T-tubules In Ventricular Myocytes Research Articles

T-Tubular Electrical Defects Contribute to Blunted β-Adrenergic Response in Heart Failure.

Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-tubules are hallmarks of heart failure (HF). Here, we assess the effect of β-adrenoceptor activation on local Ca2+ release in electrically coupled and uncoupled T-tubules in ventricular myocytes from HF rats. We employ an ultrafast random access multi-photon (RAMP) microscope to simultaneously record action potentials and Ca2+ transients from multiple T-tubules in ventricular cardiomyocytes from a HF rat model of coronary ligation compared to sham-operated rats as a control. We confirmed that β-adrenergic stimulation increases the frequency of Ca2+ sparks, reduces Ca2+ transient variability, and hastens the decay of Ca2+ transients: all these effects are similarly exerted by β-adrenergic stimulation in control and HF cardiomyocytes. Conversely, β-adrenergic stimulation in HF cells accelerates a Ca2+ rise exclusively in the proximity of T-tubules that regularly conduct the action potential. The delayed Ca2+ rise found at T-tubules that fail to conduct the action potential is instead not affected by β-adrenergic signalling. Taken together, these findings indicate that HF cells globally respond to β-adrenergic stimulation, except at T-tubules that fail to conduct action potentials, where the blunted effect of the β-adrenergic signalling may be directly caused by the lack of electrical activity.

Postnatal Development of Calcium Signaling in Rat Cardiomyocytes

During development, cardiac myocytes undergo rapid growth and structural remodeling. With increased cell size and increased requirements for calcium for contraction, excitation-contraction coupling becomes increasingly dependent on calcium-induced calcium release (CICR). Extending into the interior of the cell, t-tubules form a network securing signal transduction into the whole volume of the cell.We studied the development of L-type calcium currents and of t-tubules in ventricular myocytes of neonatal and young rats (ages 2-18 and 28 days). Calcium currents were recorded in isolated ventricular myocytes (80 ms depolarization from −50 to 0 mV) using patch-clamp. Plasma membrane-specific fluorescent probe FM 4-64 was used to observe formation of t-tubules in myocytes using isolated cells and intact myocardial tissue by laser-scanning confocal microscopy.Formation of t-tubules started around day 9 (D9) as small membrane invaginations. Invaginations formed a loose network with predominating longitudinal elements around D14, and after D17 developed into a semi-regular tubular network. Cell size, area and membrane capacitance was increasing throughout the observed age interval and around D28 reached values typical for cells from adult rat hearts. During early development, calcium current density increased, saturating around D11. The rate of Ca-current decay increased with age, reaching values typical for adult myocytes after D10.These data suggest that functional local calcium signaling is present already at D11, when the t-tubules only start to form. This is supported especially by the dynamics of calcium current decay, which at D11 becomes as fast as in adult cardiac myocytes.Support: VEGA 2/0095/15 and VEGA 2/0147/14.

PIP2 Modulates T-Tubule Remodeling During Heart Failure by Working as a Binding Substrate for BIN1

The membrane curvature-forming protein BIN1 is known to be involved in T-tubule formation. Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to anchor BIN1 and maintain its localization to the sarcolemma. Since heart failure (HF) is characterized by T-tubule remodeling and dyssynchronous Ca2+ release, we hypothesized that PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of HF and is responsible for T-tubule remodeling. We developed an in-vitro model using HL1 cells that developed tubule-like extensions during BIN1 overexpression. These exogenous BIN1-induced tubules were continuous with the sarcolemma (RH-237 staining) similar to T-tubules in ventricular myocytes. To further understand if the BIN1/PIP2 complex is disrupted in HF, we depleted PIP2 in HL1 cells by inhibition of PI4K or activation of PLCβ1. We treated transfected HL1 cells with wortmannin, endothelin-1 (Et-1), m-3M3FBS and a combination of Et-1 + m-3M3FBS. Treated cells showed significantly more tubule degeneration compared to non-treated cells and membrane PIP2 levels decreased as measured by transfecting HL1 cells with a fluorescent PIP2 reporter (PLCδ1-PH-GFP construct). To further determine whether depletion of PIP2-mediated T-tubules loss is correlated with defects in Ca2+ cycling, we isolated adult rat ventricular myocytes, treated them with the same PIP2 depletion agents, then stained them with di-4-ANEPPS in order to assess T-tubule organization. All treated cells showed significantly reduced T-tubule organization than control cells. We also measured Ca2+ cycling in these cells during basal (2000ms) and rapid (1000ms) pacing using flou-2AM. Ca2+ release was delayed in certain cellular regions in treated myocytes and rise time, time to peak and decay time were significantly increased at both pacing rates. These results show that depletion of PIP2 can lead to T-tubule disruption and suggests that PIP2 is required for T-tubule maintenance and function.

T Tubule Remodeling: Lack of PIP2 as a Binding Substrate for Bin1 during Hypertrophy and Heart Failure

