Lixia Yue

Ionic Remodeling Underlying Action Potential Changes in a Canine Model of Atrial Fibrillation

Rapid electrical activation, as occurs during atrial fibrillation (AF), is known to cause reductions in atrial refractoriness and in adaptation to heart rate of the atrial refractory period, which promote the maintenance of AF, but the underlying ionic mechanisms are unknown. In order to determine the cellular and ionic changes caused by chronic atrial tachycardia, we studied right atrial myocytes from dogs subjected to 1, 7, or 42 days of atrial pacing at 400/min and compared them with myocytes from sham-operated dogs (pacemaker inserted but not activated). Rapid pacing led to progressive increases in the duration of AF induced by bursts of 10-Hz stimuli (from 3 +/- 2 seconds in sham-operated dogs to 3060 +/- 707 seconds in dogs after 42 days of pacing, P < .001) and reduced atrial refractoriness and adaptation to rate of the atrial refractory period. Voltage-clamp studies showed that chronic rapid pacing did not alter inward rectifier K+ current, rapid or slow components of the delayed rectifier current, the ultrarapid delayed rectifier current, T-type Ca2+ current, or Ca(2+)-dependent Cl- current. In contrast, the densities of transient outward current (Ito) and L-type Ca2+ current (ICa) were progressively reduced as the duration of rapid pacing increased, without concomitant changes in kinetics or voltage dependence. In keeping with in vivo changes in refractoriness, action potential duration (APD) and APD adaptation to rate were decreased by rapid pacing. The response of the action potential and ionic currents flowing during the action potential (as exposed by action-potential voltage clamp) to nifedipine in normal canine cells and in cells from rapidly paced dogs suggested that the APD changes in paced dogs were largely due to reductions in ICa. We conclude that sustained atrial tachycardia reduces Ito and ICa, that the reduced ICa decreases APD and APD adaptation to rate, and that these cellular changes likely account for the alterations in atrial refractoriness associated with enhanced ability to maintain AF in the model.

Evidence for two components of delayed rectifier K+ current in human ventricular myocytes

Previous voltage-clamp studies have suggested that the delayed rectifier current (IK) is small or absent in the human ventricle and, when present, consists only of the rapid component (IKr); however, molecular studies suggest the presence of functionally important IK in the human heart, specific IKr blockers are known to delay ventricular repolarization and cause the long QT syndrome in humans, and we have shown that the expression of IK is strongly influenced by cell isolation techniques. The present experiments were designed to assess the expression of IK in myocytes obtained by arterial perfusion of right ventricular tissue from explanted human hearts. Of 35 cells from three hearts, 33 (94%) showed time-dependent currents typical of IK. The envelope-of-tails test was not satisfied under control conditions but became satisfied in the presence of the benzenesulfonamide E-4031 (5 micromol/L). E-4031 suppressed a portion of IK in 32 of 33 cells, with properties of the drug-sensitive and -resistant components consistent with previous descriptions of IKr and the slow component (IKs), respectively. Action potential duration to 95% repolarization at 1 Hz was prolonged by E-4031 from 336+/-16 (mean +/- SEM) to 421 +/- 19ms (n = 5, P < .01), indicating a functional role for IK. Indapamide, a diuretic agent previously shown to inhibit IKs selectively, suppressed E-4031-resistant current. The presence of a third type of delayed rectifier, the ultrarapid delayed rectifier current (IKur), was evaluated with the use of depolarizing prepulses and low concentrations (50 micromol/L) of 4-aminopyridine. Although these techniques revealed clear IKur in five of five human atrial cells, no corresponding component was observed in any of five human ventricular myocytes. We conclude that a functionally significant IK, with components corresponding to IKr and IKs, is present in human ventricular cells, whereas IKur appears to be absent. These findings are important for understanding the molecular, physiological, and pharmacological determinants of human ventricular repolarization and arrhythmias.

The TRPM7 channel is inactivated by PIP(2) hydrolysis.

TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase. The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC). Here, we show that in native cardiac cells and heterologous expression systems, Gαq-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity. Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2), the substrate of PLC, is a key regulator of TRPM7. We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP2, leading to inactivation of the TRPM7 channel.

Molecular Mechanisms Underlying Ionic Remodeling in a Dog Model of Atrial Fibrillation

