Adult cardiac tissue undergoes a rapid process of dedifferentiation when cultured outside the body. The in vivo environment, particularly constant electromechanical stimulation, is fundamental to the regulation of cardiac structure and function. We investigated the role of electromechanical stimulation in preventing culture-induced dedifferentiation of adult cardiac tissue using rat, rabbit and human heart failure myocardial slices. Here we report that the application of a preload equivalent to sarcomere length (SL) = 2.2 μm is optimal for the maintenance of rat myocardial slice structural, functional and transcriptional properties at 24 h. Gene sets associated with the preservation of structure and function are activated, while gene sets involved in dedifferentiation are suppressed. The maximum contractility of human heart failure myocardial slices at 24 h is also optimally maintained at SL = 2.2 μm. Rabbit myocardial slices cultured at SL = 2.2 μm remain stable for 5 days. This approach substantially prolongs the culture of adult cardiac tissue in vitro. Cultured adult cardiac tissue undergoes rapid dedifferentiation, which hinders chronic in vitro studies. Here the authors investigate biomimetic electromechanical stimulation of adult myocardial slices applying different preload conditions, identifying the optimum sarcomere length for prolonged culturing, and investigating transcriptional profiles associated with functional preservation. An in vitro model of intermediate complexity, with preserved cardiac multicellularity, architecture and physiology that can be maintained in a stable manner in culture and bridges the gap between cellular and in vivo studies is greatly needed.Conventional in vitro cell culture systems are relatively accessible and have been indispensable in clarifying and corroborating mechanisms, but their oversimplified nature only distantly recapitulates the in vivo environment3. They are a platform of intermediate complexity, allowing the modulation of the myocardium at the macroscopic level , while studying cellular responses within a native cardiac environment. Our group has recently optimised the technique, resulting in a preparation with 97% viable cardiomyocytes, robust contractile function and physiological Ca2+ handling and conduction velocities3. It is also possible to produce many slices from the same heart, thus reducing the number of animals/biopsies required in studies, and slices can be produced from human biopsies providing a clinically relevant human model in vitro system6. The thinness of myocardial slices ensures the diffusion of oxygen and other small molecules to all cells, which is very difficult to achieve with other preparations and is crucial for chronic culture8. However, our group and others have demonstrated that current culture techniques, utilising a liquid–air interface, cannot prevent tissue dedifferentiation in vitro9, limiting their applicability and usefulness for chronic studies.Myocardial slices are highly viable, ultrathin (300-μm) slices of the ventricular myocardium that retain the native multicellularity, architecture and physiology of the heart11), that characterise beating, adult cardiomyocytes13. While they remain bona fide cardiac muscle cells, their use as a representative model becomes debatable, with significant implications for the relevance of medium-term/chronic studies.When extracted from the beating adult heart, cardiomyocytes retain their structural and functional properties and have been widely used for acute experiments in every field of cardiovascular biology. However, within hours from isolation, cardiomyocytes undergo a process of structural and functional remodelling. This process results in the rapid loss of the highly specialised and sophisticated structure/function relationships . Cell–cell interactions, via direct contact and/or paracrine secretions, contribute to cardiomyocyte function and regulation22. The ECM provides transmembrane regulation and controls membrane stiffness, again with important functional consequences23. However, cardiac multicellular preparations, including myocardial slices, suffer from similar culture-induced changes9. The final aspect, and one that is specifically associated with cardiac muscle behaviour, is the relentless cyclic electromechanical stimulation experienced by the myocardium. Electrical stimulation exerts positive effects but, in isolation, despite reducing cardiomyocyte remodelling, is still insufficient24. In addition, neonatal and pluripotent stem cell-derived cardiomyocytes possess intrinsic pacemaker activity and are continuously stimulated, yet they remain poorly differentiated. The factor that is absent in culture and may play a major role in regulating cardiomyocyte structure and function is sustained mechanical load. We and others have shown that structure and function of cardiomyocytes in vivo are modulated by changes in load, that the changes are graded and that they are reversible28. Chronic mechanical unloading of the heart induces atrophic remodelling, despite an unchanged humoral milieu, multicellularity, ECM and electrical stimulation29. This is not surprising, given that heart function reacts to changes in mechanical load and that a profound increase in haemodynamic load is essential for postnatal cardiac development30.The precise factors responsible for the development and maintenance of the adult cardiac phenotype, including those lacking in culture, are unknown. While the regulation of cardiac phenotype is almost certainly multifactorial, there are three major aspects that are likely implicated. The first is the presence of adequate humoral stimulation and availability of metabolic substrates. Several culture protocols have been developed for isolated cardiomyocytes, particularly for gene transfections, but with limited success31. However, only a single preload was tested, and myocardial slices were electrically stimulated at substantially sub-physiological rates (0.2 Hz). A detailed characterisation of the structure, function and transcriptional properties of cultured myocardial slices was not conducted and the mechanisms regulating the preservation/dedifferentiation of myocardial slices in vitro remain unresolved. We investigated the role of biomimetic electromechanical stimulation with physiological preload to regulate the adult cardiac phenotype of myocardial slices in vitro and the gene set expression patterns involved. We used an inexpensive, simple and user-friendly platform that allows the maintenance and manipulation of adult myocardial slices in vitro. We and others observe significant dedifferentiation of myocardial slices produced from all species, with the fastest rates observed in slices produced from small mammals, such as rats. We therefore performed this study with freshly prepared rat myocardial slices and subjected them to different electromechanical stimulation protocols for 24 h. Optimised protocols were then applied to human heart failure myocardial slices for 24 h and healthy rabbit myocardial slices for 5 days (120 h).In the beating heart in vivo, mechanical load plays an essential role in the regulation of acute and chronic cardiac performance. Despite the known interplay between mechanical load and cardiac function, the prolonged effects of physiological mechanical load on adult cardiac tissue in vitro remain under-investigated. A recent publication has highlighted the importance of continuous electromechanical stimulation for the long-term culture of myocardial slices2+ handling and t-tubule structure, which is likely mediated via the MLP-TCAP-Titin stretch-sensor complex. When cultured at other SLs for 24 h, rat myocardial slices display adaptive responses compatible with dedifferentiation  without changes in viability or energy status. The maximum contractility of human heart failure myocardial slices cultured for 24 h is also optimally maintained at SL = 2.2 μm. When