase-domain, which in turn phosphorylates sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (SERCA2a) and accelerates calcium re-uptake into the SR. Cardiac-specific deletion of PKBα/β or a high fat diet inhibits insulin-induced phosphorylation of SPEG and SERCA2a, prolongs SR re-uptake of calcium, and impairs cardiac function. Mice bearing a Speg^3A mutation to prevent its phosphorylation by PKB display cardiac dysfunction. Importantly, the Speg^3A mutation impairs SERCA2a phosphorylation and calcium re-uptake into the SR. Collectively, these data demonstrate that insulin resistance impairs this PKB-SPEG-SERCA2a signal axis, which contributes to the development of diabetic cardiomyopathy.

Fig. 1. Effects of PKBα/β deletion in the heart on cardiac function and calcium transients in primary cardiomyocytes.

a PKBα, β and γ protein expression in the heart of male PKBα^f/f;MCM;PKBβ^−/− mice at 8 weeks after tamoxifen induction. b–e Ejection fraction (EF) (b), fractional shortening (FS) (c), systolic left ventricular volume (LV Vol;s) (d), and diastolic left ventricular volume (LV Vol;d) (e) were measured via echocardiography in the male PKBα^f/f; PKBβ^−/− and PKBα^f/f;MCM;PKBβ^−/− mice before and after tamoxifen induction. n = 14 (0 week), 12 (2 week), 12 (4 week), and 12 (6 week) for PKBα^f/f;PKBβ^−/− mice. n = 12 (0 week), 9 (2 week), 8 (4 week), and 7 (6 week) for PKBα^f/f;MCM;PKBβ^−/− mice. p = 0.147 (0 week), 1.60e-7 (2 week), 2.15e-8 (4 week) and 5.33e-10 (6 week) for EF. p = 0.165 (0 week), 8.56e-8 (2 week), 1.16e-8 (4 week), and 3.03e-10 (6 week) for FS. p = 0.229 (0 week), 1.05e-6 (2 week), 9.84e-7 (4 week), and 6.36e-8 (6 week) for LV Vol;s. p = 0.387 (0 week), 9.36e-5 (2 week), 2.70e-5 (4 week), and 1.23e-6 (6 week) for LV Vol;d. f Calcium transients elicited by electrical stimulation in primary cardiomyocytes isolated from the male PKBα^f/f;PKBβ^−/− and PKBα^f/f;MCM;PKBβ^−/− mice at 4 weeks after tamoxifen induction. Quantitation of amplitude, full duration at half maximum (FDHM) and time constant Tau of calcium transients was shown. 76 cells from 3 PKBα^f/f;PKBβ^−/− mice and 34 cells from 2 PKBα^f/f;MCM;PKBβ^−/− mice were analyzed. p = 0.446 (amplitude), 5.96e-5 (FDHM), and 3.99e-3 (tau). The data are given as the mean ± SEM. Statistical analyses for b–f were carried out using two-sided t-test. Two-asterisk indicates p < 0.01, and three-asterisk indicates p < 0.001. Source data are provided as a Source Data file.

Fig. 2. Identification of SPEG as a phosphoprotein regulated by the insulin–PKB pathway.

a PAS-reactive protein phosphorylation in lysates of hearts from mice that were intraperitoneally injected with or without insulin. b Immunoprecipitation of potential PKB substrates from lysates of mouse hearts stimulated with or without insulin using the PAS antibody. Immunoprecipitated proteins were identified via mass-spectrometry and summarized in Supplementary Data 1. c Immunoprecipitation of known PKB substrates from lysates of WT and PKBα/β KO (PKBα^f/f;αMHC-Cre;PKBβ^−/−) mouse hearts stimulated with or without insulin using the PAS antibody. RalGAPα1, RalGAPα2, AS160, and TSC2 in the PAS immunoprecipitates were detected with the corresponding antibodies via western blot. d Immunoprecipitation of phosphorylated SPEG from lysates of WT and PKBα/β KO (PKBα^f/f;αMHC-Cre;PKBβ^−/−) mouse hearts stimulated with or without insulin using the PAS antibody. SPEG in the PAS immunoprecipitates and heart lysates was detected via western blot. e, f. Full-length SPEG was tagged with GFP and expressed in HEK293 cells that were treated with the PI 3-kinase inhibitor PI-103 and PKB inhibitor Akti1/2 in the presence or absence of insulin. PAS-reactive phosphorylation of SPEG was detected on immunoprecipitated SPEG using the PAS antibody. e representative blots. f Quantitation of PAS-reactive phosphorylation of GFP-SPEG. n = 3. The data are given as the mean ± SEM. Statistical analysis was carried out using one-way ANOVA. One-asterisk indicates p < 0.05, and three-asterisk indicates p < 0.001. Source data are provided as a Source Data file.

