Terry B. Rogers

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

Phorbol ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes.

The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomltantly. In contrast, 4-α-phorbol-12, 13-didecanoate (α-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin n enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism.

1. The regulation of the cardiac Ca2+ release channel‐ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)‐calmodulin‐dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1‐20 U ml‐1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)‐dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/‐ 0.85 and 3.9 +/‐ 0.65 nM; Bmax (the maximal receptor density), 1.45 +/‐ 0.14 and 1.47 +/‐ 0.12 pmol mg‐1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were ‘dormant’ due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non‐hydrolysable ATP analogue adenylyl[beta, gamma‐methylene]‐diphosphate (AMP‐PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation‐dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca2+ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca2+ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca2+ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca2+ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca2+ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca2+ and the role of the Ca2+ ATPase in intact myocytes as well as in genetically altered heart cells.

Ca2+ Channel Modulation by Recombinant Auxiliary β Subunits Expressed in Young Adult Heart Cells

Abstract—L-type Ca2+ channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca2+ channel auxiliary β1–4 subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study β-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant β subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-l-lysine, and expression plasmids encoding β subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on gre...

Dynamic modulation of excitation‐contraction coupling by protein phosphatases in rat ventricular myocytes.

1. The effects of the serine/threonine protein phosphatases (PP) type 1 and 2A on L‐type Ca2+ current (ICa) and the intracellular [Ca2+]i transient were examined in rat ventricular myocytes. ICa was measured under voltage clamp using patch‐type microelectrodes in the whole‐cell mode with the cells in a steady state of sarcoplasmic reticulum (SR) Ca2+ loading. [Ca2+]i transients were measured simultaneously using the fluorescent Ca2+ indicator indo‐1 (50 microM) which was added to the pipette filling solution along with PP‐1 or PP‐2A (4 units ml‐1). 2. PP‐1 had no effect on the ICa‐V relationship but decreased the [Ca2+]i‐voltage relationship (by 43% at 0 mV). PP‐2A decreased both ICa‐V (by 26% at 0 mV) and the [Ca2+]i transient‐voltage (by 65% at 0 mV). Excitation‐contraction coupling gain, defined as (delta [Ca2+]i/ICa), was decreased to 43% of control by PP‐1 and to 29% of control by PP‐2A at‐28 mV. 3. Diastolic [Ca2+]i (i.e.[Ca2+]i measured immediately before each voltage clamp pulse) was not altered by PP‐1 or PP‐2A and neither phosphatase changed steady‐state SR Ca2+ content, as measured with caffeine. 4. We conclude that the reduced [Ca2+]i transient following the application of PP‐1 was due to reduced SR Ca2+ release channel activity. The effects of PP‐2A, while more broadly based, were still consistent with a decrease in SR Ca2+ release channel activity. 5. Our experiments, combined with recent experiments by others, suggest that the basal state of contractility in heart is dynamically regulated by dephosphorylation and phosphorylation of the SR Ca2+ release channel.

Effect of the immunosupressant FK506 on excitation—contraction coupling and outward K+ currents in rat ventricular myocytes

1 We examined the effects of the immunosupressant drug FK506 on excitation–contraction coupling in isolated rat ventricular myocytes. [Ca2+]i transients were recorded using the intracellular Ca2+ indicators fluo‐3 and indo‐1 while action potentials (APs) or membrane currents were recorded using patch‐type microelectrodes in the whole cell mode. 2 FK506 (25 (μm) rapidly and reversibly increased the magnitude of the [Ca2+]i transient in intact cells without changing resting [Ca2+]i or the kinetics of the [Ca2+]i transient, a finding consistent with previous reports that investigated the actions of FK506 on the sarcoplasmic reticulum Ca2+ release channel. 3 The 36% increase in the [Ca2+]i transient produced by FK506 was accompanied by a 293% increase in AP duration (by 293%). Importantly, the addition of FK506 had no effect on the [Ca2+]i transient when the depolarizing duration was controlled in voltage clamp experiments. The increased AP duration could be explained by a marked inward shift in the net membrane current that was observed in these experiments. 4 The net inward current change was not directly responsible for a change in Ca2+ influx, since no change in L‐type Ca2+ current (ICa) was observed. Instead, FK506 inhibited both the transient outward K+ current (Ito) and the delayed rectifier K+ current (IK). 5 We conclude that FK506 increases the [Ca2+]i transient during normal contractions by an indirect action: it prolongs the action potential. This action does not appear to depend on the established action of FK506 on the ryanodine receptor. Instead, the inhibition of outward K+ currents prolongs the AP which secondarily increases Ca2+ influx and/or decreases Ca2+ efflux.

Angiotensin-induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level of the receptor.

Previous studies show that angiotensin II (Ang II) increases phosphoinositide turnover in cultured neonatal heart cells. Ang II has also been shown to transiently increase spontaneous beating behavior in these cells. In this study we seek to identify the molecular mechanism underlying this rapid (3-5-minute) desensitization. Time-course studies on the accumulation of [3H]inositol phosphates indicate that the loss in functional responsiveness correlates with reduced efficacy of Ang II to activate the phosphoinositide path. Binding studies with 125I-Ang II revealed that there was no change in surface receptor binding capacity during the time in which desensitization developed. Normal phosphoinositide and functional responses are observed when desensitized cells are treated with probes that activate the cardiac phosphoinositide pathway at discrete steps. These studies reveal that the functional status of the major components of the phosphoinositide signaling pathway, including G proteins, phospholipase C, and protein kinase C (PKC), are normal during maintained Ang II desensitization. To study the potential role of PKC in Ang II desensitization, the cells are treated with TPA for 24 hours, which downregulates PKC activity. PKC-depleted cells rapidly desensitize after Ang II application. We conclude that the selective Ang II-evoked biochemical/functional desensitization involves inhibition at the level of the receptor, rather than at a component downstream in the path, and that this process is independent of PKC and loss of surface binding capacity.

Protein phosphatase 1 and an opposing protein kinase regulate steady‐state L‐type Ca2+ current in mouse cardiac myocytes

Studies have suggested that integration of kinase and phosphatase activities maintains the steady‐state L‐type Ca2+ current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L‐type ICa, the goal of this study was to identify the counteracting kinase and phosphatase that determine ‘basal’ICa in isolated mouse ventricular myocytes. Whole‐cell voltage‐clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased ICa at test potentials between −42 and +49mV (44% at 0mV) from a holding potential of −80mV. It also shifted the V0.5 (membrane potential at half‐maximal) of both activation (from −17 to −25mV) and steady‐state inactivation (from −32 to −37mV) in the hyperpolarizing direction. The broad‐spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on ICa when added after calyculin A. However, by itself, staurosporine decreased ICa throughout the voltage range examined (50% at 0mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitors effects depend upon an opposing kinase activity. The PKA inhibitors Rp‐cAMPs (100 μm in the pipette) and H89 (1 μm) failed to reduce basal ICa or to block the calyculin A‐evoked increase in ICa. Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba2+ was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin‐dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31‐8220 (1 μm) and Gö 6976 (200 nm) decreased steady‐state ICa and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10–100 nm okadaic acid or 500 nm fostriecin failed to increase ICa. However, PP1 is important, as dialysis with 2 μm okadaic acid or 500 nm inhibitor‐2 mimicked the increases in ICa seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor‐mediated signalling cascades, as regulatory components that determine the steady‐state level of cardiac L‐type ICa.

An Andersen-Tawil Syndrome Mutation in Kir2.1 (V302M) Alters the G-loop Cytoplasmic K+ Conduction Pathway

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.