Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart

Figure 1. In vivo cardiac function, cell size, and gene expression of hypertrophy markers in VEGF-B mice. (A) In vivo echocardiography data of left ventricle (LV): ejection fraction (LV EF%, upper left), diastolic anterior wall thickness (LVAW;d, upper right), diastolic volume (LV Vol;d, lower left), and mass (LV Mass, lower right, n = 10 for both groups). (B) Size of the isolated cardiomyocytes. Left panel: representative Coulter Counter histograms. Middle panel: average of means and medians from populations of cells isolated from hearts (WT; six hearts, 4152 ± 1770 cells/analysis TG; seven hearts, 6,040 ± 1,533 cells/analysis). Right panel: means of cell capacitances measured in whole-cell experiments (WT n = 8/51 and TG n = 10/58; animals/cells). (C) Gene markers of cardiac hypertrophy [mRNA to WT ratio: natriuretic peptide A (Nppa), natriuretic peptide B (Nppb), skeletal actin (Acta1), myosin heavy polypeptide 6 (Myh6), and myosin heavy polypeptide 7 (Myh7); WT n = 7 and TG n = 6]. *P < 0.05, #P < 0.001.

Figure 2. Cell energy metabolism and ex vivo cardiac function. (A) Extracellular acidification and oxygen consumption in isolated cardiomyocytes supplemented with either glucose (Gluc) or palmitate (Palm). Top panels: representative plots of extracellular acidification rate (ECAR, left) and oxygen consumption rate (OCR, right). Bottom panels: basal and FCCP-induced maximal ECAR (left) and OCR [right, n = 35 wells with ~2,000 cells each, from seven (7 WT and 7 TG mice) independent paired assays]. (B) Ex vivo systolic heart function in glucose- (Gluc) and palmitate-based (Palm) solutions. Systolic pressure (LVP, upper left), diastolic pressure (Diast.press., upper right), systole rise slope (dP/dT, lower left), and systole decay slope (−dP/dT, lower right, n = 6 in both groups). (C) Ventricular expression of the genes (normalized to WT) related to energy metabolism: fatty acid transporter (Slc27a1), glucose transporter (Slc2a4), carnitine palmitoyltransferase 1B (Cpt1b), mitochondrial transcription factor A (Tfam), muscle phosphofructokinase (Pfkm), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a) (WT n = 7 and TG n = 6) *P < 0.05.

Figure 3. Ca2+ handling in VEGF-B TG ventricular myocytes. (A) Representative [Ca2+]i transients (line-scans) and their profiles in Fluo4-loaded cardiomyocytes upon pacing at different stimulus frequencies in WT and TG mice (upper), frequency dependence of amplitudes (lower left), and time constants (lower right) of the [Ca2+]i transients (n = 16 myocytes for WT and n = 22 myocytes for TG, at least three animals were used for both groups). (B) Representative line-scans and profiles of stimulus- and caffeine-induced [Ca2+]i pulses in WT (upper left) and TG mice (upper right). Amplitudes (lower left) and time constant of decays (lower middle) of stimulus- (Stim) and caffeine-induced (Caff) transients, and fractional release (lower right, WT n = 23 myocytes and TG n = 26 myocytes, at least three animals were used for both groups). *P < 0.05, ¤P < 0.01 and #P < 0.001.

Figure 4. Electrophysiological properties of VEGF-B TG hearts and isolated cardiomyocytes. (A) Representative ECG recordings from WT (black line) and VEGF-B TG (red line) mice. (B) Enlarged superimposed ECG complexes (left panel) with summary of the ECG parameters (right panel, n = 10 for both groups). (C) Representative superimposed AP traces from isolated myocytes (left) and means of the AP maximum rise slope (upper right) and rise time (lower right). (D) AP durations at different percentages of repolarization (WT n = 17 and TG n = 34; 4 animals for both group). *P < 0.05, ¤P < 0.01 and #P < 0.001.

Figure 5. Depolarizing currents in VEGF-B TG myocytes. (A) I–V curves of ICaL densities (left panel, WT n = 9 and TG n = 11, myocytes from three animals in both groups), voltage-dependent activation/inactivation of ICaL (middle panel, Boltzmann fitting) and gene expression of α-subunit calcium channel (Cacna1c, right panel). (B) Typical sodium channel current traces (left panel) and I–V curves (right panel) (WT black line, n = 19 and TG red line n = 14 myocytes from 3 mice were used for both groups). (C) Gene expression of sodium channel subunits Scn5a and Scn4b (left, WT n = 7 and TG n = 6) and Western blot of Nav1.5 from membrane protein fraction. Nav1.5 western blot signal normalized to Ponceau stain (middle, WT n = 3, TG n = 4). Representative SCN5a bands and Ponceau stain from the same region of the membrane (right). (D) Voltage-dependent activation and inactivation of sodium channels (left panel) and recovery time from inactivation (right panel) (see Table S3 for fitting results, WT n = 19 myocytes and TG n = 15 myocytes from three animals in both groups). Refer to insets for voltage-step protocols. *P < 0.05, ¤P < 0.01 and #P < 0.001.

Figure 6. Voltage-gated potassium currents. Representative superimposed original traces (upper, obtained at + 50 mV) and I–V curves (lower) of (A) total potassium (Ipeak), (B) transient outward (Ito), and (C) ultra-rapid (IKur) currents. (D) I–V curves of K1 and steady state currents (IK1/IKss, WT black and TG red lines, WT n = 8 and TG n = 17; from four animals). Refer to insets for voltage-step protocols (upper without and lower with inactivation prepulse). (E) Gene expressions of potassium channel subunits (WT n = 7 and TG n = 6). *P < 0.05, ¤P < 0.01 and #P < 0.001.

Figure 7. Effect of VEGF-B-related electrophysiological alterations explored in silico. From top to the bottom: (A,E) [Ca2+]i transients, (B,F) action potentials, (C,G) currents introduced into model INa, Ito, INCX (solid red lines), and (D,H) resulting currents IKss, ICaL, IKur (dotted red lines) in simulated pacing experiments, in the presence (A–D) and absence (E–H) of EGTA. Note, the different time scale in [Ca2+]i transients panels. The VEGF-B TG model variant (red lines) accounts for the (1) decreased INa (−18%), (2) reduced Ito (conductance −49%, and inactivation rate +59%), and (3) increased NCX current (+36%) as found in experiments.