Cellular Biology

Dependence of Cardiac Transverse Tubules on the BAR Domain Protein Amphiphysin II (BIN-1)Novelty and Significance

Jessica L. Caldwell, Charlotte E.R. Smith, Rebecca F. Taylor, Ashraf Kitmitto, David A. Eisner, Katharine M. Dibb, Andrew W. Trafford
https://doi.org/10.1161/CIRCRESAHA.116.303448
Circulation Research. 2014;115:986-996
Originally published October 20, 2014

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Abstract

Rationale: Transverse tubules (t-tubules) regulate cardiac excitation–contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear.

Objective: To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient.

Methods and Results: T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA–induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient.

Conclusions: AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.

Introduction

The synchronous rise of the systolic Ca2+ transient in mammalian ventricular myocytes requires the presence of an extensive and regular transverse (t)-tubular system.1 These t-tubules ensure close apposition of L-type Ca2+ channels (LTCCs) and sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors [RyRs]) forming dyads or couplons where excitation–contraction coupling commences.2,3 The t-tubules are also surrounded by a continuous network of SR, which is thought to assist with amplification of the initial Ca2+ entry during the action potential and contribute to the synchronous rise of systolic Ca2+.4,5 The t-tubule and SR networks are, however, labile with disorganization and loss commonly observed in heart failure (HF).5–9 In such circumstances the loss of t-tubules leads to dyssynchronous Ca2+ release patterns, a smaller systolic Ca2+ transient, and altered β-adrenergic (β-adrenergic receptor) signaling.6–10 Conversely, recovery from HF is associated with restoration of the t-tubule network along with normalization of β-adrenergic receptor signaling and resynchronization of the systolic Ca2+ transient.9,11

In This Issue, see p 961

More extensive differences in t-tubule organization and density than those occurring in the ventricle during HF are known to exist between the atrium and the ventricle. For example, small mammals (mouse, rat, rabbit, etc) completely lack or possess only a rudimentary, predominantly axially arranged, t-tubule network.2–14 Conversely, some studies have suggested that limited numbers of atrial cells from smaller laboratory species such as the rat have a more ventricular-like t-tubule pattern,15 although these particular cells may be of different lineage and a feature of the pulmonary vein sleeve region.16 The poorly developed t-tubule network in these atrial myocytes leads to the characteristic early peripheral and delayed central Ca2+ transient.12,17 More recently, however, a well-developed t-tubule system has been noted in atrial myocytes of larger species including man.18–20 Although remaining less extensive than in the corresponding ventricle,19 the t-tubule system in atrial myocytes of these larger species substantially reduces the spatial heterogeneity of the systolic Ca2+ transient.19 Moreover, as in the ventricle, the atrial t-tubule network is disrupted in HF and atrial fibrillation resulting in increased Ca2+ transient heterogeneity and dyssynchronous Ca2+ release.18,19

Several proteins have been implicated in the biogenesis and maintenance of t-tubules including titin cap protein (telethonin), junctophilin 2 (JPH2), and the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII or BIN-1).21,22 Of these, the Bin/Amphiphysin/Rvs domain proteins are ubiquitously expressed, highly conserved in eukaryotes and have pleiotropic roles including sensing membrane curvature, endocytosis, and regulation of actin filament function.23,24 Certain splice variants of amphiphysin, AmpII (BIN-1),25–27 seem not to be involved in the formation of clathrin-coated vesicles and endocytosis.28,29 They are, however, highly expressed in striated muscles, localize to t-tubules, and gene deletion leads to fatal perinatal cardiomyopathy.28,30 More recently, these BIN-1 splice variants have been identified, in the mouse heart, as being involved in both t-tubule formation and causing extensive folding of the inner t-tubule membrane.27 However, the lack of a densely folded inner t-tubule membrane in other species, for example, sheep and rat as used in the present study,5 suggests that the particular BIN-1 (AmpII) splice variants responsible may be a feature of the murine myocardium.

Mutations in AmpII also lead to the inherited condition centronuclear myopathy,31 which is characterized by a severe cardiomyopathy, myocyte disarray,32 and arrhythmias.33 Moreover Drosophila AmpII mutants show flight muscle t-tubule disarray that is reversed by AmpII cDNA transfection.29 Finally, AmpII transfection induces tubule formation in Chinese hamster ovary (CHO) and the liver hepatocellular carcinoma cell line (HepG2), cell types that do not ordinarily possess t-tubules26,34,35 and, using the 13+17 splice variant, causes t-tubule rescue in cultured BIN-1 (AmpII) heterozygous knockout cardiac myocytes.27

In cardiac muscle, AmpII protein levels are decreased in HF, which as noted above is associated with t-tubule loss and disorganization.11,25 In addition, AmpII directs LTCC expression to the t-tubule26 and thereby provides a potential link between t-tubule loss and the decrease in L-type Ca2+ current observed in some models of HF.25,36 In the recently developed BIN-1 knockout mouse model, the heterozygote shows a decreased intensity of t-tubule staining although t-tubules in the ventricle seem to be localized correctly. However, no information was presented on how such perturbations in BIN-1 (AmpII) levels affect cellular Ca2+ homeostasis or interchamber differences in t-tubule density. This study therefore sought to determine (1) whether interchamber differences in t-tubule density are related to AmpII protein levels, (2) whether AmpII protein levels and t-tubule density change in HF, (3) whether AmpII gene silencing reduces t-tubule density, and (4) how AmpII gene silencing influences the synchronicity of the systolic Ca2+ transient. We demonstrate that (1) the amount of AmpII protein is lower in the atria, (2) AmpII protein levels and t-tubule density are lower in HF, (3) transfection of adult rat ventricular myocytes with small interfering RNA (siRNA) decreases both AmpII protein levels and t-tubule density, and (4) loss of AmpII increases heterogeneity (dyssynchrony) of the systolic Ca2+ transient. We conclude therefore that AmpII is required for the maintenance of t-tubules in cardiac muscle, and loss of AmpII is responsible for t-tubule disruption and increases the heterogeneity of the systolic Ca2+ transient. In contrast, using the same experimental approaches, we show that JPH2 is present, with sarcomeric intracellular distribution, in rat atrial and sheep HF atrial cells (which lack t-tubules) and also that JPH2 has an important role in determining t-tubule orientation rather than the overall density of t-tubules.

