BackgroundHeart 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 ResultsWe 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×.ConclusionsThis 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.

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BackgroundMyocardial 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.MethodsA 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.Results3D 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.ConclusionEmploying 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.

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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 nanoarchitectural 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 resultsWe 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.