3-Hydroxybutyrate Prevents Pulmonary Vein Arrhythmogenesis by Regulating Calcium/Calmodulin-Dependent Protein Kinase II Signaling and Oxidative Stress.

AimsMacrophage migration inhibitory factor (MIF), a pleiotropic inflammatory cytokine, is highly expressed in patients with atrial fibrillation (AF). Inflammation increases the risk of AF and is primarily triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether MIF can modulate the electrical activity of the PV and examined the underlying mechanisms of MIF.Methods and resultsA conventional microelectrode, a whole-cell patch clamp, western blotting, and immunofluorescent confocal microscopy were used to investigate electrical activity, calcium (Ca2+) regulation, protein expression, ionic currents, and cytosolic reactive oxygen species (ROS) in rabbit PV tissue and isolated single cardiomyocytes with and without MIF incubation (100 ng/mL, treated for 6 h). The MIF (100 ng/mL)-treated PV tissue (n = 8) demonstrated a faster beating rate (1.8 ± 0.2 vs. 2.6 ± 0.1 Hz, P < 0.05), higher incidence of triggered activity (12.5 vs. 100%, P < 0.05), and premature atrial beat (0 vs. 100%, P < 0.05) than the control PV tissue (n = 8). Compared with the control PV cardiomyocytes, MIF-treated single PV cardiomyocytes had larger Ca2+ transients (0.6 ± 0.1 vs. 1.0 ± 0.1, ΔF/F0, P < 0.05), sarcoplasmic reticulum Ca2+ content (0.9 ± 0.20 vs. 1.7 ± 0.3 mM of cytosol, P < 0.05), and cytosolic ROS (146.8 ± 5.3 vs. 163.7 ± 3.8, ΔF/F0, P < 0.05). Moreover, MIF-treated PV cardiomyocytes exhibited larger late sodium currents (INa-Late), L-type Ca2+ currents, and Na+/Ca2+ exchanger currents than the control PV cardiomyocytes. KN93 [a selective calcium/calmodulin-dependent protein kinase II (CaMKII) blocker, 1 μM], ranolazine (an INa-Late inhibitor, 10 μM), and N-(mercaptopropionyl) glycine (ROS inhibitor, 10 mM) reduced the beating rates and the incidence of triggered activity and premature captures in the MIF-treated PV tissue.ConclusionMacrophage migration inhibitory factor increased PV arrhythmogenesis through Na+ and Ca2+ dysregulation through the ROS activation of CaMKII signalling, which may contribute to the genesis of AF during inflammation. Anti-CaMKII treatment may reverse PV arrhythmogenesis. Our results clearly reveal a key link between MIF and AF and offer a viable therapeutic target for AF treatment.

Atrial fibrillation (AF) is the most common form of arrhythmia and increases the risk of stroke and heart failure (HF). Pulmonary veins (PVs) are important sources of triggers that generate AF, and calcium (Ca2+ ) overload participates in PV arrhythmogenesis. Neurohormonal activation is an important cause of AF. Higher atrial natriuretic peptide (ANP) level predicts paroxysmal AF occurrence in HF patients. However, it is not clear if ANP directly modulates electrophysiological characteristics and Ca2+ homeostasis in the PVs. Conventional microelectrodes, whole-cell patch-clamp, and the Fluo-3 fluorimetric ratio technique were performed using isolated rabbit PV preparations or single isolated PV cardiomyocytes before and after ANP administration. We found that ANP (1, 10, and 100nmol/L) concentration-dependently decreased spontaneous activity in PV preparations. ANP (100nmol/L) decreased isoproterenol (1μmol/L)-induced PV spontaneous activity and burst firing. AP811 (100nmol/L, NPR-C agonist), H89 (1μmol/L, PKA inhibitor) decreased isoproterenol-induced PV spontaneous activity or burst firing, but successive administration of ANP had no further effect on PV activity. KT5823 (1μmol/L, PKG inhibitor) decreased isoproterenol-induced PV spontaneous activity but did not change isoproterenol-induced PV burst firing, whereas successive administration of ANP did not change isoproterenol-induced PV burst firing. ANP decreased intracellular Ca2+ transient and sarcoplasmic reticulum Ca2+ content in single PV cardiomyocytes. ANP decreased the late sodium current, L-type Ca2+ current, but did not change nickel-sensitive Na+ -Ca2+ exchanger current in single PV cardiomyocytes. In conclusion, ANP directly regulates PV electrophysiological characteristics and Ca2+ homeostasis and attenuates isoproterenol-induced arrhythmogenesis through NPR-C/cAMP/PKA signal pathway.