Methods

A detailed Methods section is available in the Online Data Supplement.

All procedures involving animals accord to The United Kingdom, Animals (Scientific Procedures) Act of 1986 and have been approved by The University of Manchester Ethical Review Board.

Myocyte Isolation, T-Tubule Quantification, and Animal Models

Single cardiac myocytes were isolated from the left ventricle and left atria of sheep and rats using collagenase and protease digestion methods described in detail previously.19,36–38 Myocytes and paraformaldehyde-fixed tissue sections (ferret) were stained with the voltage-sensitive aminonapthylenylpyridium dye di-4-ANEPPS, the styryl dye N-(3-trethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl) pyridinium dibromide (FM4-64), or Alexa Fluor 488–conjugated wheat germ agglutinin (WGA) and imaged confocally on a Leica SP2 microscope to visualize t-tubules, which were then quantified after image processing as described previously.19,20 HF was induced using either right ventricular tachypacing in the sheep19,36,39 or ascending aortic coarctation in the ferret.40,41

Myocyte Culture, siRNA-Mediated Gene Silencing and [Ca2+]i Measurements

Single rat ventricular myocytes were maintained in myocyte growth medium (Promocell, UK) and transfected following manufacturer’s recommendations (Santa Cruz, USA) with 10 µmol/L siRNA (Sigma Mission siRNA or Santa Cruz scrambled siRNA) targeting AmpII, JPH2, or using a scrambled (control) siRNA. Cells were transfected for 24 hours and then imaged as described above or processed for immunoblotting or immunolabeling. Changes in [Ca2+]i were monitored using Fluo-3 acetoxymethyl ester (AM)–loaded cells on a Leica SP2 confocal microscope.

Statistics

All data are presented as mean± SEM from n observations/N experiments. To account for multiple observations (n) from the same animal (N) linear mixed modeling was performed (SPSS Statistics; IBM, USA). Where multiple observations or technical replicates were not performed, Student t test, paired t test, or Mann–Whitney rank-sum tests (used when the data was neither normally distributed nor had unequal variance) were used. Pearson coefficient was performed on paired data points using GraphPad Prism 5.0. Data were considered significant when P<0.05.

Results

Interchamber Differences in T-Tubule Density and AmpII or BIN-1 Expression

The presence of t-tubules was examined in the ventricles and atria of the rat, ferret, and sheep (Figure 1A). In agreement with previous work,1,19,20 a well-developed t-tubule network was found in all of the ventricular samples studied. In the rat atrium, we found essentially no t-tubules, whereas in the ferret atrium a rudimentary t-tubule network was present in some cells (Figure 1Ab). To determine that the cell isolation process had not led to t-tubule loss in rat atrial cells, we also performed confocal imaging of intact hearts stained with FM4-6442; both right and left ventricular myocytes had a well-developed t-tubule network, whereas t-tubules were again essentially absent in both the left and right atria with the occasional short potential membrane invagination present in few cells (Online Figure III). However, in the sheep atrium, a well-developed t-tubule network was present (Figure 1Ac). As a major role of the surface membrane and t-tubules is to facilitate Ca2+ flux in and out of the cell, we next assessed interchamber and species differences in t-tubule occurrence on the distance any point is inside the cell to the surface membrane or t-tubule membrane. This was assessed using 2 approaches; first, as described previously,19,20 by calculating distance maps (Figure 1Aaii–1Acii) profiling the distance any voxel is within the cell (in either the vertical or horizontal planes) from a membrane (surface sarcolemma or t-tubule) and calculating the distance which 50% of voxels are from a membrane (referred to as the half-distance, Figure 1Ba–1Bc; Online Figure II). The second approach provides a measure of t-tubule density independent of the effects of cell width on half-distance measurements. Here, we determined the fraction of all voxels within the central 2-µm-thick section of the cell (excluding surface sarcolemma) occupied by t-tubules identified using either di-4-ANEPPS or Alexa Fluor 488–conjugated WGA staining (referred to as the t-tubule fractional area; Figure 1Ca–1Cc). It is clear that, in all species, there are more t-tubules in the ventricular samples than the corresponding atrial samples (Figure 1B and 1C; P<0.01). Moreover, in agreement with previous studies,19,20 the fractional area occupied by t-tubules in the atrium was greatest in the sheep (Figure 1C).

Figure 1.
Figure 1.

Quantification of species and interchamber differences in transverse tubule (t-tubule) density. A, Representative examples showing membrane staining (upper) and distance maps (lower) in ventricular and atrial myocytes from rat (a), ferret (b), and sheep (c). Cells have been stained with either di-4-ANEPPS (a and c) or WGA (b). Scale bars=10 µm. B, Mean data summarizing the distance 50% of voxels (half-distances) are from the nearest cell membrane (t-tubule or surface sarcolemma) in ventricular and atrial cells from (a–c) rat, ferret, and sheep. C, Mean data summarizing the fraction of intracellular pixels occupied by t-tubules in ventricular and atrial cells from (a–c) rat, ferret, and sheep. **P<0.01; ***P<0.001. Rat ventricle, 20 cells/5 hearts; rat atria, 15 cells/3 hearts; ferret ventricle, 8 cells/4 hearts; ferret atria, 8 cells/4 hearts; sheep ventricle, 20 cells/5 hearts; and sheep atria, 21 cells/5 hearts.

