Alvin Shrier

Effects of Experimental Heart Failure on Atrial Cellular and Ionic Electrophysiology

BACKGROUND Congestive heart failure (CHF) is frequently associated with atrial fibrillation (AF), but little is known about the effects of CHF on atrial cellular electrophysiology. METHODS AND RESULTS We studied action potential (AP) properties and ionic currents in atrial myocytes from dogs with CHF induced by ventricular pacing at 220 to 240 bpm for 5 weeks. Atrial myocytes from CHF dogs were hypertrophied (mean+/-SEM capacitance, 89+/-2 pF versus 71+/-2 pF in control, n=160 cells per group, P<0.001). CHF significantly reduced the density of L-type Ca(2+) current (I(Ca)) by approximately 30%, of transient outward K(+) current (I(to)) by approximately 50%, and of slow delayed rectifier current (I(Ks)) by approximately 30% without altering their voltage dependencies or kinetics. The inward rectifier, ultrarapid and rapid delayed rectifier, and T-type Ca(2+) currents were not altered by CHF. CHF increased transient inward Na(+)/Ca(2+) exchanger (NCX) current by approximately 45%. The AP duration of atrial myocytes was not altered by CHF at slow rates but was increased at faster rates, paralleling in vivo refractory changes. CHF created a substrate for AF, prolonging mean AF duration from 8+/-4 to 535+/-82 seconds (P<0.01). CONCLUSIONS Experimental CHF selectively decreases atrial I(to), I(Ca), and I(Ks), increases NCX current, and leaves other currents unchanged. The cellular electrophysiological remodeling caused by CHF is quite distinct from that caused by atrial tachycardia, highlighting important differences in the cellular milieu characterizing different clinically relevant AF substrates.

N‐linked glycosylation sites determine HERG channel surface membrane expression

1 Long QT syndrome (LQT) is an electrophysiological disorder that can lead to sudden death from cardiac arrhythmias. One form of LQT has been attributed to mutations in the human ether‐a‐go‐go‐related gene (HERG) that encodes a voltage‐gated cardiac K+ channel. While a recent report indicates that LQT in some patients is associated with a mutation of HERG at a consensus extracellular N‐linked glycosylation site (N629), earlier studies failed to identify a role for N‐linked glycosylation in the functional expression of voltage‐gated K+ channels. In this study we used pharmacological agents and site‐directed mutagenesis to assess the contribution of N‐linked glycosylation to the surface localization of HERG channels. 2 Tunicamycin, an inhibitor of N‐linked glycosylation, blocked normal surface membrane expression of a HERG‐green fluorescent protein (GFP) fusion protein (HERGGFP) transiently expressed in human embryonic kidney (HEK 293) cells imaged with confocal microscopy. 3 Immunoblot analysis revealed that N‐glycosidase F shifted the molecular mass of HERGGFP, stably expressed in HEK 293 cells, indicating the presence of N‐linked carbohydrate moieties. Mutations at each of the two putative extracellular N‐linked glycosylation sites (N598Q and N629Q) led to a perinuclear subcellular localization of HERGGFP stably expressed in HEK 293 cells, with no surface membrane expression. Furthermore, patch clamp analysis revealed that there was a virtual absence of HERG current in the N‐glycosylation mutants. 4 Taken together, these results strongly suggest that N‐linked glycosylation is required for surface membrane expression of HERG. These findings may provide insight into a mechanism responsible for LQT2 due to N‐linked glycosylation‐related mutations of HERG.

Bifurcation and chaos in a periodically stimulated cardiac oscillator

Abstract Periodic stimulation of an aggregate of spontaneously beating cultured cardiac cells displays phase locking, period-doubling bifurcations and aperiodic “chaotic” dynamics at different values of the stimulation parameters. This behavior is analyzed by considering an experimentally determined one-dimensional Poincare or first return map. A simplified version of the experimentally determined Poincare map is proposed, and several features of the bifurcations of this map are described.

Prediction of complex atrioventricular conduction rhythms in humans with use of the atrioventricular nodal recovery curve.

Theoretical considerations indicate that complex patterns of atrioventricular conduction produced by rapid atrial stimulation can be predicted from changes in atrioventricular conduction produced by premature stimulation of the atrium. The purpose of this study was to evaluate the validity of this theoretical approach in seven patients undergoing electrophysiologic investigation. The atrioventricular nodal recovery curve was determined at two different basic cycle lengths. Subsequently, periodic atrial stimulation was delivered for 30 sec periods over a range of frequencies giving 11, Wenckebach, reverse Wenckebach, and 21 rhythms. The recovery curve data was then used to compute the response to periodic stimulation by an iterative technique. The conduction patterns actually seen during periodic atrial stimulation showed close agreement with the computed patterns. This work thus provides a unified explanation for the appearance of Wenckebach, reverse Wenckebach, alternating Wenckebach, and high grade block rhythms.

Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node.

