Thomas Sowa

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late INa Augmentation Leading to Cellular Na and Ca Overload

Rationale: In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective: We tested whether CaMKII&dgr; is required for ROS-dependent late INa regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results: 40 &mgr;mol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 &mgr;mol/L), the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKII&dgr;−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKII&dgr;−/− myocytes (versus WT). CaMKII&dgr;−/− myocytes developed significantly less H2O2-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKII&dgr; in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 &mgr;mol/L KN93 or 0.1 &mgr;mol/L AIP [autocamtide 2–related inhibitory peptide]). Conclusions: Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKII&dgr; enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

Oxidized Ca 2+ /Calmodulin-Dependent Protein Kinase II Triggers Atrial Fibrillation

Background —Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results —We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions —Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.Background— Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Angiotensin II and reactive oxygen species are validated risk factors for AF in patients, but the molecular pathways connecting reactive oxygen species and AF are unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a reactive oxygen species–activated proarrhythmic signal, so we hypothesized that oxidized CaMKII&dgr; could contribute to AF. Methods and Results— We found that oxidized CaMKII was increased in atria from AF patients compared with patients in sinus rhythm and from mice infused with angiotensin II compared with mice infused with saline. Angiotensin II–treated mice had increased susceptibility to AF compared with saline-treated wild-type mice, establishing angiotensin II as a risk factor for AF in mice. Knock-in mice lacking critical oxidation sites in CaMKII&dgr; (MM-VV) and mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, were resistant to AF induction after angiotensin II infusion. Conclusions— Our studies suggest that CaMKII is a molecular signal that couples increased reactive oxygen species with AF and that therapeutic strategies to decrease oxidized CaMKII may prevent or reduce AF.

Oxidized CaMKII Triggers Atrial Fibrillation

Background —Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results —We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions —Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.Background— Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Angiotensin II and reactive oxygen species are validated risk factors for AF in patients, but the molecular pathways connecting reactive oxygen species and AF are unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a reactive oxygen species–activated proarrhythmic signal, so we hypothesized that oxidized CaMKII&dgr; could contribute to AF. Methods and Results— We found that oxidized CaMKII was increased in atria from AF patients compared with patients in sinus rhythm and from mice infused with angiotensin II compared with mice infused with saline. Angiotensin II–treated mice had increased susceptibility to AF compared with saline-treated wild-type mice, establishing angiotensin II as a risk factor for AF in mice. Knock-in mice lacking critical oxidation sites in CaMKII&dgr; (MM-VV) and mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, were resistant to AF induction after angiotensin II infusion. Conclusions— Our studies suggest that CaMKII is a molecular signal that couples increased reactive oxygen species with AF and that therapeutic strategies to decrease oxidized CaMKII may prevent or reduce AF.

Ca/Calmodulin Kinase II Differentially Modulates Potassium Currents

Background—Potassium currents contribute to action potential duration (APD) and arrhythmogenesis. In heart failure, Ca/calmodulin-dependent protein kinase II (CaMKII) is upregulated and can alter ion channel regulation and expression. Methods and Results—We examine the influence of overexpressing cytoplasmic CaMKII&dgr;C, both acutely in rabbit ventricular myocytes (24-hour adenoviral gene transfer) and chronically in CaMKII&dgr;C-transgenic mice, on transient outward potassium current (Ito), and inward rectifying current (IK1). Acute and chronic CaMKII overexpression increases Ito,slow amplitude and expression of the underlying channel protein KV1.4. Chronic but not acute CaMKII overexpression causes downregulation of Ito,fast, as well as KV4.2 and KChIP2, suggesting that KV1.4 expression responds faster and oppositely to KV4.2 on CaMKII activation. These amplitude changes were not reversed by CaMKII inhibition, consistent with CaMKII-dependent regulation of channel expression and/or trafficking. CaMKII (acute and chronic) greatly accelerated recovery from inactivation for both Ito components, but these effects were acutely reversed by AIP (CaMKII inhibitor), suggesting that CaMKII activity directly accelerates Ito recovery. Expression levels of IK1 and Kir2.1 mRNA were downregulated by CaMKII overexpression. CaMKII acutely increased IK1, based on inhibition by AIP (in both models). CaMKII overexpression in mouse prolonged APD (consistent with reduced Ito,fast and IK1), whereas CaMKII overexpression in rabbit shortened APD (consistent with enhanced IK1 and Ito,slow and faster Ito recovery). Computational models allowed discrimination of contributions of different channel effects on APD. Conclusion—CaMKII has both acute regulatory effects and chronic expression level effects on Ito and IK1 with complex consequences on APD.