cultured at SL = 2.2 μm for 5 days (120 h), the contractility of the rabbit myocardial slices remains stable throughout. This approach is a step towards a chronically stable in vitro preparation, which is a significant unmet need in cardiovascular science.Here, we report that a preload equivalent to sarcomere length (SL) = 2.2 μm is optimal for the maintenance of rat myocardial slice structural, functional and transcriptional properties at 24 h. Rat myocardial slices cultured at SL = 2.2 μm for 24 h have significant enrichment of gene sets associated with cardiac structure/function and transcription/translation, while gene sets associated with inflammation/cytokine signalling and extracellular matrix organisation are significantly downregulated. This results in the optimal maintenance of maximum contractility, Ca9. To quantify the tissue stretch required to differentially preload slices, laser diffraction was used to assess the average slice sarcomere length  within the physiological range SL = 1.8–2.4 μm) whilst field stimulated in culture. Electromechanically stimulated slices were compared with fresh myocardial slices (0 h) and with the most common slice culture technique, where slices are cultured without electromechanical stimulation (unloaded and non-stimulated) on a liquid–air interface (unloaded)gth Fig. . The aligth Fig. . The resgth Fig.  and the gth Fig. . A 633-ngth Fig. . Rat myogth Fig. . Stretchgth Fig. . Unloade. 9 Fig. . Immunoh.8–2.4 μmCSRP3 (muscle LIM protein) was significantly higher in slices cultured at SL = 2.2 μm, compared with both SL = 1.8 μm and unloaded . Ca2+ handling of myocardial slices was assessed with Fluo-4-AM and optical mapping  was measured using a force transducer after the slices had been cultured for 24 h and then removed from their stretchers. Rat myocardial slices cultured at SL = 2.2 μm had the best-preserved contractility, with only a small non-significant reduction compared with 0 h. This was significantly greater than unloaded and SL = 2.0-μm myocardial slices Fig. . We alsoing Fig. . Slices ces Fig. , as wellise Fig.  and signcay Fig. . Slices ces Fig. .Fig. 2St4. Conduction velocity was measured both longitudinal  to 0 h slices  on the surface of unloaded rat myocardial slices at 24 h  and structural (t-tubular structure) differences occur in electromechanically stimulated slices cultured at different SLs in the absence of changes in ATP concentration, it is likely that mechanisms not related to cardiac energy status are involved in the modulation of myocardial slice phenotype in vitro.It is well established that energy status is one of the key drivers of cardiac tissue differentiation, with increased energy production and efficient energy utilisation associated with the adult cardiac phenotypeons Fig. . No diffons Fig. . Howeverons Fig. . Increasons Fig. . AMP, NAons Fig. . The red2+ release, conduction and cardiomyocyte repolarisation, the A and I-bands, t-tubules, the intercalated disc and the extracellular matrix.To investigate the effects of electromechanical stimulation on the transcriptome, RNA sequencing was performed. Rat myocardial slices cultured for 24 h were used. We chose to sequence four of our conditions  to answer two specific questions: 1) what are the genes involved in dedifferentiation of cardiac tissue in vitro (0 h vs. unloaded)? and 2) what genes are involved in the regulation of cardiac structural and functional plasticity with different preloads (SL = 1.8 μm vs. SL = 2.2 μm)? When we compared the unloaded slices to 0 h slices Fig. , we founTCAP expression in electromechanically stimulated slices compared with unloaded. This indicates that the mechanotransduction of physiological electromechanical stimulation is mediated via the MLP-TCAP-Titin stretch sensor and that this is essential for the maintenance of the adult cardiac phenotype in vitro. As expected and when compared with 0 h, slices cultured at SL = 2.2 μm did appear to suffer some degree of dedifferentiation at the transcriptome level. However, when interpreted in the context of our structural and functional findings, the process of dedifferentiation is significantly delayed when slices are cultured with biomimetic electromechanical stimulation with a physiological preload (SL = 2.2 μm).Our structural and functional data indicate that providing a preload of SL = 2.2 μm significantly improves the structure and function of myocardial slices compared with SL = 1.8 μm and is beneficial for the maintenance of the adult cardiac phenotype in vitro. RNA sequencing provided further evidence of this at the transcriptome level Fig. . Slices 9.The use of human tissue in vitro is vital for translational research. Our group received explanted human heart failure left ventricular specimens, collected during cardiac transplantation. We applied the same electromechanical stimulation with physiological preload to human heart failure (HF) slices and measured their contractility after 24 h. A very similar trend to that seen in rat myocardial slices was observed, with human slices cultured at SL = 2.2 μm having the best-preserved contractility at 24 h Fig. . This wa2 and other essential nutrients. However, as we have previously shown, culturing myocardial slices using currently published techniques (liquid–air interface—unloaded) results in the rapid loss of contractile function amongst other structural/functional parameters, suggesting that this methodology is not representative of active, working cardiac tissue. Prolonged studies were performed to see if the improvements in myocardial slice structure and function when cultured at SL = 2.2 μm could be extended to 5 days  and basal (measured every 2 h) contractility of rabbit slices cultured at SL = 2.2 μm increased over the first 24–48 h, as the tissue adapted to the in vitro environment and completely recovered from slicing (including the high [K+] used for cardioplegic arrest). From 48 to 120 h, the tissue remained in a steady state, with only a subtle decrease in function. At 5 days, both the basal and maximum contractility of rabbit myocardial slices were almost completely preserved compared with 0 h  facilitates the diffusion of Oays Fig. . Rabbit 0 h Fig. .Our data demonstrate that the application of electromechanical stimulation with physiological preload significantly improves the preservation of myocardial structure and function in culture and provides a step towards a stable in vitro model of the adult myocardium. More specifically, we have demonstrated that the application of a preload equivalent to SL = 2.2 μm significantly reduces culture-induced remodelling compared with other preloads within the physiological range or unloaded. We have demonstrated that almost all structural and functional parameters of the cardiac phenotype are regulated by changes in mechanical load in vitro and that the regulation of this phenotypic plasticity is multifactorial.9. We have demonstrated that the loss of the adult cardiac phenotype can be significantly delayed in culture, with rabbit myocardial slices remaining highly functional for 5 days. Further to Fischer et al.31, we have demonstrated that mechanical load must be applied within a very specific biomimetic range (~SL = 2.2 μm) in order to be reproducible and prevent culture-induced dedifferentiation. Myocardial slices have preserved cardiac multicellularity and the extracellular matrix, uniquely allowing the study of all cardiac cell populations in a representative environment. This is fundamental to a deeper understanding of cardiac physiology, as the non-myocyte and non-cellular components of the myocardium play vital roles in the regulation of cardiomyocyte structure and function20. In this study, we confirmed that electromechanical stimulation not only preserves cardiomyocyte function, but also maintains non-myocyte populations in a physiological state. We have confirmed that electromechanical stimulation prevents the massive