We next sought to determine whether the interchamber differences in t-tubule density were associated with changes in AmpII protein levels. Representative immunoblots from rat, ferret, and sheep samples are shown in Figure 2A. Despite the absence or limited presence of t-tubules in the atria of rat and ferret, AmpII protein is detectable, although at lower levels compared with the respective ventricle (lower by 61±4.0%; P<0.001 and 38.2±8.1%; P<0.05 respectively). In the sheep myocardium, as is observed in skeletal muscle30 and mouse ventricle,27 2 major isoforms were detected and densitometric analysis of each band alone or both combined yielded the same qualitative result (not shown); we therefore quantified both bands. In the sheep atrium, where the t-tubule network is relatively well developed, AmpII levels were still lower than in the corresponding ventricle. However, the extent of the decrease (26.7±6.7%; P<0.05) is less than that in rat and ferret where t-tubules are absent.

Figure 2.
Figure 2.

Interchamber differences in amphiphysin II (AmpII) and junctophilin 2 (JPH2) protein levels in the rat, ferret, and sheep. A, Representative Western blots for AmpII and GAPDH (upper) and mean data for ventricular and atrial tissues from rat (a), ferret (b), and sheep (c; note: GAPDH cannot be used for sheep samples). B, Representative Western blots for JPH2 and GAPDH (upper) and summary data (lower) for ventricular and atrial tissues from rat (a), ferret (b), and sheep (c). *P<0.05; ***P<0.001. For Western blots data have been normalized to the respective internal control (IC) relative to GAPDH (where used). n=6, rat ventricle; 5, rat atria; 5, ferret ventricle; 6, ferret atria; 7, sheep ventricle; and 7, sheep atria.

The membrane bridging protein JPH2 has also been implicated in determining t-tubule orientation and formation.43,44 We therefore examined whether the abundance of JPH2 protein differs between cardiac chambers in line with the changes in t-tubule density and AmpII protein expression noted above. These data are summarized in Figure 2B and show that, irrespective of the presence or absence of t-tubules, there are no interchamber differences in JPH2 in the rat and ferret. For example, in the rat atrium, JPH2 levels are 96% of those in the ventricle (Figure 2Ba; P=0.87). Conversely, in the sheep atrium where t-tubules are well developed, JPH2 levels are lower than in the ventricle (by 32.1±6.5%; P<0.001).

Reduced AmpII or BIN-1 Expression in HF and T-Tubule Loss

HF is known to lead to loss of t-tubules in the ventricle8 and atrium19 and therefore we next determined whether changes in AmpII or JPH2 protein levels occur in parallel to t-tubule loss. First, we examined t-tubule density in ventricular and atrial cells in an ovine tachypacing model of HF. Clinical signs of HF including breathlessness and lethargy were present after 50.5±4.3 days of right ventricular tachypacing. As reported previously,36,39 left ventricular internal diastolic dimension increased (prepacing, 2.41±0.14 cm; HF, 3.87±0.09 cm; P<0.001) and fractional shortening decreased (prepacing, 0.68±0.02; HF, 0.27±0.02; P<0.001) with the development of HF (Online Table I). Isolated myocytes were stained with di-4-ANEPPS to visualize the t-tubule network and it is clear that in both ventricular (Figure 3A) and atrial cells (Figure 3B) there is t-tubule loss in HF. We characterized the extent of t-tubule loss by determining the half-distance value and fractional area occupied by t-tubules (Figure 3Aa and 3Bb). The original images and voxel distance maps show that in the ventricle t-tubule loss was evident at the cell end and to varying extents throughout the cell. Conversely, in the atrium there was an almost complete loss of t-tubules in HF and correspondingly larger increase in the half-distance and smaller fractional area occupied by t-tubules compared with the ventricle. The half-distance increased from 0.38±0.01 to 0.45±0.01 µm in the ventricle (P<0.001) and 0.78±0.07 to 1.94±0.12 µm in the atrium (P<0.01) and the fractional area occupied by t-tubules decreasing from 0.22±0.01 in control to 0.15±0.01 in HF in the ventricle (Figure 3Abiii; P<0.001) and from 0.075±0.01 in control to 0.014±0.003 in HF in the atrium (Figure 3Bbiii; P<0.05). The reduction/loss of t-tubules in the ventricle and atrium in HF was associated with a reduction in AmpII protein levels in both chambers (Figure 3Ca and 3Cb; ventricle by 24.1±5.7% and atrium by 34.5±6.9%; both P<0.05). However, despite the loss of t-tubules in HF, there was no change in JPH2 protein levels in either the ventricle or the atrium (Figure 3D).

Figure 3.
Figure 3.

Decreased transverse tubule (t-tubule) density and amphiphysin II (AmpII) protein levels but unchanged junctophillin-2 (JPH2) protein levels in ventricular and atrial cells in ovine tachypacing induced heart failure (HF). A, Representative membrane staining (a), distance maps (bi) and mean data summarizing half-distances (bii), and t-tubule fractional occupation (biii) from control sheep (left) and HF sheep (right) ventricular myocytes. B, Representative membrane staining (a), distance maps (bi), and mean data summarizing half-distances (bii) and t-tubule fractional occupation (biii) from control sheep (left) and HF sheep (right) atrial myocytes. Data from (cells/hearts); ventricle, 67/6 control, 56/7 HF; atria, 21/5 control, 18/2 HF. C, Representative Western blots (upper) and summary data (lower) showing changes in AmpII protein levels in ventricular (a) and atrial (b) tissues. D, Representative Western blots (upper) and summary data (lower) showing JPH2 protein levels in ventricular (a) and atrial (b) tissues. *P<0.05; **P<0.01; ***P<0.001. For Western blots data are normalized to the respective internal control (IC) sample used in each experiment. For Western blotting n=7 control ventricle, 7 HF ventricle, 7 control atria, and 6 HF atria. Scale bars=10 µm.

