T-tubule Network Research Articles

T-Tubule Loss is a Prominent Feature of HFrEF but not HFpEF

T-tubules are invaginations of the sarcolemmal membrane which are important for effective excitation-contraction coupling. Accumulating data indicate that cardiomyocyte t-tubular structure is disrupted during heart failure with reduced ejection fraction (HFrEF), resulting in impaired Ca2+ release and contractility. Much less is known about t-tubule structure during heart failure with preserved ejection fraction (HFpEF). Based on our recent data, indicating that increased wall stress promotes t-tubule degradation during HFrEF (Frisk et al. Cardiovasc Res, 2016), we hypothesized that t-tubule structure would be safeguarded by maintained wall stress during HFpEF. In HFpEF patients with mild diastolic dysfunction (E/e’ = 10.9±2.7 and deceleration time < 235ms), we observed that t-tubule density was maintained compared to healthy patient hearts. Interestingly, patients with more severe diastolic dysfunction (E/e’ = 13.4±2.7 and deceleration time > 305ms) showed increased t-tubule density. Closer examination of t-tubule architecture revealed that the observed rise in t-tubule density was a result of maintained transversely-oriented tubules and increased levels of longitudinally-oriented tubules. In contrast, t-tubules were lost and disorganized in patients with dilated cardiomyopathy. To investigate if t-tubule maintenance or proliferation are general features of non-dilated hypertrophic hearts we investigated the t-tubule network in patients with hypertrophic cardiomyopathy or aortic stenosis. In both conditions, t-tubule density was increased due to a larger fraction of transversely-oriented tubules. We thus conclude that t-tubule density is maintained or even increased in the non-dilated hypertrophic human ventricular myocardium. These findings are consistent with a key role of increased wall stress, a hallmark of dilated ventricular remodeling, in triggering t-tubule degradation in HFrEF.

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation-contraction coupling, and inefficient Ca-induced Ca release remain major limitations. Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation-contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation-contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation-contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

MG53 is dispensable for T-tubule maturation but critical for maintaining T-tubule integrity following cardiac stress

The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca2+ handling properties, including decreased Ca2+ transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca2+ handling properties and cardiac function under pathological cardiac stress.

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200-400nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart.

Cardiac T-Tubule Microanatomy and Function.

Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organelles that consist of sarcolemma penetrating into the myocyte interior, forming a highly branched and interconnected network. Mature t-tubule networks are found in mammalian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomyocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the current knowledge in gross morphology and structure, membrane and protein composition, and function of the cardiac t-tubule network. We also review in detail current knowledge on the formation of functional membrane subdomains within t-tubules, with a particular focus on the cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts. Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and therapeutic opportunities to better treat patients with failing hearts.

Regional distribution of T-tubule density in left and right atria in dogs

The peculiarities of transverse tubule (T-tubule) morphology and distribution in the atrium-and how they contribute to excitation-contraction coupling-are just beginning to be understood. The objectives of this study were to determine T-tubule density in the intact, live right and left atria in a large animal and to determine intraregional differences in T-tubule organization within each atrium. Using confocal microscopy, T-tubules were imaged in both atria in intact, Langendorf-perfused normal dog hearts loaded with di-4-ANEPPS. T-tubules were imaged in large populations of myocytes from the endocardial surface of each atrium. Computerized data analysis was performed using a new MatLab (Mathworks, Natick, MA) routine, AutoTT. There was a large percentage of myocytes that had no T-tubules in both atria with a higher percentage in the right atrium (25.1%) than in the left atrium (12.5%) (P < .02). The density of transverse and longitudinal T-tubule elements was low in cells that did contain T-tubules, but there were no significant differences in density between the left atrial appendage, the pulmonary vein-posterior left atrium, the right atrial appendage, and the right atrial free wall. In contrast, there were significant differences in sarcomere spacing and cell width between different regions of the atria. There is a sparse T-tubule network in atrial myocytes throughout both dog atria, with significant numbers of myocytes in both atria-the right atrium more so than the left atrium-having no T-tubules at all. These regional differences in T-tubule distribution, along with differences in cell width and sarcomere spacing, may have implications for the emergence of substrate for atrial fibrillation.

Heterogeneity of T-Tubules in Pig Hearts.