proliferation of cardiac fibroblasts in vitro previously observed9, while also maintaining a more physiological gene expression profile. The ultrathin nature of myocardial slices combines the benefits of a complex multicellular 3D preparation with the simplicity of acquiring structural and functional data from a 2D monolayer. In this study, we have fully characterised both the structure and function of myocardial slices in culture using several high-sensitivity assays. We have measured direct force generation and contractile reserve, the most relevant parameters of cardiac function, and have demonstrated that both remain stable for 5 days. In addition, we have demonstrated that cultured (24 h) myocardial slices responded to isoproterenol stimulation in a comparable manner to gold-standard drug-screening models36, with both 0 h and cultured myocardial slices (SL = 1.8–2.4 μm) displaying an EC50 in the nanomolar (×10–8 M) range. The model can also be used to investigate cardiac remodelling in response to both unloading and overload, which we have sensitively quantified using laser diffraction.The ability to maintain myocardial slices in a functional state in vitro, which is not possible with current published techniques, provides a significantly improved and much needed platform for cardiovascular research. The specific benefits of this improved model include prolonged preservation of the adult cardiac phenotype in vitro, intact native cardiac multicellularity and tissue architecture, acquisition of high-quality sensitive data on cardiac structure and function in real time and an ability to investigate representative responses to pharmacological and mechanical load stimuli in vitro. In vitro adult cardiac models undergo a rapid process of tissue dedifferentiation and loss of the adult cardiac properties (phenotype and function) in culture. Our group and others have previously shown that this also occurs when myocardial slices are cultured in unloaded conditions37. The primary function of the sarcomere is to generate force; however, several of its molecules have been shown to have load sensing and mechanotransduction capabilities. Titin and muscle LIM protein (MLP) have been previously implicated in mechanotransduction and hypertrophy signalling pathways39. In our study, the MLP-TCAP-Titin complex was upregulated with electromechanical stimulation and physiological preload, particularly at SL = 2.2 μm, where MLP (CSRP3 gene) had significantly higher expression compared with both SL = 1.8 μm and unloaded slices. TCAP was also more highly expressed in both SL = 1.8 μm and SL = 2.2-μm slices. Costameres are known to be important in the lateral transmission of force40 and genes associated with them were also significantly enriched in slices cultured at SL = 2.2 μm compared with SL = 1.8 μm. Our data support the existence of multiple mechanisms to regulate load-induced changes in cardiac phenotype.Myocardial slices display remarkable plasticity to changes in preload, with changes occurring at the structural, functional and transcriptional level within 24 h. Our data suggest that these changes are adaptive, as the viability assays performed did not demonstrate significant changes in whole-slice viability and gene sets associated with apoptosis/necrosis were not enriched in any condition. Multiple mechanisms can detect changes in mechanical load34. We found that slices produced from small mammals, including rats, dedifferentiated at faster rates than large animals due to their higher metabolic rates41. Rat myocardial slices were useful in identifying optimal electromechanical conditions for prolonged culture (SL = 2.2 μm) and are adequate for 24-h experiments. The preparation of mouse myocardial slices is challenging due to the small and curved geometry of the ventricle. This results in an inability to isolate discrete sheets of myocardial fibres, resulting in reduced viability3. Larger mammal slices have a lower metabolic rate and subsequent rate of dedifferentiation, making them more amenable to prolonged experiments. Rabbit myocardial slices were cultured at SL = 2.2 μm with only a very subtle decline in contractility observed over 5 days. Real-time monitoring of myocardial slice force generation was used for 5-day experiments to confirm the stability of the preparation in vitro. Human cardiac tissue is of great value to translational studies, and in this study, we were able to optimally maintain human HF myocardial slices at SL = 2.2 μm for 24 h. It is likely that human myocardial slices can be cultured for substantially longer periods, but in this study, we focussed on the prolonged culture of healthy tissue, as there is often significant variability in the pathology and quality of human HF specimens. Future studies applying experimental translational strategies to human myocardial slices cultured for prolonged periods should be conducted, but this was outside of the remit of this study.When conducting prolonged in vitro experiments with myocardial slices, several considerations should be made. Several species differences exist in terms of cardiac function42, and the actual application of physiological afterload is technically very challenging and has only been achieved for acute studies so far. Another aspect that can be improved is the humoral stimulation of the slice in culture. The media used in this study did not contain hormones, including noradrenaline and thyroid hormones, which are known to be essential for cardiac structural and function remodelling45. In addition, the media used lacked binding proteins. Finally, we utilised field stimulation to excite the myocardial slices and physiological stimulation may be better recreated using a point/line stimulation. It was possible to point stimulate slices, as in our arrhythmia provocation studies, but practically it is not as robust as field stimulation for prolonged use in sterile conditions. Despite these limitations, our data show substantial improvements in myocardial slice structure and function following the application of electromechanical stimulation with physiological preload. It is likely that optimisation and application of the variables previously mentioned will lead to further small and incremental improvements in cultured myocardial slices, as we move closer to recreating the physiological in vivo environment. Further experiments with longer time points are now required to confirm the suitability of the approach for chronic experiments with rat and human heart failure tissues.While the application of physiological electromechanical stimulation significantly improves the maintenance of the mature adult cardiac phenotype in vitro, we still observed some dedifferentiation occurring. In our study, myocardial slices were preloaded at physiological sarcomere lengths. However, the mechanical load experienced by the myocardium in vivo consists of both preload and afterload. We focussed on preload as it directly influences the contractility of the myocardium via the Frank–Starling relationship2+ handling and t-tubules. This approach represents a significant step towards the unmet need of a stable adult cardiac in vitro preparation, which may open avenues in the testing of pharmacological agents and experimental therapeutic approaches.We have developed a platform to culture and manipulate living, beating adult myocardial slices in vitro and have demonstrated that electromechanical stimulation with specific physiological preload is required for the maintenance of adult cardiac tissue in vitro. This is a significant advance compared with current gold-standard cardiac models. We have shown that electromechanical stimulation affects structure, function and many gene set expression circuits, which regulate many aspects of the cardiac phenotype, including sarcomeres, CaAll animal experiments complied with institutional and national regulations. Our use of living cardiac tissue was approved by