To establish that the changes noted above were not restricted to the tachypacing model used in the sheep, we also examined the t-tubule network, AmpII and JPH2 protein levels in a thoracic aortic coarctation/pressure overload model of HF in the ferret.40,41 Clinical signs of HF took 39±2 days to develop after aortic coarctation and resulted in an increase in left ventricular end-diastolic dimensions from 1.99±0.03 to 2.25±0.06 cm and decrease in ejection fraction from 0.41±0.04 to 0.11±0.02 (both P<0.005; Online Table II). In the HF ventricle, t-tubule loss was evident (Figure 4A) resulting in an increase in the voxel half-distance from 0.294±0.008 µm in sham-operated hearts to 0.353±0.007 µm in HF (P<0.01). The increase in half-distance was accompanied by a decrease in the fractional area occupied by t-tubules in the ferret ventricle in HF from 0.236±0.007 in control to 0.186±0.005 in HF (Figure 4Abiii; P<0.001). As in the sheep tachypacing model of HF, AmpII protein levels were decreased in the ferret aortic coarctation model (Figure 4Ba, by 61.3±5.6%; P<0.05) but no change in JPH2 protein levels was observed (Figure 4Bb). Figure 5A examines the relationship between t-tubule density and AmpII protein levels across each of the species, cardiac chambers, and disease models studied thus far. Because different loading controls were used in each experiment, we have normalized the AmpII protein levels to those in the appropriate control ventricular sample. A significant correlation exists between t-tubule half-distance (density) and AmpII protein levels (P<0.01) indicating that, in common with Hong et al,27 t-tubule density depends on AmpII protein levels. Importantly, although the relationship between t-tubule density (half-distance) and AmpII protein seems to diverge at low AmpII levels, this significant correlation is maintained if the sheep is examined in isolation or if the rat atria, which lacks t-tubules, are excluded from the analysis (both P<0.05; data not shown).

Figure 4.
Figure 4.

Decreased transverse tubule (t-tubule) density and amphiphysin II (AmpII) protein levels but unchanged junctophillin-2 (JPH2) protein levels in ventricular cells after aortic coarctation induced heart failure (HF) in the ferret. A, Representative images showing membrane staining (a), distance maps (bi), half-distance summary data (bii), and t-tubule fractional area summary data (biii) from control (left) and HF ferret ventricular myocytes (right). Data from (cells/hearts); control, 22/7; HF, 15/5. B, Representative Western blots (upper) and summary data for AmpII (a) and JPH2 (b) in control and HF ventricular samples. *P<0.05; **P<0.01; ***P<0.001. For Western blots data are presented normalized to the internal control (IC) sample. Data from 5 control and 6 HF animals. Scale bars=10 µm.

Figure 5.
Figure 5.

Transverse tubule (t-tubule) density correlates with amphiphysin II (AmpII) protein levels across tissues, differing disease states and after small interfering RNA (siRNA)–mediated gene silencing. A, Dependence of t-tubule half-distances on AmpII protein levels in tissues and species indicated. Data are presented as mean±SEM and the solid line through the data is a best-fit linear regression (Pearson correlation coefficient, #P<0.01). Half-distances and AmpII protein levels are expressed relative to the respective control samples (eg, ventricular samples when comparing with atrial expression and, control atrial or ventricular samples when comparing with changes in heart failure, which are plotted at coordinate 1,1). B, Representative images showing immunolocalization of AmpII (a) and di-4-ANEPPS membrane staining (b) in freshly isolated (left), scrambled siRNA–treated (center), and AmpII siRNA–treated (right) rat ventricular cells. C, Representative Western blot (upper) for AmpII and β-actin and summary data showing reduced AmpII protein abundance after siRNA treatment. ***P<0.001. n=18 experiments. D. Mean data summarizing half-distances (solid bars) and t-tubule fractional area (open bars) in freshly isolated, scrambled siRNA–treated and AmpII (target)-treated rat ventricular myocytes (scrambled vs freshly isolated; $P<0.05: target vs scrambled; ***P<0.001: target vs freshly isolated; †P<0.001). Data from (cells/experiments); freshly isolated, 20/5; scrambled, 199/20; target, 196/20. E, Dependence of half-distance on AmpII protein levels after siRNA-mediated AmpII gene silencing. The solid line through the data is a best-fit linear regression (#P<0.01; n=18). Scale bars=10 µm.

AmpII or BIN-1 Gene Silencing Reduces T-Tubule Density and Increases the Heterogeneity of the Systolic Ca2+ Transient

We next sought, using siRNA-mediated gene silencing, to determine the role AmpII plays in t-tubule maintenance and synchronization of the systolic Ca2+ transient. The immunolocalization in Figure 5Ba shows that AmpII has a sarcomeric (≈2-µm spacing) distribution in freshly isolated and scrambled siRNA–transfected rat ventricular cells. However, in AmpII-targeted siRNA (target)–transfected cells the distribution of AmpII is markedly altered becoming noticeably punctate and heterogeneous. The altered AmpII immunolocalization after siRNA gene silencing is associated with disorganized t-tubule staining (Figure 5Bb). Consistent with previous studies showing some degree of t-tubule loss in cultured ventricular cells,45,46 there was a slight increase in half-distance and decrease in fractional area occupied by t-tubules in scrambled siRNA–treated cells compared with freshly isolated cells (Figure 5D; half-distances: freshly isolated, 0.32±0.02 µm; scrambled siRNA treated, 0.38±0.01 µm, P<0.05; fractional areas: freshly isolated, 0.21±0.01; scrambled siRNA, 0.17±0.01, P<0.05). However, siRNA gene silencing had a more pronounced effect on half-distance and fractional occupation with the siRNA-mediated 12±3.2% decrease in AmpII protein abundance (Figure 5C; P<0.001) resulting in an increase in the voxel half-distance from 0.37±0.01 to 0.57±0.01 µm (Figure 5D; P<0.001) and a decrease in the fractional area occupied by t-tubules from 0.17±0.01; target, 0.08±0.01 (Figure 5D; P<0.001). Importantly, siRNA-mediated gene silencing did not alter JPH2 protein abundance (Online Figure IVA).

