Normand Leblanc

Cellular Mechanisms of Atrial Contractile Dysfunction Caused by Sustained Atrial Tachycardia

BACKGROUNDnTransient atrial contractile dysfunction (atrial stunning) follows conversion of atrial fibrillation (AF) to sinus rhythm and has significant clinical implications; however, the underlying mechanisms are poorly understood. We investigated the hypothesis that rapid atrial activation (as during AF) impairs cellular contractility and affects cellular Ca2+ handling.nnnMETHODS AND RESULTSnEdge detection and indo 1 fluorescence techniques were used to measure unloaded cell shortening and intracellular Ca2+ transients in atrial myocytes from control (Ctl) dogs and dogs subjected to atrial pacing at 400 bpm for 7 (P7) or 42 (P42) days. Atrial tachycardia reduced fractional cell shortening (0.1 Hz) from 7.3+/-0.4% (Ctl) to 4.3+/-0.3% and 2.0+/-0.3% in P7 and P42 dogs, respectively (P<0.01 for each). Resting [Ca2+]i was not altered in paced dogs, but the systolic Ca2+ transient was significantly reduced. Furthermore, cells from paced dogs showed slowed relaxation and use-dependent decreases of Ca2+ transients and cell shortening compared with cells from Ctl dogs. To determine whether changes in Ca2+ transients account fully for alterations in contractility, we varied [Ca2+]o to evaluate the relation between Ca2+ transients and cell shortening. Reductions in Ca2+ transients in Ctl cells reduced shortening to the level of paced cells; however, when Ca2+ transients in P42 cells were elevated to the range of Ctl cells, a significant reduction in cell shortening remained. Similar results were obtained in dogs that maintained 1:1 capture throughout the monitoring period and dogs that developed sustained AF over the course of the study.nnnCONCLUSIONSnSustained atrial tachycardia causes important reductions in cellular contractility, in part by impairing cellular Ca2+ handling and decreasing systolic Ca2+ transients. These results provide direct evidence for the concept that AF induces atrial contractile dysfunction by causing a tachycardia-induced atrial cardiomyopathy.

Age and gender differences in excitation-contraction coupling of the rat ventricle

1 The objective of this study was to determine potential post‐pubertal gender‐specific differences in the contractility of papillary muscles, the electrophysiological properties and Ca2+ transients of freshly dissociated ventricular myocytes from the rat heart. 2 The contractions of rat papillary muscles from 2‐ to 14‐month‐old male and female rats were studied under isometric and isotonic conditions (29 °C). While the hearts of young (2–4 months) male and female rats displayed a similar contractile profile, papillary muscles of female rats aged 6 months and older exhibited smaller isometric and isotonic contractions, smaller maximal rates of tension and shortening development and decline (±DT/dt and ±DL/dt) velocities during both the onset and relaxation phases, and shorter contractions than age‐matched males. 3 To explore the possible cellular basis accounting for these differences, action potentials and macroscopic currents were recorded from freshly dissociated myocytes using the whole‐cell patch clamp technique (35 °C). Action potentials from male and female myocytes of 3‐ and 9‐month‐old rats did not vary as a function of age or gender. Consistent with these results, the magnitude (expressed in pA pF−1), voltage‐dependence and kinetics of the inward rectifier (IK1), transient outward (Ito) and sustained (IK) K+ currents displayed little, if any dependence on age or gender. 4 L‐type Ca2+ current (ICa(L)) measured in caesium‐loaded myocytes (35 °C) from male and female rats of 3, 6 and 9 months of age exhibited similar characteristics. In contrast, while Ca2+ transients measured with indo‐1 were similar between 3‐month‐old male and female rat myocytes, Ca2+ transients of 10‐month‐old female myocytes were significantly reduced and showed a diminished rate of relaxation in comparison with those recorded in male rats of similar age. 5 These results suggest that important gender‐related changes in excitation‐contraction coupling occur following puberty, probably due to differences in Ca2+ handling capabilities at the level of the sarcoplasmic reticulum.