Imperial College London. The procedures we describe here were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed following guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).Sprague Dawley rats, with a body mass between 300 and 500 g, were sedated with isoflurane (4% isoflurane and 4 L/min O2) and sacrificed using cervical dislocation and dissection of the carotid arteries.The heart and lungs were rapidly removed from the chest and placed in warm (37 °C) heparinized Tyrode’s solution (see Table The heart and lungs were then placed in cold (4 °C) Tyrode’s solution + BDM and transferred to the laboratory.A left ventricular tissue block was prepared by dissecting off the lungs and right ventricle. An incision was made down the interventricular septum and the left ventricle was opened and flattened.The tissue block was then mounted on an agarose-coated specimen holder using surgical glue  epicardial surface facing down.The specimen holder was placed in the bath of a high-precision vibratome  submerged in oxygenated cold (4 °C) Tyrode’s solution + BDM.The tissue block was sliced using a ceramic blade, longitudinal to myocardial fibre orientation and from the endocardium down to the epicardium. The vibratome was set to vibrate at 80 Hz, with an amplitude of 2 mm and a Z-axis error < 1.0 μm.Once cut and prior to being used in acute studies or being put in culture, slices were kept in oxygenated cold (4 °C) Tyrode’s solution + BDM under mesh holders.Male New Zealand White, 2.5–3.0 kg obtained from Envigo Laboratories, Huntington, UK) were sedated with ketamine (15 mg/kg) and medetomidine hydrochloride (0.25 mg/kg), intubated with a 3-mm endotracheal tube and hearts explanted under terminal anaesthesia .Rabbits  of 150–200 mL of cold (4 °C) heparinised (500 units/kg) cardioplegia solution  via a 20 G Leadercath Arterial catheter  into the ascending aorta was used to arrest the heart. Just prior to infusion, the inferior vena cava and abdominal aorta distal to the catheter were clamped, and an incision made in the inferior vena cava  to allow blood to expel during cardioplegia administration.The heart was then rapidly excised and immersed in cold (4 °C) cardioplegia solution and transferred to the laboratory.Preparation of Rat myocardial slices); however, low calcium (0.9 mM CaCl2) oxygenated cold (4 °C) Tyrode’s solution + 30 mM BDM was used.The tissue block was prepared and sliced in the same manner as for rats  were perfused with cold (4 °C) cardioplegia solution and arrested in situ prior to being explanted. The inferior 1/3rd of explanted hearts were obtained from patients undergoing cardiac transplantation.The specimens were placed in a 1-L container of cold cardioplegia, placed on ice and transported to the laboratory.2 tissue block was dissected out of the left ventricular free wall by making an incision through the full thickness of the ventricular wall using a scalpel.In order to prepare a left ventricular tissue block, the left ventricle of the specimen was identified and isolated from the rest of the tissue. A 1.5-cmThe tissue was mounted epicardial surface down and sliced in the same manner as both rat and rabbit myocardial slices.Human HF samples were provided by the NIHR Cardiovascular Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. The study performed conformed to the principles outlined in the Declaration of Helsinki and the investigation was approved by a UK institutional ethics committee  and Imperial College London. Informed consent was obtained from each patient involved in this study.Only aligned areas, where parallel myofibrils run in the same direction, were used in this study. A macroscope was used to assess global myofibril orientation and the slice was then trimmed using a razor blade to isolate the most highly aligned area Fig. .Custom-made rings were printed in biocompatible T-glase plastic  using an Ultimaker Original + 3D printer . The rings were then attached to opposite ends of the slice, perpendicular to myofibril direction, using surgical glue .Each myocardial slice was then stretched to the desired average tissue SL by turning the screw mechanism.Once the desired preload had been applied, the myocardial slice stretcher was placed into the culture chamber.Aligned myocardial slices were mounted on stretchers, as described above, and were placed in a glass Petri dish filled with Tyrode’s solution on a micromanipulator.The beam of a 6333-nm helium–neon laser  was directed at the slice surface and the position of the slice was adjusted laterally, until a clear diffraction pattern was visualised.The laser was positioned 2 cm above the slice surface and the diffraction pattern was projected on a grid 20.5 cm below.46.The diffraction pattern was captured digitally using a camera  and the distance between the zero-order and first-order bands was calculated using ImageJ . This distance was used to calculate sarcomere length using trigonometryDiffraction patterns could not be achieved for SL < 2.0 μm. We measured the % stretch  for SL = 2.0–2.4 μm at 0.1-μm intervals.A linear regression was used to calculate the % stretch required for SL = 1.8 μm.The preload applied to myocardial slices was quantified using laser diffraction.The % stretch calculation takes both the slice length and a small portion of the ring into account, as the distance between the posts was measured, instead of the tissue length, for practicality issues. This explains why a 6% stretch is required for SL = 1.8 μm, despite the resting SL being ≈1.8 μm. Slices were stretched to the desired average slice sarcomere length, measured using calipers, directly prior to being put in culture.Myocardial slice stretchers, culture chambers and tools required for putting slices in culture were autoclaved prior to experiments.During myocardial slice preparation, culture chambers were assembled in a sterile laminar flow cabinet.Superfusion tubing and air filters were connected to the chamber and the chamber was filled with 250 mL of culture medium  + 0.1% ITS   + 3% Penicillin/Streptomycin ).2/5% CO2  at 1 L/min and recirculated using a peristaltic pump at 15 mL/min.The culture medium was continuously bubbled with 95% OImmediately after each slice had been prepared, custom-made 3D-printed T-Glase rectangular rings were mounted at opposite ends of the aligned portion of a slice orthogonal to myocardial fibre direction, as described above .The length and width of each mounted myocardial slice were measured and recorded.Mounted myocardial slices were transferred to the sterile laminar flow cabinet.In total, 1.5 mL of cold Tyrode’s solution was pipetted onto the myocardial slice, so that it was floating in the Petri dish.Using a pair of tweezers, myocardial slices were mounted on myocardial slice stretchers, as described above .Each myocardial slice was then stretched to the desired average tissue SL by turning the screw mechanism (SL = 1.8–2.4 μm).The myocardial slice stretcher was then carefully placed into the culture chamber and its position noted. It took less than 5 min to put each myocardial slice in culture.Once four myocardial slices had been placed in culture, the chamber was sealed.The culture chamber was transferred to an incubator and slices were cultured at 37 °C.Rat myocardial slices were stimulated at 1 Hz  and human heart failure myocardial slices were stimulated at 0.5 Hz . Due to the large culture medium volume, the actual voltage experienced by myocardial slices was equivalent to 5–10 V.The voltage used was equivalent to 200% of the voltage required to capture the slice and as voltage was increased recruitment was observed.In order to apply electromechanical stimulation and physiological preload to rat and human HF myocardial slices in culture, myocardial slice stretchers Fig.  and cust2 in an incubator.Electromechanically stimulated slices were compared with slices cultured in the absence of electromechanical stimulation or preload. Slices