Fig. 3. Identification of PKB-mediated phosphorylation sites on SPEG.

a–c SPEG fragments were expressed in HEK293 cells that were treated with indicated inhibitors and insulin. PAS-reactive phosphorylation of SPEG was detected on immunoprecipitated SPEG using the PAS antibody. d, e GFP-SPEG^P2227-S2583 WT and mutant proteins were expressed in HEK293 cells. PAS-reactive phosphorylation of SPEG was detected on immunoprecipitated proteins using the PAS antibody. f GFP-SPEG WT and mutant proteins were expressed in HEK293 cells. PAS-reactive phosphorylation of SPEG was detected on immunoprecipitated proteins using the PAS antibody. g In vitro phosphorylation of GST-SPEG^P2227-S2583 WT and mutant recombinant proteins by PKB. Source data are provided as a Source Data file.

Fig. 4. Effects of PKB-mediated phosphorylation of SPEG on the activity of its second kinase-domain.

a, b Flag-SERCA2a was co-expressed with GFP-SPEG WT or mutant proteins in HEK293 cells. After immunoprecipitation, phosphorylation of Flag-SERCA2a was detected using the pSer/Thr antibody. a representative blots. b Quantitation of SERCA2a phosphorylation. n = 8 (GFP and SPEG^3A) and 7 (SPEG and SPEG^D3098A). c, d Flag-SERCA2a was co-expressed with GFP-SPEG WT or mutant proteins in HEK293 cells. Oligomerization of SERCA2a was determined via western blot and subsequently quantified. Representative blots were shown in c and quantitative data shown in d. n = 6. E-F. CFP-SERCA2a and YFP-SERCA2a were co-expressed with HA-SPEG WT or mutant proteins in HEK293 cells. Inter-molecular interaction of SERCA2a was measured via FRET. E, quantitative data on FRET efficiency. f representative images for FRET. n = 28 (vector), 30 (SPEG), 35 (SPEG^D3098A), and 29 (SPEG^3A). Bars indicate 5 μm in length. g, h HA-SERCA2a was co-expressed with Flag-SERCA2a in the presence of GFP-SPEG WT and mutant proteins in HEK293 cells. Flag-SERCA2a was immunoprecipitated, and the abundance of HA-SERCA2a in the immunoprecipitates was detected via immunoblotting. g representative blots. h Quantitative data. n = 11 (GFP), 10 (SPEG), and 9 (SPEG^D3098A and SPEG^3A). i, j Calcium transients in HEK293 cells expressing mCherry-SERCA2a together with HA-SPEG WT or mutant proteins. Calcium transients were recorded using a confocal microscopy in cells that were stimulated with ATP. Full duration at half maximum (FDHM, i), and time constant Tau (j) of calcium transients were subsequently determined. n = 68 (SERCA2a WT + SPEG WT), 43 (SERCA2a WT + SPEG^D3098A), 64 (SPEG + SERCA2a^T484A), and 20 (SERCA2a WT + SPEG^3A). k Calcium transients in neonatal rat cardiomyocytes expressing vector, mCherry-SPEG WT or mCherry-SPEG^3A mutant proteins upon field stimulation. Amplitudes, FDHM and Tau of calcium transients were quantified from 30 (vector), 12 (mCherry-SPEG WT) or 18 (mCherry-SPEG^3A) cells. The data are given as the mean ± SEM. Statistical analyses were carried out using one-way ANOVA. One-asterisk indicates p < 0.05, two-asterisk indicates p < 0.01, and three-asterisk indicates p < 0.001. Source data are provided as a Source Data file.