Mechanism of inhibition of delayed rectifier K+ current by 4‐aminopyridine in rabbit coronary myocytes.

1. The mechanisms involved in the 4‐aminopyridine (4‐AP)‐induced block of delayed rectifier K+ current (IK(V)) in vascular smooth muscle cells were studied in cells enzymatically isolated from the rabbit coronary artery. 2. 4‐AP inhibited slowly inactivating IK(V) in a dose‐dependent manner (concentration producing half‐maximal inhibition, K1/2, = 1.37 mM), and shifted the steady‐state activation and inactivation curves of IK(V) by +9 and +16 mV, respectively. 3. The time constant of activation was significantly increased by 4‐AP at +20 mV; deactivation kinetics were unaffected upon repolarization to ‐40 mV. The fast (tau f approximately 1 s) and slow (tau s approximately 5 s) time constants of inactivation (0 and +20 mV), and the recovery kinetics (tau r approximately 6 s) at ‐60 mV were not significantly affected by 0.5 mM 4‐AP. However, tau f disappeared in the presence of 2 mM 4‐AP while tau s remained unaffected. 4. Use‐dependent unblock of IK(V) was revealed at potentials > or = ‐10 mV from analyses of the voltage dependence of 4‐AP‐sensitive currents and the frequency‐dependent changes (‘reverse use dependence’) of IK(V) during the application of repetitive steps (‐60 to +20 mV for 250 ms at a rate of 0.25 Hz) in control conditions, in the presence of 0.5 mM 4‐AP, and after washout of the drug. These results suggested that 4‐AP preferentially binds to the channel in the closed state, and unbinding is promoted by transitions to the open state. 5. The channel was modelled as a simple three‐state mathematical loop model incorporating single closed, open and inactivated states. The block by 4‐AP was modelled as a state‐dependent interaction with 4‐AP primarily binding to the closed state. Computer simulations support the hypothesis that 4‐AP‐induced block of the delayed rectifier K+ (KV) channel in the closed state is relieved during membrane depolarization. 6. Closed state binding of 4‐AP to the KV channel depolarizes vascular smooth muscle cells by shifting the activation curve of these channels to more positive potentials.

Ca2+-activated Cl− current can be triggered by Na+ current-induced SR Ca2+ release in rabbit ventricle

The Ca2+-activated Cl- current [ I Cl(Ca)] contributes to the repolarization of the cardiac action potential under physiological conditions. I Cl(Ca) is known to be primarily activated by Ca2+release from the sarcoplasmic reticulum (SR). L-type Ca2+ current [ I Ca(L)] represents the major trigger for Ca2+ release in the heart. Recent evidence, however, suggests that Ca2+ entry via reverse-mode Na+/Ca2+exchange promoted by voltage and/or Na+ current ( I Na) may also play a role. The purpose of this study was to test the hypothesis that I Cl(Ca) can be induced by I Na in the absence of I Ca(L). Macroscopic currents and Ca2+transients were measured using the whole cell patch-clamp technique in rabbit ventricular myocytes loaded with Indo-1. Nicardipine (10 μM) abolished I Ca(L)at a holding potential of -75 mV as tested in Na+-free external solution. In the presence of 131 mM external Na+and in the absence of I Ca(L), a 4-aminopyridine-resistant transient outward current was recorded in 64 of 81 cells accompanying a phasic Ca2+ transient. The current reversed at -42.0 ± 1.3 mV ( n = 6) and at +0.3 ± 1.4 mV ( n = 6) with 21 and 141 mM of internal Cl-, respectively, similar to the predicted reversal potential with low intracellular Cl- concentration ([Cl-]i) (-47.8 mV) and high [Cl-]i(-1.2 mV). Niflumic acid (100 μM) inhibited the current without affecting the Ca2+ signal ( n = 8). Both the current and Ca2+ transient were abolished by 10 mM caffeine ( n = 6), 10 μM ryanodine ( n = 3), 30 μM tetrodotoxin ( n = 9), or removal of extracellular Ca2+( n = 6). These properties are consistent with those of I Cl(Ca)previously described in mammalian cardiac myocytes. We conclude that 1) I Cl(Ca) can be recorded in the absence of I Ca(L), and 2) I Na-induced SR Ca2+ release mechanism is also present in the rabbit heart and may play a physiological role in activating the Ca2+-sensitive membrane Cl- conductance.