Given the potential role of AmpII in trafficking the LTCC to the t-tubule,26 we also examined the cellular distribution of the LTCC, AmpII, and t-tubules in freshly isolated, scrambled siRNA and AmpII-targeted siRNA–transfected cells (Online Figure V). In freshly isolated cells (Online Figure VA), AmpII and the LTCC have regular striated intracellular distribution. The LTCC is also present to some extent on the surface sarcolemma; nevertheless, there is strong colocalization of the LTCC and AmpII. In scrambled siRNA–treated cells t-tubules were visualized by WGA staining and are clearly well maintained as is the cellular distribution of, and colocalization with, the LTCC (Online Figure VB). After AmpII siRNA–mediated gene silencing there is loss of t-tubules and LTCC predominantly from the center of the cell, whereas at the cell edges colocalization of the t-tubule and the LTCC is maintained (Online Figure VC).

Because transient transfection techniques have limited efficiency and can be variably successful, we examined the post-transfection relationship between AmpII protein levels and the voxel half-distance as a measure of t-tubule density. These data are summarized in Figure 5E and show an inverse correlation between voxel half-distance and AmpII protein levels (P<0.01). Thus, the density of t-tubules depends on the level of AmpII within cardiac myocytes.

Finally, we sought to determine the effect that AmpII gene silencing–mediated t-tubule loss had on the systolic Ca2+ transient. Cells were field stimulated and changes in [Ca2+]i measured using xt scanning confocal microscopy (Figure 6A). In scrambled siRNA–transfected cells the systolic Ca2+ transient rose synchronously along the length of the cell, whereas in the target siRNA–treated cell there are areas where the rise of [Ca2+]i was delayed. On average the time for the systolic Ca2+ transient to reach 50% of its peak at each point along the linescan image (F50) increased from 17.3±0.5 ms in scrambled siRNA–treated cells to 20.2±0.8 ms in target siRNA–treated cells (Figure 6B; P<0.01). To assess the degree of spatial heterogeneity of the systolic rise of [Ca2+]i the dyssynchrony index was calculated from the SD of the F50 times along each linescan image.7 The dyssynchrony of the systolic Ca2+ transient increased by 163±33% (Figure 6C; scrambled, 13.9±1.3 ms; target, 36.9±3.2 ms; P<0.001). In addition to the delayed and dyssynchronous rise of [Ca2+]I, the amplitude of the systolic Ca2+ transient was also reduced in the target siRNA–treated cells, in this instance by 20.6±4.1% (Figure 6D; P<0.001).

Figure 6.
Figure 6.

Amphiphysin II (AmpII) gene silencing–mediated changes to the systolic Ca2+ transient. A, Confocal xt line-scans (left) and line-by-line systolic Ca2+ rise times (right) from a representative scrambled (upper) and AmpII siRNA–targeted (lower) rat ventricular cell. B, Mean data showing delayed rise time (F50) of the systolic Ca2+ transient in AmpII gene–silenced rat ventricular cells. C, Mean data showing increased dyssynchrony index (SD of rise times) in AmpII-silenced rat ventricular cells. D, Mean data showing reduced Ca2+ transient amplitude after AmpII gene silencing in rat ventricular cells. **P<0.01; ***P<0.001. Data from (cells/experiments); scrambled, 80/12; target, 94/12.

JPH2 Gene Silencing Does Not Affect T-Tubule Density but Does Alter T-Tubule Orientation

The previous experiments point to a key role for AmpII in regulating t-tubule maintenance in cardiac muscle. However, several additional proteins have also been implicated in controlling t-tubule maintenance/formation. We investigated a potential role for the membrane bridging protein JPH2, which is thought to be responsible for tethering the SR to the t-tubule and maintaining t-tubule orientation.42,47 First, the cellular localization of JPH2 was examined (Figure 7Aa). In both freshly dissociated and scrambled siRNA–transfected cells JPH2 has a primarily sarcomeric distribution with some surface membrane staining. In JPH2 siRNA–transfected cells there is a 26±5.4% decrease in JPH2 protein levels (Figure 7B; P<0.01) with no changes in AmpII protein abundance detected (Online Figure IVB). Notably however, in JPH2 siRNA–targeted cells the cellular distribution of JPH2 appears more diffuse throughout the cell. The altered JPH2 cellular distribution is reflected in a less organized t-tubule network in JPH2 siRNA–transfected cells (Figure 7Ab). However, despite the disorganization of the t-tubule network the voxel half-distance and fractional area occupied by t-tubules are unaltered by JPH2 gene silencing (Figure 7C).

Figure 7.
Figure 7.

Junctophilin 2 (JPH2) gene silencing does not reduce transverse tubule (t-tubule) density in rat ventricular cells. A, Representative images showing immunolocalization of JPH2 (a) and membrane staining with di-4-ANEPPS (b) in freshly isolated (left), scrambled siRNA–treated (center) and JPH2 siRNA–treated (right) rat ventricular cells. B, Example Western blots of JPH2 and β-actin in scrambled and JPH2 target–treated rat ventricular cells (upper) and mean data showing reduced JPH2 protein abundance in JPH2 gene–silenced cells (lower). **P<0.01. n=4 experiments. C, Mean data showing no change in half-distance (solid bars) or fractional t-tubule area (open bars) measurements after JPH2 gene silencing in adult rat ventricular myocytes. *P<0.05 vs freshly isolated myocytes. Data from (cells/experiments); freshly isolated, 20/5; scrambled, 48/5; target, 51/5 Scale bars=10 µm.

