Dr Patrizia Camelliti


Lecturer in Cardiovascular Biology
MSc, DPhil, FHEA
+44 (0)1483 682451

Biography

Biography

Dr Patrizia Camelliti is a Lecturer in Cardiovascular Biology in the School of Biosciences and Medicine, Faculty of Health and Medical Sciences. Patrizia graduated in Biological Sciences (magna cum laude) from the University of Milan, Italy. She then moved to the University of Oxford, to undertake a DPhil under the joint supervision of Prof Peter Kohl (Department of Physiology, Anatomy and Genetics, Oxford) and Prof Colin Green (University of Auckland, New Zealand). After completing her DPhil, Patrizia was awarded a Junior Research Fellowship at Christ Church College, University of Oxford, and an EP Abraham Cephalosporin research grant, to pursue research at the interface between cardiac physiology and bioengineering. She then received a Fellowship from Imperial College and moved to the Myocardial Function section at the National Heart and Lung Institute, Imperial College London, where she extensively validated the cardiac slice preparation, a representative in vitro model of the heart suitable for a wide range of biological and pharmacological applications.

Patrizia's research has been published in high impact journals, with 3 Journal Covers and over 2,850 citations. She has presented her work at many international scientific conferences, including the AHA Scientific Sessions, Heart Rhythm, and the European Society of Cardiology, and as invited speaker at several Universities, including Tianjin Medical University (China), University of Tübingen (Germany), Nara Medical School (Japan), Dresden University of Technology (Germany), Southwestern Medical Center (Dallas, US), and Montreal Heart Institute (Canada).

Patrizia is a review editor for Frontiers in Cardiac Electrophysiology, and a member of the UK Physiological Society, the British Society for Cardiovascular Research, the European Society of Cardiology, the International Society for Heart Research and the Biophysical Society.

During her career Patrizia has developed and validated a number of novel research methods and tools, including advanced structured cell cultures, myocardial slices and state of the art imaging techniques (multiphoton microscopy, optical mapping, electron tomography and advanced immunohistochemistry). Her current research continues to develop and utilize these methods and she is keen to develop new collaborations with likeminded scientists.

Research interests

I have two major research interests:

(1) Characterise the multicellular mechanisms involved in the functional and structural remodelling that occurs in ischaemic, non-ischaemic and genetic heart diseases, for the development of novel pharmacological, cell and gene therapies.This research uses living cardiac tissue slices and a combination of structure-function assays, including multi-electrode arrays, optical mapping (voltage and calcium), confocal and multiphoton microscopy, and molecular biology.

(2) Elucidate the active role that cardiac fibroblasts - the largest cell population in the heart - play in the co-ordination and regulation of heart function under physiological and pathological conditions. Specifically, we are investigating how fibroblast-myocyte cross-talk affects myocyte electrophysiology and fibroblast phenotype via direct cell coupling and/or paracrine communication.

 

Fundraising and Research Grants• BHF MBPhD Studentship (co-applicant; PI: Dr Terracciano, Imperial College) 2013-2016• Imperial College Fellowship, Imperial College London (PI) 2010-2013• EP Abraham Cephalosporin Fund (PIs: Camelliti P & Kohl P) 2008-2010• Junior Research Fellowship, Christ Church College Oxford (PI) 2005-2009

 

Awards• Imperial College Fellowship, Imperial College London, 2010.• British Heart Foundation Reflections of Research image competition (finalist), 2010.• Young Investigator Award, Working Group on Cardiac Cellular Electrophysiology; Spain, 2008.• Poster of Excellence Award, Gordon Research Conference, Cardiac Arrhythmia Mechanisms; Ventura, USA; 2007.• Poster Presentation Award, Oxford Postdoc Network; Oxford, 2006.• Christ Church College Junior Research Fellowship, Christ Church College, Oxford, 2005.• British Heart Foundation Reflections of Research image competition (2nd place), 2005.• Blue Riband Prize, The UK Physiological Society.• Erasmus Scholarship, University of Milan (to spend 6 months in the laboratory of Dr Allan Levi at the University of Bristol).

Research collaborations

Dr Cesare Terracciano, Imperial College London, UK

Dr Ken MacLeod and Hsiang-Yu Yang, Imperial College London, UK

Prof Nicholas Peters, Dr Fu Siong Ng & Dr Rasheda Chowdhury, Imperial College London, UK

Prof Peter Kohl, Imperial College London & University of Oxford, UK

Dr David Connolly and Dr Jay Dudhia, Royal Veterinary College, UK

Dr Daniel Stuckey, University College London, UK

Dr Tom Smolenski, Medical University of Gdansk, Poland

Prof Haifang Yin, Tianjin-Oxford Laboratory of Gene Therapy, Tianjin Medical University, China

Dr Arun Sridhar, Safety Pharmacology, GlaxoSmithKline, UK

Affiliations

• The Physiological Society• The British Society of Cardiovascular Research• European Society of Cardiology• Biophysical Society• International Society for Heart Research• Imperial Cardiovascular Technology Network• London Matrix Group• London Regenerative Medicine Network

Public Engagement

Royal Society Pairing SchemeImperial Festival (science public event)• Research featured on the Guardian science news 2011• Research featured on the Guardian science news 2010• Contribution to the children science book “Instructions for ME”, Magic World Media Edition• British Heart Foundation Reflections of Research Exhibition (The Royal Society of Arts, London)• Research featured on the BBC science news