Heat stress-induced responses reduce the occurrence of atrial fibrillation (AF). Pulmonary vein (PV) cardiomyocytes with pacemaker activity play a critical role in the pathophysiology of AF. In this study, we examined whether heat-stress responses alter the electrophysiological characteristics of PV cardiomyocytes and protect the PV against angiotensin II- or isoproterenol-induced arrhythmogenesis. We used whole-cell patch clamp techniques to investigate the spontaneous activity and ionic currents in single isolated rabbit PV pacemaker cardiomyocytes with or without (control) exposure to heat stress (43°C, 15 minutes) 5 ± 1 hours before the experiments. Compared to control cardiomyocytes, heat-stressed PV cardiomyocytes had slower beating rates. Heat-stressed PV cardiomyocytes had larger L-type calcium currents, transient outward currents, smaller inward rectifier potassium currents, but similar sodium-calcium exchanger currents. Additionally, heat-stressed PV cardiomyocytes had a lower incidence of pacemaker currents than control PV cardiomyocytes. Moreover, isoproterenol increased the beating rate of control cardiomyocytes but not heat-stressed PV cardiomyocytes. Similarly, angiotensin II also increased the beating rate of control cardiomyocytes, but not heat-stressed PV cardiomyocytes, in association with decreased expression of the angiotensin II type 1 receptor. Heat-stress responses altered the electrophysiological characteristics of PV cardiomyocytes and attenuated the effects of isoproterenol and angiotensin II on PV arrhythmogenesis, which may play a role in the protective potential of heat-stress responses.

BackgroundChronic kidney disease (CKD) increases the occurrence of atrial fibrillation and pulmonary vein (PV) arrhythmogenesis. Calcium dysregulation and reactive oxygen species (ROS) enhance PV arrhythmogenic activity. The purposes of this study were to investigate whether CKD modulates PV electrical activity through dysregulation of calcium homeostasis and ROS.Methods and ResultsBiochemical and electrocardiographic studies were conducted in rabbits with and without CKD (induced by 150 mg/kg per day neomycin sulfate and 500 mg/kg per day cefazolin). Confocal microscopy with fluorescence and a whole‐cell patch clamp were applied to study calcium homeostasis and electrical activities in control and CKD isolated single PV cardiomyocytes with or without treatment with H89 (1 μmol/L, a protein kinase A inhibitor) and MPG (N‐[2‐mercaptopropionyl]glycine; 100 μmol/L, a ROS scavenger). The ROS in mitochondria and cytosol were evaluated via intracellular dye fluorescence and lipid peroxidation. CKD rabbits had excessive atrial premature captures over those of control rabbits. Compared with the control, CKD PV cardiomyocytes had a faster beating rate and larger calcium transient amplitudes, sarcoplasmic reticulum calcium contents, sodium/calcium exchanger currents, and late sodium currents but smaller L‐type calcium current densities. CKD PV cardiomyocytes had a higher frequency and longer duration of calcium sparks and more ROS in the mitochondria and cytosol than did controls. Moreover, H89 suppressed all calcium sparks in CKD PV cardiomyocytes, and H89‐ and MPG‐treated CKD PV cardiomyocytes had similar calcium transients compared with control PV cardiomyocytes.ConclusionsCKD increases PV arrhythmogenesis with enhanced calcium‐handling abnormalities through activation of protein kinase A and ROS.

Characterization of constitutively active GIRK ion channels in rat pulmonary vein

Introduction Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a crucial regulator of Ca2+-handling and excitation-contraction coupling in cardiomyocytes. Lately, an oxidative activation of CaMKII has been described. Oxidative stress is an integral component of ischemia/reperfusion (IR) injury which results in a complex of subcellular and molecular changes leading to impaired cardiac function and cell death. As AT1 receptor activation promotes reactive oxygen species production through NADPH oxidase system it may be hypothesized that this signaling cascade might play an important role in oxidative activation of CaMKII under IR conditions. Methods To test this, the isolated Langendorff-perfused hearts of Wistar rats treated with losartan (20 mg/kg, p.o., 14 days) as well as control ones were subjected to ischemia (30 min) followed by reperfusion (40 min). A role of CaMKII was studied by using a specific CaMKII inhibitor (KN-93, 0,5 µmol/dm3) which was present in the perfusion solution 10 min before onset of ischemia, during ischemia and first 10 min of reperfusion. During the whole IR protocol, myocardial parameters were being continuously recorded. Protein content of total, oxidized CaMKII, oxidative stress markers, such as pro-oxidant NADPH oxidase (NOX2) and antioxidant superoxide dismutase (MnSOD) and catalase (CAT) as well as apoptotic markers (Bax, Bcl-2, cyt c) was evaluated in left ventricles by Western blotting. Results In the IR hearts, inhibition of AT1 receptors or CaMKII alone normalized the protein content of antioxidant proteins MnSOD and CAT to the values of the non-ischemic hearts. Likewise, the protein content of oxidized CaMKII was modified. Interestingly, the protein content of NOX2 was significantly increased in IR-subjected group treated with losartan, suggesting a negative feedback due to a permanent AT1 inhibition. Moreover, the protein content of anti-apoptotic Bcl-2 was not changed upon losartan treatment nor CaMKII inhibition in the ischemic hearts. On the other hand, pro-apoptotic Bax and cytochrome C were upregulated in the non-ischemic losartan group. Simultaneous inhibition of AT1 receptors and CaMKII had no additive effect on content of any protein studied. On the other hand, although AT1 receptor inhibition or CaMKII inhibition improved postischemic recovery of left-ventricular developed pressure (LVDP), the simultaneous inhibition of AT1 receptors and CaMKII abolished this beneficial effect. Conclusion In conclusion, it is likely that AT1 receptor signaling cascade plays a role in oxidative activation of CaMKII documented by normalized anti-oxidant protein content and by improved contractile function of heart after AT1 inhibition. However, a simultaneous inhibition of CaMKII and AT1 receptors resulted in worsened LVDP recovery suggesting that excessive inhibition of CaMKII activation and therefore diminished activation of CaMKII target proteins of Ca2+-handling may be deleterious. Figure 1 Figure 2

