Alexey V. Glukhov

Microdomain-Specific Modulation of L-type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure

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Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes

Background— Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). Methods and Results— Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈40% greater current. Optical mapping of Ca2+ transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca2+ release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca2+ transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-&bgr;-cyclodextrin, with an associated ≈30% whole-cell ICa,L reduction. Heart failure (16 weeks post–myocardial infarction) in rats resulted in a T-tubule degradation (by ≈40%) and significant elevation of spontaneous Ca2+ release events. Although heart failure did not affect LTCC occurrence, it led to ≈25% decrease in T-tubule LTCC amplitude. Conclusions— We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.

T-tubule remodelling disturbs localized β2-adrenergic signalling in rat ventricular myocytes during the progression of heart failure

Aims Cardiomyocyte β2-adrenergic receptor (β2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors’ subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), β2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon β2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown. Methods and results Rat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), β2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local β2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the β2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell β2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve β2AR-mediated signal compartmentation or reduce cAMP diffusion. Conclusion Although changes in T-tubule structure and β2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of β2AR-mediated cAMP is temporarily reversed. Complete disorganization of β2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.

Electrophysiological Characteristics, Rhythm, Disturbances and Conduction Discontinuities Under Autonomic Stimulation in the Rat Pulmonary Vein Myocardium

Despite the importance of neurogenic initiation of rapid firing from pulmonary veins (PVs), the mechanism of autonomic modulation of electrophysiological properties of the PV myocardium to form a substrate for atrial arrhythmia remains poorly understood.

Distribution and Regulation of L-Type Ca 2+ Channels in Cardiomyocyte Microdomains

Cardiac excitation involves action potential generation by individual cells and its conduction from cell to cell through intercellular gap junctions. Excitation of the cellular membrane results in opening of the voltage-gated L-type Ca2+ channels, which allow a small amount of Ca2+ to enter the cell. This triggers the release of a much greater amount of Ca2+ from the intracellular Ca2+ store, the sarcoplasmic reticulum, and gives rise to the systolic Ca2+ transient and contraction. These processes are highly regulated by the autonomic nervous system, which ensures the acute and reliable contractile function of the heart and the short-term modulation of this function upon changes in heart rate or workload. Recently, it became evident that discrete clusters of L-type Ca2+ channels exist in the sarcolemma, where they form an interacting network with regulatory proteins and receptors. It allows the specificity, reliability, and accuracy of autonomic modulation of the excitation-contraction processes by a variety of neurohormonal pathways. Disruption in subcellular targeting of calcium channels and associated signaling pathways may contribute to the pathophysiology of a variety of cardiac diseases including heart failure and certain arrhythmias. This chapter reviews the emerging understanding of microdomain-specific distribution, functioning, regulation, and remodeling of L-type Ca2+ channels in atrial and ventricular myocytes and their contributions to the cellular signaling and cardiac pathology.