My publications

Publications

Lei M, Cooper PJ, Camelliti P, Kohl P (2002) Role of the 293b-sensitive, slowly activating delayed rectifier potassium current, i(Ks), in pacemaker activity of rabbit isolated sino-atrial node cells., Cardiovasc Res 53 (1) pp. 68-79
OBJECTIVES: (i) to characterize the electrophysiological properties of the slowly activating delayed rectifier potassium current, i(Ks), defined as the 293b-sensitive current, during the action potential (AP) of rabbit sino-atrial node (SAN) pacemaker cells; (ii) to evaluate the contribution of i(Ks) to the pacemaker AP under physiological conditions and during beta-adrenergic stimulation. METHODS: Rabbit SAN pacemaker cells were studied using the perforated patch clamp technique in voltage-, AP- and current-clamp modes. RESULTS: Voltage-clamp findings. Block of i(Ks) by 293b is dose-dependent, with an IC(50) (half block) in rabbit SAN cells of 1.35 microM and an IC(80) (sub-maximal block) of 5 microM. Sub-maximal concentrations of 293b have no significant effects on long-lasting and transient inward calcium currents, i(Ca,L) and i(Ca,T), inward hyperpolarization activated current, i(f), and transient outward current, i(to). AP-clamp experiments. The 293b-sensitive current activates near the peak of the SAN pacemaker action potential, reaches a mean maximal current density of 1.0+/-0.3 pA/pF (n=8, cell capacitances 27 to 62 pF, mean 35+/-4.0 pF) during late repolarization, and inactivates towards the end of repolarization. Additionally, in two smaller cells (cell capacitances 15 and 23 pF), no discernible 293b-sensitive current component was detected. Current-clamp data. In spontaneously beating SAN cells under control conditions, sub-maximal block of i(Ks) by 5 microM 293b has negligible effects on action potential characteristics and does not change average cycle length (n=11). In contrast, after pre-treatment with 10 nM isoprenaline to mimic beta-adrenergic stimulation, cells showed a 293b-induced depolarization of maximum diastolic potential by 2.2+/-1%, a decrease in diastolic depolarization rate by 9.9+/-4%, and a slowing of late action potential repolarization by 28.7+/-10.2%, resulting in a prolongation of spontaneous cycle length by 9.8+/-3.0% (P
Lei M, Cooper PJ, Camelliti P, Kohl P (2002) Contribution of the fast sodium inward current, iNa, to murine sino-atrial node pacemaking., Biophysical Journal 82 (1)
Bussek A, Wettwer E, Christ T, Lohmann H, Camelliti P, Ravens U (2009) Tissue slices from adult mammalian hearts as a model for pharmacological drug testing, Cellular Physiology and Biochemistry 24 (5-6) pp. 527-536
Aim: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. Method: Vibratome cut ventricular slices (350 ¼m thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). Results: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD 90, 169.9 ± 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD 90 with a half maximal effective concentration (EC 50) of 4.5 ¼M. The I Kr blocker E4031 and neuroleptic drug risperidone prolonged APD 90 with EC 50 values of 31 nM and 0.67 ¼M, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m s -1. Conclusion: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research. Copyright © 2009 S. Karger AG, Basel.
Camelliti P, Green CR, Kohl P (2004) Fibroblast-myocyte coupling: fact or fiction?, Z Kardiol 93 (Suppl 3)
Camelliti P, Green C, Holloway H, Kohl P (2001) Effects of acute ventricular volume manipulation on cardiomyocyte membrane organisation in rabbit heart.,
Lei M, Cooper PJ, Camelliti P, Kohl P (2002) Variability in mechanically induced changes in cardiac action potentials: effect of probe attachment., Proceedings of the 3rd International Workshop on Cardiac Mechano-Electric Feedback
Quinn TA, Camelliti P, Rog-Zielinska EA, Siedlecka U, Poggioli T, O'Toole ET, Knoepfel T, Kohl P (2016) Electrotonic coupling of excitable and nonexcitable cells in the heart revealed by optogenetics, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (51) pp. 14852-14857 NATL ACAD SCIENCES
Kohl P, Camelliti P, Burton FL, Smith GL (2006) Fibroblast-Myocyte interrelation in the mammalian heart: experiments and models., FASEB Journal 20 (5)
Prieto CJ, Revol-Muller C, Camelliti P, Odet C (2012) Modeling 3D realistic organic tissues from 2D digital microscopy images,
Camelliti P, Gallagher JO, Kohl P, McCulloch AD (2006) Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium., Nat Protoc 1 (3) pp. 1379-1391
We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.
Cartledge JE, Clark LA, Ibrahim M, Siedlecka U, Navaratnarajah M, Yacoub MH, Camelliti P, Terracciano CM, Chester AH (2011) Cardiac fibroblasts regulate adult cardiomyocyte excitation contraction coupling by soluble mediators in co-culture., Circulation 124
Prieto J-C, Revol-Muller C, Peyrin F, Camelliti P, Odet C (2012) 3D Texture Synthesis for Modeling Realistic Organic Tissues., VISAPP (2) pp. 60-65 SciTePress
Virtual anatomy models show in detail characteristics of the human body systems. These models are based in surface representation of the structures and lack information from the interior of the object. Creating models that represent the surface, the interior of the object and are able to provide pathological information is the current challenge of research in life sciences. We present a method to synthesize realistic three-dimensional organic tissues starting from bidimensional textured multi-channel samples. The method relies on an energy function that measures the difference between the reference texture and the synthesized object, through a distance metric that compares perpendicular neighborhoods in the object to neighborhoods in the sample. When this function is minimized by IRLS, the result is a solid object that resembles the sample at every slice. In some cases, the optimization might be aided by adding the feature distance transform, calculated from a given binary mask. This allows to code large textured areas. Multiple textures can also be provided to the optimization in order to create anistropic textures. We apply our method starting from various micrometric images such as histology images or slices of Synchrotron Radiation Computed Micro-Tomography (SR¼CT) images. A major advantage of our method is to extend 2D histological information to a 3D representation. We demonstrate the accuracy of the generated texture by comparing statistical and morphological parameters computed from the synthetic object with those obtained from the real object underlying the reference images.
Lei M, Jones S, Lancaster M, Fung S, Liu J, Dobrzynski H, Camelliti P, Maier SKG, Boyett M, Noble D (2004) Requirement of brain and cardiac-type sodium channels for murine sinoatrial node pacemaking., Biophysical Journal 86 (1) pp. 424A-425A
Alayoubi S, Al-Aidarous S, Pinto Ricardo C, Dias P, Zaman J, Kane C, Camelliti P, Peters N, Yacoub M, Terracciano C (2014) P379Slowed conduction velocity in spontaneously hypertensive rat hearts is due to disease related remodelling., Cardiovasc Res 103 Suppl 1