Although the recent identification of the mitochondrial Ca2+ uniporter (MCU) has resolved a long-standing mystery as to how Ca2+ freely enters the mitochondria, it has also evoked additional questions such as its mode of regulation and the identity of other associated factors. In an article recently published in Nature , Joiner et al1 provide data demonstrating that in the heart, matrix-localized Ca2+/calmodulin-dependent protein kinase II (CaMKII) can upregulate MCU activity in a manner requiring phosphorylation of the channel N terminus. They showed that inhibition of CaMKII-dependent MCU activity protected the heart from ischemic injury by presumably reducing Ca2+ influx and desensitizing the mitochondrial permeability transition pore (MPTP) to opening. Although these results demonstrate convincingly that CaMKII plays an important role in MCU regulation and subsequent response to cardiac injury, several questions remain unanswered. The ability of mitochondria to take up and sequester Ca2+ plays an important role in the buffering of cytosolic Ca2+, regulation of ATP production via the citric acid cycle, and regulation of apoptotic and necrotic cell death pathways.2 Although mitochondrial Ca2+ uptake was first described in the 1960s3 and the electrophysiological properties of the MCU were reported in 2004,4 it was not until 2011 that 2 articles were published revealing the genetic identity of the MCU.5,6 This pioneering work has initiated a search for additional members of the MCU complex (such as MICU17 and the recently discovered MCUR1),8 as well as an attempt to understand how MCU-mediated Ca2+ influx participates in the regulation of whole-cell Ca2+ signaling and whether well-described pathways that regulate other Ca2+ handling processes can similarly modulate MCU-dependent Ca2+ uptake. In the featured article, Joiner et al1 show that CaMKII serves …

Extracellular matrix of collagen modulates arrhythmogenic activity of pulmonary veins through p38 MAPK activation

Temperature plays an important role in the electrophysiology of cardiomyocytes. Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. The effects of temperature on the arrhythmogenic activity of rabbit single PV and atrial cardiomyocytes were assessed using the whole-cell clamp technique. PV cardiomyocytes had different beating rates at low (22–25°C), normal (38–39°C) and high (40–41°C) temperatures (0.9 ± 0.1, 3.2 ± 0.4, 6.4 ± 0.6 Hz, respectively; p < 0.001). There were different action potential durations and incidences of delayed afterdepolarization in PV cardiomyocytes with pacemaker activity (31, 59, 63%; p < 0.05), PV cardiomyocytes without pacemaker activity (16, 47, 60%; p < 0.001), and atrial myocytes (0, 0, 21%; p < 0.05). However, oscillatory afterpotentials were only found in PV cardiomyocytes with pacemaker activity at normal (50%) or high (68%) temperatures, but not at low temperatures (p < 0.001). Both PV and atrial cardiomyocytes had larger transient inward currents and inward rectified currents at high temperatures. Additionally, PV cardiomyocytes with and without pacemaker activity had larger pacemaker currents at higher temperatures. This study demonstrated that PV cardiomyocytes have an increase in arrhythmogenic activity at high temperatures because of enhanced automaticity, induced triggered activity, or shortening of action potential duration.

