Yin Hua Zhang

A Myocardial Nox2 Containing NAD(P)H Oxidase Contributes to Oxidative Stress in Human Atrial Fibrillation

Human atrial fibrillation (AF) has been associated with increased atrial oxidative stress. In animal models, inhibition of reactive oxygen species prevents atrial remodeling induced by rapid pacing, suggesting that oxidative stress may play an important role in the pathophysiology of AF. NAD(P)H oxidase is a major source of superoxide in the cardiovascular system; however, whether this enzyme contributes to atrial oxidative stress in AF remains to be elucidated. We investigated the sources of superoxide production (using inhibitors and substrates of a range of oxidases, RT-PCR, immunofluorescence, and immunoblotting) in tissue homogenates and isolated atrial myocytes from the right atrial appendage (RAA) of patients undergoing cardiac surgery (n=54 in sinus rhythm [SR] and 15 in AF). A membrane-bound gp91phox containing NAD(P)H oxidase in atrial myocytes was the main source of atrial superoxide production in SR and in AF. NADPH-stimulated superoxide release from RAA homogenates was significantly increased in patients with AF in the absence of changes in mRNA expression of the p22phox and gp91phox subunits of the NAD(P)H oxidase. In contrast with findings in SR patients, NO synthases (NOSs) contributed significantly to atrial superoxide production in fibrillating atria, suggesting that increased oxidative stress in AF may lead to NOS “uncoupling.” These findings indicate that a myocardial NAD(P)H oxidase and, to a lesser extent, dysfunctional NOS contribute significantly to superoxide production in the fibrillating human atrial myocardium and may play an important role in the atrial oxidative injury and electrophysiological remodeling observed in patients with AF.

Stretch-activated and background non-selective cation channels in rat atrial myocytes

1 Stretch‐activated channels (SACs) were studied in isolated rat atrial myocytes using the whole‐cell and single‐channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. 2 In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from ‐63·6 ± 0·58 mV (n= 19) to ‐54·6 ± 2·4 mV (n= 13) and prolonged the action potential duration (APD) by 32·2 ± 8·8 ms (n= 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current‐voltage (I–V) relationship of the stretch‐activated current (ISAC) was linear and reversed at ‐6·1 ± 3·7 mV (n= 7). 3 The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl− channel blocker DIDS or removal of external Cl−. The permeability ratio for various cations (PCs:PNa:PLi= 1·05:1:0·98) indicated that the SAC current was a non‐selective cation current (ISAC,NC). The background current was also found to be non‐selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi= 1·49:1:0·70) was different from that of ISAC,NC. 4 Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration‐dependent manner with an IC50 value of 46·2 ± 0·8 μM (n= 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (‐71·1 ± 0·26 mV, n= 9). In the presence of Gd3+ (0·1 mM), stretch still induced ISAC,NC and diastolic depolarisation. 5 Single‐channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside‐out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 μM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 μM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21·27 ± 0·24 pS; n= 7), which was distinct from background activity. 6 We concluded from the above results that longitudinal stretch in rat atrial myocytes induces the activation of non‐selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.

Reduced Phospholamban Phosphorylation Is Associated With Impaired Relaxation in Left Ventricular Myocytes From Neuronal NO Synthase–Deficient Mice

Stimulation of nitric oxide (NO) release from the coronary endothelium facilitates myocardial relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity. Recent evidence suggests that NO released by a neuronal NO synthase (nNOS) in the myocardium can also hasten left ventricular relaxation; however, the mechanism underlying these findings is uncertain. Here we show that both relaxation (TR50) and the rate of [Ca2+]i transient decay (tau) are significantly prolonged in field-stimulated or voltage-clamped left ventricular myocytes from nNOS−/− mice and in wild-type myocytes (nNOS+/+) after acute nNOS inhibition. Disabling the sarcoplasmic reticulum abolished the differences in TR50 and tau, suggesting that impaired sarcoplasmic reticulum Ca2+ reuptake may account for the slower relaxation in nNOS−/− mice. In line with these findings, disruption of nNOS (but not of endothelial NOS) decreased phospholamban phosphorylation (P-Ser16 PLN), whereas nNOS inhibition had no effect on TR50 or tau in PLN−/− myocytes. Inhibition of cGMP signaling had no effect on relaxation in either group whereas protein kinase A inhibition abolished the difference in relaxation and PLN phosphorylation by decreasing P-Ser16 PLN and prolonging TR50 in nNOS+/+ myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR50 and increased P-Ser16 PLN in nNOS−/− but not in nNOS+/+ myocytes, in agreement with data showing increased protein phosphatase activity in nNOS−/− hearts. Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes left ventricular relaxation by regulating the protein kinase A–mediated phosphorylation of PLN and the rate of sarcoplasmic reticulum Ca2+ reuptake via a cGMP-independent effect on protein phosphatase activity.

