N Malik

Apoptosis and Cell Proliferation After Porcine Coronary Angioplasty

BACKGROUND Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.

Intravascular stents: a new technique for tissue processing for histology, immunohistochemistry, and transmission electron microscopy

Background Study of the vascular response to stent implantation has been hampered by difficulties in sectioning metal and tissue without distortion of the tissue stent interface. The metal is often removed before histochemical processing, causing a loss of arterial architecture. Histological and immunohistochemical sections should be 5 μm with an intact tissue stent interface. Objectives To identify the most suitable cutting and grinding equipment, embedding resin, and slides for producing thin sections of stented arteries with the stent wires in situ for histological, immunohistochemical, and transmission electron microscopic (TEM) analyses. Methods 20 balloon stainless steel stents were implanted in the coronary arteries of 10 pigs. Twenty eight days later the stented arterial segments were excised, formalin fixed, embedded in five different resins (Epon 812, LR white, T9100, T8100, and JB4), and sectioned with two different high speed saws and a grinder for histological, immunohistochemical, and TEM analyses. Five stented human arteries were obtained at necropsy and processed using the best of the reported methods. Results The Isomet precision saw and grinder/polisher unit reliably produced 5 μm sections with most embedding resins; minimum section thickness with the horizontal saw was 400 μm. Resin T8100, a glycol methacrylate, enabled satisfactory sectioning, grinding, and histological (toluidine blue, haematoxylin and eosin, and trichromatic and polychromatic stains) and immunohistochemical analyses (α smooth muscle actin, von Willebrand factor, vimentin, proliferating cell nuclear antigen, and CD68 (mac 387)). T9100 and T8100 embedded stented sections were suitable for ultrastructural examination with TEM. Stented human arterial sections showed preserved arterial architecture with the struts in situ. Conclusion This study identified the optimal methods for embedding, sawing, grinding, and slide mounting of stented arteries to achieve 5 μm sections with an intact tissue metal interface, excellent surface qualities, histological and immunohistochemical staining properties, and suitability for TEM examination. The technique is applicable to experimental and clinical specimens.

Encapsulated Glucagon-Like Peptide-1-Producing Mesenchymal Stem Cells Have a Beneficial Effect on Failing Pig Hearts

Stem cell therapy is an exciting and emerging treatment option to promote post‐myocardial infarction (post‐MI) healing; however, cell retention and efficacy in the heart remain problematic. Glucagon‐like peptide‐1 (GLP‐1) is an incretin hormone with cardioprotective properties but a short half‐life in vivo. The effects of prolonged GLP‐1 delivery from stromal cells post‐MI were evaluated in a porcine model. Human mesenchymal stem cells immortalized and engineered to produce a GLP‐1 fusion protein were encapsulated in alginate (bead‐GLP‐1 MSC) and delivered to coronary artery branches. Control groups were cell‐free beads and beads containing unmodified MSCs (bead‐MSC), n = 4–5 per group. Echocardiography confirmed left ventricular (LV) dysfunction at time of delivery in all groups. Four weeks after intervention, only the bead‐GLP‐1 MSC group demonstrated LV function improvement toward baseline and showed decreased infarction area compared with controls. Histological analysis showed reduced inflammation and a trend toward reduced apoptosis in the infarct zone. Increased collagen but fewer myofibroblasts were observed in infarcts of the bead‐GLP‐1 MSC and bead‐MSC groups, and significantly more vessels per mm2 were noted in the infarct of the bead‐GLP‐1 MSC group. No differences were observed in myocyte cross‐sectional area between groups. Post‐MI delivery of GLP‐1 encapsulated genetically modified MSCs provided a prolonged supply of GLP‐1 and paracrine stem cell factors, which improved LV function and reduced epicardial infarct size. This was associated with increased angiogenesis and an altered remodeling response. Combined benefits of paracrine stem cell factors and GLP‐1 were superior to those of stem cells alone. These results suggest that encapsulated genetically modified MSCs would be beneficial for recovery following MI.

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.

A novel porcine model of early left ventricular dysfunction for translational research