cultured in the absence of electromechanical stimulation were kept on a liquid–air interface using Transwell membranes. Slices were placed on the Transwell membranes and excess solution was removed from the slice surface and edges. One millilitre of culture medium was added to the well of a six-well plate. The cultures were kept in humidified air at 37 °C with 5% COThe Electroforce culture chamber, superfusion tubing and medium bottle were autoclaved prior to experiments.The entire Electroforce system was assembled in an incubator and kept at 37 °C for the entire experiment.The force transducer, linear motor, superfusion and electrical stimulation were all tested prior to experiments.2/5% CO2 at 3 L/min.The medium bottle was filled with 1.5 L of culture medium and oxygenated with 95% ORabbit myocardial slices were prepared, mounted and measured as described above .The mounted myocardial slice was then loaded into the culture chamber of the Electroforce system.The culture chamber was sealed and filled with culture medium using the peristaltic pump. The medium was recirculated at 15 mL/min throughout the culture.The myocardial slice was field stimulated using bipolar stimulation with platinum electrodes at 1 Hz .The myocardial slice was slowly stretched over 30 min, until the slice was progressively stretched, until a preload equivalent to SL = 2.2 μm was applied . Myocardial slice length was digitally monitored using a camera and 1-mm graph paper attached to the back of the culture chamber throughout stretching.Myocardial slices were cultured for 5 days and culture medium was completely replaced after 72 h.During culture, the electrical stimulation had to be occasionally increased , when contractile alternans occurred.A 10-s recording of myocardial slice contractility was taken every 15 min for the duration of the experiment.At 120 h, myocardial slice maximum contractility was measured by the Electroforce. Slices were stretched in a stepwise manner, until maximum isometric contraction was recorded. Myocardial slice contractility was normalised to slice width.For cultures longer than 24 h, a Bose Electroforce 5100 was used. This device was able to provide a uniaxial mechanical load to myocardial slices, whilst continuously recording myocardial slice contractility. Continuous monitoring of myocardial slice performance was required in order to optimise culture conditions during prolonged cultures.Rat myocardial slices cultured at SL = 2.2 μm using myocardial slice stretchers and the Bose Electroforce have a similar contractility at 24 h . To assess the contractility of fresh slices (0 h) and those cultured without electromechanical stimulation (unloaded), the areas with the most highly aligned myofibrils were selected and custom-made rings were attached as described above. Slices cultured with electromechanical stimulation  were carefully removed from the culture chamber and then removed from stretchers. The slices were then attached to the force transducer using the attached rings. Rat myocardial slices were field stimulated at 1 Hz, 10 ms and 10–30 V and human HF and donor slices were field stimulated at 0.5 Hz, 10 ms and 10–30 V. The slices were continuously superfused in 37 °C oxygenated Tyrode’s solution. The slices were progressively stretched in a stepwise manner, until maximum isometric contraction was obtained. Data were recorded using AxoScope software and peak amplitude analyses were conducted using Clampfit software . A video of the contraction of human HF and rat myocardial slices can be found here: 2+ handling, myocardial slices were loaded with Fluo-4 AM (5.56 μg/mL)  in medium-199 and 0.001% Pluronic F-127 . Zero-hour and unloaded myocardial slices were loaded in a six-well dish with a circular gauze and washer placed over the slice to prevent contraction during loading. Electromechanically stimulated slices were loaded in a six-well dish and kept on their stretchers at their respective sarcomere lengths. Rat myocardial slices were loaded for 10 min at 37 °C and then directly transferred to the optical mapping setup. Slices were placed in a chamber and superfused with 37 °C Tyrode’s solution + BDM. BDM is required to prevent movement artefact during calcium transient acquisition. Slices were field stimulated at 1 Hz, 10 ms and 20 V. Changes in Fluo-4 fluorescence were detected using an ORCA-Flash4.0 LT CMOS camera (Hamamatsu) at 154 frames/second and data were acquired using HCImage Live software (Hamamatsu). Ca2+ transient amplitude, time to peak, time to 50% decay, time to 90% decay, rate of Ca2+ rise and Ca2+ decay rate were measured using Clampfit software (Molecular Devices). A video of the Fluo-4 signal recorded from the myocardial slice surface can be found here: https://images.nature.com/original/nature-assets/nprot/journal/v12/n12/extref/nprot.2017.139-sv9.mp4To assess CaMyocardial slice viability was assessed using two assays:ueous One Solution Cell Proliferation Assay (Promega)CellTiter 96 AQueous One solution reagent were added to each well. The plate was incubated at 37 °C for 20 min in a humidified, 5% CO2 atmosphere. Following incubation, the absorbance at 490 nm was immediately recorded using a 96-well plate reader (Labtech). The absorbance value for each slice was normalised to slice mass.Following culture, slices were removed from the stretchers and the rings were detached using a razor blade. For 0 h and unloaded slices, the aligned area was selected and used. Slices were blotted dry and their mass measured using an electronic balance. Slices were put into individual wells of a 96-well plate and 200-μl medium-199 + 40 μl of CellTiter 96 AQLive/Dead viability assay (Molecular Probes) and wide-field microscopy2 atmosphere. Following incubation, slices were washed in phosphate- buffered solution (PBS) and then fixed in 4% formaldehyde solution for 15 min at room temperature. Circular gauzes and washers were again used to make sure that the slice was fixed as flat as possible. Tiled images of the whole myocardial slice were automatically collected using a ×10 objective on a Zeiss AxioObserver equipped with a motorised stage. To assess slice surface viability, we measured the percentage of area stained with fluorescent calcein-AM signal using ImageJ.Following culture, slices were removed from the stretchers and the rings were detached using a razor blade. For 0 h and unloaded slices, the aligned area was selected and used. The stock reagents were diluted as per the manufacturer’s instructions to produce a solution with 2 μM calcein-AM and 4 μM ethidium homodimer-1. Two millilitres of solution was added to each well of a six-well plate and slices were placed in the individual wells and covered with a circular gauze and washer. Slices were incubated at 37 °C for 45 min in a humidified, 5% CO2. Slices were positioned over the microelectrode array and held in contact using a plastic slice holder. Slices were continuously superfused with oxygenated Tyrode’s solution at 37 °C. Electrical stimulation  was applied via one of the MEA microelectrodes. Field potential data were acquired simultaneously from all 60 microelectrodes with a sampling frequency of 50 kHz. Slices were stimulated longitudinal and transverse to known myofibril direction. A custom MATLAB (MathWorks) software was used to locate field potentials from recording electrodes, produce activation maps and calculate conduction velocities, using time of maximum amplitude. A ratio of the longitudinal and transverse conduction velocities of each slice (CVL:CVT) was used as a measure of CV anisotropy.The conduction velocity of myocardial slices was assessed using a multielectrode array (MEA) system (Multichannel Systems), which allows noninvasive synchronous multifocal recording of extracellular field potentials. The MEA consists of 60 microelectrodes arranged in an 8 × 8 matrix, with a 100-μm electrode diameter and an inter-electrode distance of 700 μm, providing a