Temperature plays an important role in the electrophysiology of cardiomyocytes. Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. The effects of temperature on the arrhythmogenic activity of rabbit single PV and atrial cardiomyocytes were assessed using the whole-cell clamp technique. PV cardiomyocytes had different beating rates at low (22-25 degrees C), normal (38-39 degrees C) and high (40-41 degrees C) temperatures (0.9 +/- 0.1, 3.2 +/- 0.4, 6.4 +/- 0.6 Hz, respectively; p < 0.001). There were different action potential durations and incidences of delayed afterdepolarization in PV cardiomyocytes with pacemaker activity (31, 59, 63%; p < 0.05), PV cardiomyocytes without pacemaker activity (16, 47, 60%; p < 0.001), and atrial myocytes (0, 0, 21%; p < 0.05). However, oscillatory afterpotentials were only found in PV cardiomyocytes with pacemaker activity at normal (50%) or high (68%) temperatures, but not at low temperatures (p < 0.001). Both PV and atrial cardiomyocytes had larger transient inward currents and inward rectified currents at high temperatures. Additionally, PV cardiomyocytes with and without pacemaker activity had larger pacemaker currents at higher temperatures. This study demonstrated that PV cardiomyocytes have an increase in arrhythmogenic activity at high temperatures because of enhanced automaticity, induced triggered activity, or shortening of action potential duration.

Heart failure (HF) plays a critical role in the genesis of atrial fibrillation (AF). A high B-type natriuretic peptide (BNP) level occurs in patients with HF and in patients with AF. However, the role of BNP in the pathophysiology of AF is not clear. The purposes of this study were to evaluate the effects of BNP on pulmonary vein (PV) arrhythmogenesis. Whole-cell patch clamp and fluorescence were used to study the action potential, ionic currents, and calcium homeostasis in isolated single rabbit PV cardiomyocytes before and after a BNP infusion, with or without ODQ (10 μM), milrinone (50 μM), or ouabain (1 μM). BNP increased PV spontaneous activity by 28.2 ± 7.5% at 100 nM and by 23.8 ± 9.1% at 300 nM. Similar to those with BNP, milrinone 50 μM increased the PV beating rate from 3.0 ± 0.2 to 3.6 ± 0.3 Hz (P < 0.0005, n = 7). In the presence of ODQ application, BNP didn't change PV spontaneous activity. BNP (100 nM) increased calcium transients (F/F0 from 1.6 ± 0.1 to 1.9 ± 0.2, n = 20, P < 0.05) and increased the pacemaker current (0.4 ± 0.1 to 1.0 ± 0.2 pA/pF, n = 17, P < 0.0005) in PV cardiomyocytes. Moreover, BNP (100 nM) increased the transient inward current, sodium currents, sodium-calcium exchanger currents, and L-type calcium current; but reduced late sodium currents and the Na-K pump in PV cardiomyocytes. BNP increases PV arrhythmogenesis, which may contribute to the genesis of atrial tachyarrhythmogenesis in HF. Cyclic GMP activation, phosphodiesterase 3 inhibition and Na+ /K+ -ATPase inhibition might participate in the BNP modulation of PV electrophysiology.

Endothelin-1 has important cardiovascular effects and is activated during atrial fibrillation. Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation. The aim of this study was to evaluate whether endothelin-1 affects PV arrhythmogenic activity. Conventional microelectrodes were used to record the action potentials (APs) and contractility in isolated rabbit PV tissue specimens before and after the administration of endothelin-1 (0.1, 1, 10 nM). The ionic currents of isolated PV cardiomyocytes were investigated before and after the administration of endothelin-1 (10 nM) through whole-cell patch clamps. In the tissue preparation, endothelin-1 (1, 10 nM) concentration dependently shortened the AP duration and decreased the PV firing rates. Endothelin-1 (10 nM) decreased the resting membrane potential. Endothelin-1 (0.1, 1, 10 nM) decreased the contractility and increased the resting diastolic tension. In single PV cardiomyocytes, endothelin-1 (10 nM) decreased the PV firing rates from 2.7 +/- 1.0 Hz to 0.8 +/- 0.5 Hz (n = 16). BQ-485 (100 microM, endothelin-1 type A receptor blocker) reversed and prevented the chrono-inhibitory effects of endothelin-1 (10 nM). Endothelin-1 (10 nM) reduced the L-type calcium currents, transient outward currents, delayed rectifier currents, transient inward currents, and sodium-calcium exchanger currents in the PV cardiomyocytes with and without pacemaker activity. Endothelin-1 (10 nM) increased the inward rectifier potassium current, hyperpolarization-induced pacemaker current, and the sustained outward potassium current in PV cardiomyocytes with and without pacemaker activity. Endothelin-1 may have an antiarrhythmic potential through its direct electrophysiological effects on the PV cardiomyocytes and its action on multiple ionic currents.

Eicosapentaenoic acid reduces the pulmonary vein arrhythmias through nitric oxide

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca(2+)/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca(2+)-independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.