BackgroundT-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in mammalian cardiac myocytes. Although t-tubules were generally considered to be effectively absent in atrial myocytes, recent studies on atrial cells from larger mammals suggest that t-tubules may be more numerous than previously supposed. However, the degree of heterogeneity between cardiomyocytes in the extent of the t-tubule network remains unclear. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular tissue.MethodsCardiac tissue was obtained from young female Landrace White pigs (45–75 kg, 5–6 months old). Cardiomyocytes were isolated by arterial perfusion with a collagenase-containing solution. Ca2+ transients were examined in field-stimulated isolated cells loaded with fluo-4-AM. Membranes of isolated cells were visualized using di-8-ANEPPS. T-tubules were visualized in fixed-frozen tissue sections stained with Alexa-Fluor 488-conjugated WGA. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated. A distance mapping approach was used to calculate half-distance to nearest t-tubule (HDTT).Results & ConclusionThe spatio-temporal properties of the Ca2+ transient appeared to be consistent with the absence of functional t-tubules in isolated atrial myocytes. However, t-tubules could be identified in a sub-population of atrial cells in frozen sections. While all ventricular myocytes had TTD >3% (mean TTD = 6.94±0.395%, n = 24), this was true of just 5/22 atrial cells. Mean atrial TTD (2.35±0.457%, n = 22) was lower than ventricular TTD (P<0.0001). TTD correlated with cell-width (r = 0.7756, n = 46, P<0.0001). HDTT was significantly greater in the atrial cells with TTD ≤3% (2.29±0.16 μm, n = 17) than in either ventricular cells (1.33±0.05 μm, n = 24, P<0.0001) or in atrial cells with TTD >3% (1.65±0.06 μm, n = 5, P<0.05). These data demonstrate considerable heterogeneity between pig cardiomyocytes in the extent of t-tubule network, which correlated with cell size.

Fluorescent Dextran Diffusion Assay to Study Cardiac T-Tubules

T-tubules are membrane invaginations of cardiac myocytes critical for effective excitation-contraction coupling and are significantly altered in various diseases. However, the mechanisms underlying t-tubule remodeling remain largely unknown, particularly due to a number of limitations of currently available technologies. Therefore, we have developed an assay that employs the diffusion of fluorescent 3 KD dextran out of the t-tubule network of live intact isolated ventricular cardiomyocytes. To significantly improve the quality of measurements, dextran is applied locally using a patch-clamp pipette (∼2-3 μm diameter) under constant flow of washing solution through 0.5 mm ID capillary glass, which limits dextran filling of t-tubules to a small area of the cardiomyocyte. With 40X objective and small pinholes in the light paths, excitation is limited to ∼9-10 μm diameter area and fluorescence emission is measured from ∼5-6 μm diameter region using a photomultiplier. Upon cessation of dye application, fluorescence decline displays 2 major components: a fast component (τ ∼5-50 ms) corresponding to dye washout from the solution surrounding the cell and a slow component corresponding to dye diffusion out of the t-tubule system. In healthy mouse ventricular myocytes the slow component had a time constant of 1304 ± 98 ms (n=24). In contrast, “detubulation” by acute hyposmotic stress significantly decreased the time constant to 848 ± 99 ms (n=27, p<0.01) presumably due to shorter remaining t-tubule segments. In HEK293 cells, which lack t-tubules, the slow component has time constant of 210 ± 51 ms (n=5), suggesting that dextran unbinding from cellular membrane is significantly faster than its diffusion out of t-tubules. This technique allows for rapid (within seconds) repetitive measures of the diffusional constraints imposed by the t-tubule network and provides a novel tool for studying dynamics of t-tubule remodeling in various experimental conditions.

Postnatal Development of T-Tubules in Sheep Atrial Myocytes

Background: Cardiac t-tubules are essential in ensuring systolic calcium rises synchronously to enable contraction. The t-tubule network is absent or very sparse at birth in the ventricles of small mammals and develops postnatally with subsequent changes in calcium handling. Nothing is currently known about the development of t-tubules in the atria.Methods and Results: Newborn, 1 month, 3 month, 6 month and adult sheep (∼18 months) were euthanased and cells isolated from the left atrial appendage. T-tubule density assessed using di-4-ANEPPS and confocal microscopy showed partially developed t-tubules were present at birth in sheep atrial myocytes but at 30±9% of adult density (p<0.001). T-tubule density increased at 1 month and by 3 months t-tubules appeared fully developed despite decreased cell width compared to adult (p<0.01). In current clamp experiments, performed at 37°C with fluo-3 AM loaded cells, APD10, 25 and 50 were shorter in newborn, 1 month and 3 months compared to adult (p<0.01). There was no difference in calcium transient amplitude between newborn, 1 month and 3 month cells but the rate of decay of systolic calcium was slower in newborn compared to both 1 month and 3 months (p<0.05).Conclusion: T-tubules are present at birth in sheep atrial myocytes and develop over the first 3 months of life. Both action potential duration and rate of calcium transient decay increase during development. We hypothesise that this may be due to increased L-type calcium current as t-tubules develop and increased SERCA function as the sarcoplasmic reticulum matures. Further investigation is required to determine how the expression of t-tubule regulatory proteins and activity of calcium handling proteins changes throughout development.