recording area of about 4.9 × 4.9 mm–9 to 3 × 10–6 M (–)–isoproterenol hydrochloride (Sigma) in half-log increments + 0.1 mM ascorbic acid (Sigma). Isoproterenol concentrations were added cumulatively, once changes in contractility had stabilised following the administration of the previous concentration. Arrhythmia was assessed using two parameters, the rate of aftercontractions at each concentration of isoproterenol and the concentration of isoproterenol required to stimulate sustained tachyarrhythmia. The threshold for tachyarrhythmia was converted into a linear arrhythmogenicity score such that tachyarrhythmias triggered by lower concentrations of isoproterenol translated to a higher arrhythmogenicity score  and a consistent preload was applied. Slices were point stimulated  such that electrical propagation was longitudinal to fibre direction. Slices were superfused with oxygenated Tyrode’s solution at 37 °C with the addition of 1 × 10Following culture, slices were washed in PBS and then fixed in 4% formaldehyde solution for 15 min at room temperature. The samples were not subjected to freezing or sectioning in order to prevent tissue damage and to preserve cardiac structure. Slices were permeabilised in 1% Triton X-100 for 1 h at room temperature. Slices were then blocked in 10% foetal bovine serum, 5% bovine serum albumin and 10% horse serum in PBS solution for 2 h. Slices were incubated in the primary antibody in PBS at 4 °C overnight, and then washed three times for 30 min. Slices were incubated in the secondary antibody in PBS for 3 h at room temperature and kept covered with aluminum foil to prevent excessive light exposure. Slices were then washed a final time and stored in PBS at 4 °C. See Table g for 5 min at 4 °C, supernatants were neutralised with 2 M KOH. Subsequent centrifugation at 15115xg for 5 min at 4 °C removed precipitated potassium perchlorate and supernatants were used for analysis on the day of extraction. HPLC analysis of ATP, ADP, AMP, phosphocreatine, creatine, NAD and NADH (as adenosine diphosphoribose— ADPR) was carried out. The HPLC instrument was Agilent 1100 system with 150 × 4.6 mm, 3 µm BDS Hypersil column protected by Security Guard (Phenomenex).For analysis of ATP-related metabolites, cultured slices were quickly dried to culture media and were snap frozen in liquid nitrogen. Slices were freeze dried before extraction with 0.4 perchloric acid at a ratio of 1:100 using a glass homogeniser. Following centrifugation at 15115xg at 4 °C for 15 min and phase separation occurred. The top aqueous layer (containing the RNA) was then removed. An equivalent volume (to that of the aqueous layer removed) of 70% ethanol was then added and the solution mixed via pipetting. All of the solution was then transferred to an RNeasy spin column (Qiagen) and the manufacturer’s instructions were then followed. Following collection, RNA was frozen and kept at −80 °C.Following culture, myocardial slices were immediately frozen in liquid nitrogen and stored at −80 °C. A TissueLyser LT (Qiagen) was used to disrupt the sample. The frozen slices were transferred to a pre-cooled 2-mL round-bottom tube containing a 5-mm stainless-steel ball and 500 μl of TRIzol (Thermo Fisher Scientific). The TissueLyser was set to 40 Hz and left for 3–5 min, until the sample was completely lysed. One-hundred microlitres of chloroform (Sigma) was added to each tube and then vortexed for 15 s. The tube was then left in a rack for 5 min at room temperature and intermittently shaken. The tube was then centrifuged at 12879 × http://www.bioinformatics.babraham.ac.uk/projects/fastqc—version 0.11.2). Sequencing reads were aligned to Rattus Norvegicus (Rnor_6_0) reference genome by Tophat (version 2.0.10) using the set of known genes provided by Ensembl database (release 90) with the average alignment rate of 96%47. The raw number of read pairs mapped to each Ensemble gene was calculated with HTSeq (version 0.6.0) in ‘union’ mode48. Reads (or read pairs) that overlap more than one gene or mapped to multiple locations were discarded. The raw count data were normalised using DESeq2 (version 1.14.1)49. Differential gene expression analysis for various pairs of conditions was performed using DESeq2 (version 1.14.1). Genes were ranked according to their log2 fold change and a preranked gene set enrichment analysis was conducted using GSEA software from the Broad Institute (available at http://software.broadinstitute.org/gsea/index.jsp). Genes were compared with gene sets  in the Molecular Signatures Database. We only present gene sets with a normalised enrichment score q value < 0.005 and that related to our structural and functional assays. Plots were produced in R using ‘ggplot2’.TruSeq Stranded mRNA libraries were multiplexed and sequenced with the average of 70 million DNA fragments per sample (75 base pair paired-end reads). Quality control was performed using FastQC software (Andrews S (2010) FastQC: a quality control tool for high-throughput sequence data. Available online at: Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Heart failure is a complex clinical syndrome associated with a significant morbidity and mortality burden. Reductions in left ventricular (LV) function trigger adaptive mechanisms, leading to structural changes within the LV and the potential development of dyssynchronous ventricular activation. This is the substrate targeted during cardiac resynchronisation therapy (CRT); however, around 30–50% of patients do not experience benefit from this treatment. Non-response occurs as a result of pre-implant, peri-implant and post implant factors but the technical constraints of traditional, transvenous epicardial CRT mean they can be challenging to overcome. In an effort to improve response, novel alternative methods of CRT delivery have been developed and of these endocardial pacing, where the LV is stimulated from inside the LV cavity, appears the most promising. Heart failure is a complex clinical syndrome associated with a significant morbidity and mortality burden. Reductions in left ventricular (LV) function trigger adaptive mechanisms aimed at regulating the cardiac output; however, over time these processes cause structural changes within the LV. This remodelling can result in the development of dyssynchronous ventricular activation, typically manifested by left bundle branch block on the 12-lead ECG. This is the substrate targeted during cardiac resynchronisation therapy; however, between 30 and 50% of patients do not experience significant benefits from this treatment [In response to the progressive haemodynamic stress associated with heart failure, compensatory structural changes occur within the heart. The left ventricular myocardial mass and composition exhibit evidence of change. As this occurs, the heart becomes less elliptical and more spherical as the geometry and volume of the LV change. This process is called remodelling and initially enables the heart to increase cardiac output by increasing both contractility and stroke volume. Whilst stroke volume initially increases, this process eventually becomes deleterious. Progressive enlargement of the ventricle leads to hypertrophy, causing increased wall tension and ultimately results in the myocardium becoming fibrotic, impairing contractility. As the left ventricle dilates and remodels, it exhibits greater contractile dyssynchrony further reducing efficiency.Dyssynchronous electrical conduction can manifest as abnormal heart rate modulation, disruption to the sequence of atrio-ventricular systole and overt ventricular systolic discoordination which typically manifests as bundle branch block on the 12-lead ECG. The development of left bundle branch block is thought to occur as a result of ventricular remodelling and/or fibrogenic damage to the cardiac condition system –6. Left In the context of left bundle branch block, the right anterior septal region is initially activated via