Although the decrease in JPH2 protein abundance (Figure 7B) does not alter the distance any point within the cell is from either the surface or t-tubule membrane, the decrease in JPH2 expression does alter the orientation of t-tubules. We quantified changes in t-tubule orientation by skeletonizing the di-4-ANEPPS–stained cells and calculating the probability distribution of the angular orientation of t-tubules48 (Figure 8A). In scrambled siRNA–transfected cells t-tubules are predominantly oriented perpendicular to the long axis of the cell, whereas in JPH2 siRNA–transfected cells t-tubules have both perpendicular and horizontal orientation such that the ratio of transverse (90±15° to long axis) to longitudinal (0±15° to long axis) t-tubules decreased by 31±7.8% in JPH2 siRNA–transfected cells (Figure 8B; P<0.01). T-tubule orientation was also examined in ventricular cells from the ovine tachypacing and ferret pressure overload models of HF (Online Figure VI). In the sheep, but not the ferret, longitudinal t-tubules were more evident in HF.

Figure 8.
Figure 8.

Junctophilin 2 (JPH2) gene silencing leads to transverse tubule (t-tubule) reorientation in adult rat ventricular cells but no change in t-tubule density. A, Representative images showing di-4-ANEPPS membrane staining (a), skeletonized images (b) and t-tubule orientation (c) from a scrambled siRNA–treated (left) and JPH2 siRNA (right)–targeted rat ventricular myocytes. B, Mean data showing a reduction in the ratio of transverse (perpendicular) to longitudinal (axial) t-tubules in JPH2 gene–silenced rat ventricular cells. **P<0.01. Data from (cells/experiments); scrambled 48/5; target, 51/5. Scale bars=10 µm.

We also examined the cellular distribution of JPH2 and the RyR in those atrial tissues where t-tubules are essentially absent (HF sheep atria and rat atria; Online Figure VII). A similar colocalizing JPH2 and RyR sarcomeric distribution is observed in rat ventricular tissue (Online Figure VIIBb) where t-tubules are known to be present. Conversely, AmpII and WGA (cell membrane) staining are present around the cell surface rat atrial myocytes but colocalize with sarcomeric distribution throughout the cytoplasm of rat ventricular myocytes (Online Figure VIIC).

Discussion

Five main findings are presented in this article: (1) within a given species there are interchamber differences in t-tubule density that are paralleled by differences in AmpII but not JPH2 protein levels; (2) t-tubule density and AmpII protein levels, but not JPH2 protein levels, are decreased in 2 distinct models of HF; (3) AmpII gene silencing in adult rat ventricular cells decreased t-tubule density, AmpII protein levels, Ca2+ transient amplitude and increased the dyssynchrony of the systolic Ca2+ transient, (4) JPH2 gene silencing did not reduce the overall density of t-tubules in rat ventricular cells but did alter t-tubule orientation, and (5) JPH2 has sarcomeric intracellular distribution colocalizing with RyRs in cells lacking t-tubules. Taken together these findings indicate that AmpII has a major role in t-tubule maintenance in cardiac muscle. Conversely, however, JPH2 seems to be more important for controlling t-tubule orientation and localization of nonjunctional RyRs rather than controlling t-tubule density in cardiac muscle.

Differences in T-Tubule Density, AmpII or BIN-1, and JPH2 Protein Levels Between Atrial and Ventricular Cardiac Myocytes

In accordance with many,1,49 but not all studies,15,42 we found that rat atrial myocytes lack a discernible t-tubule network. We also found that the ferret atrium like that of the cat50 and rabbit13 had only a sparse t-tubule network present in some cells. However, as described previously,18–20 the sheep atrium has a well-developed t-tubule network. Functionally, where the t-tubule network is absent in atrial myocytes the systolic Ca2+ transient rises initially at the cell periphery and then propagates as a wave of Ca2+-induced Ca2+ release to the cell center.1,49 However, the relatively well-developed t-tubule network in the sheep atrium ensures that the systolic Ca2+ transient rises synchronously at the cell periphery and the cell center.18,19 Despite the interspecies differences in t-tubule density in the atria we noted that all ventricular cells from the rat, ferret, and sheep possessed a regular t-tubule network throughout the entire volume of the cell and in all species the density of the ventricular t-tubule network was greater than that in the atrium.

Given the interchamber differences in t-tubule density one of the first aims of this study was to elucidate which proteins may be involved in t-tubule formation. To this end several candidates exist and we have studied 2 of these in detail, AmpII and JPH2. Expression of the Bin/Amphiphysin/Rvs domain protein bridging integrator-1 (BIN-1), or AmpII, in nonmuscle CHO cells is sufficient to induce tubule formation.34 AmpII has also been shown to be responsible for trafficking of the LTCC to the cell membrane in cardiac myocytes.26 JPH2, however, tethers the SR/RyR to the sarcolemma/Z-line maintaining the geometry of the dyad.4,47,51 Deletion of either AmpII and JPH2 results in perinatal lethality with evidence of cardiac failure and structural disorganization.28,44,47 It is also noteworthy that the recently described murine cardiac conditional BIN-1 (AmpII) knockout heterozygote heart retains a regular, albeit less intensely stained, t-tubule network.27 However in the same study, short hairpin RNA gene silencing of AmpII caused t-tubule loss as was observed herein and moreover, adenoviral-mediated expression of the 13+17 splice variant of BIN-1 increased t-tubule intensity in cultured heterozygote cells. In addition, in the BIN-1 knockout heart the main secondary effect of BIN-1, after maintaining t-tubule density, seems to be the generation of a tightly folded inner t-tubule membrane. Interestingly, in agreement with several other studies,52,53 we do not note inner t-tubule membrane folding in the rat or sheep ventricular myocardium by serial block face scanning electron microscopy5 and the ≈250 nm resolving capability of confocal microscopy is insufficient to visualize such structures or subtle changes in t-tubule lumen diameter. As such the inner t-tubule membranes noted by Hong et al27 may be features specific to the mouse or reflect the role of species differences in BIN-1 (AmpII) isoform expression.