Correspondence: Cathy M Holt Institute for Cardiovascular Science, University of Manchester, 3.31b Core Technology Facility, 46 Grafton St, Manchester, M13 9NT, United Kingdom Tel +44 161 275 5671 Fax +44 161 275 1183 Email cathy.holt@manchester.ac.uk Background: The early stages of left ventricular (LV) dysfunction account for a much larger proportion of the population with heart disease than that with clinical heart failure. However, LV dysfunction is more difficult to diagnose than established heart failure, and because of this it is not usually treated. Research on LV dysfunction is commonly conducted in small animal models in which the cardiac pathophysiology is dissimilar to that in humans, thereby restricting translation. This study aimed to use a novel pig model of mild to moderate early ischemic LV dysfunction to assess the effects of such dysfunction in the myocardium. Methods: Multiple areas of controlled microinfarcts were created via microembolization using embolization beads, with invasive hemodynamic and transthoracic echocardiographic assessment of LV function. Four weeks after intervention, the hearts were explanted for determination of the infarcted surface area and analysis of calcium regulatory proteins. Results: In vivo hemodynamic measurements confirmed a .25% decrease in LV dP/dt (maximum and minimum) with creation of microinfarcts compared with baseline, whilst echocardiography showed mild to moderate LV dysfunction. Perioperative mortality was 10%–15%. In surviving pigs, morphometry at 4 weeks confirmed that up to 20% of the total LV surface area contained microinfarcts. Western blot analysis showed alterations in levels of the calcium regulatory proteins, sarcoplasmic reticulum Ca2+ ATPase and sodium-calcium exchange, in infarcted areas, compared with normal LV tissue from the same animals. Conclusion: These results demonstrate the usefulness of this model for investigation of the precise molecular and cellular changes associated with early mild to moderate LV dysfunction from ischemic injury, and its potential use for modulating these changes with the aim of achieving functional reversibility or regeneration of the myocardium.

Combined MSC and GLP-1 Therapy Modulates Collagen Remodeling and Apoptosis following Myocardial Infarction

Background. Mesenchymal stem cells (MSCs) and glucagon-like peptide-1 (GLP-1) are being tested as treatment strategies for myocardial infarction (MI); however, their mechanisms in the heart are not fully understood. Methods. We examined the effects of MSCs, either native, or engineered to secrete a GLP-1 fusion protein (MSCs ± GLP-1), on human cardiomyocyte apoptosis in vitro. The effect on cardiac remodeling when encapsulated in alginate beads (CellBeads-MSC and CellBeads-MSC + GLP-1) was also evaluated in a pig MI model, whereby pigs were treated with Empty Beads, CellBeads-MSC, or CellBeads-MSC + GLP-1 and sacrificed at one or four weeks following MI. Results. MSC + GLP-1 conditioned media demonstrated antiapoptotic effects on ischaemic human cardiomyocytes in vitro. In vivo, qRT-PCR revealed large changes in the expression of several genes involved in extracellular matrix remodeling, which were altered following MSC ± GLP treatment. After four weeks, infarcted areas were imaged using atomic force microscopy, demonstrating significant alterations between groups in the structure of collagen fibrils and resulting scar. Conclusions. These data demonstrate that MSCs ± GLP-1 exhibit modulatory effects on healing post-MI, affecting both apoptosis and collagen scar formation. These data support the premise that both MSCs and GLP-1 could be beneficial in MI treatment.

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.

P39 Post-myocardial infarction extracellular matrix remodelling in the pig is associated with altered expression of micro-rnas

Extracellular matrix remodelling is a key component of myocardial repair after ischaemic injury, and contributes to fibrotic changes leading to heart failure. MicroRNAs, short non-coding RNAs which regulate gene expression, have been shown to be dysregulated in a number of rodent models of ischaemia and heart failure, yet limited data is available in large translational animal models. The expression of a number of fibrosis-associated microRNAs were investigated in a relevant porcine model of mild to moderate left ventricular (LV) dysfunction induced by coronary microembolisation (n=3 MI and n=3 control pigs). Samples of myocardial tissue were obtained 4 weeks post-MI and classified into infarct, border and remote (normal) regions. The infarct area was histologically mostly acellular with dense inflammatory infiltrate, extracellular matrix expansion and collagen deposition as seen with Masson’s trichrome and Picrosirius red staining. qRT-PCR analysis of microRNA expression demonstrated downregulation of miR-133a (p=0.0085), whereas miR-214 was upregulated (p=0.0027) in the infarct region, compared to normal/control myocardium. miR-133a has previously been shown to protect against cardiac fibrosis, and is downregulated in the heart in numerous small-animal models of heart failure. miR-214 is upregulated in the fibrotic heart, and its inhibition has been shown to be protective against hepatic fibrosis. Therefore, our porcine data supports the hypothesis that pathological myocardial remodelling may be influenced by altered expression of microRNAs, and confirms the pattern of microRNA expression observed in other species. Future work is aimed at testing therapeutic approaches targeting these specific microRNAs.

171 Nano-structural and Molecular Remodelling Extends to the Remote Region in the Post Myocardial Infarcted Porcine Heart — A Basis for Heart Failure Development?