The spontaneously hypertensive rat (SHR) is a well-characterised model for studies of hypertension and atrial arrhythmias but little is known about the electrophysiological properties of the left ventricle (LV) and their relation with ventricular arrhythmias in the development of this disease. To investigate the mechanisms behind electrophysiological abnormalities in the LV we used myocardial slices which allow the investigation of functional and structural properties in the same tissue location. Myocardial slices (300¼m thick) were prepared from young (3months) and old (20months) SHR and age-matched control LVs. Slices were point-stimulated and analysed using a multi-electrode array system; longitudinal conduction velocity (CVL) was measured. CVL was unchanged between the young and old control groups. However, CVL was significantly reduced in the old SHR group compared to the corresponding age-matched control and young SHR groups (20months: 27±2 cm/s, n=29 slices/3 hearts vs control 39±4 cm/s, n=22 slices/4hearts and 3months: 37±3 cm/s, n=18 slices/3 hearts; p
Camelliti P, Kohl P (2007) Fibroblasts and cardiac electrophysiology., Proc Physiol Soc 8
Camelliti P, Kohl P (2004) Interrelation of cardiac fibroblasts and myocytes: New tools and insights, Microscopy and Microanalysis 10 (SUPPL. 2) pp. 1398-1399
Camelliti P, Abou Al-Saud S, Smolenski RT, Al-Ayoubi S, Banner NR, Bowles CT, Yacoub MH, Terracciano CM (2011) Human heart slices are a novel multicellular system for electrophysiological, pharmacological and metabolic studies of the heart., Biophysical Journal
Camelliti P, Bub G, Stuckey DJ, Bollensdorff C, Tyler DJ, Helmes M, Clarke K, Kohl P (2008) Two photon microscopy measurements of sub-epicardial sarcomere length in perfused rat hearts., Circulation 118
Camelliti P, Morphew MK, Mason F, Hoenger A, Kohl P (2007) 3D reconstruction of microtubules, SR and T-tubular membrane systems organisation in rat ventricular myocytes with high resolution EM tomography.,
Camelliti P, Kohl P (2003) Myocyte/fibroblast 2D structured cardiac tissue models., J Physiol 552P
Forlani G, Camelliti P, Balestrini M, Peres A, Zippel R, Giovannardi S (2001) Modulation of the inward rectifier potassium channel IRK1 by the Ras signaling pathway, J Physiol 531
Lei M, Jones SA, Liu J, Lancaster MK, Fung SS, Dobrzynski H, Camelliti P, Maier SK, Noble D, Boyett MR (2004) Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking., J Physiol 559 (Pt 3) pp. 835-848
The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2-6 microm) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (KD, 1-10 nm) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na+ currents under physiological conditions and the expression of cardiac and neuronal Na+ channel isoforms. We identified two distinct Na+ current components, TTX resistant and TTX sensitive. At 37 degrees C, TTX-resistant iNa and TTX-sensitive iNa started to activate at approximately -70 and approximately -60 mV, and peaked at -30 and -10 mV, with a current density of 22 +/- 3 and 18 +/- 1 pA pF(-1), respectively. TTX-sensitive iNa inactivated at more positive potentials as compared to TTX-resistant iNa. Using action potential clamp, TTX-sensitive iNa was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Nav1.5, and the TTX-sensitive neuronal isoform, Nav1.1, were observed: Nav1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Nav1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nm) of TTX, which block TTX-sensitive iNa, slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 microm) of TTX, which block TTX-resistant iNa as well as TTX-sensitive iNa, slowed both pacemaking and SA node conduction. It is concluded that two Na+ channel isoforms are important for the functioning of the SA node: neuronal (putative Nav1.1) and cardiac Nav1.5 isoforms are involved in pacemaking, although the cardiac Nav1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.
Camelliti P, McCulloch AD, Kohl P (2005) Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue., Microsc Microanal 11 (3) pp. 249-259
Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-microm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.
Camelliti P, Iribe G, Fink M, Burton RA, Kohl P (2007) Co-localization of ryanodine receptors and microtubules in rat ventricular cardiomyocytes: functional and structural evidence.,
Camelliti P, Kohl P (2005) Stretch-induced Cx43 remodelling in a 2D in vitro model of myocardium., Heart Rhythm 5 (2)
Camelliti P (2009) Heart tissue slices as a simple, yet physiologically relevant 3D model of myocardium for cardiac research and development.,
Swietach P, Camelliti P, Kohl P, Vaughan-Jones RD (2007) Generating local acid-loads in multicellular strands of cultured neonatal myocytes using flash-photolysis., Biophysical Journal
Cartledge JE, Dias P, Ibrahim M, Alayoubi S, Yacoub MH, Camelliti P, Terracciano CM (2013) Freshly isolated adult fibroblasts from normal and overloaded hearts affect viability, cell volume and calcium cycling of cardiac myocytes in co-culture by paracrine TGF-² signalling.,
Camelliti P, Devlin G, Kohl P, Green C (2003) Gap junction expression following myocardial ischemia suggests a bystander effect in progressive infarction., American Society for Cell Biology Congress Proceedings
Camelliti P, Alayoubi S, Cartledge J, Yacoub MH, Sridhar A, Terracciano CM (2013) Mapping of Repolarisation Gradients in the Canine Left Ventricular Free Wall Using Myocardial Slices,
Camelliti P, Al-Ayoubi S, Terracciano C (2014) MAPPING REGIONAL REPOLARISATION GRADIENTS IN THE FAILING HUMAN VENTRICLE USING CARDIAC SLICES, Heart 100
Camelliti P, Abou Al-Saud S, Prashar R, Yacoub MH, Terracciano CM (2010) Dog and human ventricular tissue slices are a novel multicellular system suitable for physiological and pharmacological studies.,
Trantidou T, Rao C, Barrett H, Camelliti P, Pinto K, Yacoub MH, Athanasiou T, Toumazou C, Terracciano CM, Prodromakis T (2014) Selective hydrophilic modification of Parylene C films: A new approach to cell micro-patterning for synthetic biology applications, Biofabrication 6 (2)
We demonstrate a simple, accurate and versatile method to manipulate Parylene C, a material widely known for its high biocompatibility, and transform it to a substrate that can effectively control the cellular microenvironment and consequently affect the morphology and function of the cells in vitro. The Parylene C scaffolds are fabricated by selectively increasing the material's surface water affinity through lithography and oxygen plasma treatment, providing free bonds for attachment of hydrophilic biomolecules. The micro-engineered constructs were tested as culture scaffolds for rat ventricular fibroblasts and neonatal myocytes (NRVM), toward modeling the unique anisotropic architecture of native cardiac tissue. The scaffolds induced the patterning of extracellular matrix compounds and therefore of the cells, which demonstrated substantial alignment compared to typical unstructured cultures. Ca2+ cycling properties of the NRVM measured at rates of stimulation 0.5-2 Hz were significantly modified with a shorter time to peak and time to 90% decay, and a larger fluorescence amplitude (p
Giovannardi S, Forlani G, Camelliti P, Filippini L, Bossi E, Balestrini M, Zippel R, Peres A (2000) Modulation of the inward rectifier potassium channel IRK1 by the Ras pathway, Pflugers Archiv 5 (440)
Bub G, Camelliti P, Bollensdorff C, Stuckey DJ, Picton G, Burton RAB, Clarke K, Kohl P (2010) Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro, American Journal of Physiology - Heart and Circulatory Physiology 298 (5)
Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 ¼m (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 ¼m, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 ¼m, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data. Copyright © 2010 the American Physiological Society.
