Gap Junction Channels Exhibit Connexin-specific Permeability to Cyclic Nucleotides

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (Ih) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in Ih current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K+ permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were ∼10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 ≥ Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell

Alternans in atria: Mechanisms and clinical relevance

Atrial fibrillation is the most common sustained arrhythmia and its prevalence is rapidly rising with the aging of the population. Cardiac alternans, defined as cyclic beat-to-beat alternations in contraction force, action potential (AP) duration and intracellular Ca2+ release at constant stimulation rate, has been associated with the development of ventricular arrhythmias. Recent clinical data also provide strong evidence that alternans plays a central role in arrhythmogenesis in atria. The aim of this article is to review the mechanisms that are responsible for repolarization alternans and contribute to the transition from spatially concordant alternans to the more arrhythmogenic spatially discordant alternans in atria

Metabolic Inhibition Induces Transient Increase of L-type Ca²⁺ Current in Human and Rat Cardiac Myocytes

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-adrenergic receptors. The whole-cell patch–clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs

Metabolic inhibition reduces cardiac L-type Ca²⁺ channel current due to acidification caused by ATP hydrolysis

<div><p>Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca²⁺ channels (LTCCs) in frog ventricular myocytes. Metabolic inhibition induced by the protonophore FCCP (as well as by 2,4- dinitrophenol, sodium azide or antimycin A) resulted in a dose-dependent reduction of LTCC current (I_Ca,L) which was more pronounced during β-adrenergic stimulation with isoprenaline. I_Ca,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in I_Ca,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on I_Ca,L was strongly attenuated when the mitochondrial F₁F₀-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on I_Ca,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F₁F₀-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of I_Ca,L. Limiting ATP break-down by F₁F₀-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.</p></div

Effect of intracellular ATP on I_Ca,L amplitude.

<p>(A) A typical experiment representing the effect of ISO (1 μM) on basal I_Ca,L in the absence of ATP in pipette solution. The current traces shown on the top of the panel were recorded at the times indicated by the corresponding letters on the main graph. (B) Mean basal and ISO-stimulated I_Ca,L amplitudes recorded in cells dialyzed with 3 mM ATP and ATP-free internal solutions. <b>*</b>P<0.05, <b>**</b>P<0.01 for the number of cells indicated in parentheses.</p