T tubule remodeling is one of the prevalent characteristics of failing heart. The structural protein Bin1 has been reported to be involved in t-tubule formation. The interaction of exon10 domain of Bin1 with phosphatidylinositol-4,5-bisphosphate [PIP2] appears to be crucial for localization of Bin1 to t-tubules. Molecular basis for t-tubule disruption during transition from hypertrophy to heart failure (HF) has not been identified yet. We hypothesized that PLCβ1-induced hydrolysis of PIP2 reduces the targeting of Bin1 to t-tubules and leads to t-tubule disruption. Through co-localization experiments in normal rat ventricular myocardium, we found that Bin1, PIP2 and PLCβ1 were all located in t-tubules. Next, we focused our attention on the importance of Bin1 for t-tubule formation by overexpression of Bin1 in HL1 cells. These HL1 cells normally do not have t-tubules and lack Bin1. After 24 hours of Bin1 overexpression, cells showed tubular structures that were absent in control (empty vector) cells. We also found that these tubular structures were continuous with plasma membrane like normal t-tubules in ventricular myocytes. To further understand the mechanism of t-tubules loss, Bin1 overexpressed cells were treated with 200nM endothelin1 (Et1). Et1 significantly increased the activity of PLCβ1. After 48 hours, Et1 treated HL1 cells start showing tubule degeneration and significantly fewer cells with tubules compared to the non-treatment cells. Overall our data indicate that Bin1, PIP2 and PLCβ1 may form a signaling complex at t-tubules and that hydrolysis of PIP2 through PLCβ1 might be crucial for t-tubule degeneration during HF progression. In addition, the HL1 cell line can be a useful experimental model especially for cardiac studies of t-tubule formation and remodeling in disease.

Evoked Centripetal Ca 2+ Activation in Cardiac Purkinje Cells: CICR or Ca 2+ Diffusion?

In large mammals, cardiac Purkinje cells (Pcells) lack transverse tubules (T-tubules). However, Pcells respond to stimulations by massive Ca-releases from the sarcoplasmic reticulum (SR) similar to those of ventricular myocytes (VMs) with T-tubules. Ca-imaging revealed a wave of elevated Ca-concentration (Cai) propagating from the sarcolemma (SL) toward the centre of Pcells upon stimulation. Ca-diffusion was proposed to explain this centripetal propagation. A thin layer of SR expressing RyR3 was found ∼5µm below the SL while other SR-regions expressed RyR2. Ca-Induced Ca-Release (CICR) from RyR3-SR-region could relay Ca-signal from Ca-entry to RyR2-Ca-release sites in the core, playing equivalent role as T-tubules in VMs. Our objective wasto examine whether centripetal Ca-signaling results exclusively from Ca-diffusion or involved intermediate RyR3-CICR. Methods:Pcells were prepared from Yucatan swine (30-40kg) and incubated with Fluo4-AM. Ca-dynamics was assessed by 2D-confocal microscopy (30-100fps); pH7.3,35oC. 26 cells were field stimulated at various external Ca-concentrations (Cao). Ca-data were compared with those from a model of Ca-propagation across 3 virtual adjacent SR-Ca-release regions. Results: In stimulated Pcells, Ca-increase first happened under SL with amplitude increasing from 0.1 to 0.5µmol/L with Cao from 1 to 4mmol/L. A Ca-wave then propagated uniformly from the SL toward cell center at 100-250µm/s. An overall Ca-transient appeared when Ca-wave reached the core region. When the model included intermediate CICR-region, predicted data were consistent with Pcells observations: minimal amplitude (minAmp) of peripheral Ca-increase mediating a central Ca-transient similar to that of Pcells, was 130nmol/L; this generated Ca-propagation with velocity (propVel) of 160µm/s; propVel was independent on Cao. In contrast, minAmp and propVel were 220nmol/L and 40µm/s respectively, and propVel increased with Cao when only diffusion was considered. Conclusion: Our data were consistent with an intermediate CICR-mechanism which “boosts” Ca-propagation between SL and core of Pcells.

Spatiotemporal Profiles of Sarcoplasmic Reticulum Ca2+ Release in Mouse Atrial Cardiomyoctes in situ

Previous studies in atrial cardiomyocytes isolated from various mammalian species have demonstrated poorly developed transverse(t)-tubules and inhomogeneities of sarcoplasmic reticulum (SR) Ca2+ release, but little is known of mice. Here, we examined the t-tubular organization and action potential-evoked Ca2+ release in in situ myocytes, using confocal microscopy of Langendorff-perfused hearts. Imaging of ANNINE-6plus-stained sarcolemmal membranes revealed paucity of t-tubules in atrial, but densely and regularly spaced t-tubules in ventricular myocytes. However, both myocyte types exhibited regular, striated appearance of type 2 ryanodine receptor distribution. Line-scans (2 ms/line) across multiple myocytes obtained during sinus rhythm from fluo-4 loaded hearts revealed homogeneous [Ca2+]i increases in ventricular myocytes, whereas atrial myocytes exhibited areas with delayed transients (see Figure). Histograms of F50values (the time to 50% of peak F/F0 [where F indicates fluorescence intensity, and F0 indicates F at rest]) for the subsarcolemmal (SS) and central (C) compartments in ventricular myocytes were largely congruent, whereas the corresponding atrial histograms did not superimpose and exhibited multiple peaks. Thus, major myocyte-to-myocyte differences in the spatial organization of SR Ca2+ release exist among in situ mouse atrial myocytes, likely reflecting non-uniform t-tubule distribution.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes.

We examined the role of the Ca(2+)-regulated protein phosphatase calcineurin in controlling Ca(2+) signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch-clamp technique. Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was measured in cells loaded with the fluorescent Ca(2+) indicators fluo-4 or fura-2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 nM) or the calcineurin auto-inhibitory peptide (CiP, 100 microM), increased the amplitude and rate of decay of the evoked [Ca(2+)](i) transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 nM) and 100 microM CiP on the [Ca(2+)](i) transient and AP were not additive. Calcineurin inhibition did not modify the K(+) currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca(2+) current (I(Ca)) and the evoked calcium transient normalized to the I(Ca). Calcium sparks, which underlie the [Ca(2+)](i) transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co-localize near the T-tubules of ventricular myocytes. We propose that calcineurin and PKA are co-localized to control Ca(2+) influx through calcium channels and calcium release through ryanodine receptors.