BIN1 regulates dynamic t-tubule membrane

Cardiac transverse tubules (t-tubules) are specific membrane organelles critical in calcium signaling and excitation–contraction coupling required for beat-to-beat heart contraction. T-tubules are highly branched and form an interconnected network that penetrates the myocyte interior to form junctions with the sarcoplasmic reticulum. T-tubules are selectively enriched with specific ion channels and proteins crucial in calcium transient development necessary in excitation–contraction coupling, thus t-tubules are a key component of cardiac myocyte function. In this review, we focus primarily on two proteins concentrated within the t-tubular network, the L-type calcium channel (LTCC) and associated membrane anchor protein, bridging integrator 1 (BIN1). Here, we provide an overview of current knowledge in t-tubule morphology, composition, microdomains, as well as the dynamics of the t-tubule network. Secondly, we highlight multiple aspects of BIN1-dependent t-tubule function, which includes forward trafficking of LTCCs to t-tubules, LTCC clustering at t-tubule surface, microdomain organization and regulation at t-tubule membrane, and the formation of a slow diffusion barrier within t-tubules. Lastly, we describe progress in characterizing how acquired human heart failure can be attributed to abnormal BIN1 transcription and associated t-tubule remodeling. Understanding BIN1-regulated cardiac t-tubule biology in human heart failure management has the dual benefit of promoting progress in both biomarker development and therapeutic target identification. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

166 T-tubule remodelling and formation of super-tubules in the border zone of cardiac myocytes in the infarcted pig heart

Background Myocardial infarction (MI) is a common cause of death, with approximately 175000 inpatient episodes of acute myocardial infarction in the UK in 2012. Following an MI the loss of cardiac myocytes triggers a remodelling process depositing extracellular matrix in the infarct region. Hypocontractile, damaged cardiac myocytes surround the infarct, forming a border zone. One of the key structural components regulating excitation-contraction coupling in the heart is the t-tubule network. Conventional confocal microscopy (resolution ˜100 nm) of isolated cardiac myocytes from the border zone post-MI has shown a loss of t-tubules. Here we apply, for the first time to our knowledge, serial block face scanning electron microscopy (SBF-SEM), to investigate the morphology of the t-tubule network within the infarct border zone cardiac myocytes to provide nano-scale structural details of the remodelling process. Methods A porcine model of MI was employed for this study. All animal work was approved by the University of Manchester local ethics committee and was covered by the necessary UK Home Office project and personal licences. Tissue (˜0.5 mm 3 ) was collected from the border and remote zones of the MI pigs and corresponding regions were also taken from control animals and processed for SBF-SEM. Blocks were imaged using an FEI Quanta 250 FEG SEM equipped with a Gatan 3View system. Serial images were collected at different magnifications ranging from 5.4 to 90.0 nm per pixel in the X-Y plane, while the cutting depth along the Z-axis was fixed at 50 nm for all the datasets. Images were segmented and rendered in Fiji or IMOD. Results 3D reconstruction of the t-tubule network from the left ventricle of control animals revealed a spoke-like arrangement, similar to that observed in human cardiac myocytes and other large mammals e.g. the sheep. Cardiac myocytes within the remote region of the infarcted heart have a t-tubule network indistinguishable from that of the control myocytes. In contrast, border zone myocytes showed large areas that were devoid of t-tubules. 3D modelling revealed that the surviving transverse tubules presented gross deformations, appearing to be the result of t-tubules fusing with each other to form a large complex that adopts a variety of orientations within the cardiac myocyte. Conclusion Employing SBF-SEM we have collected 3D datasets of cardiac myocytes in situ within the left ventricle of infarcted pigs at magnifications corresponding to ˜5 nm per pixel in the X-Y plane. This has allowed the resolution of nano-scale details of the remodelled t-tubules, including features such as the basal lamina. Together the loss of t-tubules within parts of the cell, coupled with the formation of super-tubule networks, provide novel structural insights towards unravelling the hypocontractile properties of the border zone cardiac myocytes.