Picton GK, Camelliti P, Bub G, Bussek A, Wettwer E, Ravens U, Kohl P (2008) Rat left ventricular wall thin slices as a 3-dimensional cardiac tissue model., Proc Physiol Soc 11 pp. 107-108
Camelliti P, Al-Saud SA, Smolenski RT, Al-Ayoubi S, Bussek A, Wettwer E, Banner NR, Bowles CT, Yacoub MH, Terracciano CM (2011) Adult human heart slices are a multicellular system suitable for electrophysiological and pharmacological studies, Journal of Molecular and Cellular Cardiology 51 (3) pp. 390-398
Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human myocardium. The aim of this study was to establish and characterise adult human myocardial slices from small patients' heart biopsies as a simple, reproducible and relevant preparation suitable for the study of human cardiac tissue at the multicellular level.Vibratome-cut myocardial slices were prepared from left ventricular biopsies obtained from end-stage heart failure patients undergoing heart transplant or ventricular assist device implantation, and from hearts of normal dogs. Multiple slices were prepared from each biopsy. Regular contractility was observed at a range of stimulation frequencies (0.1-2 Hz), and stable electrical activity, monitored using multi-electrode arrays (MEA), was maintained for at least 8 h from slice preparation. ATP/ADP and phosphocreatine/creatine ratios were comparable to intact organ values, and morphology and gap junction distribution were representative of native myocardium. MEA recordings showed that field potential duration (FPD) and conduction velocity (CV) in human and dog slices were similar to the values previously reported for papillary muscles, ventricular wedges and whole hearts. Longitudinal CV was significantly faster than transversal CV, with an anisotropic ratio of 3:1 for human and 2.3:1 for dog slices. Importantly, slices responded to the application of E-4031, chromanol and 4-aminopyridine, three potassium channel blockers known to affect action potential duration, with an increase in FPD.We conclude that viable myocardial slices with preserved structural, biochemical and electrophysiological properties can be prepared from adult human and canine heart biopsies and offer a novel preparation suitable for the study of heart failure and drug screening. © 2011 Elsevier Ltd.
Wang Q, Yin H, Camelliti P, Betts C, Moulton H, Lee H, Saleh AF, Gait MJ, Wood MJA (2010) In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy, Journal of Gene Medicine 12 (4) pp. 354-364
Background: Targeted splice modulation of pre-mRNA transcripts by antisense oligonucleotides (AOs) can correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) arises as a result of mutations that interrupt the open-reading frame in the DMD gene encoding dystrophin such that dystrophin protein is absent, leading to fatal muscle degeneration. AOs have been shown to correct this dystrophin defect via exon skipping to yield functional dystrophin protein in animal models of DMD and also in DMD patients via intramuscular administration. To advance this therapeutic method requires increased exon skipping efficiency via an optimized AO sequence, backbone chemistry and additional modifications, and the improvement of methods for evaluating AO efficacy. Methods: In the present study, we establish the conditions for rapid in vitro AO screening in H2K muscle cells, in which we evaluate the exon skipping properties of a number of known and novel AO chemistries [22-O-methyl, peptide nucleic acid, phosphorodiamidate morpholino (PMO)] and their peptide-conjugated derivatives and correlate their in vitro and in vivo exon skipping activities. Results: The present study demonstrates that using AO concentrations of 300 nM with analysis at a single time-point of 24 h post-transfection allowed the effective in vitro screening of AO compounds to yield data predictive of in vivo exon skipping efficacy. Peptide-conjugated PMO AOs provided the highest in vitro activity. We also show for the first time that the feasibility of rapid AO screening extends to primary cardiomyocytes. Conclusions: In vitro screening of different AOs within the same chemical class is a reliable method for predicting the in vivo exon skipping efficiency of AOs for DMD. Copyright © 2010 John Wiley & Sons, Ltd.
Yin H, Saleh AF, Betts C, Camelliti P, Seow Y, Ashraf S, Arzumanov A, Hammond S, Merritt T, Gait MJ, Wood MJA (2011) Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice, Molecular Therapy 19 (7) pp. 1295-1303
Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics. © The American Society of Gene & Cell Therapy.
Kohl P, Camelliti P (2007) Cardiac myocyte-nonmyocyte electrotonic coupling: implications for ventricular arrhythmogenesis., Heart Rhythm 4 (2) pp. 233-235
Stuckey DJ, Carr CA, Camelliti P, Tyler DJ, Davies KE, Clarke K (2012) In vivo MRI characterization of progressive cardiac dysfunction in the mdx mouse model of muscular dystrophy., PLoS One 7 (1)
The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice.
Bub G, Camelliti P, Bollensdorff C, Kohl P (2007) Sarcomere length measurement in intact resting Guinea pig heart., Proceedings of the 4th International Workshop on Cardiac Mechano-Electric Feedback
Xie Y, Garfinkel A, Camelliti P, Kohl P, Weiss JN, Qu Z (2009) Effects of fibroblast-myocyte coupling on cardiac conduction and vulnerability to reentry: A computational study, Heart Rhythm 6 (11) pp. 1641-1649
Background: Recent experimental studies have documented that functional gap junctions form between fibroblasts and myocytes, raising the possibility that fibroblasts play roles in cardiac electrophysiology that extend beyond acting as passive electrical insulators. Objective: The purpose of this study was to use computational models to investigate how fibroblasts may affect cardiac conduction and vulnerability to reentry under different fibroblast-myocyte coupling conditions and tissue structures. Methods: Computational models of two-dimensional tissue with fibroblast-myocyte coupling were developed and numerically simulated. Myocytes were modeled by the phase I of the Luo-Rudy model, and fibroblasts were modeled by a passive model. Results: Besides slowing conduction by cardiomyocyte decoupling and electrotonic loading, fibroblast coupling to myocytes elevates myocyte resting membrane potential, causing conduction velocity to first increase and then decrease as fibroblast content increases, until conduction failure occurs. Fibroblast-myocyte coupling can also enhance conduction by connecting uncoupled myocytes. These competing effects of fibroblasts on conduction give rise to different conduction patterns under different fibroblast-myocyte coupling conditions and tissue structures. Elevation of myocyte resting potential due to fibroblast-myocyte coupling slows sodium channel recovery, which extends postrepolarization refractoriness. Owing to this prolongation of the myocyte refractory period, reentry was more readily induced by a premature stimulation in heterogeneous tissue models when fibroblasts were electrotonically coupled to myocytes compared with uncoupled fibroblasts acting as pure passive electrical insulators. Conclusions: Fibroblasts affect cardiac conduction by acting as obstacles or by creating electrotonic loading and elevating myocyte resting potential. Functional fibroblast-myocyte coupling prolongs the myocyte refractory period, which may facilitate induction of reentry in cardiac tissue with fibrosis. © 2009 Heart Rhythm Society.