the intact right bundle in juxtaposition to the left basal posterolateral region, which slowly propagates via cell-to-cell, intra-myocardial conduction. Contraction of the anterior right septum is imbalanced due to delayed activation of the lateral free wall and occurs unopposed. This has the effect of delaying elevation in the intra-cavity pressure gradient (dP/dtmax) as septal activation merely results in pre-stretch of the inactive lateral wall. When the lateral wall finally contracts, the septum is now in diastole, generating an energy sink which further reduces the overall ejection of blood via the left ventricular outflow tract. Cardiac mechano-energetic efficiency is further exacerbated by delayed activation of the posterolateral papillary muscle, causing sub-optimal closure of the mitral valve and ultimately mitral regurgitation.Over time, left bundle branch block (LBBB) activation causes molecular and cellular remodelling leading to alterations in glucose uptake, regional perfusion and calcium transport . These fCardiac resynchronisation therapy (CRT) aims to eliminate the dyssynchrony which results from bundle branch block activation and restore the mechano-energetic efficiency of the heart. During CRT, both the left and right ventricles are stimulated in an attempt to re-coordinate cardiac electrical activation and produce a synchronous and efficient contraction. Several large randomised controlled trials of biventricular (BiV) pacing have been conducted which have established the efficacy of this therapy, which yields both reductions in morbidity and mortality, see Fig. Around 30–50% of patients fail to respond to transvenous, epicardial CRT. This group are classified as non-responders although no unifying definition of response to CRT exists. Response can be measured in a variety of different clinical, functional and structural endpoints and patients can fail to respond for a variety of different reasons.The process of defining response to CRT is challenging as no universally accepted definition of CRT response exists. Response rates tend to be higher when clinical measures, such as subjective assessments of symptoms are used but are much lower when remodelling or outcome measurements are employed, see Fig. Additionally, symptomatic improvements do not always correlate with echocardiographic or functional classification improvements. There is also no consensus as to the optimal timeframe to re-assess LV function when gauging remodelling. The most widely accepted definition of response involves an assessment of left ventricular reverse remodelling (LVRR) 6 months after implantation, with reductions in LV end-systolic volume (LVESV) the most useful measure , 13. RemThe reasons why some patients fail to adequately respond to transvenous, epicardial CRT are multifactorial and involve several pre-, peri- and post implant factors, see Fig. Pre-implant factors associated with non-response to CRT include appropriate patient selection, baseline ECG morphology and QRS duration, gender, aetiology, presence of myocardial scar and disease severity.Optimal patient selection is critical when looking to maximise response to CRT. In both European  and AmerThe presence of left bundle branch block morphology is a strong predictor of response to CRT . Whilst The evidence for CRT in patients with non-specific intraventricular conduction delay (NICD) who possess a wide QRS without the appearance of left or right bundle branch block is also sparse. This ECG abnormality is present in between 3.8 and 5.8% of patients with impaired LV function , 23 and Conflicting outcomes following the use of CRT in patients with NICD have been reported with some studies appearing to show benefits in quality of life , whilst Women have been consistently under-represented in nearly all large-scale trials of CRT and yet gender appears to play a key part in determining response to CRT –30. FemaIn roughly 50% of patients receiving CRT, the aetiology of their heart failure will be ischaemic in origin ; howeverAround 25% of patients undergoing CRT implantation are in permanent atrial fibrillation (AF) . It is cGiven the importance of maximising the percentage of BiV pacing, it has been postulated that all patients with AF should undergo atrio-ventricular (AV) junctional ablation and this approach does appear to enhance the effects of CRT with the same magnitude as those seen in patients with sinus rhythm .Patients with a bradycardia pacing system in situ who go on to develop heart failure symptoms account for around 23–28% of CRT implants. The deleterious effects of chronic RV pacing have been well established –42 and rPeri-implant factors associated with non-response to CRT include utilising appropriate LV lead technology and optimisation of the LV lead stimulation site.Transvenous, epicardial CRT was traditionally performed by implanting a bipolar pacing lead in a tributary of the coronary sinus, facilitating depolarisation of the LV. Multisite pacing (MSP) where stimulation is delivered from two or more sites within the LV would intuitively appear preferable to single-site stimulation. In early pilot work, MSP was associated with greater reverse remodelling , 45; howMSP can also be delivered via multipoint pacing (MPP). This technique utilises quadripolar LV pacing leads which have four integrated pacing cathodes along the course of the lead, allowing greater customisation from any of the of the 10–17 MPP vectors available. The increased choice for the implanting physician means it is possible to programme around frequently occurring issues including high pacing thresholds–potentially caused by areas of scar- or phrenic nerve stimulation, where LV depolarisation causes diaphragmatic twitching. The use of MPP has been shown to yield greater improvements in acute haemodynamic response  and in aThe optimal LV pacing site displays large inter and intra-patient variability amongst patients with both DCM  and ICM Post implant factors associated with non-response to CRT include remote monitoring, frequency of biventricular pacing, device programming and optimisation.Almost all modern cardiac implanted electronic devices (CIEDs) have the capability to allow remote device follow up. Large, randomised studies have consistently shown that remote device monitoring is both feasible and reduces the need for ambulatory outpatient clinic attendance . The useWhilst CIEDs can analyse fluid status by calculating thoracic impedance as part of a multiparametric assessment, monitoring-only implantable technologies have also been devised. The CardioMEMS Heart Failure system  is a wireless pulmonary artery haemodynamic monitor, which provides an accurate assessment of real time pulmonary artery (PA) pressure, allowing the physician to optimise pharmacotherapy. Use of this system was associated with a 33% reduction in hospitalisations when compared to standard of care . Such syIn order for a CRT system to function effectively, it is essential that it is able to deliver consistent biventricular pacing. Frequent ventricular ectopics and atrial tachyarrhythmias can reduce the frequency of biventricular pacing and were identified in up to a third of non-responders to CRT . There aVentricular extra systolic beats can similarly disrupt the efficient function of a CRT system, reducing the frequency of effective biventricular pacing and as such, contributing to non-response. Again, the therapeutic target is to minimise the occurrence of such events either through the use of pharmacological therapy or catheter ablation .Appropriate programming can increase the frequency of biventricular pacing but is also essential in order to ensure the optimal mechanical functioning of the heart, which facilitates greater response. Arguably, the most important settings requiring optimisation are the pacing mode, upper and lower rate limits, the LV capture voltage, the stimulation vector and A-V and V-V intervals. Programming