Phosphodiesterase-1 inhibitor modulates Ca2+ regulation in sirtuin 1-deficient mouse cardiomyocytes.

BACKGROUND: Phosphodiesterase inhibitors can be used to enhance second messenger signaling to regulate intracellular Ca2+ cycling. This study investigated whether ITI-214, a selective phosphodiesterase-1 inhibitor, modulates intracellular Ca2+ regulation, resulting in a positive inotropic effect in sirtuin 1 (Sirt1)-deficient cardiomyocytes. METHODS: Mice with cardiac-specific Sirt1 knockout (Sirt1-/-) were used, with Sirt1flox/flox mice serving as controls. Electromechanical analyses of ventricular tissues were conducted, and we monitored intracellular Ca2+ using Fluo-3 as well as reactive oxygen species production in isolated cardiomyocytes. RESULTS: Sirt1-/- ventricles showed prolonged action potential duration at 90% repolarization and increased contractile force after treatment with ITI-214. The rates and sustained durations of burst firing in ventricles were higher and longer, respectively, in Sirt1-/- ventricles than in controls. ITI-214 treatment decreased the rates and shortened the durations of burst firing in Sirt1-/- mice. Sirt1-/- cardiomyocytes showed reduced Ca2+ transient amplitudes and sarcoplasmic reticulum (SR) Ca2+ stores compared to those in control cardiac myocytes, which was reversed after ITI-214 treatment. SR Ca2+ leakage was larger in Sirt1-/- cardiac myocytes than in control myocytes. ITI-214 reduced SR Ca2+ leakage in Sirt1-/- cardiac myocytes. Increased levels of reactive oxygen species in Sirt1-/- cardiomyocytes compared to those in controls were reduced after ITI-214 treatment. Levels of Ca2+ regulatory proteins, including ryanodine receptor 2, phospholamban, and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a were not affected by ITI-214 administration. CONCLUSIONS: Our results suggest that ITI-214 improves intracellular Ca2+ regulation, which in turn exerts inotropic effects and suppresses arrhythmic events in Sirt1-deficient ventricular myocytes.

Concurrent increases in post-pacing action potential duration and contractility predict occurrence of ventricular arrhythmia.

Excitation-contraction coupling from the integration of action potential duration (APD) and muscle contractility plays an important role in arrhythmogenesis. We aimed to determine whether distinctive excitation-contraction coupling contributes to the genesis of ventricular tachycardias (VTs). Action potential (AP) and mechanical activity were simultaneously recorded under electrical pacing (cycle lengths from 1000 to 100 ms) in the tissue model created from isolated rabbit right ventricular outflow tracts treated with NS 5806 (10 µM, transient outward potassium current enhancer), pinacidil (2 µM, ATP-sensitive potassium channel opener), and pilsicainide (5 µM, sodium channel blocker). There were 15 (9.9%) inducible VT episodes (group 1) and 136 (90.1%) non-inducible VT episodes (group 2) in our tissue model. Group 1 had greater post-pacing increases of the first occurrence of AP at 90% repolarization (ΔAPD90, p < 0.001) and contractility (ΔContractility, p = 0.003) compared with group 2. Triggered VT episodes were common (72.7%) in cases with a ΔAPD90 > 15% and a ΔContractility > 270%, but were undetectable in those with a ΔAPD90 < 15% and a ΔContractility < 270%. In those with pacing-induced VTs, KB-R7943 (10 µM, a Na+-Ca2+ exchanger inhibitor, NCX inhibitor) significantly reduced the occurrence of VTs from 100.0 to 20.0% (15/15 to 3/15 episodes, p < 0.001). Concurrent increases in both post-pacing APD and contractility resulted in the occurrence of ventricular arrhythmias. NCX inhibition may be a potential therapeutic strategy for ventricular arrhythmias.

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes.

