CD36 protein influences myocardial Ca2+ homeostasis and phospholipid metabolism: conduction anomalies in CD36-deficient mice during fasting

Abstract

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca(2+) flux and activation of Ca(2+)-dependent phospholipases A(2) that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca(2+) handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18-22 h) CD36(-/-) and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36(-/-) mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36(-/-) mice documented prolongation of Ca(2+) transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca(2+). Hearts of CD36(-/-) mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36(-/-) hearts. Abnormal Ca(2+) homeostasis in the CD36(-/-) myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A(2) activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca(2+) homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.

FIGURE 1.

Cardiac hypertrophy and impaired metabolic response of working CD36^−/− hearts to a hemodynamic challenge. A, body weight of WT and CD36^−/− mice. B, wet heart weight. C, dry biventricular weight (n = 32). D, mRNA for atrial natriuretic factor (ANF) determined by quantitative real time PCR (n = 5). E, fractional shortening evaluated by transthoracic ECG in conscious fed or fasted mice (n = 8–9). F, working heart preparation showing oxidation of glucose and palmitate in WT and CD36^−/− hearts at low (5 mm Hg) and high (35 mm Hg) workloads (LW and HW, n = 5). Data are means ± S.E. *, p < 0.05.

FIGURE 2.

Effect of fasting on surface ECG parameters in WT and CD36^−/− mice. Mice (n = 7) were fasted overnight and anesthetized to record surface ECGs. A and B, representative records from fasted CD36^−/− mice showing anomalous conduction. A, sinus rhythm with alternating QRS amplitude. B, high degree AV block with premature ventricular complexes (PVCs, in detail). A and B, P indicates P-waves. C, mean ± S.E. HR in fed and fasted mice. D, mean ± S.E. QTc intervals; E, mean ± S.E. PR intervals (see “Experimental Procedures” for explanations of PR interval, QT interval, and QTc interval measurements). The data are representative of three independent experiments (n = 15 per genotype *, p < 0.05).

FIGURE 3.

Changes in ECG parameters during fasting-refeeding. A, time course of changes in ECG parameters obtained from unanesthetized, unrestrained mice (n = 7 WT and 6 CD36^−/−) at base line, during fasting (dashed lines), and refeeding; mean ± S.E. HR, QTc, and PR intervals are plotted (*, p < 0.05). B–E, representative recordings of electrical anomalies observed in unanesthetized CD36^−/− mice during fasting, including altered RR intervals (B and C, two representative tracings), ventricular escape beats (VEB) (D), PVC, and 2nd degree AV block with long sinus pauses (E).

FIGURE 4.

AV-node ERP and ventricular FRP in fasted WT and CD36^−/− hearts. After an overnight fast, isolated hearts from WT or CD36^−/− mice were optically mapped. A, summary of AV-node ERP (n = 10 per genotype). B, representative atrial (teal) and ventricular (blue) optical signals from CD36^−/− hearts. C, representative atrial (gray) and ventricular (black) signals from WT hearts. Left, S2 cycle length (underlined) at which AV-node transmits signal. Right, S2 cycle length (underlined) at which AV-node fails to transmit signal. D, summary of ventricular FRP (n = 10 per genotype). E and F, representative ventricular transmembrane potential with calcium transients in CD36^−/− (E) and WT (F) hearts. RFU, relative fluorescence units; V_m, transmembrane potentials; CaT, calcium transients.

FIGURE 5.

Prolongation of ventricular APD and CaD in fasted WT and CD36^−/− hearts. After an overnight fast, isolated hearts from WT or CD36^−/− mice were optically mapped. A, calculation of ventricular APD at 80% repolarization (APD80) (top) and CaD at 80% relaxation (CaD80) (bottom). B, representative APD80 (top) and CaD80 (bottom) color maps of the posterior ventricular epicardial surface. C, summary of APD80 (top) (n = 10 per genotype) and CaD80 (bottom) (n = 5 per genotype) calculated at pacing cycle lengths of 160, 140, 120, and 100 ms. LA, left atrium; RA, right atrium; RV, right ventricle; LV, left ventricle.

FIGURE 6.

Delayed clearance of cytosolic Ca²⁺ with CD36 knockdown in cardiomyocytes. A, densitometry results from immunoblots verifying knockdown of CD36 in HL-1 cardiomyocytes using two siRNA sequences directed against CD36 (CD36-1 and CD36-2), n = 4 independent determinations of knockdown efficiency; *, p < 0.05. B, representative tracings of caffeine-evoked (4 mm) Ca²⁺ transients from HL-1 cells transfected with GFP or CD36 siRNA and loaded with 5 μm Fluo4-AM. C, half-life of intracellular Ca²⁺ computed using MATLAB curve fitting. CTRL, control. Data are means ± S.E. (n = 59–64 tracings) and representative of three experiments. *, p < 0.05.

FIGURE 7.

Changes in SERCA2a, phospholamban, cyclic AMP levels, and PKA activity in fed and fasted WT and CD36^−/− hearts. A, levels of SERCA2a, SERCA2, phospho(Ser-16) and total PLN proteins from hearts of WT and CD36^−/− fed and fasted mice. B, immunoblot densitometric analysis with samples normalized either to the control protein Ran (Serca2a, total phospholamban) or to total phospholamban (Ser-16(P)-phospholamban). AU, arbitrary units. C, cytosolic cyclic AMP levels from WT and CD36^−/− fed and fasted hearts determined by ELISA. Data are expressed relative to WT fed. D, Western blotting documenting increased PKA activity (PKA-specific phosphorylated substrates RRX(S*/T*)) in CD36^−/− hearts. Data are means ± S.E. *, p < 0.05, n = 3, representative of three experiments.

FIGURE 8.

Remodeling of myocardial phospholipid in CD36^−/− and WT myocardium during fasting. Myocardial lipids from fed or fasted WT and CD36^−/− hearts were analyzed using shotgun lipidomics mass spectrometry. A, lyso-PC; B, lyso-PE; C, ratio of DHA/AA content in PC species. Data are means ± S.E. from two experiments each with n = 5/genotype/condition, *, p < 0.05; **, p < 0.01. D–F, PCA analysis was used to identify lipid species that discriminate the most between the two genotypes in the fed or fasted states. D, score plot showing genotype separation in the fed state and a much wider separation in the fasted state. E, loading bi-plot highlighting increased lysolipid (PC, green and PE, blue) species in CD36^−/− hearts, and F, bi-plot showing increased content of acyl 22:6 containing phospholipids (blue points) in CD36^−/− hearts. The bi-plots display both the loadings and the observations (n = 5/genotype/condition).