Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Regulates Basal Cardiac Pacemaker Function: Pros and Cons

Figure 1. (A) Schematic illustration of the coupled-clock pacemaker system and active P-CaMKII in SANCs. (A) Schematic presentation of ion channels “membrane clock” (including the most important currents, i.e., hyperpolarization-activated “funny” current (If), L-type Ca2+ current (ICa,L), carried via Cav1.2 or Cav1.3 [3], T-type Ca2+ current (ICa,T), delayed rectifier potassium current (IK), Na+/Ca2+ exchange current (INCX), sustained current (Ist), etc.) and “Ca2+ clock” in cardiac pacemaker cells. (B) Top: Illustration of spontaneous SANC action potentials, Ca2+ transients, LCRs, and schematic illustration of several major ion currents involved in generation of the diastolic depolarization (DD) and interactions of molecules comprising the full coupled-clock pacemaker system. Bottom: the restitution process that defines the LCR period which is regulated by the rate of Ca2+ pumping into the SR and SR Ca2+ load required for activation of spontaneous release from RyRs. LCR-induced increase in local [Ca2+] beneath sarcolemma activates an inward INCX current creating exponential increase in the DD rate (nonlinear DD). The LCR period represents the essence of the “coupled-clock” pacemaker…

Figure 2. Regulation of spontaneous Ca2+ releases by cytosolic Ca2+ differs in permeabilized SANCs and ventricular myocytes (VMs). (A) Representative images and Ca2+ waveforms from bands (indicated by arrows) of SANCs and (B) of VMs exposed to different concentrations of cytosolic-free Ca2+. (a) FFT of Ca2+ waveforms of SANCs in (A) and (b) FFT of the Ca2+ waveforms of VMs from bands indicated by the color-matched arrows in (B). (C) Comparison of total Ca2+ signal mass released by either SANCs or VMs at different cytosolic [Ca2+]c. (D) Average total SR Ca2+ content in SANCs and VMs. (E) Elevation of [Ca2+]c increases phosphorylation of PLB at Thr17 site in SANCs, but not in VMs. Top: Representative confocal images of permeabilized SANCs at 0 nmol/L and 150 nmol/L of [Ca2+]c, total PLB (red), and PLB phosphorylated at Thr17 (green); (bottom) relative changes of phosphorylated PLB at Thr17 normalized to total PLB in SANCs or VMs at different free cytosolic [Ca2+]c. * p < 0.05. (F) Suppression of basal CaMKII activity decreases the SR Ca2+ content in permeabilized SANCs. Effects of a rapid application of 20 mmol/L caffeine on representative permeabilized rabbit SANCs in the absence or…

Figure 3. Inhibition of CaMKII suppresses spontaneous, periodic LCRs in permeabilized SANCs. (A) Left: Confocal line-scan images of a representative SANC bathed in 200 nmol/L [Ca2+]c before (top) and after (bottom) superfusion with 10 μmol/L AIP. Right: AIP treatment resulted in decreased total Ca2+ signal mass released by SANCs. (B) Spatial size of LCRs (control, yellow squares) was markedly decreased after treatment with AIP (blue squares), * p < 0.05. (C) Left: FFT of Ca2+ oscillations (from bands indicated by arrows) in (A), before and after AIP treatment. Right: relative number of SANCs that generated periodic LCRs under control conditions and after AIP treatment. Logistic regression analysis demonstrated a significant difference between the two curves (p < 0.002). Modified from [12].

Figure 4. Inhibition of CaMKII activity suppresses L-type Ca2+ current but has no effect on hyperpolarization-activated funny current If. (A) Average current–voltage relationships of ICa,L in the presence or absence of the CaMKII inhibitor KN-93 (1 μmol/L); inset shows representative control recordings of ICa,L. (B) Immunofluorescence intensity of autophosphorylated, active P-CaMKII in control conditions (control) and after treatment with KN-93 or AIP. * p < 0.05. (C) Activation (triangles) and steady-state inactivation (squares) curves of ICa,L measured from a holding potential of –70 mV. Filled symbols show control data, and open symbols represent data recorded after 5 min treatment with 1 μmol/L KN-93. (D) Inhibition of CaMKII by KN-93 or AIP, but not KN-92, slows the recovery of ICa,L from inactivation. (A–D) modified from [7]. (E) (a) Traces of If current in control (left) and following exposure to 1 μmol/L KN-93 (right). Cell capacitance is 34 pF. (b) Conductance–voltage relationship on If current in control (filled squares) and in KN-93 (open squares). Current was normalized to max control current. Inset: Peak ICa,L current (holding potential −40 mV, step to 0 mV) in the absence…