1. Experiments were conducted using the whole‐cell patch clamp technique to determine the electrophysiological properties and ionic currents of ovoid and rod‐shaped single isolated calcium‐tolerant rabbit atrioventricular (AV) nodal cells. 2. Action potential morphologies observed in these cells were similar to those obtained previously from intracellular recordings of intact atrioventricular nodal preparations: ovoid cells had N‐ or NH‐like action potential configurations (see below), whereas rod‐shaped cells had AN‐like configurations. 3. Action potential restitution in AV nodal cells was characterized by a progressive increase in overshoot potential, maximal upstroke velocity (Vmax) and action potential duration, as well as a decrease in latency from stimulus to Vmax. In rod‐shaped cells, premature stimuli could induce regenerative membrane responses before full action potential repolarization, whereas ovoid cells showed only post‐repolarization refractoriness. In ovoid cells stimulated at the low stimulus intensities there was no shortening of the action potential duration and the most premature action potentials were often prolonged. 4. The quasi‐steady‐state current‐voltage relationship of ovoid cells was significantly steeper, at both depolarized and hyperpolarized potentials, than that of either the rod‐shaped AV nodal cells or atrial cells. The rod‐shaped AV nodal cells and the atrial cells had similar current‐voltage (I‐V) relationships in the positive potential range, but the I‐V curves crossed over at potentials of about‐90 mV. 5. A hyperpolarization‐activated inward current (I(f)) was apparent in the range between ‐60 and ‐90 mV in 95% of the ovoid cells (n = 75), whereas in 88% of rod‐shaped cells (n = 16) I(f) was activated at more negative potentials. The magnitude of I(f) in ovoid cells, measured at ‐100 mV, was approximately 25 times that in rod‐shaped cells. 6. A rapid inward current (INa) greater than 1 nA was found in all rod‐shaped cells (n = 16) but in only 30% of ovoid cells (n = 75). A transient outward current (I(to)) was found in 93% of rod‐shaped cells (n = 14) and in 42% of ovoid cells (n = 54). The combination of I(to) and INa was found in 93% of rod‐shaped cells but in only 24% of ovoid cells. 7. These results suggest that there are at least two populations of isolated AV nodal cells with distinct action potentials and ionic current profiles that may contribute to the complex electrophysiological properties observed in the intact AV node.

Co-chaperone FKBP38 Promotes HERG Trafficking

The Long QT Syndrome is a cardiac disorder associated with ventricular arrhythmias that can lead to syncope and sudden death. One prominent form of the Long QT syndrome has been linked to mutations in the HERG gene (KCNH2) that encodes the voltage-dependent delayed rectifier potassium channel (IKr). In order to search for HERG-interacting proteins important for HERG maturation and trafficking, we conducted a proteomics screen using myc-tagged HERG transfected into cardiac (HL-1) and non-cardiac (human embryonic kidney 293) cell lines. A partial list of putative HERG-interacting proteins includes several known components of the cytosolic chaperone system, including Hsc70 (70-kDa heat shock cognate protein), Hsp90 (90-kDa heat shock protein), Hdj-2, Hop (Hsp-organizing protein), and Bag-2 (BCL-associated athanogene 2). In addition, two membrane-integrated proteins were identified, calnexin and FKBP38 (38-kDa FK506-binding protein, FKBP8). We show that FKBP38 immunoprecipitates and co-localizes with HERG in our cellular system. Importantly, small interfering RNA knock down of FKBP38 causes a reduction of HERG trafficking, and overexpression of FKBP38 is able to partially rescue the LQT2 trafficking mutant F805C. We propose that FKBP38 is a co-chaperone of HERG and contributes via the Hsc70/Hsp90 chaperone system to the trafficking of wild type and mutant HERG potassium channels.

Subcellular localization of the Na+/H+ exchanger NHE1 in rat myocardium

The Na+/H+ exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na+-K+-ATPase alpha1-subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (e.g., ryanodine receptor), thereby influencing impulse conduction and excitation-contraction coupling.The Na+/H+exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na+-K+-ATPase α1-subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (e.g., ryanodine receptor), thereby influencing impulse conduction and excitation-contraction coupling.

Nonlinear dynamics, chaos and complex cardiac arrhythmias

Periodic stimulation of a nonlinear cardiac oscillator in vitro gives rise to complex dynamics that is well described by one-dimensional finite difference equations. As stimulation parameters are varied, a large number of different phase locked and chaotic rhythms is observed. Similar rhythms can be observed in the intact human heart when there is interaction between two pacemaker sites. Simplified models are analysed, which show some correspondence to clinical observations.

Identification of the cyclic-nucleotide-binding domain as a conserved determinant of ion-channel cell-surface localization

Mutations of a putative cyclic-nucleotide-binding domain (CNBD) can disrupt the function of the hyperpolarization-activated cyclic-nucleotide-gated channel (HCN2) and the human ether-a-go-go-related gene potassium channel (HERG). Loss of function caused by C-terminal truncation, which includes all or part of the CNBD in HCN and HERG, has been related to abnormal channel trafficking. Similar defects have been reported for several of the missense mutations of HERG associated with long QT syndrome type 2 (LQT2). Thus, we postulate that normal processing of these channels depends upon the presence of the CNBD. Here, we show that removal of the entire CNBD prevents Golgi transit, surface localization and function of HERG channel tetramers. This is also true when any of the structural motifs of the CNBD is deleted, suggesting that deletion of any highly conserved region along the entire length of the CNBD can disrupt channel trafficking. Furthermore, we demonstrate that defective trafficking is a consequence of all LQT2 mutations in the CNBD, including two mutations not previously assessed and two others for which there are conflicting results in the literature. The trafficking sensitivity of the CNBD might be of general significance for other ion channels because complete deletion of the CNBD or mutations at highly conserved residues within the CNBD of the related ERG3 channel and HCN2 also prevent Golgi transit. These results broadly implicate the CNBD in ion-channel trafficking that accounts for the commonly observed loss of function associated with CNBD mutants and provides a rationale for distinct genetic disorders.

Hsp40 Chaperones Promote Degradation of the hERG Potassium Channel

Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.