Calcium-Activated Chloride Channels

This chapter defines the calcium-activated chloride channels mediated by a Ca 2+ -activated Clˉ conductance. It also illustrates two voltage clamp protocols used to elicit and characterize I Cl(Ca) mediated by Cl Ca channels. It shows the recordings of membrane potential (upper traces) and membrane current (lower traces) and the activation of I Cl(Ca) by increases in intracellular Ca 2+ ([Ca 2+ ] i ) mediated by voltage-gated Ca 2+ channels in the plasma membrane of rat dorsal root ganglion (DRG) neurons in culture. The chapter explains the wide variety of cells expressing large I Cl(Ca) which is regulated and often changes during development and in response to injury suggests that a Ca 2+ -activated Clˉ conductance provides a broadly useful function (or functions). Finally, the advances include interesting but observational studies in neurons that correlate I Cl(Ca) and cation-chloride cotransport expression in development and injury as well as the important characterization of the regulation of I Cl(Ca) in smooth muscle myocytes by kinases and phosphatases.

Influence of the extracellular matrix and integrins on volume-sensitive osmolyte anion channels in C2C12 myoblasts.

The purpose of this study was to determine whether extracellular matrix (ECM) composition through integrin receptors modulated the volume-sensitive osmolyte anion channels (VSOACs) in skeletal muscle-derived C2C12 cells. Cl(-) currents were recorded in whole cell voltage-clamped cells grown on laminin (LM), fibronectin (FN), or in the absence of a defined ECM (NM). Basal membrane currents recorded in isotonic media (300 mosmol/kg) were larger in cells grown on FN (3.8-fold at +100 mV) or LM (8.8-fold at +100 mV) when compared with NM. VSOAC currents activated by cell exposure to hypotonic solution were larger in cells grown on LM (1.72-fold at +100 mV) or FN (1.75-fold at +100 mV) compared with NM. Additionally, the kinetics of VSOAC activation was approximately 27% quicker on FN and LM. These currents were tamoxifen sensitive, displayed outward rectification, reversed at the equilibrium potential of Cl(-) and inactivated at potentials >+60 mV. Specific knockdown of beta(1)-integrin by short hairpin RNA interference strongly inhibited the VSOAC Cl(-) currents in cells plated on FN. In conclusion, ECM composition and integrins profoundly influence the biophysical properties and mechanisms of onset of VSOACs.

Concepts for Patch-Clamp Recording of Whole-Cell and Single-Channel K+ Currents in Cardiac and Vascular Myocytes

The development of the patch-clamp technique (Hamill et al., 1981; Neher and Sakmann, 1976) in the mid-1970s and early 1980s by Bert Sakmann and Erwin Neher, the Nobel laureates for physiology and medicine of 1991, in parallel with the development of methods to disperse single cells from complex organs, has revolutionized our knowledge about the proteins that regulate transmembrane flux of ions across biological membranes. Its widespread implementation in the 1980s initially served to identify and characterize the various ion channels present in the membrane of various cell types. In the 1990s, molecular identification of several genes encoding for ion channel and transporter proteins provided electrophysiologists with new tools to unravel protein behavior at an unprecedented level of resolution. With the explosion of new information regarding their encoding sequences at the nucleotide and amino acid levels, these proteins can now be expressed and functionally studied in different cell types (Xenopus oocytes, mammalian cell lines, etc.), mutated, and even crystallized (Doyle et al., 1998) for investigation of the molecular domains responsible for their behavior in their normal environment.