Figure 5. CaMKII inhibition suppresses slowly activating delayed rectifier potassium current IKs and spontaneous beating of cardiac pacemaker cells. (A) Ca2+-dependent inhibitory effect of CaMKII inhibitor KN-93 on IKs. (a) Time course of normalized IKs tail currents in response to KN-93 (1 μmol/L) in cells dialyzed with Ca (+) (pCa 7) pipette solution. The inset shows original current traces recorded at the indicated time points. (b) IKs in the presence of KN-93 under Ca (+) or Ca (−) (pCa 10) conditions. (c) Pulse protocol. Depolarizing pulses of 100 ms were applied to +30 mV from a holding potential of −50 mV at a rate of 240 pulses/min. After a cell was subjected to a train of depolarizing pulses (240 pulses), KN-93 was added to the bath solution. (d) IKs at P1 (first pulse), P2, P3, P240, and P720. The dashed line indicates the zero-current level. (e) The summary of KN-93 effect on IKs induced by simulated pacemaker potentials, ** p < 0.05. (B) Effects of CaMKII inhibition on spontaneous APs in guinea pig SANCs. (a) Recordings of APs in the absence (left) or presence (right) of CaMKII inhibitor KN-93 or (b) inactive KN-93 analog KN-92; (c) recordings of spontaneous APs in the absence…

Figure 6. CaMKII inhibition decreases phosphorylation of Ca2+-cycling proteins PLB and RyRs in intact rabbit SANCs, suppresses LCRs, and prolongs the T-90 and LCR period. (A) Top: representative western blots of PLB phosphorylated at Thr17 site and total PLB at baseline and after treatment with 1 μmol/L KN-93 or 1 μmol/L KN-92; bottom: average values of phosphorylated PLB normalized to total PLB in basal conditions, after treatment with KN-93 or KN-92. (B) Top: representative western blots of RyRs phosphorylated at Ser2815 site and total RyRs in SA node tissue at baseline and in response to CaMKII inhibition by 1 μmol/L KN-93; bottom: average values of phosphorylated RyRs normalized to total RyRs in basal conditions and after treatment with KN-93; data are presented as % control. * p < 0.05, by t-test. (C) Representative western blots of total PLB and PLB phosphorylated at PKA-dependent Ser16 site (left) or CaMKII-dependent Thr17 (right) in response to inhibition of PKA by selective PKA inhibitor peptide PKI; bottom: average values of PLB phosphorylated at Ser16 site or Thr17 site and normalized to total PLB after treatment with PKI; data are presented as % control. (D) Confocal line-scan…

Figure 7. Bradycardia during heart failure in mice is linked to reduced CaMKII phosphorylation, decrease in Ca2+ spark frequency, and amplitude of pre-transient Ca2+ release. (A) Representative western blots and quantification of total and phosphorylated CaMKII (normalized by total CaMKII) in SAN tissues from sham and HF mice. (B) Representative western blots and quantification of phosphorylated PLB-T17 normalized by total PLB. (C) Representative western blots and quantification of RYR2-S2814 normalized to total RYR2 in SAN tissues from sham and HF mice. Blue circles in (A), (B), (C) highlight, respectively, basal level of CaMKII activity, PLB and RyR phosphorylation in sham operated mice. (D) Left: Representative line-scan images (0.25 ms per line) of spontaneous beating cells from intact SA node; arrows indicate Ca2+ sparks. Right: Quantification of Ca2+ spark frequency (number of sparks/s/100 μm) in sham and HF mice SAN tissues. (E) Quantification of the fluorescence of the fluorescence ramp (pre-transient Ca2+) was measured in SA nodes from sham and HF mice. (F) Representative examples of telemetric ECG traces (daytime) under basal conditions and after atropine and propranolol…

Figure 8. SANCs from wild-type mice, employed for transgenic CaMKII inhibition, have no CaMKII activity in the basal state. (A) Representative immunofluorescence images show absence of activated (pCaMKII) in SANCs from wild-type mice (blue arrows). ISO activates CaMKII in SANCs isolated from wild-type (WT) and AC3-C mice, but not from AC3-I mice with SA node CaMKII inhibition. Columns are as follows: 1, eGFP (expressed in AC3-C and AC3-I SANCs); 2, Thr287 autophosphorylated, activated CaMKII (pCaMKII, red); 3, merge; 4, magnified images from column 2. (Scale bar, 10 μm). (B) Top: ECGs recorded from Langendorff-perfused hearts at baseline and after 1 μmol/L ISO. Bottom: Heart rates recorded from ECG-telemetered mice (in vivo) at rest and Langendorff-perfused hearts (ex vivo). Langendorff-perfused hearts from AC3-I and control mice (n = 5–6/group) beat at equivalent rates in the basal conditions (p = 0.318). (C) Consistent with the absence of basal CaMKII activity, SANC isolated from WT, AC3-C or AC3-I mouse SA nodes have the same beating rates in the basal conditions (blue oval). In response to a range of ISO concentrations, AC3-I SANC beating rates were significantly (* p < 0.05, ** p <…