a high upper tracking rate ensures biventricular pacing is maintained during exercise. Similarly, the LV pacing output should include an adequate safety margin to ensure continual BiV pacing, although some modern devices can automatically adjust this parameter.Iterative optimisation of the A-V and V-V intervals using doppler echocardiography is the established reference method of achieving optimal programming by ensuring optimal diastolic filling of the LV , 71. RecThe persistent rate of non-response to transvenous, epicardial CRT has led to the development of novel forms of undertaking CRT. Of these, biventricular endocardial pacing (BiV ENDO), where LV stimulation occurs from within the LV cavity, holds a great deal of promise. This technique is associated with several advantages over epicardial activation. BiV ENDO pacing can be achieved using several different approaches including the use of novel, leadless pacing.A large body of evidence exists highlighting the potential benefits of BiV ENDO CRT.Failure to implant an LV lead in a tributary of the coronary sinus during a transvenous, epicardial CRT procedure occurs in around 5–15% of cases , 75, 76.BiV ENDO pacing is not reliant on the CS anatomy and instead, operators can choose to deliver stimulation at any site within the LV cavity. This allows for customisation of the stimulation site based on the patient’s pathology and physiology. In addition, whilst lead-based pacing systems may still encounter issues such as central venous stenosis and occlusion, particularly in the upgrade population, newer wireless pacing systems which can be delivered via a retrograde aortic approach eliminate the need for central venous access .Clinically relevant phrenic nerve stimulation (PNS) can complicate LV lead deployment during transvenous, epicardial CRT implantation and this complication is encountered in over 20% of patients at implant or at follow up . UnfortuEndocardial stimulation of the LV facilitates more rapid myocardial depolarisation as wave front propagation occurs along the shorter endocardial surface of the heart. Several studies have also demonstrated enhanced conduction velocities associated with stimulation of endocardial tissue –88. One Hyde et al. set out to establish a greater understanding of fast endocardial conduction (FEC). Non-Purkinje sub-endocardial tissue conducts impulses faster than either mid-myocardial or epicardial fibres . InsteadTraditional CRT delivers electrical stimulation to the epicardial surface of the LV wall. This pattern of activation is juxtaposed to the sequence exhibited during intrinsic myocardial activation which follows an endocardial to epicardial course. Reversing the normal depolarisation sequence is thought to promote repolarisation heterogeneity, which can prolong the QTc interval in susceptible patients and result in malignant arrhythmias –97. In cIncreasing evidence supports the hypothesis that BiV ENDO pacing has the capability to achieve greater electrical resynchronisation and consequently, maximise response to CRT. Traditionally, BiV ENDO pacing has been performed in a relatively small number of patients, typically those with failure to achieve conventional CRT or those that have been non-responders. The recently published ALSYNC study was the first multicentre study of LV endocardial pacing and demonstrated the feasibility and safety of LV endocardial CRT delivered through the atrial trans-septal approach . In 138 The acute haemodynamic benefits of BiV ENDO appear superior to those achievable utilising BiV EPI, even amongst a cohort of ischaemic, non-responders to conventional epicardial CRT . When a Chronic BiV ENDO CRT has traditionally been delivered via a pacing lead delivered via either a transventricular-septal or atrial-septal puncture. Due to the risk of thrombus formation resulting in systemic embolization, lead-based LV endocardial pacing requires lifelong anti-coagulation. In a recent meta-analysis, the stroke rate was found to be 2.5 per 100 patient years [Following a trans-septal implant, the potential also exists for an adverse interaction between the mitral valve and an LV lead which passes through it. The presence of a pacing lead can worsen valvular regurgitation and this phenomenon has been acknowledged with right-sided leads. Reassuringly in the largest series of trans-septal LV endocardial pacing to date, no system-related valvular complications were recorded. Instead, echocardiography showed reductions in mitral regurgitation as a result of improved LV function . The othLead-based BiV ENDO is predominantly delivered via the atrial trans-septal approach . SuperioVentricular trans-septal techniques allow the deployment of the lead using only superior access, eliminating the need for femoral punctures . This apA hybrid surgical/percutaneous approach has also been described where the LV apex is accessed via a mini-thoracotomy facilitating the deployment of a lead at the LV apex, which is then tunnelled up to a generator, implanted in the conventional sub-clavicular position . Whilst Whilst chronic BiV ENDO delivered via a pacing lead currently requires ongoing anti-coagulants due to the risk of thrombus formation, newer wireless technology almost entirely eliminated this risk. Formal anti-coagulation is not required and this development increases the likelihood of endocardial pacing becoming more widespread.A leadless BiV ENDO CRT pacing system has been developed, which eliminates the need for any form of pacing lead to be present within the left ventricle. Instead stimulation is delivered to the endocardial surface of the heart acoustically from an ultrasound (USS) pulse generator, implanted subcutaneously in an intercostal space, see Fig. Whilst co-axial alignment of the USS pulse generator and pacing electrode is desirable, the USS transmitter uses beam-forming to direct the energy towards the pacing electrode, minimising the amount of energy required. Refinements of this generator mean it can now be implanted via a small horizontal incision and is attached to a dedicated battery, typically placed in the mid-axillary line via traditional lead tunnelling techniques. Battery life of the current system is typically quoted at 3 years, but newer battery technology currently nearing release is expected to double this longevity to over 6 years.Early clinical studies evaluating this system confirmed implantation was safe and capable of achieving consistent biventricular capture  whilst mThe most commonly encountered complication associated with the use of the WiSE CRT system is femoral arterial access complications. In addition, not every patient has suitable anatomy to allow sufficient arterial access to allow the insertion of the electrode delivery catheter. Ischaemic heart disease is common in this population and as such, concurrent peripheral vascular disease is frequently encountered.A method of transvenously accessing the LV cavity via an atrial trans-septal puncture has been described to facilitate deployment of the pacing electrode . WiSE CRNon-response to CRT is a multifactorial issue. Improving patient selection and post implant device troubleshooting remain the cornerstone of optimising patient outcomes. Whilst newer lead technology including multipoint pacing and a greater focus on the importance of site selection are likely to be beneficial, the limitations of transvenous, epicardial CRT are widely acknowledged. In patients where implanting a transvenous lead is not possible, consideration should be given to endocardial pacing. Newer leadless LV endocardial systems no longer mandate lifelong anti-coagulation. In addition, endocardial pacing appears capable of yielding more effective electromechanical resynchronisation. Given the ability to stimulate the LV from any location, this technique would appear to offer a significant benefit to patients where transvenous lead implantation is restricted to inferior sites.