The rapid atrial rate during atrial fibrillation (AF) decreases the ionic current density of transient outward K+ current, L-type Ca2+ current, and Na+ current, thereby altering cardiac electrophysiology and promoting arrhythmia maintenance. To assess possible underlying changes in cardiac gene expression, we applied competitive reverse transcriptase-polymerase chain reaction to quantify mRNA concentrations in dogs subjected to 7 (group P7 dogs) or 42 (group P42 dogs) days of atrial pacing at 400 bpm and in sham controls. Rapid pacing reduced mRNA concentrations of Kv4.3 (putative gene encoding transient outward K+ current; by 60% in P7 and 74% in P42 dogs; P<0.01 and P<0.001, respectively, versus shams), the alpha1c subunit of L-type Ca2+ channels (by 57% in P7 and 72% in P42 dogs; P<0.01 versus shams for each) and the alpha subunit of cardiac Na+ channels (by 18% in P7 and 42% in P42; P=NS and P<0.01, respectively, versus shams) genes. The observed changes in ion channel mRNA concentrations paralleled previously measured changes in corresponding atrial ionic current densities. Atrial tachycardia did not affect mRNA concentrations of genes encoding delayed or Kir2.1 inward rectifier K+ currents (of which the densities are unchanged by atrial tachycardia) or of the Na+,Ca2+ exchanger. Western blot techniques were used to quantify protein expression for Kv4.3 and Na+ channel alpha subunits, which were decreased by 72% and 47%, respectively, in P42 dogs (P<0.001 versus control for each), in a manner quantitatively similar to measured changes in mRNA and currents, whereas Na+,Ca2+ exchanger protein concentration was unchanged. We conclude that chronic atrial tachycardia alters atrial ion channel gene expression, thereby altering ionic currents in a fashion that promotes the occurrence of AF. These observations provide a potential molecular basis for the self-perpetuating nature of AF.

CaT1 manifests the pore properties of the calcium-release-activated calcium channel

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.

Functional characterization of homo- and heteromeric channel kinases TRPM6 and TRPM7.

TRPM6 and TRPM7 are two known channel kinases that play important roles in various physiological processes, including Mg2+ homeostasis. Mutations in TRPM6 cause hereditary hypomagnesemia and secondary hypocalcemia (HSH). However, whether TRPM6 encodes functional channels is controversial. Here we demonstrate several signature features of TRPM6 that distinguish TRPM6 from TRPM7 and TRPM6/7 channels. We show that heterologous expression of TRPM6 but not the mutant TRPM6S141L produces functional channels with divalent cation permeability profile and pH sensitivity distinctive from those of TRPM7 channels and TRPM6/7 complexes. TRPM6 exhibits unique unitary conductance that is 2- and 1.5-fold bigger than that of TRPM7 and TRPM6/7. Moreover, micromolar levels of 2-aminoethoxydiphenyl borate (2-APB) maximally increase TRPM6 but significantly inhibit TRPM7 channel activities; whereas millimolar concentrations of 2-APB potentiate TRPM6/7 and TRPM7 channel activities. Furthermore, Mg2+ and Ca2+ entry through TRPM6 is enhanced three- to fourfold by 2-APB. Collectively, these results indicate that TRPM6 forms functional homomeric channels as well as heteromeric TRPM6/7 complexes. The unique characteristics of these three channel types, TRPM6, TRPM7, and TRPM6/7, suggest that they may play different roles in vivo.

Transmural heterogeneity of action potentials andIto1 in myocytes isolated from the human right ventricle

Limited information is available about transmural heterogeneity in cardiac electrophysiology in man. The present study was designed to evaluate heterogeneity of cardiac action potential (AP), transient outward K+ current ( I to1) and inwardly rectifying K+ current ( I K1) in human right ventricle. AP and membrane currents were recorded using whole cell current- and voltage-clamp techniques in myocytes isolated from subepicardial, midmyocardial, and subendocardial layers of the right ventricle of explanted failing human hearts. AP morphology differed among the regional cell types. AP duration (APD) at 0.5-2 Hz was longer in midmyocardial cells (M cells) than in subepicardial and subendocardial cells. At room temperature, observed I to1, on step to +60 mV, was significantly greater in subepicardial (6.9 ± 0.8 pA/pF) and M cells (6.0 ± 1.1 pA/pF) than in subendocardial cells (2.2 ± 0.7 pA/pF, P < 0.01). Slower recovery of I to1 was observed in subendocardial cells. The half-inactivation voltage of I to1 was more negative in subendocardial cells than in M and subepicardial cells. At 36°C, the density of I to1 increased, the time-dependent inactivation and reactivation accelerated, and the frequency-dependent reduction attenuated in all regional cell types. No significant difference was observed in I K1 density among the regional cell types. The results indicate that M cells in humans, as in canines, show the greatest APD and that a gradient of I to1 density is present in the transmural ventricular wall. Therefore, the human right ventricle shows significant transmural heterogeneity in AP morphology and I to1properties.

Molecular determinants of cardiac fibroblast electrical function and therapeutic implications for atrial fibrillation.

