Anita Alvarez-Laviada

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.

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor

Flecainide is a use‐dependent blocker of cardiac Na+ channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na+ channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca2+‐release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na+ and/or RyR2 channels.

Age and strain related aberrant Ca2+ release is associated with sudden cardiac death in the ACTC E99K mouse model of hypertrophic cardiomyopathy

Patients with hypertrophic cardiomyopathy, particularly young adults, can die from arrhythmia, but the mechanism underlying abnormal rhythm formation remains unknown. C57Bl6 × CBA/Ca mice carrying a cardiac actin ( ACTC) E99K (Glu99Lys) mutation reproduce many aspects of human hypertrophic cardiomyopathy, including increased myofilament Ca2+ sensitivity and sudden death in a proportion (up to 40%) of young (28-40 day old) animals. We studied the hearts of transgenic (TG; ACTC E99K) mice and their non-TG (NTG) littermates when they were in their vulnerable period (28-40 days old) and when they were adult (8-12 wk old). Ventricular myocytes were isolated from the hearts of TG and NTG mice at these two time points. We also examined the hearts of mice that died suddenly (SCD). SCD animals had approximately four times more collagen compared with age-matched NTG mice, yet myocyte cell size was normal. Young TG mice had double the collagen content of NTG mice. Contraction and Ca2+ transients were greater in cells from young TG mice compared with their NTG littermates but not in cells from adult mice (TG or NTG). Cells from young TG mice had a greater propensity for Ca2+ waves than NTG littermates, and, despite similar sarcoplasmic reticulum Ca2+ content, a proportion of these cells had larger Ca2+ spark mass. We found that the probability of SCD in young TG mice was increased when the mutation was expressed in animals with a CBA/Ca2+ background and almost eliminated in mice bred on a C57Bl6 background. The latter TG mice had normal cellular Ca2+ homeostasis. NEW & NOTEWORTHY Mice with the actin Glu99Lys hypertrophic cardiomyopathy mutation ( ACTC E99K) are prone to sudden cardiac death around 40 days, associated with increased Ca2+ transients, spark mass, and fibrosis. However, adult survivors have normal Ca2+ transients and spark density accompanied by hypertrophy. Penetrance of the sudden cardiac death phenotype depends on the genetic background of the mouse. Listen to this articles corresponding podcast at http://ajpheart.podbean.com/e/calcium-regulation-in-e99k-mouse-heart/ .

The adhesion function of the sodium channel beta subunit (β1) contributes to cardiac action potential propagation

Computational modeling indicates that cardiac conduction may involve ephaptic coupling – intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) –mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

Cardiomyocyte Membrane Structure and cAMP Compartmentation Produce Anatomical Variation in β2AR-cAMP Responsiveness in Murine Hearts

Summary Cardiomyocytes from the apex but not the base of the heart increase their contractility in response to β2-adrenoceptor (β2AR) stimulation, which may underlie the development of Takotsubo cardiomyopathy. However, both cell types produce comparable cytosolic amounts of the second messenger cAMP. We investigated this discrepancy using nanoscale imaging techniques and found that, structurally, basal cardiomyocytes have more organized membranes (higher T-tubular and caveolar densities). Local membrane microdomain responses measured in isolated basal cardiomyocytes or in whole hearts revealed significantly smaller and more short-lived β2AR/cAMP signals. Inhibition of PDE4, caveolar disruption by removing cholesterol or genetic deletion of Cav3 eliminated differences in local cAMP production and equilibrated the contractile response to β2AR. We conclude that basal cells possess tighter control of cAMP because of a higher degree of signaling microdomain organization. This provides varying levels of nanostructural control for cAMP-mediated functional effects that orchestrate macroscopic, regional physiological differences within the heart.

STORM Localizations - Part 1/3

Zip file containing single molecule localization data from STORM. The zip file is divided into 3 volumes. Volume 1/3

Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains

Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (βAR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific βAR-cAMP signaling within cardiomyocyte membrane microdomains. Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and βAR-cAMP signaling. Results: β2AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM ∗p < 0.05). There was no effect of PMU on β2AR-cAMP signaling in RII_Protein Kinase A domains. β1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that β2AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001 vs. loaded TT). By comparison β1AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (Po) were reduced by PMU (loaded TT Po 0.073 ± 0.011% vs. loaded crest Po 0.027 ± 0.006% N,n 5/18-26 mean ± SEM ∗p < 0.05) (unloaded TT 0.0350 ± 0.003% vs. unloaded crest Po 0.025 N,n 5/20-30 mean ± SEM NS #p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2. Discussion: PMU suppresses’ β2AR-cAMP and LTCC activity. When activated, the signaling of β2AR-cAMP and LTCC become more far-reaching after PMU. We suggest that a situation of ‘suppression/decompartmentation’ is elicited by the loss of refined cardiomyocyte structure following PMU. As PMU is a component of modern device therapy for heart failure this study has clinical ramifications and raises important questions for regenerative medicine.

Ursodeoxycholic acid prevents ventricular conduction slowing and arrhythmia by restoring T-type calcium current in fetuses during cholestasis

Background Increased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated. Methods High-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes. Results TC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (−10.2±1.5 versus −5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (−22.3±1.1 versus −9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L. Conclusion Our findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA.

Effect of Flecainide Derivatives on Sarcoplasmic Reticulum Ca2+ Release Confirms a Lack of Direct Action on the Cardiac Ryanodine Receptor

Flecainide is a use-dependent blocker of the cardiac Na+ channel. It has been shown to be effective in the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), however, its mechanism of action is contentious. The mechanisms involved in block have been elucidated by using fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide, establishing that Na+ channel block requires entry of the cationic form of the molecule into the cytosolic vestibule of the open channel. We investigated how flecainide derivatives influence RyR2-mediated Ca2+-release from the sarcoplasmic reticulum and whether this correlates with their effectiveness as blockers of Na+ channels and/or RyR2. We compared the effects of flecainide, QX-FL and NU-FL applied extracellulary or via intra-pipette, on the properties of Ca2+ sparks in intact adult rat cardiac myocytes with their ability to block recombinant human RyR2. Intracellular flecainide or QX-FL reduced Ca2+ spark frequency whereas NU-FL did not. Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 (resulting in different residual currents) but were significantly less effective than flecainide. None of the compounds influenced luminal to cytosol cation flux through RyR2. Given QX-FL inability to block physiologically-relevant cation flux through RyR2, and its comparative lack of efficacy in blocking the cytosolic-to-luminal current through the channel, the effect of QX-FL on Ca2+ sparks is likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal important differences in the nature of molecular interaction of flecainide with sites in the cytosolic vestibules of Na+ and RyR2 channels.Research supported by the British Heart Foundation.