AMP-Activated Protein Kinase Phosphorylates Cardiac Troponin I and Alters Contractility of Murine Ventricular Myocytes

Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory &ggr;2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. Objective: The &ggr; isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of &ggr;2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of &ggr;2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca2+ sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca2+ transient and prolonged relaxation despite shortening the time constant of Ca2+ transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca2+ sensitivity. Conclusions: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Inhibition of HERG K+ Current and Prolongation of the Guinea‐Pig Ventricular Action Potential by 4‐Aminopyridine

4‐Aminopyridine (4‐AP) has been used extensively to study transient outward K+ current (ITO,1) in cardiac cells and tissues. We report here inhibition by 4‐AP of HERG (the human ether‐à‐go‐go‐related gene) K+ channels expressed in a mammalian cell line, at concentrations relevant to those used to study ITO,1. Under voltage clamp, whole cell HERG current (IHERG) tails following commands to +30 mV were blocked with an IC50 of 4.4 ± 0.5 mm. Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 mm 4‐AP inhibited peak IHERG during an applied action potential clamp waveform by ∼59 %. It also significantly prolonged action potentials and inhibited resurgent IK tails from guinea‐pig isolated ventricular myocytes, which lack an ITO,1. We conclude that by blocking the α‐subunit of the IKr channel, millimolar concentrations of 4‐AP can modulate ventricular repolarisation independently of any action on ITO,1.

A mathematical model of the murine ventricular myocyte: a data-driven biophysically based approach applied to mice overexpressing the canine NCX isoform.

Mathematical modeling of Ca(2+) dynamics in the heart has the potential to provide an integrated understanding of Ca(2+)-handling mechanisms. However, many previous published models used heterogeneous experimental data sources from a variety of animals and temperatures to characterize model parameters and motivate model equations. This methodology limits the direct comparison of these models with any particular experimental data set. To directly address this issue, in this study, we present a biophysically based model of Ca(2+) dynamics directly fitted to experimental data collected in left ventricular myocytes isolated from the C57BL/6 mouse, the most commonly used genetic background for genetically modified mice in studies of heart diseases. This Ca(2+) dynamics model was then integrated into an existing mouse cardiac electrophysiology model, which was reparameterized using experimental data recorded at consistent and physiological temperatures. The model was validated against the experimentally observed frequency response of Ca(2+) dynamics, action potential shape, dependence of action potential duration on cycle length, and electrical restitution. Using this framework, the implications of cardiac Na(+)/Ca(2+) exchanger (NCX) overexpression in transgenic mice were investigated. These simulations showed that heterozygous overexpression of the canine cardiac NCX increases intracellular Ca(2+) concentration transient magnitude and sarcoplasmic reticulum Ca(2+) loading, in agreement with experimental observations, whereas acute overexpression of the murine cardiac NCX results in a significant loss of Ca(2+) from the cell and, hence, depressed sarcoplasmic reticulum Ca(2+) load and intracellular Ca(2+) concentration transient magnitude. From this analysis, we conclude that these differences are primarily due to the presence of allosteric regulation in the canine cardiac NCX, which has not been observed experimentally in the wild-type mouse heart.

The role of nitric oxide and reactive oxygen species in the positive inotropic response to mechanical stretch in the mammalian myocardium.