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late I Na Augmentation Leading to Cellular Na and Ca Overload

Rationale: In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective: We tested whether CaMKII&dgr; is required for ROS-dependent late INa regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results: 40 &mgr;mol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 &mgr;mol/L), the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKII&dgr;−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKII&dgr;−/− myocytes (versus WT). CaMKII&dgr;−/− myocytes developed significantly less H2O2-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKII&dgr; in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 &mgr;mol/L KN93 or 0.1 &mgr;mol/L AIP [autocamtide 2–related inhibitory peptide]). Conclusions: Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKII&dgr; enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

Tubulin polymerization disrupts cardiac β-adrenergic regulation of late INa

AIMS The anticancer drug paclitaxel (TXL) that polymerizes microtubules is associated with arrhythmias and sinus node dysfunction. TXL can alter membrane expression of Na channels (NaV1.5) and Na current (INa), but the mechanisms are unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can be activated by β-adrenergic stimulation and regulates INa gating. We tested whether TXL interferes with isoproterenol (ISO)-induced activation of CaMKII and consequent INa regulation. METHODS AND RESULTS In wild-type mouse myocytes, the addition of ISO (1 µmol/L) resulted in increased CaMKII auto-phosphorylation (western blotting). This increase was completely abolished after pre-treatment with TXL (100 µmol/L, 1.5 h). The mechanism was further investigated in human embryonic kidney cells. TXL inhibited the ISO-induced β-arrestin translocation. Interestingly, both knockdown of β-arrestin2 expression using small interfering RNA and inhibition of exchange protein directly activated by cAMP (Epac) blocked the ISO-induced CaMKII auto-phosphorylation similar to TXL. The generation of cAMP, however, was unaltered (Epac1-camps). CaMKII-dependent Na channel function was measured using patch-clamp technique in isolated cardiomyoctes. ISO stimulation failed to induce CaMKII-dependent enhancement of late INa and Na channel inactivation (negative voltage shift in steady-state activation and enhanced intermediate inactivation) after pre-incubation with TXL. Consistent with this, TXL also inhibited ISO-induced CaMKII-specific Na channel phosphorylation (at serine 571 of NaV1.5). CONCLUSION Pre-incubation with TXL disrupts the ISO-dependent CaMKII activation and consequent Na channel regulation. This may be important for patients receiving TXL treatments, but also relevant for conditions of increased CaMKII expression and enhanced β-adrenergic stimulation like in heart failure.

ROS-activated Ca/calmodulin kinase IIδ is required for late INa augmentation leading to cellular Na and Ca overload

Rationale: In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective: We tested whether CaMKII&dgr; is required for ROS-dependent late INa regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results: 40 &mgr;mol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 &mgr;mol/L), the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKII&dgr;−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKII&dgr;−/− myocytes (versus WT). CaMKII&dgr;−/− myocytes developed significantly less H2O2-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKII&dgr; in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 &mgr;mol/L KN93 or 0.1 &mgr;mol/L AIP [autocamtide 2–related inhibitory peptide]). Conclusions: Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKII&dgr; enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

Inhibition of PI3K improves contractility in alpha1-adrenergically stimulated myocardium

Recent studies have demonstrated that phosphoinositide 3-kinases (PI3Ks) play a fundamental role in regulating myocardial contractility. However, even though alpha1-adrenergic receptor stimulation is known to activate PI3Ks, the impact of this pathway on the inotropic effects of alpha1-stimulation is unclear. Isolated rabbit ventricular myocytes were preincubated with the PI3K inhibitor wortmannin (WM, 0.1 micromol/L). The alpha1 agonist phenylephrine (PE, 10 micromol/L) induced a significantly stronger increase in contractility in WM-treated versus control myocytes (Fractional shortening in percent of resting cell length: 6.14+/-0.33 percent; n=26 versus 4.85+/-0.33 percent; n=26, P less than 0.05). Furthermore, pretreatment with WM significantly increased the positive inotropic effect of PE in intact muscle strips from rabbit hearts. Mechanistically, we demonstrate that in WM-treated myocytes PE increased phospholamban (PLN) phosphorylation and intracellular Ca2+ transients to a significantly greater extent than in control myocytes. In summary, this is the first study to demonstrate that inhibition of PI3K by increasing PLN phosphorylation and Ca2+ transients significantly improves contractility in alpha1-adrenergically stimulated myocardium. This may have clinical implications for the treatment of decreased cardiac function in acute heart failure.

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late INa Augmentation Leading to Cellular Na and Ca OverloadNovelty and Significance

Rationale: In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective: We tested whether CaMKII&dgr; is required for ROS-dependent late INa regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results: 40 &mgr;mol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 &mgr;mol/L), the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKII&dgr;−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKII&dgr;−/− myocytes (versus WT). CaMKII&dgr;−/− myocytes developed significantly less H2O2-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKII&dgr; in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 &mgr;mol/L KN93 or 0.1 &mgr;mol/L AIP [autocamtide 2–related inhibitory peptide]). Conclusions: Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKII&dgr; enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.