We find that AmpII protein levels are lower in the atrium compared with the ventricle, which therefore parallels the observed differences in t-tubule density between the atrium and ventricle. Conversely, in the rat and ferret atria where the t-tubule network is either absent or sparse, we do not observe a corresponding decrease in JPH2 protein levels compared with that seen in the ventricle. Paradoxically however in the sheep atrium, where t-tubules are prevalent, JPH2 protein levels are less than that in the corresponding ventricle.

An unresolved question arising from this work is what is the basis of the constancy of JPH2 expression in the rat and ferret atria and ventricle despite the marked differences in t-tubule density? Although we do not have a definitive answer, in the rat atrium where t-tubules are virtually absent, RyRs and LTCCs form dyads at the cell surface.54 In addition, at least in some studies, the L-type Ca2+ current density in the rat atrium and ventricle is the same37,55 suggesting that the surface density of LTCCs and thus dyads may be greater in the atrium than the ventricle. Furthermore, RyRs are distributed throughout the cell as part of the nonjunctional or corbular SR.17,56 Thus, JPH2 may still ensure dyad alignment and nonjunctional SR alignment in these cells. In support of this we find that RyR and JPH2 have a regular sarcomeric intracellular distribution and colocalize in sheep HF atrial myocytes and rat atrial myocytes where t-tubules are absent.

A subsidiary question is why, despite the marked difference in t-tubule density between the atria and ventricles in the rat and ferret, is there only slightly less AmpII protein in the atrium compared with the ventricle? We propose that the explanation for this observation is possibly because of the plurality of roles of AmpII. In a recent study by Hong et al27 and in Drosophila indirect flight muscle,29 loss of AmpII is associated with t-tubule loss. Conversely, AmpII also seems to be required for trafficking of the LTCC to the cell membrane26 and, at least in the mouse heart, multiple nontubule-forming AmpII isoforms are expressed.27 Therefore, the existence of LTCCs in atrial cells lacking t-tubules taken together with the other roles known to be performed by various AmpII isoforms would suggest a maintained requirement for AmpII expression even in tissues where t-tubules are lacking. Determining the potential roles for changes in AmpII isoform expression in different tissues and disease settings and the impact that this has on t-tubule formation, maintenance and function are worthy of future elucidation.

Remodeling of T-Tubules in HF

Further evidence for the importance of AmpII in t-tubule maintenance is provided by the observation that in the 2 different models of HF used in the present study there is a reduction in t-tubule density paralleled by a decrease in AmpII but not JPH2 levels. Several previous studies have reported t-tubule loss and disorganization in cardiac disease states,7–9,18,19,48 whereas some, in line with the present study, have reported reductions in AmpII11,25,26 and others have also reported reductions in JPH2 in HF.11,42,48 It is noteworthy however that, in response to SR Ca2+-ATPAse (SERCA) gene delivery11 or mechanical unloading9 as treatments for HF, t-tubule density9,11 and AmpII (BIN-1) protein levels11 increased toward control levels but those for JPH2 remained reduced. Similarly, in a pulmonary hypertension model of right ventricular failure sildenafil treatment commenced when ventricular dysfunction was evident leads to an increase in t-tubule density but not of JPH2 protein levels.57 Conversely, β-blocker therapy commenced after myocardial infarction was associated with an increase in t-tubule density and JPH2 protein relative to failing tissues.58 However, these discrepant findings most likely reflect differences in study design and commencement of β-blocker therapy before ventricular dysfunction was evident and hence attenuation of the progression of HF rather than recovery from HF; a situation distinct from those noted above for the effects of mechanical unloading and SERCA gene therapy.9,11

Gene Silencing Approaches Highlight an Important Role for AmpII or BIN-1 in T-Tubule Maintenance in Cardiac Muscle

The interchamber and HF differences in t-tubule density and AmpII expression described above, while indicating an important role for AmpII in t-tubule maintenance in cardiac muscle, remain as only associative observations. We therefore sought to define more precisely if reductions in AmpII expression directly influence t-tubule density in cardiac muscle and adopted a siRNA transient transfection approach in adult rat ventricular myocytes. Here, we observed a direct relationship between AmpII protein and t-tubule density even after a relatively short period of ≈24-hour gene silencing. A similar linear dependence of t-tubule intensity on AmpII protein in cardiac myocytes has also been reported recently using lentiviral-mediated short hairpin RNA gene silencing during an extended 4-day culture period27; however, whether the extended culture period also influences these latter observations is unclear given the propensity of adult cardiac cells to dedifferentiate relatively rapidly when maintained in culture conditions.46 The reported-half life of AmpII is ≈2 hours59 and therefore amenable to transient transfection techniques and short-term culture of adult ventricular myocytes was therefore used in the present study. Given the low transfection efficiency of adult cardiac myocytes when using nonviral approaches,60 the reduction in AmpII protein levels after siRNA treatment (≈12%) is likely an underestimate of the reduction in AmpII in individual cells that have been successfully transfected; although cells were randomly studied, identifying which cells had been successfully transfected using fluorescently labeled siRNA was not successful in the present study.

Although AmpII gene silencing leads to a loss of t-tubules and therefore an increase in the distance any point within the cell is from a t-tubule or surface membrane, we found that both the half-distance and the fraction of the cell volume occupied by t-tubules were unaltered by siRNA-mediated JPH2 gene silencing. However, JPH2 gene silencing did result in an alteration to the spatial arrangement of the t-tubules from a predominantly transverse (perpendicular to the long axis of the cell) orientation to one where axially arranged t-tubules were frequently observed. A similar t-tubule reorientation as a consequence of JPH2 gene silencing has been noted previously.42 More recently, it has also been suggested that, during postnatal development, JPH2 may have an important role in determining the formation of transversely oriented t-tubules rather than axially arranged t-tubules as the transversely oriented t-tubules were found to persist after JPH2 gene silencing.61 Similarly, in those models of HF where JPH2 levels are decreased, an increase in the proportion of axially arranged t-tubule elements has been noted.48,62 Although in the present study JPH2 was not reduced in either the sheep tachypacing or ferret pressure overload models of HF, there was a change in t-tubule orientation in the sheep tachypacing model. This implies that in addition to JPH2, AmpII, potentially even specific AmpII isoforms or other factors may also be responsible for maintaining t-tubule orientation. However, it seems that our findings are most consistent with AmpII being required for t-tubule maintenance and that JPH2 has a role in ensuring the correct spatial alignment of t-tubules and RyRs in the heart.

