Cellular Mechanisms of the Anti-Arrhythmic Effect of Cardiac PDE2 Overexpression

Figures

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  Figure 1. PDE2 OE display diminished cellular arrhythmogenic events. (A) Representative traces recorded in whole-cell patch clamp mode from WT (upper) and PDE2 OE (lower) ventricular cardiomyocytes upon stimulation at 0.125 Hz following stimulation at 4 Hz. Stars represent the stimulated action potentials; the following signals are spontaneous. Measurements were performed under basal conditions (Ctrl, black symbols) and upon β-adrenergic stimulation with isoprenaline (ISO, 100 nM, 10 min, orange symbols) and simultaneous PDE2 inhibition with BAY60-7550 (BAY, 100 nM). Quantification of (B) delayed afterdepolarizations (DAD, ≥4 mV), (C) spontaneous action potentials (sAP), (D) early afterdepolarizations (EAD), and (E) total number of arrhythmic events per cell in WT (circles) and PDE2 OE (squares). * p < 0.05, ** p < 0.01, *** p < 0.001 (N = 8 animals/genotype, 20 ≤ n ≤ 26 cells/group).

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  Figure 2. Similar basal action potential properties in PDE2 OE and WT. (A) Representative voltage traces of action potentials (AP) at 1 Hz from left ventricular myocytes of WT (circles) and PDE2 OE (squares) mice under basal conditions (Ctrl, black symbols) and upon β-adrenergic stimulation with isoprenaline (ISO, 100 nM, 10 min, orange symbols) and simultaneous PDE2 inhibition with BAY60-7550 (BAY, 100 nM, dark blue symbols). (B) Average resting membrane potential Vm (mV), (C) maximum depolarization potential (mV), (D) maximal upstroke velocity dV/dt (V/s), (E–G) action potential duration at 20%, 50%, and 90% repolarization (APD20, APD50, APD90). n.s. not significant, * p < 0.05 (N = 8 animals/genotype, 11≤ n ≤ 22 cells/group).

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  Figure 3. β-AR stimulation does not increase Calciumspark frequency upon β-adrenergic stimulation in PDE2 OE. Ventricular myocytes were loaded with fluo-4 AM and monitored for spontaneous intracellular rises in Ca2+ levels (Ca2+ sparks) by line scan confocal microscopy (A,B) under basal conditions (black) and after 10 min stimulation with ISO (100 nM, orange), ISO (100 nM) and AIP (1 μmol/L, blue), ISO (100 nM) and PKI (5 μmol/L, green); (C,D) 8-CPT (10 μmol/L, orange), 8-CPT (10 μmol/L) and AIP (1 μmol/L, blue). (A,C) Representative original registrations and (B,D) quantification of Ca2+ spark frequency (CaSpF) normalized to cell width and scan rate from WT (circles) and PDE2 OE (squares). * p < 0.05, ** p < 0.01 *** p < 0.001. (N = 4 animals/genotype, 45 ≤ n ≤ 79 cells/group).

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  Figure 4. Ca2+/calmodulin-dependent kinase II (CaMKII)-mediated increase in INaL and cellular arrhythmogenic events upon β-AR stimulation is abolished in PDE2 OE. Late sodium current (INaL) was measured in isolated ventricular cardiomyocytes from PDE2 OE (squares) and WT (circles) mice at −35 mV (1 s) under basal conditions (black symbols) and after 10 min stimulation with ISO (100 nM, orange), ISO (100 nM) and AIP (1 µM, blue), ISO (100 nM) and PKI (5 µM, green), 8-CPT (10 µM, red), 8-CPT (10 µM) and AIP (1 µM, dark blue). (A,B) Representative current traces and (C,D) quantification of INaL estimated by integrating INa between 100 and 500 ms and normalized to the membrane capacitance. *** p < 0.001 vs. control (Ctrl), (N = animals/genotype, 6 ≤ n ≤ 17 cells per group). (E,F) Isolated ventricular cardiomyocytes were subjected to an arrhythmia provocation protocol under basal conditions (black), after 10 min stimulation with ISO (100 nM, orange) or ISO (100 nM) and AIP (1 µM, blue). Arrhythmogenic events (DADs, EADs, and sAPs) were quantified. *** p < 0.001; 8≤ n ≤ 13, (N = 5 animals/group).

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  Figure 5. Basal expression and phosphorylation of CaMKII is affected by PDE2 overexpression. (A) Representative immunoblots and (B) quantification of protein kinase expression normalized to GAPDH, CaMKII phosphorylation at Threonine 286 normalized to total CaMKIIδ in lysates of isolated cardiomyocytes from PDE2 OE (orange) and WT (black) mice. (N = 6 animals/genotype. * p < 0.05, *** p < 0.001 vs. WT).

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  Figure 6. Specific PDE2 inhibition tends to increase the number of total arrhythmic events in ex vivo perfused hearts after reperfusion injury. (A) Representative traces of ECG recordings after reperfusion injury with 30 min of LAD ligation in ex vivo perfused hearts with and without PDE2 inhibitor BAY 60-7550 (BAY, 100 nM). (B) Evans blue and TTC staining of heart slices after reperfusion injury and quantification of infarcted area at risk (AAR, %). (C) Quantification of ventricular extrasystoles (VES), bigeminy, couplets and total number of arrhythmic events. Statistical p-values vs. Ctrl as indicated (N = 3–6 hearts/group).