Lei M, Camelliti P, Cooper P, Linz K, Kohl P (2001) Stretch induced whole cell currents during the action potential of guinea-pig ventricular myocytes., J Physiol 533
Camelliti P, Kohl P, Green C (2003) Functional coupling of fibroblasts in rabbit sino-atrial node., Biophysical Journal 84 (2)
Rao C, Chaudhry UA, Camelliti P, Darzi A, Yacoub MH, Athanasious T, Prodromakis T, Terracciano CM (2011) Structured culture scaffolds force maturation of calcium transients in rat neonatal ventricular myocytes., Circulation 124
Wen Q, Gandhi K, Wu J, Capel R, Ni HB, Camelliti P, Zhang H, Faggian G, Terrar D, Lei M (2015) Transmural 2D living cardiac tissue slice model for investigating spatial heterogeneity of intracellular calcium handling in the heart, EUROPEAN HEART JOURNAL 36 pp. 1122-1123 OXFORD UNIV PRESS
Camelliti P, Abou Al-Saud S, Smolenski RT, Al-Ayoubi S, Lee P, Bollensdorff C, Yacoub MH, Terracciano CM, Kohl P (2010) Ventricular tissue slices: a near-physiological multicellular system for cardiac research.,
Dias P, Navaratnarajah M, Sarathchandra P, Latif N, Camelliti P, Yacoub MH, Terracciano CM (2012) Ivabradine reverses the extracellular matrix changes in heart failure., Circulation 126
Camelliti P, Garny A, Kohl P (2002) Mechanically controlled, morphologically determined 2D cardiac tissue models in vitro., Proceedings of the 3rd International Workshop on Cardiac Mechano-Electric Feedback
Cartledge JE, Clark LA, Ibrahim M, Siedlecka U, Navaratnarajah M, Yacoub MH, Camelliti P, Terracciano CM, Chester AH Cardiac fibroblasts regulate adult cardiomyocyte calcium handling in co-culture by paracrine signalling mediated by endothelin-1., Cardiovascular Research 93
Camelliti P, Devlin GP, Matthews KG, Kohl P, Green CR (2004) Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction., Cardiovasc Res 62 (2) pp. 415-425
OBJECTIVES: Myocardial infarction leads to extensive changes in the organization of cardiac myocytes and fibroblasts, and changes in gap junction protein expression. In the immediate period following ischemia, reperfusion causes hypercontraction, spreading the necrotic lesion. Further progressive infarction continues over several weeks. In reperfusion injury, the nonspecific gap junction channel uncoupler heptanol limits necrosis. We hypothesize that gap junction coupling and fibroblast invasion provide a substrate for progressive infarction via a gap junction mediated bystander effect. METHODS: A sheep coronary occlusion infarct model was used with samples collected at 12, 24 and 48 h, and 6, 12 and 30 d (days) post-infarction. Immunohistochemical labelling of gap junction connexins Cx40, Cx43, and Cx45 was combined with cell-specific markers for fibroblasts (anti-vimentin) and myocytes (anti-myomesin). Double and triple immunolabelling and confocal microscopy were used to follow changes in cardiac myocyte morphology, fibroblast content and gap junction expression after myocardial infarction. Gap junction protein levels and fibroblast numbers were quantified. RESULTS: Within 12 h of ischemia, myocyte viability is impaired within small islands in the ischemic region. These islands spread and fuse into larger infarct zones until 12 d post-infarction. Thereafter, surviving myocytes within the infarct and in the border-zone appear to become stabilized. Distant from the infarct, continuing myocyte disruption is regularly observed, even after 30 d. Cx43 becomes redistributed from intercalated discs to the lateral surface of structurally compromised myocytes within 12 d. Cx45 expressing fibroblasts infiltrate the damaged region within 24 h, becoming most numerous at 6-12 d post-infarction, with peak Cx45 levels at 6 d. Later, Cx43 expressing fibroblasts are observed, and the related Cx43 label increases over the 30 d observation period, even though fibroblast numbers decline after 12 d. Cx40 was only seen in vascular endothelium. CONCLUSIONS: Progressive infarction, identified by myocyte sarcomere disruption and subsequent cell loss, occurs in parallel with fibroblast invasion and gap junction remodeling. Two fibroblast phenotypes occur within infarcts, expressing either Cx43 or Cx45. Coupled fibroblasts may play a number of roles in tissue remodeling following myocardial infarction, including provision of a possible substrate for progressive infarction via a ga
Burton RA, Picton GK, Fink M, Camelliti P, Mansoori T, Bollensdorff C, Sheldon J, Bub G, Kohl P (2008) Caveolar remodelling in rabbit left ventricular myocytes after cell isolation., Proc Physiol Soc 11
Camelliti P, Green CR, Kohl P (2006) Structural and functional coupling of cardiac myocytes and fibroblasts., Adv Cardiol 42 pp. 132-149
Cardiac myocytes and fibroblasts form extensive networks in the heart, with numerous anatomical contacts between cells. Fibroblasts, obligatory components of the extracellular matrix, represent the majority of cells in the normal heart, and their number increases with aging and during disease. The myocyte network, coupled by gap junctions, is generally believed to be electrically isolated from fibroblasts in vivo. In culture, however, the heterogeneous cell types form functional gap junctions, which can provide a substrate for electrical coupling of distant myocytes, interconnected by fibroblasts only. Whether similar behavior occurs in vivo has been the subject of considerable debate. Recent electrophysiological, immunohistochemical, and dye-coupling data confirmed the presence of direct electrical coupling between the two cell types in normal cardiac tissue (sinoatrial node), and it has been suggested that similar interactions may occur in post-infarct scar tissue. Such heterogeneous cell coupling could have major implications on in vivo electrical impulse conduction and the transport of small molecules or ions in both the normal and pathological myocardium. This review illustrates that it would be wrong to adhere to a scenario of functional integration of the heart that does not allow for a potential active contribution of non-myocytes to cardiac electrophysiology, and proposes to focus further research on the relevance of non-myocytes for cardiac structure and function.
Kohl P, Camelliti P, Burton FL, Smith GL (2005) Electrical coupling of fibroblasts and myocytes: relevance for cardiac propagation., J Electrocardiol 38 (4 Suppl) pp. 45-50