Consequences of T-Tubule Loss on the Systolic Ca2+ Transient

In the present study, we show that AmpII gene silencing–mediated depletion of t-tubules leads to a reduced Ca2+ transient amplitude and dyssynchrony of the systolic Ca2+ transient. Previous studies have also shown that t-tubule disorientation after JPH2 gene silencing in various cell types also increases the heterogeneity of the systolic Ca2+ transient.44,61,63 Our findings are also in line with earlier studies showing that the systolic Ca2+ transient amplitude is reduced and becomes dyssynchronous in HF.7,9,11 In HF, there is also a reduction in the t-tubule density, although the loss of t-tubules has not been previously shown to be causative of the dyssynchronous Ca2+ transient. However, acute formamide–induced detubulation of cardiac myocytes and extended culture of adult cardiac myocytes also lead to a reduction in L-type Ca2+ current and reduced synchronicity of the systolic Ca2+ transient, suggesting a causal link between t-tubule disruption and Ca2+ transient heterogeneity.1,46,64,65

In summary, we show that the Bin/Amphiphysin/Rvs domain protein AmpII (BIN-1) is intricately involved in the maintenance of cardiac t-tubules and thus ensuring the synchronicity of the systolic Ca2+ transient. Our data also support a role for JPH2 in maintaining the normal orientation of t-tubules. It is therefore possible that AmpII and JPH2, along with several other proteins implicated in t-tubule formation, for example, titin cap protein (telethonin) and phosphoinositide 3-kinase (PI3K), may form a signaling nexus along the z-line to regulate t-tubule formation, maintenance, and orientation. However, from the present study it is clear that t-tubule maintenance depends on AmpII levels and that changes in t-tubule density correlate strongly with AmpII in both the healthy and diseased heart. Thus, AmpII may be an attractive target for restoring t-tubules and thus systolic Ca2+ and contractility in HF.

Sources of Funding

This work was supported by grants from The British Heart Foundation (FS/12/57/29717, RG/11/2/28701, CH/2000/04, FS/10/52/28678, FS/14/4/30532, and PG/12/89/29970), The European Union Framework 6 programme (Normacor), Wellcome Trust Institutional Strategic Support Fund (097280) to The University of Manchester and The Manchester Biomedical Research Centre (George Lancashire Award).

Disclosures

None.

Footnotes

  • In September, 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.29 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.303448/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    AmpII
    amphiphysin II (BIN-1)
    BIN-1
    bridging integrator-1 (AmpII)
    HF
    heart failure
    JPH2
    junctophilin 2
    LTCC
    L-type Ca2+ channel
    RyR
    ryanodine receptor
    siRNA
    small interfering RNA
    SR
    sarcoplasmic reticulum

  • Received January 22, 2014.
  • Revision received October 14, 2014.
  • Accepted October 17, 2014.

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Novelty and Significance

What Is Known?

  • Transverse tubules (t- tubules) are surface membrane invaginations and are found in all mammalian ventricular myocytes.

  • Key ion channels linking the action potential to the systolic rise of calcium are located on t-tubules.

  • In different cardiac diseases t-tubules density is decreased and this leads to heterogeneity of the systolic rise of calcium and reduced contractility.

What New Information Does This Article Contribute?

  • We demonstrate that the bridging integrator protein, amphiphysin II (AmpII), is vital for maintaining t-tubules in ventricular myocytes.

  • Differences in t-tubule density between normal and heart failure myocytes and between atrial and ventricular chambers are correlated with AmpII but not junctophillin-2 protein expression.

  • Gene silencing of AmpII in adult ventricular myocytes leads to t-tubule loss, heterogeneity, and reduced amplitude of the systolic calcium transient and restricts expression of the L-type calcium channel to the cell surface.

T-tubules have a pivotal role in regulating cardiac excitation–contraction coupling. Differences in t-tubule distribution and density in cardiac disease or between atrial and ventricular mycoytes have a substantial effect on the synchronicity of the systolic rise of calcium. Despite the importance of t-tubules, the factors that are responsible for the formation and maintenance of t-tubules remain largely unknown. We show that differences in t-tubule density between atrial and ventricular myocytes and with progression to heart failure correlate with expression of the bridging integrator protein AmpII. We also, using small interfering RNA approaches, demonstrate that loss of AmpII causes t-tubule depletion in adult ventricular myocytes. This loss of t-tubules is associated with a decrease in calcium transient amplitude and reduced synchronicity of the rise of calcium. Conversely, expression of the membrane spanning protein junctophillin-2 does not correlate with interchamber differences in t-tubule density nor does it alter in failing ventricular myocytes where t-tubule density is reduced. Our work indicates that AmpII has a vital role in maintaining t-tubules in adult cardiac myocytes and suggests that it could be used to restore t-tubules and the systolic calcium transient in heart failure.

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  • Dependence of Cardiac Transverse Tubules on the BAR Domain Protein Amphiphysin II (BIN-1)Novelty and Significance
    Jessica L. Caldwell, Charlotte E.R. Smith, Rebecca F. Taylor, Ashraf Kitmitto, David A. Eisner, Katharine M. Dibb and Andrew W. Trafford
    Circulation Research. 2014;115:986-996, originally published October 20, 2014
    https://doi.org/10.1161/CIRCRESAHA.116.303448
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