In situ single photon confocal imaging of cardiomyocyte T-tubule system from Langendorff-perfused hearts.

Transverse tubules (T-tubules) are orderly invaginations of the sarcolemma in mammalian cardiomyocytes. The integrity of T-tubule architecture is critical for cardiac excitation–contraction coupling function. T-tubule remodeling is recognized as a key player in cardiac dysfunction. Early studies on T-tubule structure were based on electron microscopy, which uncovered important information about the T-tubule architecture. The advent of fluorescent membrane probes allowed the application of confocal microscopy to investigations of T-tubule structure. Studies have now been extended beyond single cardiomyocytes to examine the T-tubule network in intact hearts through in situ confocal imaging of Langendorff-perfused hearts. This technique has allowed visualization of T-tubule organization in their natural habitat, avoiding the damage induced by isolation of cardiomyocytes. Additionally, it is possible to obtain T-tubule images in different subepicardial regions in a single intact heart. We review how this state-of-the-art imaging technique has provided important mechanistic insights into maturation of T-tubules in developing hearts and defined the role of T-tubule remodeling in development and progression of heart failure.

Mineralocorticoid receptor blockade prevents Western diet-induced diastolic dysfunction in female mice.

Overnutrition/obesity predisposes individuals, particularly women, to diastolic dysfunction (DD), an independent predictor of future cardiovascular disease. We examined whether low-dose spironolactone (Sp) prevents DD associated with consumption of a Western Diet (WD) high in fat, fructose, and sucrose. Female C57BL6J mice were fed a WD with or without Sp (1 mg·kg(-1)·day(-1)). After 4 mo on the WD, mice exhibited increased body weight and visceral fat, but similar blood pressures, compared with control diet-fed mice. Sp prevented the development of WD-induced DD, as indicated by decreased isovolumic relaxation time and an improvement in myocardial performance (<Tei index) and septal annular velocity (<E'-to-A' ratio), as assessed by echocardiography, as well as decreased diastolic relaxation time/increased diastolic initial filling rate, as assessed by MRI. The relationship between passive sarcomere length of cardiac myocytes and ventricular pressure was monitored using di-8-ANEPPS staining of the t-tubule network in hearts ex vivo. Sp administration led to longer sarcomere lengths at each pressure indicative of improved ventricular compliance in WD-fed mice. Sp also prevented left ventricular hypertrophy, interstitial fibrosis, and oxidative stress. Sp prevented the WD-induced increased expression of myocardial proinflammatory M1 macrophage markers monocyte chemoattractant protein-1 and CD11c and increased the expression of the anti-inflammatory M2 macrophage marker CD206. These findings demonstrate that WD-induced DD is associated with increased oxidant stress, fibrosis, and immune dysregulation. Mineralocorticoid receptor antagonism enhanced M2 macrophage polarization and ameliorated oxidant stress and fibrosis. This work supports a novel blood pressure-independent effect of MR antagonism as a strategy to prevent diet-induced DD in women. Mineralocorticoid antagonism; low-dose spironolactone; aldosterone;high-fat diet; high-fructose diet; oxidative stress; inflammation; cardiac hypertrophy; myocardial compliance.

EC Coupling for Muscle Aficionados: Abnormal Contraction and Disrupted Excitability in Some Enzymatically Dissociated Skeletal Muscle Fibers

Enzymatically dissociated skeletal muscle fibers are routinely used in studies of the excitation-contraction coupling (ECC) process. Here we show that skeletal muscle fibers enzymatically dissociated and cultured for 1-2 days are in general useful for experimentation; however a subset of fibers, despite their normal appearance, exhibit abnormal contractility and calcium transient alterations. We monitored the contractile response of fibers to electric field stimulation using transmitted light microscopy, and the spatial distribution of the calcium transients using Rhod-2, a non-ratiometric calcium indicator, and ultra-fast confocal microscopy. A variable fraction of fibers expressed abnormal ECC properties; they were unable to contract homogenously in response to electrical stimuli, and most notably exhibited alternating local contractions at fiber ends in response to stimuli of alternating polarity. These alternating (ALT) response fibers exhibit minor gross morphological differences from normal (WT) fibers, which contract uniformly and respond to both polarity electrical pulses. In some ALT fibers, di-8-anepps staining shows a partial disruption of the T-tubule network at the center of the fibers. This might indicate that T-tubule network disruption accompanies changes in excitability observed in ALT fibers. We hypothesize that changes in the function and/or expression of voltage gated channels, responsible for action potential generation, or voltage gated calcium channels, essential for ECC, could explain our results. Regardless of the mechanism(s) responsible for lack of excitability, we encourage the careful monitoring of the contractile response of the fiber (and if available, the evaluation of the calcium transients) to determine that normal behavior of skeletal muscle fibers be implemented when choosing fibers for physiological experiments, and recommend avoiding the use of muscle fibers that display only local contractile activity to alternating polarity. Supported by NIH-NIAMS award R37-AR055099.