Introduction A common consequence of coronary artery disease is myocardial infarction (MI). Following an MI a sequence of pathological events occur with necrosis and acute inflammation leading to the formation of a stable fibrous scar. However, although many patients now survive an MI many also go on to develop heart failure (HF), with cellular remodelling implicated as a precipitating factor. However, the nano-architectural changes and associated molecular remodelling remains poorly understood. Mitochondria occupying between 30–40% of the cardiomyocyte volume, play a central role in cardiac energetics, with evidence to indicate dysfunction in the post-MI heart.1 Here we have combined 3-D electron microscopy with biochemical and quantitative mass spectrometry methods, to investigate morphological changes to mitochondria within both the peri-infarct and remote regions. We have interrogated structural changes in the context of protein, molecular, level remodelling. Methods and results We have employed a translationally relevant porcine model of MI, presenting mild to moderate left ventricular dysfunction (n = 3, control and MI).2 Animals were studied 1 month post-MI when the scar region has stabilised. Tissue was sampled from the peri-infarct and remote regions and a corresponding region from the control hearts, fixed and prepared for serial block face scanning electron microscopy as previously described.3 Tissue was also lysed and analysed by quantitative mass spectrometry and western blotting. 3-D reconstruction of the mitochondria within both the peri-infarct region and remote area determined that they were smaller in terms of volume (no. of mitochondria = 389, P < 0.01; no. of mitochondria =262 P < 0.05 respectively) compared to control. There was also a change to the distribution of the subsarcolemmal and inter-fibrillar mitochondria in both regions post-MI. Quantitative mass spectrometry identified between 1400–2000 proteins within each tissue sample with alterations (both up and down regulation) of proteins associated with β oxidation and OXPHOS. Conclusion These data reveal that mitochondrial structural rearrangements are accompanied by expression level changes to proteins regulating cardiac energetics. Importantly, the data also indicate that while morphological remodelling is more acute within the peri-infarct region the remote areas of the infarcted heart are also undergoing cellular maladaptations. Currently, there are no treatments that specifically target cellular structural remodelling post-MI. Here we show that mitochondrial remodelling is a feature of the post-MI heart; targeting these changes at the structural and molecular level may represent a novel treatment strategy for improved outcomes. References Dodd, M. S., Atherton, H. J., Carr, C. A., Stukey, D. J., West, J. A., Griffin, G. K., Radda, K., Clarke, K., Heather, L. C., Tyler, D. J. Impaired in-vivo mitochondrial Krebs cycle activity after myocardial infarction assessed using hyperpolarized magnetic resonance spectroscopy. Circ Cardiovasc Imaging. 2014;7(6);895-–904 Malik N, Farrell KA, Withers SB, Wright EJ, Holt CM. A novel porcine model of early left ventricular dysfunction for translational resaerch. Research Reports in Clinical Cardiology. 2013;4:1–7 Pinali C, Bennett H, Davenport JB, Trafford AW, Kitmitto A. Three-dimensional reconstruction of cardiac sarcoplasmic reticulum reveals a continuous network linking transverse-tubules: this organisation is perturbed in heart failure. Circ Res. 2013;113:1219–30

53 Effects Of GLP-1 eluting stem cell therapy on collagen remodelling in a porcine model of myocardial infarction

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with cardioprotective effects. Human stem cells (hMSCs) secreting a GLP-1 fusion protein and encapsulated in alginate (GLP-1 CellBeads) have been developed as a novel therapeutic. This study investigated the effects of GLP-1 CellBeads on post-MI healing in a porcine model. GLP-1 CellBeads were delivered to coronary artery branches, creating micro-infarcts and mild LV dysfunction. Cell-free beads (empty) and CellBeads containing hMSCs but not secreting GLP-1 (hMSC-beads) were delivered as controls. Left ventricular (LV) function, infarct size, myocyte cross-sectional area, total collagen content (picrosirius red staining) and collagen -1 and -3 levels (qRT-PCR) were analysed to determine therapeutic potential. Four weeks post-MI, only GLP-1 CellBead treatment produced significantly improved LV function compared to 30 min post-MI (44.00%±1.29% vs 49.75%±1.03%, p<0.001). This was also associated with less infarct (3.21%±0.90%) compared to control groups (empty; 17.70%±4.69% vs hMSC-beads: 16.71%±1.60%, p<0.05). Total collagen content (infarct) was increased following delivery of hMSC-beads and GLP-1 CellBeads compared to empty beads (6.87%±2.92% vs 19.63%±1.37% vs 16.88%±6.61%, p<0.05), with increases in collagen 1 and 3 mRNA synthesis observed in all groups. Border zone myocytes were significantly larger in all groups compared to control (non-infarcted) tissue (empty: 378.4±31.3; hMSC-beads: 364.6±15.7; GLP-1 CellBeads: 344.7±38.9; control: 132.7±13.0), with no differences in remote regions. Delivery of GLP-1 CellBeads restored LV function, limited infarct size, enhanced collagen scar formation, altered collagen mRNA synthesis and decreased border myocyte size. Effects observed were due to a combination of GLP-1 and paracrine factors released from the hMSCs.