Myocytes, while giving rise to the bulk volume of normal cardiac muscle, form a "minority cell population" in the heart compared with nonmyocytes, chiefly fibroblasts. The heterogeneous cell types show very intimate spatial interrelation in situ, with virtually every myocyte in the mammalian heart bordering to 1 or more fibroblasts. Nonetheless, gap junction coupling in the heart is traditionally assumed to occur exclusively between myocytes. Yet, both freshly isolated cells and cell cultures have unambiguously shown functional heterogeneous myocyte-fibroblast coupling (mainly via connexin 43). Such coupling is sufficient, in vitro, to synchronize spontaneous beating in distant myocytes, connected over distances of up to 300 microm by fibroblasts only. More recently, functional myocyte-fibroblast coupling (via connexin 45) has been demonstrated in situ for sinoatrial node pacemaker tissue, and preliminary immunohistochemical data suggest that myocyte-fibroblast coupling may be present in postinfarct scar tissue. The functional relevance of such heterogeneous coupling for cardiac electrophysiology is only starting to emerge and has thus far mainly been assessed in theoretical studies. According to this research, fibroblasts may affect the origin and spread of excitation in several ways above and beyond formation of "passive" barriers that obstruct electrical conduction. Thus, fibroblasts may act as current sinks, contributing to the formation of unidirectional block or to the delay in atrioventricular conduction. Via short-range interaction, fibroblasts may help to smooth out propagating wave fronts, in particular in the sinoatrial node and in the cross-sheet direction of healthy ventricular myocardium, 2 tissues that might otherwise be expected to show fragmented conduction patterns. As long-distance communication lines, fibroblasts may bridge posttransplantation or ischemic scar tissue, with beneficial or detrimental effects on organ function (depending on the relation to normal conduction patterns), and explain the recruitment of myocyte islands embedded in fibrotic scar tissue. The inherent mechanosensitivity of cardiac fibroblasts could, furthermore, allow them to play a sensory role and to affect cardiac electrophysiology via mechanoelectric feedback. This article reviews the currently available experimental and theoretical evidence on the previous scenarios, and highlights areas for further research.
Bollensdorff C, Bub G, Camelliti P, Picton G, Kohl P (2008) Measurements of sarcomere length in intact resting rat heart., Biophysical Journal 94
Camelliti P, Dudhia J, Dias P, Cartledge J, Connolly DJ, Terracciano CM Electrophysiological characterisation of the porcine right and left ventricle using myocardial slices., Cardiovascular Research 93 (Suppl.1)
Camelliti P, Fink M, Burton RA, Iribe G, Kohl P (2007) Evidence for co-localization of ryanodine receptors and microtubules in rat ventricular cardiomyocytes., Heart Rhythm 4 (5) pp. S154-55
Hammond SM, Yin HF, Saleh AF, Betts C, Camelliti P, Seow Y, Ashraf S, Arzumanov A, Merritt T, Gait MJ, Wood MJA (2011) Novel cell penetrating peptides for skeletal and cardiac muscle delivery of PMO antisense oligonucleotides for the treatment of duchenne muscular dystrophy., Nucleic acid therapeutics 21
Bub G, Camelliti P, Iribe G, Burton R, Bollensdorff C, Kohl P (2006) Guinea pig ventricular sarcomere length in vitro and in situ., Proc Physiol Soc 3
Iribe G, Ward CW, Camelliti P, Bollensdorff C, Mason F, Burton RAB, Garny A, Morphew MK, Hoenger A, Lederer WJ, Kohl P (2009) Axial stretch of rat single ventricular cardiomyocytes causes an acute and transient increase in Ca2+ spark rate, Circulation Research 104 (6) pp. 787-795
We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca2+ spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01±0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca2+ spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca2+ spark rate (to 130.7±6.4%) within 5 seconds, followed by a return to near background levels (to 104.4±5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 ¼mol/L GsMTx-4), perfusion with Na+/Ca2+-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca2+ spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 ¼mol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular- sarcoplasmic reticulum complex (to within H10-8m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca2+ spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation. © 2009 American Heart Association, Inc.
Alayoubi S, Pinto Ricardo C, Zaman J, Camelliti P, Yacoub M, Terracciano C (2014) ELECTROPHYSIOLOGICAL AND STRUCTURAL LEFT VENTRICLE REMODELLING
IN SPONTANEOUSLY HYPERTENSIVE RAT HEARTS: A MULTICELLULAR STUDY,
Biophysical Journal
Swietach P, Camelliti P, Hulikova A, Kohl P, Vaughan-Jones RD (2010) Spatial regulation of intracellular pH in multicellular strands of neonatal rat cardiomyocytes, Cardiovascular Research 85 (4) pp. 729-738
AimsIntracellular pH (pHi), an important modulator of cardiac function, is normally regulated to within narrow limits (7.1-7.2). In adult ventricular cell pairs, localized cellular pHi disturbances are removed by sarcolemmal acid/base transporters, but can also be dissipated (diluted) across gap junctions, aboard mobile buffers such as CO2/HCO3- and histidine-containing dipeptides (HCDPs). In the present work, we test this model of spatial pHi regulation in multicellular strands of neonatal rat ventricular myocytes.Methods and resultsWe confocally image pHi (intracellular fluorescence emitted from the pH dye carboxy-SNARF-1) in multicellular (>500 ¼m long,
Cartledge JE, Tesfom M, Dias P, Ibrahim M, Yacoub MH, Camelliti P, Terracciano CM (2012) Freshly isolated fibroblasts from normal hearts affect cardiomyocyte structure and function via paracrine communication, and these effects are altered in fibroblasts from pressure overloaded hearts., Circulation 126
Stevens J, Camelliti P, Kohl P (2007) Hemichannel remodelling in rat ventricular cardiomyocytes after cell isolation.,
Cartledge JE, Kane C, Dias P, Tesfom M, Clarke L, Mckee B, Al Ayoubi S, Chester A, Yacoub MH, Camelliti P, Terracciano CM (2015) Functional crosstalk between cardiac fibroblasts and adult cardiomyocytes by soluble mediators., Cardiovasc Res 105 (3) pp. 260-270
Crosstalk between cardiomyocytes and fibroblasts in physiological conditions and during disease remains poorly defined. Previous studies have shown that fibroblasts and myocytes interact via paracrine communication, but several experimental confounding factors, including the use of immature myocytes and the induction of alpha-smooth muscle actin (±-SMA) expression in fibroblasts by prolonged culture, have hindered our understanding of this phenomenon. We hypothesize that fibroblasts and myofibroblasts differentially affect cardiomyocytes viability, volume, and Ca(2+) handling via soluble mediators. More specifically here: (i) we compare the effects of freshly isolated fibroblasts and cultured fibroblasts from normal rat hearts on adult cardiomyocytes; (ii) we compare the effects of (freshly isolated) normal fibroblasts and myofibroblasts from pressure-overloaded hearts; and (iii) we study the contribution of TGF-² and the importance of the crosstalk between the two cell types.
Kane C, Cartledge J, Dias P, Camelliti P, Yacoub M, Terracciano C (2014) 17 Cardiomyocytes Influence Fibroblast Proliferation and ±-Smooth Muscle Actin Expression via the Secretion of Paracrine Mediators., Heart 100 Suppl 1 pp. A6-A7
Cardiac fibroblasts are known to modulate cardiomyocyte structure and function through soluble mediators. While less studied, the ability of cardiomyocytes to influence fibroblast properties, such as proliferation and activation, is likely to be of equal importance, as myocytes are known to produce a number of soluble factors to which fibroblasts are sensitive. Further, whether disease alters this myocyte-fibroblast cross talk is an important question to address.
Kane C, Hellen N, Trantidou T, Camelliti P, Terracciano C (2014) DIRECT CONTACT BETWEEN HUMAN CARDIAC FIBROBLASTS AND HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES COUNTERACTS CHANGES IN CALCIUM CYCLING INDUCED BY SOLUBLE MEDIATORS, Biophysical Journal
Iribe G, Ward CW, Camelliti P, Lederer WJ, Kohl P (2006) Acute increase of Ca2+ spark rate by axial stretch is mediated by cytoskeleton in intact rat ventricular myocytes., Heart Rhythm 5 (3)
