Ian Findlay

Na Channels and Automatic Activity in Rat PV: Introduction: Ectopic activity arising from pulmonary veins (PV) plays a prominent role in the onset of atrial fibrillation in humans. Rat PV cardiac muscle cells have a lower resting membrane potential (RMP) than the left atria (LA) and presents in the presence of norepinephrine an automatic activity, which occurs in bursts. This study investigated the role of Na channels upon the RMP and the catecholaminergic automatic activity (CAA) in PV cardiac muscle.Methods and Results: RMP and CAA experiments were performed in male Wistar rat PV. Whole‐cell I_Na was recorded in isolated PV and LA cardiomyocytes. PV has a higher tetrodotoxin (TTX)‐sensitive basal Na⁺ permeability than the LA, due to a ∼5 mV more negative Na window current in the former tissue. TTX, quinidine, and ranolazine (1 to 10 μM each) decreased CAA incidence and arrhythmias by increasing burst intervals because of a reduction of the slope of slow depolarization between bursts. TTX and ranolazine also reduced burst duration. At 1 Hz, 10 μM quinidine, ranolazine, and TTX inhibited peak I_Na by 33%, 28%, and 98%, respectively. Each reduced the Na window current. There was no evidence for a TTX‐ or ranolazine‐sensitive late Na current.Conclusion: Na channels confer a TTX‐sensitive basal Na⁺ permeability to rat PV cardiac muscle cells and contribute to the slope of slow depolarization between bursts of CAA. Na channel blockers act mostly via reduction of the Na window current. Ranolazine also has an anti‐α₁ adrenergic effect, which contributed to its antiarrhythmic effect.

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na_v1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na_v1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na_v1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β₁-subunit was essential for this dominant negative effect. Indeed, the absence of the β₁-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β₁-subunit was present. Our findings reveal a new role for β₁-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.

Inwardly rectifying K⁺(Kir) channels allow K⁺to move more easily into rather than out of the cell. They have diverse physiological functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are regulated by G protein-coupled receptors, ATP-sensitive K⁺channels (Kir6.x) are tightly linked to cellular metabolism, and K⁺transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular substances such as Mg²⁺and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH₂and COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening the way to understanding the structure-function relationships of this simple but diverse ion channel family.

Ectopic activity in cardiac muscle within pulmonary veins (PVs) is associated with the onset and the maintenance of atrial fibrillation in humans. The mechanism underlying this ectopic activity is unknown. Here we investigate automatic activity generated by catecholaminergic stimulation in the rat PV. Intracellular microelectrodes were used to record electrical activity in isolated strips of rat PV and left atrium (LA). The resting cardiac muscle membrane potential was lower in PV [−70 ± 1 (SE) mV, n = 8] than in LA (−85 ± 1 mV, n = 8). No spontaneous activity was recorded in PV or LA under basal conditions. Norepinephrine (10⁻⁵ M) induced first a hyperpolarization (−8 ± 1 mV in PV, −3 ± 1 mV in LA, n = 8 for both) then a slowly developing depolarization (+21 ± 2 mV after 15 min in PV, +1 ± 2 mV in LA) of the resting membrane potential. Automatic activity occurred only in PV; it was triggered at approximately −50 mV, and it occurred as repetitive bursts of slow action potentials. The diastolic membrane potential increased during a burst and slowly depolarized between bursts. Automatic activity in the PV was blocked by either atenolol or prazosine, and it could be generated with a mixture of cirazoline and isoprenaline. In both tissues, cirazoline (10⁻⁶ M) induced a depolarization (+37 ± 2 mV in PV, n = 5; +5 ± 1 mV in LA, n = 5), and isoprenaline (10⁻⁷ M) evoked a hyperpolarization (−11 ± 3 mV in PV, n = 7; −3 ± 1 mV in LA, n = 6). The differences in membrane potential and reaction to adrenergic stimulation lead to automatic electrical activity occurring specifically in cardiac muscle in the PV.

Ca(2+) dynamics underlying cardiac excitation-contraction coupling are essential for heart functions. In this study, we constructed microstructure-based models of Ca(2+) dynamics to simulate Ca(2+) influx through individual L-type Calcium channels (LCCs), an effective Ca(2+) diffusion within the cytoplasmic space and in the dyadic space, and the experimentally observed calcium-dependent inactivation (CDI) of the LCCs induced by local and global Ca(2+) sensing. The models consisted of LCCs with distal and proximal Ca(2+) (Calmodulin-Ca(2+) complex) binding sites. In one model, the intra-cellular space was organelle-free cytoplasmic space, and the other was with a dyadic space including sarcoplasmic reticulum membrane. The Ca(2+) dynamics and CDI of the LCCs in the model with and without the dyadic space were then simulated using the Monte Carlo method. We first showed that an appropriate set of parameter values of the models with effectively extra-slow Ca(2+) diffusion enabled the models to reproduce major features of the CDI process induced by the local and global sensing of Ca(2+) near LCCs as measured with single and two spatially separated LCCs by Imredy and Yue (Neuron. 1992;9:197-207). The effective slow Ca(2+) diffusion might be due to association and dissociation of Ca(2+) and Calmodulin (CaM). We then examined how the local and global CDIs were affected by the presence of the dyadic space. The results suggested that in microstructure modeling of Ca(2+) dynamics in cardiac myocytes, the effective Ca(2+) diffusion under CaM-Ca(2+) interaction, the nanodomain structure of LCCs for detailed CDI, and the geometry of subcellular space for modeling dyadic space should be considered.