Fig. 5. Phosphorylation of PKB-SPEG–SERCA2a axis in insulin-resistant cardiomyocytes.

a, b Phosphorylation of PKB, SPEG, and SERCA2a in WT or PKBα/β-KO primary cardiomyocytes stimulated with or without insulin. Phosphorylation of PKB, SPEG and SERCA2a was normalized with their respective total proteins, and quantitative data were shown in b. n = 3. c, d. Phosphorylation of PKB, SPEG and SERCA2a in insulin-sensitive or resistant H9C2 cardiomyocytes stimulated with or without insulin. H9C2 cardiomyocytes were differentiated for 7 days before insulin resistance was induced by prolonged treatment with 300 nM insulin for 24 h. Cells were then stimulated with or without insulin for 30 min. Representative blots were shown in c. Phosphorylation of PKB, SPEG and SERCA2a was normalized with their respective total proteins, and quantitative data were shown in d. n = 6. IS insulin sensitive, IR insulin-resistant. One-dagger (IS insulin vs IR insulin) indicates p < 0.01. One-diesis (IS basal vs IR basal) indicates p < 0.001. The data are given as the mean ± SEM. Statistical analyses were carried out using two-way ANOVA. One-asterisk indicates p < 0.05, two-asterisk indicates p < 0.01, and three-asterisk indicates p < 0.001. Source data are provided as a Source Data file.

Fig. 6. Effects of HFD on calcium homeostasis and phosphorylation of PKB-SPEG-SERCA2a axis in primary cardiomyocytes.

a, b. Ejection fraction (EF, a) and fractional shortening (FS, b) in male mice fed with chow diet (CD) or high fat diet (HFD) for 3 months. n = 6 (CD) and 5 (HFD). p = 1.29e-3 (EF) and 7.05e-4 (FS). c Calcium transients in primary cardiomyocytes isolated from male mice fed with CD or HFD upon electrical stimulation. 64 cells from CD-fed mice and 42 cells from HFD-fed mice were analyzed. p = 1.24e-2 (amplitude), 1.79e-7 (FDHM), and 2.52e-4 (Tau). D-E. Phosphorylation of PKB, SPEG and SERCA2a in primary cardiomyocytes isolated from CD- or HFD-fed mice in response to insulin. Phosphorylation of PKB, SPEG and SERCA2a was normalized with their respective total proteins. Represent blots were shown in d, and quantitative data were shown in e. n = 9. The data are given as the mean ± SEM. Statistical analyses were carried out using two-sided t-test for a–c, and two-way ANOVA for e. One-asterisk indicates p < 0.05, two-asterisk indicates p < 0.01, and three-asterisk indicates p < 0.001. One-dagger (CD insulin vs HFD insulin) indicates p < 0.001. Source data are provided as a Source Data file.

Fig. 7. Generation and basic characterization of the Speg^3A-knockin mice.

a Diagram of strategy for generation of the Speg^3A-knockin mice. The cluster of serine/threonine residues Ser²⁴⁶¹/Ser²⁴⁶²/Thr²⁴⁶³ (the surrounding sequence is LAVRRRLsstLERL, Ser²⁴⁶¹/Ser²⁴⁶²/Thr²⁴⁶³ shown in lower case, and numbering is according to NP_031489.4) on SPEG was substituted to alanine by point mutagenesis. b Oral glucose tolerance test in the male Speg^3A-knockin mice and wild-type littermates (9-month-old). n = 5 (WT) and 6 (Speg^3A). p = 0.050 (0 min), 0.799 (15 min), 0.996 (30 min), 0.718 (45 min), 0.244 (60 min), and 0.134 (120 min). c Levels of glucose, non-esterified fatty acids (NEFA), triglycerides (TG) and total cholesterol (TC) were determined in the blood of male Speg^3A-knockin mice and wild-type littermates (8-month-old) after an overnight fast. n = 15 (WT) and 11 (Speg^3A). p = 0.270 (blood glucose), 0.317 (plasma NEFA), 0.819 (plasma TG), and 0.412 (plasma TC). d SPEG expression and PAS-reactive phosphorylation in the heart of WT and Speg^3A-knockin mice in response to insulin. The data are given as the mean ± SEM. Statistical analyses were carried out using two-sided t-test. Source data are provided as a Source Data file.