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca2+ and Na+ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac-specific Sirt1 knockout (Sirt1-/- ). Sirt1flox/flox mice were served as control. Sirt1-/- mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca2+ transient amplitudes and sarcoplasmic reticulum (SR) Ca2+ stores, and increased SR Ca2+ leak were shown in the Sirt1-/- mice. Electrophysiological measurements were performed using patch-clamp method. While L-type Ca2+ current (ICa, L ) was smaller in Sirt1-/- myocytes, reverse-mode Na+ /Ca2+ exchanger (NCX) current was larger compared with those in control myocytes. Late Na+ current (INa, L ) was enhanced in the Sirt1-/- mice, alongside with elevated cytosolic Na+ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1-/- mice. Sirt1-/- cardiomyocytes showed down-regulation of L-type Ca2+ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca2+  ATPase 2a (SERCA2a), but up-regulation of Ca2+ /calmodulin-dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca2+ and Na+ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.

The AMP-activated protein kinase modulates hypothermia-induced J wave.

BACKGROUND: The mechanism underlying the occurrence of the J wave in low temperature remains unclear. However, low temperature is associated with metabolic disorder and 5' AMP-activated protein kinase (AMPK), which modulates ionic currents and cardiac metabolism. This study investigated whether AMPK regulation can modulate the occurrence of the J wave at low temperature. METHODS: Unipolar and bipolar leads were used to record monophasic action potential (the endocardium and epicardium) and pseudo-electrocardiograms (inferior leads) to study the cardiac electrical activity. Measurements were taken in isolated Langendorff rabbit hearts at both 30℃ and 37℃ before and after administration of 4-aminopyridine (an ultrarapid delayed rectifier potassium current inhibitor, IKur , 50 µmol L-1 ), PF06409577 (an AMPK activator, 1 µmol L-1 ), compound C (an AMPK inhibitor, 10 µmol L-1 ) and glibenclamide (an ATP-sensitive inward rectifier potassium channel inhibitor, IKATP , 20 µmol L-1 ). RESULTS: The amplitude of the J wave (2.46 ± 0.34 mV vs. 1.11 ± 0.23 mV, P < .01) at 30℃ (n = 15) was larger than that at 37℃ (n = 15). PF06409577 (1 µmol L-1 ) increased the J waves at both 30℃ and 37℃. In contrast, compound C (10 µmol L-1 ) reduced J wave at both 37℃ and 30℃. Low-temperature-induced J waves were individually suppressed by 4-AP (50 µmol L-1 ) and glibenclamide (20 µmol L-1 ). CONCLUSIONS: AMPK inhibition reduces low-temperature-induced J waves and possible ventricular arrhythmogenesis by modulating IKATP and IKur channels.

Cardiac calcium dysregulation in mice with chronic kidney disease.

Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca2+ ) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca2+ decay time, Ca2+ sparks, and Ca2+ leakage but lower [Ca2+ ]i transients and sarcoplasmic reticulum Ca2+ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na+ current in CKD significantly altered cardiac Ca2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.

Staphylococcal phosphatidylinositol-specific phospholipase C potentiates lung injury via complement sensitisation.

Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc- mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.

Oxidative stress on pulmonary vein and left atrium arrhythmogenesis.

BACKGROUND: Oxidative stress and pulmonary veins (PVs) play critical roles in the pathophysiology of atrial fibrillation. The purpose of the present study was to investigate whether oxidative stress and antioxidant agents can change the electrophysiological characteristics of the left atrium (LA) and PVs. METHODS AND RESULTS: Conventional microelectrodes were used to record the action potentials (APs) in isolated rabbit PV and LA specimens before and after H(2)O(2) administration with or without ascorbic acid or N-mercaptopropionyl-glycine (N-MPG, a free radical .OH scavenger). H(2)O(2) (0.02 and 0.2 mmol/L) decreased the PV spontaneous rates from 2.0+/-0.1 Hz to 1.6+/-0.1 Hz, and 1.7+/-0.1 Hz (n=10, P<0.05), but H(2)O(2) (2 mmol/L) increased PV spontaneous rates from 2.0+/-0.1 Hz to 2.8+/-0.2 Hz. H(2)O(2) easily induced PV burst firing and early afterdepolarizations, but not in the LA. H(2)O(2) shortened the AP duration and increased the contractile force to a greater extent in the LA than in PVs. In addition, the H(2)O(2)-induced PV burst firing and increasing spontaneous rates were suppressed or attenuated by pretreatment with ascorbic acid (1 mmol/L) or N-MPG (10 mmol/L). CONCLUSIONS: H(2)O(2) significantly changed the electrophysiological characteristics of PV and LA through activation of free radicals and may facilitate the occurrence of atrial fibrillation.