Bridging Integrator 1 (BIN1) Initiates T-Tubule Growth during Cardiac Development and Disease

The cardiomyocyte t-tubule network is malleable; t-tubules develop after birth and are disrupted during diseases such as heart failure (HF). In failing cells, loss of the primarily transverse orientation of tubules is accompanied by growth of longitudinal t-tubules (LTTs), which we have shown are compensatory. We presently hypothesized that bridging integrator 1 (BIN1), a membrane tubulating protein, triggers t-tubule growth during both development and disease. Confocal microscopy and di-8-ANEPPS staining revealed cardiomyocytes isolated from 10 day-old mice predominantly contained LTTs, whereas the mature transverse network began developing 10-15 days following birth. Additionally, we observed that cardiac BIN1 protein levels in mice were highest during periods of t-tubule biogenesis, suggesting that BIN1 may be involved during early stages of development. In rats examined 6 weeks following myocardial infarction, appearance of LTTs was accompanied by a 2-fold increase in BIN1 transcript levels, despite marked loss of transverse elements. Immunostaining of failing cardiomyocytes fixed in 4% paraformaldehyde confirmed the presence of BIN1 in newly formed LTTs. To directly examine whether BIN1 upregulation could underlie the observed appearance of LTTs in developing and diseased cardiomyocytes, we expressed BIN1 in cultured cardiomyocytes lacking t-tubules (HL-1 cells). Following transfection with exogenous BIN1, a dense network of BIN1-positive sarcolemmal membrane invaginations was formed in HL-1 cells as early as 12 hours after transfection. Importantly, these tubules were chaotically organized and resembled the LTTs grown prior to t-tubule maturation. Labeling of intracellular Ca2+ with fluo-4 revealed that these tubules have t-tubule-like functionality, as Ca2+ release events occurred adjacent to BIN1-positive tubules. We propose that BIN1 initiates LTT growth during development and disease, and that upregulation of BIN1 in the diseased heart may represent a return to an immature gene program.

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress.

Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca(2+) handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na(+)/Ca(2+) exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.

Bin-1 Expression in Normal Rat Cardiac Myocytes and in Myocytes with Reduced T-Tubule Density Due to Cell Culture or Heart Failure

Bridging Integrator-1 (BIN-1), member of the BAR domain protein superfamily, has been implicated in the formation and maintenance of membrane invaginations in skeletal and cardiac muscle cells. However, it is not clear whether BIN-1 expression and t-tubule density are correlated in all circumstances. The aim of the present study was to investigate the relationship between t-tubule density and BIN-1 expression in (i) fresh atrial and ventricular myocytes (ii) ventricular myocytes cultured for 24-72 hours and (iii) ventricular myocytes from animals with monocrotaline-induced right heart failure (HF). Confocal microscopy was used to assess t-tubule density and BIN1 expression in rat myocytes using di-8-ANEPPS fluorescence or immunofluorescence respectively. In fresh ventricular myocytes, a regular t-tubule network was apparent and BIN-1 exhibited a corresponding striated pattern (n=10). In atrial myocytes t-tubules were typically absent and BIN-1 immunofluorescence was only detectable in the peripheral sarcolemma (n=20). In ventricular myocytes after 24, 48 or 72 hrs cell-culture, t-tubule density was reduced to 61 ± 1.4%, 26 ± 0.6% and 11 ± 0.5% respectively (n=10, 8 and 6, P<0.05), while mean cell area did not change significantly. Correspondingly, BIN1 expression was significantly reduced in ventricular myocytes at 48 (to 17 ± 0.14%, n=7, P<0.05) and 72 hrs (to 16 ± 0.09%; n=5, P<0.05) compared with control. In right ventricular myocytes, from rats with right HF and reduced t-tubule density, BIN1 expression decreased significantly (69 ± 1.3%; n=7, P<0.05) compared with saline-injected rats. These results suggest that where t-tubules are absent (e.g. atrial cells) or in circumstances where t-tubule density is decreased (cell culture, HF) BIN-1 expression is correspondingly reduced. This supports the hypothesis that BIN-1 has an important role in formation and maintenance of t-tubules.