Fig. 8. Calcium homeostasis in Speg^3A-knockin cardiomyocytes.

a, b Phosphorylation of PKB, SPEG, and SERCA2a in WT or Speg^3A primary cardiomyocytes stimulated with or without insulin. Phosphorylation of PKB, SPEG and SERCA2a was normalized with their respective total proteins, and quantitative data were shown in b. n = 6. One-dagger (WT insulin vs Speg^3A insulin) indicates p < 0.001. c, d Thr⁴⁸⁴ phosphorylation of SERCA2a in the heart of Speg^3A mice and WT littermates (7-month-old). c representative blots. d quantitation of SERCA2a-Thr⁴⁸⁴ phosphorylation. n = 10 (WT) and 8 (Speg^3A). p = 2.81e-3. e, f. Oligomerization of SERCA2a in the heart of Speg^3A mice and WT littermates (7-month-old). e representative blots. f quantitation data. n = 12. p = 1.97e-2. G-H. SERCA2a Ca²⁺-transporting activity (n = 15 (WT) and 14 (Speg^3A), g) and ATPase activity (n = 4, h) in microsomes isolated from the heart of WT and Speg^3A-knockin mice. p = 1.98e-6 (SR calcium uptake) and 0.198 (ATPase activity). i, j Calcium transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and Speg^3A-knockin mice (3-month-old). i Representative calcium transient images and curves. j Quantitation of amplitudes, full duration at half maximum (FDHM) and time constant Tau of calcium transients. 75 cells from 7 WT mice and 68 cells from 7 Speg^3A-knockin mice were analyzed. p = 0.603 (amplitude), 1.14e-6 (FDHM), and 8.68e-7 (Tau). k Spontaneous calcium sparks in primary cardiomyocytes isolated from the WT and Speg^3A-knockin mice (2-month-old). Frequency, amplitudes, full-width at half maximum (FWHM), full duration at half maximum (FDHM) and time constant Tau of calcium sparks. 168 sparks from 35 cells of four WT mice and 194 sparks from 41 cells of four Speg^3A-knockin mice were analyzed. p = 0.839 (frequency), 0.800 (amplitude), 0.171 (FWHM), 3.27e-4 (FDHM), and 5.71e-4 (Tau). The data are given as the mean ± SEM. Statistical analyses were carried out using two-way ANOVA for b, and two-sided t-test for d, f–h, and j, k. One-asterisk indicates p < 0.05, two-asterisk indicates p < 0.01, and three-asterisk indicates p < 0.001. Source data are provided as a Source Data file.

Fig. 9. Cardiac function of the Speg^3A-knockin mice.

a Echocardiography was performed on the anaesthetized male Speg^3A-knockin mice and wild-type littermates at age of 2 and 5 months to measure EF, FS, LV Vol;s, LV Vol;d, LVAW;s, LVAW;d, LVPW;s, and LVPW;d. n = 15 (WT, 2 M), 17 (WT, 5 M), 9 (Speg^3A, 2 M), and 12 (Speg^3A, 5 M). The data are given as the mean ± SEM. Statistical analyses were carried out using two-way ANOVA. One-asterisk (WT vs Speg^3A) and one-dagger (Speg^3A 2 M vs Speg^3A 5 M) indicate p < 0.05. Two-asterisk (WT vs Speg^3A), two-dagger (Speg^3A 2 M vs Speg^3A 5 M), and two-diesis (WT 2 M vs WT 5 M) indicate p < 0.01. Three-asterisk (WT vs Speg^3A), three-dagger (Speg^3A 2 M vs Speg^3A 5 M), and three-diesis (WT 2 M vs WT 5 M) indicate p < 0.001. b A diagram represents the proposed model in which the PKB−SPEG signaling nexus links insulin signaling with calcium homeostasis in cardiomyocytes to maintain cardiac function. Upon insulin stimulation, PKB phosphorylates SPEG and activates its second kinase domain, which consequently phosphorylates SERCA2a. Phosphorylation of SERCA2a increases its dimerization that enhances its Ca²⁺-transporting activity. Impaired SPEG phosphorylation by PKB links insulin resistance with cardiac dysfunction through SERCA2a-mediated Ca²⁺ reuptake into the SR in cardiomyocytes independent of metabolic assaults. Source data are provided as a Source Data file.