J. Bernard Davenport

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules This Organization Is Perturbed in Heart Failure

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

3-D Reconstruction of the Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking T-Tubules: This Organization is Perturbed in Heart Failure

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Post‐Myocardial Infarction T‐tubules Form Enlarged Branched Structures With Dysregulation of Junctophilin‐2 and Bridging Integrator 1 (BIN‐1)

Background Heart failure is a common secondary complication following a myocardial infarction (MI), characterized by impaired cardiac contraction and t‐tubule (t‐t) loss. However, post‐MI nano‐scale morphological changes to the remaining t‐ts are poorly understood. Method and Results We utilized a porcine model of MI, using a nonlethal microembolization method to generate controlled microinfarcts. Using serial block face scanning electron microscopy, we report that post‐MI, after mild left‐ventricular dysfunction has developed, t‐ts are not only lost in the peri‐infarct region, but also the remnant t‐ts form enlarged, highly branched disordered structures, containing a dense intricate inner membrane. Biochemical and proteomics analyses showed that the calcium release channel, ryanodine receptor 2 (RyR2), abundance is unchanged, but junctophilin‐2 (JP2), important for maintaining t‐t trajectory, is depressed (−0.5×) in keeping with the t‐ts being disorganized. However, immunolabeling shows that populations of RyR2 and JP2 remain associated with the remodeled t‐ts. The bridging integrator 1 protein (BIN‐1), a regulator of tubulogensis, is upregulated (+5.4×), consistent with an overdeveloped internal membrane system, a feature not present in control t‐ts. Importantly, we have determined that t‐ts, in the remote region, are narrowed and also contain dense membrane folds (BIN‐1 is up‐regulated +3.4×), whereas the t‐ts have a radial organization comparable to control JP2 is upregulated +1.7×. Conclusions This study reveals previously unidentified remodeling of the t‐t nano‐architecture in the post‐MI heart that extends to the remote region. Our findings highlight that targeting JP2 may be beneficial for preserving the orientation of the t‐ts, attenuating the development of hypocontractility post‐MI.

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 mm3) 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.

172 Junctophilin-2 interacts with the cardiac L-Type voltage-gated calcium channel

Background Excitation contraction (E-C) coupling in cardiac muscle is regulated by the L-type voltage-gated calcium channels (LTCC) within the t-tubules, and surface membrane, and the ryanodine receptors (RyR2) localised to the junctional sarcoplasmic reticulum (jSR). The juxtaposition of the t-tubules and jSR forms a microdomain termed a dyad. It is now widely accepted that junctophilin-2 (JP2) acts as a protein bridge to maintain the dyad geometry. JP2 is anchored in the SR, via its C-terminal transmembrane domain, and is thought to span the cytosol and interact with the plasma membrane thereby constraining the dyad architecture to provide the optimal proximal distance between the LTCCs and RyR2s to facilitate effective calcium induced calcium release for cardiac contraction. More recently, data has emerged to suggest that JP2 has an additional role, influencing E-C coupling through an interaction with RyR2. Furthermore, there is evidence for an association between JP2 and the LTCC in skeletal muscle. However, the region of the LTCC involved in binding to JP2 was not established nor was whether there is an interaction between JP2 and the LTCC in the heart. Methods We employed yeast-2-hybrid (Y2H) (Hybrigenics) experiments to investigate JP2 binding partners using human heart cDNA library. Domains of the LTCC were expressed as GST-fusion proteins and JP2 full-length and domains were purified from E.coli. Pull-down experiments were employed to investigate the interaction between the JP2 and the LTCC protein domains. Results Data from Y2H experiments identified the C-terminal region within the ion channel pore of LTCCs as a JP2 binding partner. Therefore, we designed a series of constructs encompassing the C-terminal domain of the Cav1.2 subunit and expressed them as GST-fusion proteins. Employing purified JP2 we showed a direct interaction with the C-terminal tail of Cav1.2. Furthermore, we determined that the N-terminal portion of JP2 mediates this interaction. We also investigated whether this interaction is Ca2+ dependent since we have previously reported that JP2 has a cation binding domain. Our experiments revealed that the JP2-Cav1.2 interaction is independent of [Ca2+]. Conclusion This study has provided novel data revealing that not only is there a direct interaction between the cardiac LTCC and JP2 but that the binding domains involve the C-terminal region of Cav1.2 and the N-terminal domain of JP2. Significantly, the Cav1.2 C-terminal domain binds calmodulin (CaM), both apoCaM and Ca2+CaM, regulating both calcium dependent inactivation (CDI) and facilitation (CDF) of the channel. Therefore, our future studies are directed at understanding the significance of JP2 binding, for example whether JP2 influences channel function (CDI/CDF) and/or whether a JP2-Cav1.2 is important for LTCC localisation to the dyad.

185 Remodelling of Specialised Domains of the Sarcolemma in Heart Failure; Reorganisation of the Intercalted Disc Revealed by Nano-scale Imaging

Introduction The sarcolemma is a highly specialised organelle of the cardiac myocyte. In addition to forming the envelope of the cell its organisation into specialised domains (transverse tubules, TT) is central for calcium influx and facilitation of mechanical, electrical and chemical coupling (intercalated disc, ICD) between myocytes. We have previously used serial block face scanning electron microscopy (sbfSEM) for the three-dimensional reconstruction (3-D) of the TT network revealing remodelling in an ovine model of heart failure (HF). We have hypothesised that in HF there is also remodelling of the ICD. However, to-date there are no high resolution 3-D structures for an ICD. Current knowledge of the ICD organisation is derived from two-dimensional transmission electron microscopy images. In this study we have applied sbfSEM for the first 3-D morphological analysis of the ICD structure in healthy and failing cells. Methods Samples were taken from the left ventricle (LV) of tachypacing induced HF control sheep (n = 4). Sheep were killed with an overdose of pentobarbitone (200mg/kg iv). All procedures were carried out in accordance to the United Kingdom Animals (Scientific Procedures) Act of 1986 and the University of Manchester’s ethical review process. Samples were prepared as described previously. Blocks of fixed tissue were imaged using an FEI Quanta 250 FEG SEM equipped with a Gatan 3View system. 3-D information through the block was generated at 15, 15, 50 nm (X, Y, Z) resolution at the pixel level. Images were segmented in Fiji or IMOD. Frozen tissue was lysed using RIPA buffer and protein profiles analysed by western blotting. Results We report the first high resolution 3-D structure of entire ICDs in situ in the LV of healthy and failing hearts leading to the identification and quantification of structural remodelling. The amplitudes of the plicae are four-fold greater in the failing cells (P < 0.01) compared to control. These morphological adaptations are accompanied by an up-regulation of desmin and the N-cadherin/catenin complex involved in stabilising the end-to-end myocyte adhesive and mechanical properties. We also identified vacuoles within the extracellular space and larger vacuoles within the cells that associate with each side of the ICD. Conclusions Our data reveal how the failing heart adapts to the increased mechanical stress from the tachypacing by remodelling both the structural and protein properties of the ICD. We suggest that the vacuoles formed between the ICD leaflets may in part be due to the loss of gap junctions but that the larger vacuoles are a result of mitochondrial rupture. We have characterised in 3-D the features of control ICDs revealing a consistency in morphological features and therefore, we propose that the remodelling and extent of the rearrangement may have applications for differentiating between healthy and disease phenotypes and also as a measure of disease progression.

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-TubulesNovelty and Significance

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-TubulesNovelty and Significance: This Organization Is Perturbed in Heart Failure

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.