Alayoubi S, Ibrahim M, Cartledge J, Yacoub MH, Terracciano CM, Camelliti P (2013) Electrophysiological Remodelling in Response to Chronic Mechanical Load Variations: a Multicellular Study, IUPS
Kohl P, Camelliti P (2012) Fibroblast-myocyte connections in the heart, Heart Rhythm 9 (3) pp. 461-464
Rao C, Prodromakis T, Kolker L, Chaudhry UAR, Trantidou T, Sridhar A, Weekes C, Camelliti P, Harding SE, Darzi A, Yacoub MH, Athanasiou T, Terracciano CM (2013) The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells, Biomaterials 34 (10) pp. 2399-2411
Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have been widely proposed as in vitro models of myocardial physiology and disease. A significant obstacle, however, is their immature phenotype. We hypothesised that Ca2+ cycling of iPSC-CM is influenced by culture conditions and can be manipulated to obtain a more mature cellular behaviour. To test this hypothesis we seeded iPSC-CM onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds fabricated using photolithography, or onto unstructured PDMS membrane. After two weeks in culture, the structure and function of iPSC-CM were studied. PDMS microgrooved culture substrates brought about cellular alignment (p
Carr CA, Stuckey DJ, Tan JJ, Tan SC, Gomes RS, Camelliti P, Messina E, Giacomello A, Ellison GM, Clarke K (2011) Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks--an MRI study., PLoS One 6 (10)
Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart.
Camelliti P, Mason F, Morphew MK, Hoenger A, Kohl P (2008) 3D reconstruction of microtubules, sarcoplasmic reticulum, and T-tubular membrane interrelation in rat ventricular myocytes using electron tomography., Proc Physiol Soc 11 pp. 82-83
Nisbet AM, Camelliti P, Walker NL, Burton FL, Cobbe SM, Kohl P, Smith GL (2016) Prolongation of atrio-ventricular node conduction in a rabbit model of ischaemic cardiomyopathy: Role of fibrosis and connexin remodelling., Journal of Molecular and Cellular Cardiology 94 pp. 54-64
Conduction abnormalities are frequently associated with cardiac disease, though the mechanisms underlying the commonly associated increases in PQ interval are not known. This study uses a chronic left ventricular (LV) apex myocardial infarction (MI) model in the rabbit to create significant left ventricular dysfunction (LVD) 8weeks post-MI. In vivo studies established that PQ interval increases by approximately 7ms (10%) with no significant change in average heart rate. Optical mapping of isolated Langendorff perfused rabbit hearts recapitulated this result; time to earliest activation of the LV was increased by 14ms (16%) in the LVD group. Intra-atrial and LV transmural conduction times were not altered in the LVD group. Isolated AVN preparations from the LVD group demonstrated a significantly longer conduction time (by approximately 20ms) between atrial and His electrograms than sham controls across a range of pacing cycle lengths. This difference was accompanied by increased effective refractory period and Wenckebach cycle length, suggesting significantly altered AVN electrophysiology post-MI. The AVN origin of abnormality was further highlighted by optical mapping of the isolated AVN. Immunohistochemistry of AVN preparations revealed increased fibrosis and gap junction proteins (connexin43 and 40) remodelling in the AVN of LVD animals compared to sham. A significant increase in myocyte-non-myocyte connexin co-localization was also observed after LVD. These changes may increase the electrotonic load experienced by AVN muscle cells and contribute to slowed conduction velocity within the AVN.
de Boer TP, Camelliti P, Ravens U, Kohl P (2009) Myocardial tissue slices: Organotypic pseudo-2D models for cardiac research & development, Future Cardiology 5 (5) pp. 425-430
Kane C, Dias P, Helen N, Trantidou T, Camelliti P, Gorelik J, Terracciano CM (2014) 267Direct contact between human cardiac fibroblasts and human induced pluripotent stem cell-derived cardiomyocytes counteracts changes in calcium cycling induced by soluble mediators., Cardiovasc Res 103 Suppl 1
Cardiac fibroblasts influence cardiomyocyte structure and function through direct physical interaction and/or by the secretion of soluble factors. A role for cardiac fibroblasts in cardiomyopathies has been proposed but clear mechanisms are still lacking. This in vitro study set out to characterise the influence of cardiac fibroblasts from patients with dilated cardiomyopathy on cardiomyocyte Ca2+ cycling, a fundamental mechanism of cardiac function universally altered in cardiac disease. Human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) were cultured with human DCM ventricular fibroblasts at a ratio of 2:1 (120,000 fibroblasts to 60,000 iPS-CMs) for 24 hours in three groups: iPS-CMs with fibroblast conditioned medium, co-cultured in transwells to allow bi-directional paracrine communication but prevent physical contact, and iPS-CMs in direct contact with fibroblasts. iPS-CMs alone were used as a baseline. iPS-CMs were field-stimulated at 1Hz and calcium transients were recorded optically. TGF-², a cytokine previously shown to be important in fibroblast-myocyte interaction, was measured in culture supernate using an ELISA assay. Versus iPS-CMs alone, Ca2+ transient amplitude was reduced in the conditioned medium group but increased in co-culture (p
Bussek A, Wettwer E, Lohmann H, Camelliti P, Ravens U (2009) Cardiac tissue slices from guinea-pig hearts as a suitable model for pharmacological and physiological investigations.,
Camelliti P, Green CR, LeGrice I, Kohl P (2004) Fibroblast network in rabbit sinoatrial node: structural and functional identification of homogeneous and heterogeneous cell coupling., Circ Res 94 (6) pp. 828-835
Cardiomyocytes form a conducting network that is assumed to be electrically isolated from nonmyocytes in vivo. In cell culture, however, cardiac fibroblasts can contribute to the spread of excitation via functional gap junctions with cardiomyocytes. To assess the ability of fibroblasts to form gap junctions in vivo, we combine in situ detection of connexins in rabbit sinoatrial node (a tissue that is particularly rich in fibroblasts) with identification of myocytes and fibroblasts using immunohistochemical labeling and confocal microscopy. We distinguish two spatially distinct fibroblast populations expressing different connexins: fibroblasts surrounded by other fibroblasts preferentially express connexin40, whereas fibroblasts that are intermingled with myocytes largely express connexin45. Functionality of homogeneous and heterogeneous cell coupling was investigated by dye transfer in sinoatrial node tissue explants. These studies reveal spread of Lucifer yellow, predominantly along extended threads of interconnected fibroblasts (probably via connexin40), and occasionally between neighboring fibroblasts and myocytes (probably via connexin45). Our findings show that cardiac fibroblasts form a coupled network of cells, which may be functionally linked to myocytes in rabbit SAN.
Camelliti P, Kohl P, Green C (2002) Gap junction coupling of cardiac fibroblasts in situ., Biophysical Journal 82 (1)
Camelliti P, Borg TK, Kohl P (2005) Structural and functional characterisation of cardiac fibroblasts., Cardiovasc Res 65 (1) pp. 40-51