Cardiac fibroblasts account for about 75% of all cardiac cells, but because of their small size contribute only ∼10-15% of total cardiac cell volume. They play a crucial role in cardiac pathophysiology. For a long time, it has been recognized that fibroblasts and related cell types are the principal sources of extracellular matrix (ECM) proteins, which organize cardiac cellular architecture. In disease states, fibroblast production of increased quantities of ECM proteins leads to tissue fibrosis, which can impair both mechanical and electrical function of the heart, contributing to heart failure and arrhythmogenesis. Atrial fibrosis is known to play a particularly important role in atrial fibrillation (AF). This review article focuses on recent advances in understanding the molecular electrophysiology of cardiac fibroblasts. Cardiac fibroblasts express a variety of ion channels, in particular voltage-gated K(+) channels and non-selective cation channels of the transient receptor potential (TRP) family. Both K(+) and TRP channels are important determinants of fibroblast function, with TRP channels acting as Ca(2+)-entry pathways that stimulate fibroblast differentiation into secretory myofibroblast phenotypes producing ECM proteins. Fibroblasts can couple to cardiomyocytes and substantially affect their cellular electrical properties, including conduction, resting potential, repolarization, and excitability. Co-cultured preparations of cardiomyocytes and fibroblasts generate arrhythmias by a variety of mechanisms, including spontaneous impulse formation and rotor-driven reentry. In addition, the excess ECM proteins produced by fibroblasts can interrupt cardiomyocyte-bundle continuity, leading to local conduction disturbances and reentrant arrhythmias. A better understanding of the electrical properties of fibroblasts should lead to an improved comprehension of AF pathophysiology and a variety of novel targets for antiarrhythmic intervention.

Differential Distribution of Inward Rectifier Potassium Channel Transcripts in Human Atrium Versus Ventricle

BACKGROUND The inward rectifier K+ current (IK1) plays an important role in governing cardiac electrical activity and is well known to have different properties in the atrium compared with the ventricle. Several inward rectifier K+ channel (IRK) subunits (hIRK, HH-IRK1, HIR, and TWIK-1) with different properties have been cloned from human tissues, but their relative expression in cardiac tissues has not been quantified. The present study was designed to define the relative levels of mRNA for various IRKs in human atrium and in failing and nonfailing ventricle. METHODS AND RESULTS Competitive reverse transcription-polymerase chain reaction was used to quantify in human atrium and ventricle the mRNA levels of hIRK, HH-IRK1, HIR, and TWIK-1. The absence of important noncardiac contamination was confirmed by demonstrating a lack of detectable mRNA markers for neuronal (acetylcholine receptor) and vascular (maxi-K channel) tissue. mRNA of HIR was more abundant in normal atrium (7.1+/-1.3 amol/ microg total RNA) than ventricle (0.6+/-0.1 amol/ microg, P<0. 05), whereas TWIK-1 mRNA was more concentrated in ventricle (18. 1+/-4.3 amol/ microg) than atrium (1.4+/-0.3 amol/ microg, P<0.05). Concentrations of hIRK (42.7+/-6.7 amol/ microg in atrium vs 57. 1+/-9.2 amol/ microg in ventricle) and HH-IRK1 (2.0+/-0.5 amol/ microg in atrium vs 1.5+/-0.5 amol/ microg in ventricle) were comparable. No significant differences in IRK subunit transcript concentrations were found between normal and failing ventricles. CONCLUSIONS mRNAs for all 4 IRKs are detected in human atrium and ventricle, but the mRNA copy number of a low-conductance subunit (HIR) is larger in atrium and the copy number of a weakly rectifying subunit (TWIK-1) is larger in ventricle. These differences in relative message levels may provide a potential molecular basis for different properties of IK1 in human atrium compared with ventricle.

TRPM7-Mediated Ca2+ Signals Confer Fibrogenesis in Human Atrial Fibrillation

Rationale: Cardiac fibrosis contributes to pathogenesis of atrial fibrillation (AF), which is the most commonly sustained arrhythmia and a major cause of morbidity and mortality. Although it has been suggested that Ca2+ signals are involved in fibrosis promotion, the molecular basis of Ca2+ signaling mechanisms and how Ca2+ signals contribute to fibrogenesis remain unknown. Objective: To determine the molecular mechanisms of Ca2+-permeable channel(s) in human atrial fibroblasts, and to investigate how Ca2+ signals contribute to fibrogenesis in human AF. Methods and Results: We demonstrate that the transient receptor potential (TRP) melastatin related 7 (TRPM7) is the molecular basis of the major Ca2+-permeable channel in human atrial fibroblasts. Endogenous TRPM7 currents in atrial fibroblasts resemble the biophysical and pharmacological properties of heterologous expressed TRPM7. Knocking down TRPM7 by small hairpin RNA largely eliminates TRPM7 current and Ca2+ influx in atrial fibroblasts. More importantly, atrial fibroblasts from AF patients show a striking upregulation of both TRPM7 currents and Ca2+ influx and are more prone to myofibroblast differentiation, presumably attributable to the enhanced expression of TRPM7. TRPM7 small hairpin RNA markedly reduced basal AF fibroblast differentiation. Transforming growth factor (TGF)-&bgr;1, the major stimulator of atrial fibrosis, requires TRPM7-mediated Ca2+ signal for its effect on fibroblast proliferation and differentiation. Furthermore, TGF-&bgr;1–induced differentiation of cultured human atrial fibroblasts is well correlated with an increase of TRPM7 expression induced by TGF-&bgr;1. Conclusions: Our results establish that TRPM7 is the major Ca2+-permeable channel in human atrial fibroblasts and likely plays an essential role in TGF-&bgr;1–elicited fibrogenesis in human AF.