The endothelial nitric oxide synthase (eNOS) has been implicated in the rapid (Frank–Starling) and slow (Anrep) cardiac response to stretch. Our work and that of others have demonstrated that a neuronal nitric oxide synthase (nNOS) localized to the myocardium plays an important role in the regulation of cardiac function and calcium handling. However, the effect of nNOS on the myocardial response to stretch has yet to be investigated. Recent evidence suggests that the stretch-induced release of angiotensin II (Ang II) and endothelin 1 (ET-1) stimulates myocardial superoxide production from NADPH oxidases which, in turn, contributes to the Anrep effect. nNOS has also been shown to regulate the production of myocardial superoxide, suggesting that this isoform may influence the cardiac response to stretch or ET-1 by altering the NO-redox balance in the myocardium. Here we show that the increase in left ventricular (LV) myocyte shortening in response to the application of ET-1 (10 nM, 5 min) did not differ between nNOS−/− mice and their wild type littermates (nNOS+/+). Pre-incubating LV myocytes with the NADPH oxidase inhibitor, apocynin (100 μM, 30 min), reduced cell shortening in nNOS−/− myocytes only but prevented the positive inotropic effects of ET-1 in both groups. Superoxide production (O2−) was enhanced in nNOS−/− myocytes compared to nNOS+/+; however, this difference was abolished by pre-incubation with apocynin. There was no detectable increase in O2− production in ET-1 pre-treated LV myocytes. Inhibition of protein kinase C (chelerythrine, 1 μM) did not affect cell shortening in either group, however, protein kinase A inhibitor, PKI (2 μM), significantly reduced the positive inotropic effects of ET-1 in both nNOS+/+ and nNOS−/− myocytes. Taken together, our findings show that the positive inotropic effect of ET-1 in murine LV myocytes is independent of nNOS but requires NADPH oxidases and protein kinase A (PKA)-dependent signaling. These results may further our understanding of the signaling pathways involved in the myocardial inotropic response to stretch.

Gadolinium inhibits Na+‐Ca2+ exchanger current in guinea‐pig isolated ventricular myocytes

The trivalent cation, gadolinium (Gd3+) is commonly used to inhibit stretch‐activated channels. In this report, we show that Gd3+ also inhibits ionic current (INaCa), carried by the Na+‐Ca2+ exchanger protein. Under selective recording conditions, Gd3+ inhibited both outward and inward INaCa from guinea‐pig isolated ventricular myocytes in a dose‐dependent manner, with half‐maximal inhibition concentrations (IC50) of 30.0±4.0 μM at +60 mV (Hill‐coefficient, h=1.04±0.13) and 20.0±2.7 μM at −100 mV (h=1.13±0.16), respectively (P>0.05, n=5–9). Thus, inhibition was not voltage‐dependent. The time from Gd3+ application to steady‐state effect was slow compared to the divalent blocker Ni2+. The slow time course appeared to reflect gradual Gd3+ accumulation at its binding site on the exchanger, rather than a use‐dependent blocking mechanism. This study indicates that for experiments in which Gd3+ is used, its inhibitory effect on INaCa should be taken into account.

A Novel, Voltage-Dependent Nonselective Cation Current Activated by Insulin in Guinea Pig Isolated Ventricular Myocytes

&NA; —Insulin regulates cardiac metabolism and function by targeting metabolic proteins or voltage‐gated ion channels. This study provides evidence for a novel, voltage‐dependent, nonselective cation channel (NSCC) in the heart. Under voltage clamp at 37°C and with major known conductances blocked, insulin (1 nmol/L to 1 &mgr;mol/L) activated an outwardly rectifying current (Iinsulin) in guinea pig ventricular myocytes. Iinsulin could be carried by Cs+, K+, Li+, and Na+ ions but not by NMDG+. It was inhibited by the NSCC blockers gadolinium and SKF96365 but not flufenamic acid. Iinsulin was largely blocked by the insulin receptor tyrosine kinase inhibitor HNMPA‐(AM)3 and by the phospholipase C inhibitor U73122 but not by its inactive analogue U73433. Staurosporine, a potent blocker of protein kinase C, did not prevent the activation of Iinsulin. Application of an analogue of diacylglycerol, 1‐oleoyl‐2‐acetyl‐sn‐glycerol, mimicked the effect of insulin. This activated an outwardly rectifying NSCC that could be carried by Cs+, K+, Li+, or Na+ and that was blocked by gadolinium but not by flufenamic acid or staurosporine. We conclude that the intracellular pathway leading to activation of this novel cardiac NSCC involves phospholipase C, is protein kinase C‐independent, and may depend on direct channel activation by diacylglycerol. (Circ Res. 2003;92:765–768.)

Role of thromboxane A2-activated nonselective cation channels in hypoxic pulmonary vasoconstriction of rat

Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K(+) channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K(+) channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A(2) (TXA(2)) is required for induction of HPV, we hypothesize that TXA(2) receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K(+) channel (K(v)). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (I(NSC,TXA2)) with weakly outward rectifying current-voltage relation. I(NSC,TXA2) was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of I(NSC,TXA2) activation and K(v) inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA(2)-activated NSC in rat PASMC.