β-Adrenergic Regulation of Cyclic AMP and Ca Current at the T-Tubules and Surface Membrane in Rat Cardiomyocytes

β-Adrenoceptor (β-AR) signalling is severely impaired in heart failure (HF), and this is accompanied by a loss and disorganization of t-tubules. However, how the latter affects the former is unknown. Here, we examined how acute detubulation affects the β-AR response of L-type Ca2+ channel (LTCC) current (ICa,L) and membrane cAMP ([cAMP]m) in adult rat ventricular myocytes (ARVMs) and the contribution of phosphodiesterases PDE3 and PDE4 in this process. ARVMs were infected with an adenovirus encoding a mutant of the olfactory cyclic nucleotide-gated (CNG) channels α subunit to follow [cAMP]m dynamics by recording the associated cationic current. [cAMP]m was also monitored by fluorescent imaging using a FRET-based sensor encoding a plasma-membrane targeted cAMP probe (Epac2-camps). Osmotic shock treatment with 1.5M formamide during 15 min induced a loss of t-tubule network as verified by confocal imaging with di-8-ANEPPS staining. Detubulation of ARVMs reduced membrane capacitance by 40% and basal ICa,L density ∼3-fold. Short (15s) applications of isoprenaline (Iso 100nM) increased ICa,L ∼2.5-fold similarly in both conditions but had a 30% smaller effect on [cAMP]m in detubulated ARVMs. After Iso washout, ICa,L and [cAMP]m returned to basal with a ∼1.5-fold faster kinetic in detubulated ARVMs than in control, suggesting a faster cAMP degradation by PDEs. The contribution of PDE3 and PDE4 was thus tested using the inhibitors cilostamide (1µM) and Ro20-1724 (10µM), respectively. Both inhibitors had a more pronounced effect on Iso response of ICa,L and [cAMP]m in detubulated ARVMs. Thus, detubulation per se has a profound effect on the β-AR signalling cascade which may contribute to its impairment in HF. This is partly due to a change in the respective contributions of PDE3 and PDE4 to the hydrolysis of cAMP near the membrane and LTCCs.

Subcellular Origin and Tissue-Wide Synchronization of Abnormal Ca Release in the Genesis of Ca-Dependent Atrial Arrhythmia

Introduction. Abnormal diastolic Ca release (DCR) from the sarcoplasmic reticulum has been implicated in both ventricular as well as atrial fibrillation (AF). Atrial cells lack the extensive T-tubule network that facilitates Ca signaling in ventricular myocytes. How the distinct structural organization of atrial cells affects the genesis of AF is currently unknown.Methods and Results. We used rapid 2D confocal imaging to map Ca changes in atrial single myocytes and tissue preparations derived from CASQ2 knock-out (CASQ2.KO) mice manifesting Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Isolated CASQ2.KO atrial cells showed frequent local and cell-wide DCR events when exposed to isoproterenol. DCR predominantly originated nearly simultaneously at several “eager” sites localized at cell periphery. The distribution of latencies to local DCR events in CASQ2.KO cells displayed a left-ward shift consistent with abbreviated RyR2 refractoriness associated with CPVT. This abbreviated RyR2 refractoriness translated into highly synchronous spontaneous Ca oscillations across the intact atrial tissue. Conditional overexpression of SERCA2a in CASQ.KO mice exacerbated proarrhythmic Ca oscillations by increasing the proportion of interior eager release sites and enhancing DCR synchronicity in both isolated atrial cells and atrial tissue preparations.Conclusions. Our results suggest that aberrant DCR involves a certain set of eager Ca release sites that 1) only in part coincide with sites involved in normal EC coupling at cell periphery and 2) act as pacemakers for spontaneous Ca oscillations present in atrial cells. Due to abbreviated release site refractoriness, DCR is synchronized between neighboring cells thereby leading to tissue-wide Ca oscillations that may form basis for AF. SERCA2a overexpression exacerbates arrhythmic Ca oscillations by further enhancing DCR synchronicity.

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of Calcium/Calmodulin-Dependent Kinase II in the Dyadic Cleft

In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca(2+)/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca(2+)/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca(2+)/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Coupled RyRs have a distinct modulation by Ca(2+)/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/reactive oxygen species microdomain, and subject to modification with disease.