Cardiac fibroblasts form one of the largest cell populations, in terms of cell numbers, in the heart. They contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, they are often disregarded by in vivo and in vitro studies into cardiac function. This review summarizes our understanding of fibroblast origin and identity, their structural organization and role in myocardial architecture, as well as functional aspects related to cell signalling and electro-mechanical function in the heart.
Kang C, Qiao Y, Li G, Baechle K, Camelliti Patrizia, Rentschler S, Efimov IR (2016) Human Organotypic Cultured Cardiac Slices: New Platform For High Throughput Preclinical Human Trials, Scientific Reports 6 28798 pp. 1-13 Nature Publishing Group
Translation of novel therapies from bench to bedside is hampered by profound disparities between animal and human genetics and physiology. The ability to test for efficacy and cardiotoxicity in a clinically relevant human model system would enable more rapid therapy development. We have developed a preclinical platform for validation of new therapies in human heart tissue using organotypic slices isolated from donor and endstage failing hearts. A major advantage of the slices when compared with human iPSderived cardiomyocytes is that native tissue architecture and extracellular matrix are preserved, thereby allowing investigation of multi-cellular physiology in normal or diseased myocardium. To validate this model, we used optical mapping of transmembrane potential and calcium transients. We found that normal human electrophysiology is preserved in slice preparations when compared with intact hearts, including slices obtained from the region of the sinus node. Physiology is maintained in slices during culture, enabling testing the acute and chronic effects of pharmacological, gene, cell, optogenetic, device, and other therapies. This methodology offers a powerful high-throughput platform for assessing the physiological response of the human heart to disease and novel putative therapies.
Johnson R, Camelliti P (2018) Role of Non-Myocyte Gap Junctions and Connexin
Hemichannels in Cardiovascular Health and Disease:
Novel Therapeutic Targets?,
International Journal of Molecular Sciences 19 (3) 866 MDPI
The heart is a complex organ composed of multiple cell types, including cardiomyocytes and
different non-myocyte populations, all working closely together to determine the hearts properties
and maintain normal cardiac function. Connexins are abundantly expressed proteins that form
plasma membrane hemichannels and gap junctions between cells. Gap junctions are intracellular
channels that allow for communication between cells, and in the heart they play a crucial role
in cardiac conduction by coupling adjacent cardiomyocytes. Connexins are expressed in both
cardiomyocytes and non-myocytes, including cardiac fibroblasts, endothelial cells, and macrophages.
Non-myocytes are the largest population of cells in the heart, and therefore it is important to consider
what roles connexins, hemichannels, and gap junctions play in these cell types. The aim of this
review is to provide insight into connexin-based signalling in non-myocytes during health and
disease, and highlight how targeting these proteins could lead to the development of novel therapies.
We conclude that connexins in non-myocytes contribute to arrhythmias and adverse ventricular
remodelling following myocardial infarction, and are associated with the initiation and development
of atherosclerosis. Therefore, therapeutic interventions targeting these connexins represent an exciting
new research avenue with great potential.
Wang Z, Stuckey D, Murdoch C, Camelliti P, Lip G, Griffin M (2018) Cardiac fibrosis can be attenuated by blocking the activity of transglutaminase 2 using a selective small-molecule inhibitor, Cell Death & Disease 9 613 pp. 1-12 Nature Publishing Group
Cardiac fibrosis is implicit in all forms of heart disease but there are no effective treatments. In this report, we investigate the role of the multi-functional enzyme Transglutaminase 2 (TG2) in cardiac fibrosis and assess its potential as a therapeutic target. Here we describe the use a highly selective TG2 small-molecule inhibitor to test the efficacy of TG2 inhibition as an anti-fibrotic therapy for heart failure employing two different in vivo models of cardiac fibrosis: Progressively induced interstitial cardiac fibrosis by pressure overload using angiotensin II infusion: Acutely induced focal cardiac fibrosis through myocardial infarction by ligation of the left anterior descending coronary artery (AMI model). In the AMI model, in vivo MRI showed that the TG2 inhibitor 1?155 significantly reduced infarct size by over 50% and reduced post-infarct remodelling at 20 days post insult. In both models, Sirius red staining for collagen deposition and levels of the TG2-mediated protein crosslink µ(³-glutamyl)lysine were significantly reduced. No cardiac rupture or obvious signs of toxicity were observed. To provide a molecular mechanism for TG2 involvement in cardiac fibrosis, we show that both TGF²1-induced transition of cardiofibroblasts into myofibroblast-like cells and TGF²1-induced EndMT, together with matrix deposition, can be attenuated by the TG2 selective inhibitor 1?155, suggesting a new role for TG2 in regulating TGF²1 signalling in addition to its role in latent TGF²1 activation. In conclusion, TG2 has a role in cardiac fibrosis through activation of myofibroblasts and matrix deposition. TG2 inhibition using a selective small-molecule inhibitor can attenuate cardiac fibrosis.
Wen Q., Gandhi K., Capel Rebecca A., Hao G., O'Shea C., Neagu G., Pearcey S., Pavlovic D., Terrar Derek A., Wu J., Faggian G., Camelliti Patrizia, Lei M. (2018) Transverse cardiac slicing and optical imaging for analysis of transmural gradients in membrane potential and Ca2+ transients in murine heart, The Journal of Physiology 596 (17) pp. 3951-3965 Wiley
Transmural and regional gradients in membrane potential and Ca2+ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra?thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (Vm) and intracellular Ca2+ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca2+ dye Rhod?2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short?axis slices (300 ¼m thick) were prepared from the entire ventricles (from the apex to the base) by using a high?precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady?state baseline and rapid pacing. Significant transmural gradients in Vm and CaT were observed in the left ventricle, with longer AP duration (APD50 and APD75) and CaT duration (CaTD50 and CaTD75) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico?basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD50, APD75 and CaTD50 in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT in murine ventricular tissue.
He S., Wen Q., O?Shea C., Mu-u-min R., Kou K., Grassam-Rowe A., Liu Y., Fan Z., Tan X., Ou X., Camelliti P., Pavlovic D., Lei M. (2019) A Protocol for Transverse Cardiac Slicing and Optical Mapping in Murine Heart, Frontiers in Physiology 10 Frontiers Media
Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (Vm) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure Vm and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in Vm and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or ²-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular Vm and CaT in a cell type specific manner.